Fight Aging!

Today's materials from the Intraclear Biologics team may be of interest to those following the development of senolytic therapies. Since the Mayo Clinic has yet to publish results from their clinical trials of fisetin as a senolytic therapy, and may not do so for a few years yet, it is good to see even preliminary data from other sources. Senolytic therapies selectively destroy senescent cells, though only one approach (the combination of dasatinib and quercetin) has been definitely shown to destroy significant numbers of senescent cells in humans. Data has yet to be published on whether fisetin performs as well in humans as it does in mice.

The Intraclear Biologics data is an example of the Mayo Clinic's senolytic dose of fisetin applied to a single younger patient with autoimmunity - in effect a well-conducted self-experiment. The age of the patient, mid-thirties, is far too young to have any meaningful age-related accumulation of senescent cells. But various lines of work from recent years suggest that many autoimmune conditions are at least in part driven by the presence of senescent cells, a bidirectional dysfunctional relationship between the immune system and senescent cells in the tissue under attack. Type 1 diabetes, for example, and perhaps rheumatoid arthritis. Unfortunately, no assessment of senescent cell burden was carried out in this study, so it is possible that other mechanisms are involved in the lowered inflammation and other benefits experienced by the patient. Still, the results point the way to larger studies that include more comprehensive assessments.

Preliminary results of trials of Fisetin in a person with autoimmune thyroiditis

Since senescent cells also arise in the immune system, being one of the causes of autoimmune diseases, there is a hypothesis that the destruction of senescent cells will help in the prevention and treatment of many autoimmune diseases. It is important that the mechanisms of cell senescence and the effects of their destruction by senolytics are similar in mice and humans. For example, it has been shown that the combination of dasatinib (a relatively aggressive chemotherapeutic) and quercetin (a flavonoid) works in humans as well as in mice when it comes to destruction of senescent cells.

Some drugs that have established senolytic effects are available for purchase just now. However, they are usually used in much lower dosages than is required for the senolytic effect. Such substances include the readily available and cheap bioactive flavonoid fisetin. Mice experiments show that fisetin is about as effective against senescent cells as the dasatinib + quercetin combination. The advantage of fisetin, which is a plant substance, is its safety compared to many other drugs that have shown a senolytic effect.

There are currently three trials of fisetin as a senolytic in humans. They are conducted in Mayo Clinic (USA), where a special treatment protocol was developed. Mayo Protocol consists of taking 20 mg/kg of fisetin orally for two days in a row, after which a person takes the second course after a month or two months. Because of the availability and safety of fisetin, we decided to conduct our own trial of this drug in a person with autoimmune thyroiditis. It is noteworthy that, unlike most tests, we focus not on chronic inflammation, but on immune function.

In accordance with animal experiments, the inflammatory factors C reactive protein and rheumatoid factor decreased. Antibodies to thyroglobulin did not change, which means the autoimmune response does not decrease. TSH returned to normal value, which allowed the patient to reduce the dose of hormone therapy.

In this commentary, scientists note the paucity of funding for chronic kidney disease research, given the widespread suffering and death caused by this presently incurable condition. This and many other areas of medicine are seen as solved problems by the powers that be simply because there is some form of treatment, even palliative treatment, in widespread use. That the treatment does little and many people die doesn't appear to motivate those who could fund progress. There is no sense of urgency and little sense of need. We might make the same comments in the case of atherosclerosis, a condition many consider to be adequately treated and under control, due to the existence of statins and similar drugs that lower LDL cholesterol in the bloodstream, despite the fact that these treatments only somewhat reduce mortality, and that atherosclerosis still kills 27% of our species at the present time. We could indeed make much the same argument for many other primarily age-related conditions.

The uptake of the current concept of chronic kidney disease (CKD) by the public, physicians and health authorities is low. Physicians still mix up CKD with chronic kidney insufficiency or kidney failure. In a recent manuscript, only 23% of participants in a cohort of persons with CKD had been diagnosed by their physicians as having CKD while 29% has a diagnosis of cancer and 82% had a diagnosis of hypertension. For the wider public and health authorities, CKD evokes kidney replacement therapy (KRT). In Spain, the prevalence of KRT is 0.13%.

A prevalent view is that for those in whom kidneys fail, the problem is "solved" by dialysis or kidney transplantation. However, the main burden of CKD is accelerated aging and all-cause and cardiovascular premature death. CKD is the most prevalent risk factor for lethal COVID-19 and the factor that most increases the risk of death in COVID-19, after old age. Moreover, men and women undergoing KRT still have an annual mortality which is 10-100-fold higher than similar age peers, and life expectancy is shortened by around 40 years for young persons on dialysis and by 15 years for young persons with a functioning kidney graft. CKD is expected to become the fifth global cause of death by 2040 and the second cause of death in Spain before the end of the century, a time when 1 in 4 Spaniards will have CKD.

However, by 2022, CKD will become the only top-15 global predicted cause of death that is not supported by a dedicated well-funded CIBER network research structure in Spain. Leading Spanish kidney researchers aim to prevent the dire predictions for the global 2040 burden of CKD from becoming true. However, only the highest level of research funding through the CIBER will allow to adequately address the issue before it is too late.

Link: https://doi.org/10.1016/j.nefro.2021.09.004

Heart rate variability is an increasingly popular measure of cardiovascular health. Heart rate variability is known to decline with age, but to what degree is this a reflection of processes of cardiovascular aging versus the loss of physical fitness that is a feature of old age in our present, overly sedentary societies? This remains an open question, and an important one, given the range of evidence for exercise programs, and thus increased physical fitness, to be as effective as many medical interventions when it comes to improving cardiovascular health and reducing mortality in later life. Yes, aging causes an inevitable decline in physical condition, but living a sedentary lifestyle certainly speeds up that process.

Fluctuation analysis in intervals between heartbeats provides important indices related to autonomic modulation of heart rate variability (HRV). These indices are considered predictors of morbidity and mortality as they are frequently altered in patients with chronic degenerative diseases, especially in those with cardiovascular and metabolic diseases. Similarly, a reduction in HRV is common with aging. In all cases, cardiovascular fitness is often reduced to below the predicted values.

In turn, increases in cardiovascular fitness through regular physical exercise, especially aerobic exercise, represent an important therapeutic tool capable of promoting positive adjustments in cardiac autonomic modulation. These adjustments are characterized by reduced sympathetic modulatory influence and/or increased vagal modulatory influence on the heart, increasing the HRV. Therefore, several methodological tools have been used to assess the degree of impairment of autonomic modulation and the therapeutic effects of physical exercise.

In summary, there is a close relationship between cardiovascular fitness and HRV. This relationship was more evident in patients with cardiovascular and metabolic diseases and in aging, especially in those whose cardiovascular fitness and HRV were below the predicted values for age and sex. The challenge is to develop techniques and interpretation of increasingly accurate data, as well as standardizing the application of these techniques.

Link: https://doi.org/10.2147/VHRM.S279322

Senescent cells accumulate with age throughout the body, and contribute directly to the onset and progression of a wide range of age-related conditions. While never present in large numbers in comparison to normal somatic cells, senescent cells are metabolically active, secreting signals that provoke chronic inflammation, altered cell behavior, and numerous forms of tissue dysfunction. Senolytic therapies selectively target senescent cells for destruction, most by forcing these errant cells into the programmed cell death process of apoptosis. Senescent cells are primed to self-destruct, and suppressing anti-apoptosis mechanisms for a time can push them over the edge while leaving normal cells largely unaffected.

In recent years, researchers have shown that many of the detrimental effects of excess visceral fat tissue are mediated by the presence of senescent cells in that fat tissue. This isn't just age-related: excess visceral fat generates senescent cells even in youth, but it does become worse with age. Thus eliminating senescent cells on a periodic basis via the use of senolytic therapies may allow for some decoupling of excess visceral fat from poor health and accelerated aging. One of the more prominent consequences of gaining too much fat tissue is the onset of type 2 diabetes, a condition that can be reversed even at a comparatively late stage by low-calorie diets leading to weight loss. Senescent cells appear to play an important role here, causing the death and dysfunction of islet cells in the pancreas, cells that are necessary for the correct function of insulin metabolism.

Researchers here demonstrate that eliminating senescent cells in fat tissue causes a sizable improvement in the manifestations of type 2 diabetes in mice. This isn't the first time that results of this sort have been produced by the scientific community, and the data here can be added to that from similar studies conducted in last few years. Since most of these studies used the readily available senolytic treatment of dasatinib and quercetin, presently in human trials for other conditions, it is perhaps surprising to see little movement towards off-label use in humans, given the considerable size of the diabetic patient community.

Deleting Dysfunctional Cells Alleviates Diabetes

The cells in your body are constantly renewing themselves, with older cells aging and dying as new ones are being born. But sometimes that process goes awry. Occasionally damaged cells linger. Called senescent cells, they hang around, acting as a bad influence on other cells nearby. Their bad influence changes how the neighboring cells handle sugars or proteins and so causes metabolic problems. Type 2 diabetes is the most common metabolic disease in the US. Most people with diabetes have insulin resistance, which is associated with obesity, lack of exercise, and poor diet. But it also has a lot to do with senescent cells in people's body fat, according to new findings. And clearing away those senescent cells seems to stop diabetic behavior in obese mice.

Alleviating the negative effects of fat on metabolism was a dramatic result, the researchers said. If a therapy worked that well in humans, it would be a game-changing treatment for diabetes. Researchers tested the efficacy of a combination of experimental drugs, dasatinib and quercetin. Dasatinib and quercetin had already been shown to extend lifespan and good health in aged mice. In this study, they found these drugs can kill senescent cells from cultures of human fat tissue. The tissue was donated by individuals with obesity who were known to have metabolic troubles. Without treatment, the human fat tissues induced metabolic problems in immune-deficient mice. After treatment with dasatinib and quercetin, the harmful effects of the fat tissue were almost eliminated.

Targeting p21Cip1 highly expressing cells in adipose tissue alleviates insulin resistance in obesity

Insulin resistance is a pathological state often associated with obesity, representing a major risk factor for type 2 diabetes. Limited mechanism-based strategies exist to alleviate insulin resistance. Here, using single-cell transcriptomics, we identify a small, critically important, but previously unexamined cell population, p21Cip1 highly expressing (p21high) cells, which accumulate in adipose tissue with obesity. By leveraging a p21-Cre mouse model, we demonstrate that intermittent clearance of p21high cells can both prevent and alleviate insulin resistance in obese mice.

Exclusive inactivation of the NF-κB pathway within p21high cells, without killing them, attenuates insulin resistance. Moreover, fat transplantation experiments establish that p21high cells within fat are sufficient to cause insulin resistance in vivo. Importantly, a senolytic cocktail, dasatinib plus quercetin, eliminates p21high cells in human fat ex vivo and mitigates insulin resistance following xenotransplantation into immunodeficient mice. Our findings lay the foundation for pursuing the targeting of p21high cells as a new therapy to alleviate insulin resistance.

Researchers have in the past proposed links between oral bacteria and chronic inflammation, particularly in the heart and brain, proposing that bacterial toxins and bacteria themselves enter the bloodstream via damaged gums. This undoubtedly happens, but supporting data is mixed when it comes to the question of whether or not this has a meaningful effect size in comparison to other inflammatory mechanisms and contributions to age-related disease. Here, a different route for bacteria is proposed: passage into the airways and lungs, a possibly explanation as why gum disease and respiratory mortality are correlated in older patients.

The global population is aging, and elderly people have a higher incidence of lower airway diseases owing to decline in swallowing function, airway ciliary motility, and overall immunity associated with aging. Furthermore, lower airway diseases in the elderly tend to have a high mortality rate. Their prevention is important for extending healthy life expectancy and improving the quality of life of each individual.

In recent years, the relationship between chronic periodontitis and oral bacteria, especially the periodontopathic ones, and respiratory diseases (e.g., pneumonia, chronic obstructive pulmonary disease, and influenza) has become clear. In addition, the association of several periodontal pathogens with the onset and aggravation of coronavirus disease 2019 (COVID-19) is also being reported. The oral cavity is the entry point for bacteria and viruses to enter the body, and it is also the entrance to the lower airway, including the bronchi and lungs, in which inflammation occurs during lower airway diseases. Therefore, if aspiration of oral bacteria has an adverse effect on lower airway diseases, it is not difficult to imagine such an effect.

In support of these findings, oral health management has shown to reduce deaths from pneumonia and prevent influenza in nursing homes and inpatient wards. This has led to clinical and multidisciplinary cooperation between physicians and dentists, among others. However, to date, the mechanisms by which chronic periodontitis and oral bacteria contribute to lower airway diseases have not been well understood. Clarifying these mechanisms will lead to a theoretical basis for answering the question, "Why is oral health management effective in preventing lower airway diseases?"

Link: https://doi.org/10.1016/j.jdsr.2021.10.003

Exercise in the form of strength training creates such broad changes in metabolism and produces such diverse benefits to health that it is interesting to see a specific example in which it doesn't help at all. That is possibly a path to better understanding which of the results of exercise are important, versus which are not, when it comes to functional improvement of specific tissues in later life. Many of the benefit of exercise are likely secondary effects produced by changes in skeletal muscle metabolism and myokine signaling on other parts of the body. Those effects will be largely absent in a study such as this, where only one small set of muscles are trained.

Exercise-based treatment approaches for dysphagia may improve swallow function in part by inducing adaptive changes to muscles involved in swallowing. We have previously shown that both aging and progressive resistance tongue exercise, in a rat model, can induce biological changes in the genioglossus (GG); a muscle that elevates and protrudes the tongue. However, the impacts of progressive resistance tongue exercise on the retrusive muscles (styloglossus, SG; hyoglossus, HG) of the tongue are unknown. The purpose of this study was to examine the impact of a progressive resistance tongue exercise regimen on the retrusive tongue musculature in the context of aging. Given that aging alters retrusive tongue muscles to more slowly contracting fiber types, we hypothesized that these biological changes may be mitigated by tongue exercise.

Hyoglossus (HG) and styloglossus (SG) muscles of male rats were assayed in age groups of young (9 months old, n = 24), middle-aged (24 months old, n = 23), and old (32 months old, n = 26), after receiving an 8-week period of either progressive resistance protrusive tongue exercise, or sham exercise conditions. Following exercise, HG and SG tongue muscle contractile properties were assessed in vivo. HG and SG muscles were then isolated and assayed to determine myosin heavy chain isoform (MyHC) composition.

Both retrusive tongue muscle contractile properties and MyHC profiles of the HG and SG muscles were significantly impacted by age, but were not significantly impacted by tongue exercise. Old rats had significantly longer retrusive tongue contraction times and longer decay times than young rats. Additionally, HG and SG muscles showed significant MyHC profile changes with age, in that old groups had slower MyHC profiles as compared to young groups. However, the exercise condition did not induce significant effects in any of the biological outcome measures.

Link: https://doi.org/10.3389/fphys.2021.740876

Genes determine species longevity, though within a species, and particularly within our species, the estimated involvement of genetic variants in individual life expectancy is becoming ever smaller as ever more data accumulates. Nonetheless, researchers are very interested in the comparative biology of aging, the question of why long-lived species are long-lived in comparison to their closest relatives. Which of the many evolved differences tend to produce a longer life span?

A longer species life span necessarily requires a lower incidence of cancer. Cancer is a numbers game: a larger body size means that there are more cells that can suffer mutation and become cancerous; a longer life allows more time for those cells to suffer mutation and become cancerous. Thus in larger and longer-lived species there must be mechanisms that either (a) lower the rate at which cancerous mutations can occur, or (b) increase the efficiency of cancer suppression mechanisms. These mechanisms are layered, ranging from those inside cells that provoke self-destruction when damage is identified, to the ability of the immune system to detect and destroy cancerous cells.

Elephants are both large and long-lived, and yet have a lower risk of cancer than is the case for our species. In recent years, researchers identified that elephants have many duplicated copies of the TP53 cancer suppression gene. The protein p53 produced from this gene is involved in DNA repair, as well as induction of cellular senescence and programmed cell death in response to DNA damage. It is thus an important part of cellular responses to potentially cancerous mutations. In today's open access paper, researchers report on their discovery of similar duplications in genes related to longevity and cancer suppression in long-lived Galapagos tortoises, indicating that this sort of evolutionary change is probably commonplace in longer-lived species.

It is interesting to consider that continually upregulating TP53 expression in short-lived mammals such as mice does improve cancer suppression, but also shortens life span, via mechanisms that likely include a reduction in stem cell activity and increase in the burden of cellular senescence. Too much vigilance has its costs. Researchers have worked around this issue, in mice at least, via forms of intermittent upregulation that only operate when TP53 is called upon, or via combining p53 upregulation with telomerase upregulation.

Concurrent evolution of anti-aging gene duplications and cellular phenotypes in long-lived turtles

A recurring theme in lifespan and aging regulation is the critical role played by processes that promote cellular protection and maintenance, including the ability of cells to recycle materials, repair damage, and remove waste. Senescent cells, whose numbers greatly increase with age, exhibit declines in these processes, and are also associated with pro-inflammatory phenotypes that are linked to age-related diseases. At the same time, apoptosis, which is the programmed destruction of unfit or damaged cells, is reduced in older individuals. This decline in cell performance in combination with a decreased ability to remove poor-performing cells is central to the aging process. Similarly, cancer can arise from cumulative genotoxic and cytotoxic stress, and apoptosis also plays a primary role in cancer resistance by removing potentially cancerous cells. Thus, if cancer-suppressing mechanisms are similar across species, then larger, longer-lived organisms should be at greater risk of cancer than smaller, shorter-lived ones. While this correlation exists within species, for example, cancer incidence increases with increasing adult height for most cancer types in humans and overall body mass in dogs, there is no such correlation between species - an observation often referred to as "Peto's paradox".

The molecular and cellular mechanisms underlying the evolution of large bodies and long lifespans have been explored in mammals such as elephants, whales, bats, and naked mole rats, but are less well studied in other vertebrates. Reptiles are an excellent system in which to study the evolution of body size and longevity because diverse lineages have repeatedly evolved large body sizes and long lifespans. Turtles, in particular, have lower rates of neoplasia than snakes and lizards, are especially long-lived, and are "slower aging" than other reptiles. Most notably, Galapagos giant tortoises (C. niger) and Aldabra giant tortoises (Aldabrachelys gigantea) can live over 150 years (3-5 times longer than their closest relatives) and weigh over 200 kg (50-100 times heavier than their closest relatives). Galapagos giant tortoises also appear to have evolved a suite of cellular traits that may contribute to their longevity, such as a slower rate of telomere shortening and extended cellular lifespans compared to mammals.

Here, we explore the evolution of body size and lifespan in turtles by integrating several approaches: (1) phylogenetic comparative analysis of body size, lifespan, and intrinsic cancer risk in turtles; (2) gene duplication analysis of aging and cancer-related genes across available turtle genomes; (3) cell-based assays of apoptosis and necrosis in multiple turtle species varying in body size and lifespan. We show that species with remarkably long lifespans, such as Galapagos giant tortoises, also evolved reduced cancer risk. We also confirm that the Galapagos giant and desert tortoise genomes encode numerous duplicated genes with tumor suppressor and anti-aging functions. Our comparative genomic analysis further suggests that cells from large, long-lived species may respond differently to cytotoxic stress, including endoplasmic reticulum (ER) stress and oxidative stress. The combined genomic and cellular results suggest that at least some turtle lineages evolved large bodies and long lifespans, in part, by increasing the copy number of tumor suppressors and other anti-aging genes and undergoing changes in cellular phenotypes associated with cellular stress.

Regeneration from injury is an intricate dance between stem cells, somatic cells, senescent cells, and immune cells. In particular, research into the biochemistry of species such the axolotl that can regenerate limbs and organs has identified the innate immune cells known as macrophages as essential to the process. Specific differences in macrophage behavior between more regenerative species such as the axolotl and less regenerative species such as our own are still under exploration. Here, researchers uncover a greater level of complexity in axolotl regeneration, in that only some macrophages are important to scar-free regeneration, and these cells originate in the liver rather than the bone marrow.

In 2013 it was discovered that a type of white blood cell called a macrophage is essential to limb regeneration in the axolotl. Without macrophages, which are part of the immune system, regeneration did not take place. Instead of regenerating a limb, the axolotl formed a scar at the site of the injury, which acted as a barrier to regeneration, just as it would in a mammal such as a mouse or human. Now, in a new study, researchers have identified the origin of pro-regenerative macrophages in the axolotl as the liver. By providing science with a place to look for pro-regenerative macrophages in humans - the liver, rather than the bone marrow, which is the source of most human macrophages - the finding paves the way for regenerative medicine therapies in humans.

Although the prospect of regrowing a human limb may be unrealistic in the short term due to its complexity, regenerative medicine therapies could potentially be employed in the shorter term in the treatment of the many diseases in which scarring plays a pathological role, including heart, lung and kidney disease. "If axolotls can regenerate by having a single cell type as their guardian, then maybe we can achieve scar-free healing in humans by populating our bodies with an equivalent guardian cell type, which would open up the opportunity for regeneration."

Although it remains to be seen if achieving scar-free healing in mammals will allow regeneration to proceed, researchers believe that may be the case. Because mammals already possess the machinery for regeneration - young mice can regenerate, as can human newborns - mammalian regeneration may simply be a matter of removing the barrier posed by scarring. "In axolotls, macrophages act as a brake on fibrosis, or scarring. Humans may possess macrophages that are doing their hardest to repair the damage, but are being held back. If we can engineer human macrophages to promote scar-free healing, we might be able to achieve a huge improvement in repair with just a little tweak. We have the luxury in the salamander of being able to work out which macrophage functions are essential to scar suppression and regeneration, gene by gene if we have to. If we can find out what that is, then maybe we can get that interaction happening in mammals."

Link: https://mdibl.org/press-release/mdi-biological-laboratory-scientist-advances-prospect-of-regeneration-in-humans/

There have been suggested that metformin, a at best weak calorie restriction mimetic, can suppress some of the beneficial metabolic response to exercise. Metformin is in general a poor choice in comparison to mTOR inhibitors when it comes to animal evidence for an ability to modestly slow the progression of aging. The primary human evidence for metformin to be useful, and why it attracted interest in the first place, comes from a large study of diabetic patients, and the gain in life expectancy was not large. Researchers here provide evidence against any suppression by metformin of beneficial mechanisms resulting from exercise, but I can't say that this does much to make metformin an attractive option. At the end of the day, small effect sizes are just not worth chasing, given the many other lines of research and development that offer greater promise.

Metformin and exercise both improve glycemic control, but in vitro studies have indicated that an interaction between metformin and exercise occurs in skeletal muscle, suggesting a blunting effect of metformin on exercise training adaptations. Two studies (a double-blind, parallel-group, randomized clinical trial conducted in 29 glucose-intolerant individuals and a double-blind, cross-over trial conducted in 15 healthy lean males) were included in this paper. In both studies, the effect of acute exercise with or without metformin treatment on different skeletal muscle variables, previously suggested to be involved in a pharmaco-physiological interaction between metformin and exercise, was assessed. Furthermore, in the parallel-group trial, the effect of 12 weeks of exercise training was assessed.

Skeletal muscle biopsies were obtained before and after acute exercise and 12 weeks of exercise training, and mitochondrial respiration, oxidative stress, and AMPK activation was determined. Metformin did not significantly affect the effects of acute exercise or exercise training on mitochondrial respiration, oxidative stress or AMPK activation, indicating that the response to acute exercise and exercise training adaptations in skeletal muscle is not affected by metformin treatment. Further studies are needed to investigate whether an interaction between metformin and exercise is present in other tissues, e.g. the gut.

Link: https://doi.org/10.1139/apnm-2021-0194

Most of the work aimed at treating aging as a medical condition is focused on stress response upregulation, finding ways to trigger some of the regulatory pathways and mechanisms involved in beneficial cellular reactions to the mild stresses of exercise, reduced calorie intake, hypoxia, heat, cold, and so forth. Improved cell behavior leads to improved tissue function, which in turn slows the progression of degenerative aging. Many of these pathways converge on autophagy, and evidence from the study of calorie restriction suggests that improved autophagy is the largest contributing factor.

Autophagy is the name given to a collection of cellular maintenance processes that remove unwanted or damaged structures and proteins, ensuring that they are broken down in a lysosome and the raw materials returned to the cell for reuse. Autophagy becomes less effective with age, and researchers have in recent years identified a range of age-related defects in the various component parts of the autophagic process. Tracing these issues back to root causes is, as for most of the changes observed in cells in aged tissues, quite challenging and very much a work in progress.

Stress response upregulation is much more effective at extending life span in short-lived species than it is in long-lived species. Calorie restriction can increase life span by 40% in mice, but certainly doesn't do that in humans. Stress response upregulation as a strategy for the development of therapies does not seem likely to greatly improve the healthy human life span to a much greater degree than is already possible via lifestyle changes. So it is disappointing that so much of present efforts are directed towards this part of the field. Results in mice should not be taken as indicative of the benefits that these same treatments might achieve in longer-lived species such as our own.

AMPK activator O304 improves metabolic and cardiac function, and exercise capacity in aged mice

Metabolic function, cardiac capacity, and vascular flexibility decline progressively with age, which in combination reduce work capacity, mobility, and quality of life. Type 2 diabetes (T2D) and cardiovascular diseases (CVDs) incidence increase with age and are current global epidemics representing major challenges to health care systems. Regular exercise not only increases work/exercise capacity but also counteracts the development of numerous age-related diseases, including several forms of metabolic and CVDs, thus promoting healthy aging. However, aged individuals are frequently unable to engage in regular exercise/physical activity. Thus, there is a large need to develop pharmacological treatments that can increase exercise/work capacity to counteract metabolic dysfunction and improve cardiac and vascular function and thereby promote healthy aging and increase quality of life in the aging population.

Insulin resistance and associated hyperinsulinemia are predictors of age-related diseases such as T2D and CVD. Increased activity of AMP-activated protein kinase (AMPK), a key energy sensor that is activated in response to low energy and glucose levels following exercise, enhances insulin-dependent and insulin-independent skeletal glucose uptake, thus improving glucose homeostasis and insulin resistance. AMPK activity declines however with age. Thus, AMPK has emerged as a potential important link between, and mediator of, numerous positive effects of exercise including protection against age-related diseases.

We previously showed that pan-AMPK activator O304 stimulates AMPK activity and glucose uptake in both skeletal muscle and heart of diet-induced obese (DIO) mice in vivo. In DIO mice, O304 mitigated hyperglycemia, hyperinsulinemia, insulin resistance, and obesity, and in a transgenic type 2 diabetic mouse model, O304 reversed established diabetes. O304 also significantly increased stroke volume, end-diastolic volume, and reduced heart rate in DIO mice, mimicking the cardiac effects of exercise. Thus, AMPK activator O304 efficiently ameliorated obesity-provoked insulin resistance, diabetes, and cardiovascular dysfunction in obese mice. O304 is currently in clinical development, and in a short proof-of-concept phase IIa clinical trial in T2D patients O304 reduced fasting plasma glucose levels and insulin resistance, i.e., HOMA-IR, and increased microvascular perfusion in the calf muscle and reduced blood pressure.

Here we show that the pan-AMPK activator O304, which is well tolerated in humans, prevented and reverted age-associated hyperinsulinemia and insulin resistance, and improved cardiac function and exercise capacity in aged mice. These results provide preclinical evidence that O304 mimics the beneficial effects of exercise. Thus, as an exercise mimetic in clinical development, AMPK activator O304 holds great potential to mitigate metabolic dysfunction, and to improve cardiac function and exercise capacity, and hence quality of life in aged individuals.

Researchers here mine patient data in order to demonstrate that type 2 diabetes combines with the age-related stiffening of blood vessels to produce greater structural damage in the brain, leading to a more rapid cognitive decline. This correlation between stiffening and damage was not observed in non-diabetic patients. This is an interesting result, as a reasonable view of the consequence of blood vessel stiffening with age is that it will produce increased blood pressure and consequence pressure damage to delicate tissues regardless of other factors. The authors conclude that the most likely explanation is that diabetes makes this process significantly worse, and thus more easily identified in patient data.

Cerebrovascular dysfunction has been proposed as a possible mechanism underlying cognitive impairment in the context of type 2 diabetes mellitus (DM). Although magnetic resonance imaging (MRI) evidence of cerebrovascular disease, such as white matter hyperintensities (WMH), is often observed in DM, the vascular dynamics underlying this pathology remain unclear. Thus, we assessed the independent and combined effects of DM status and different vascular hemodynamic measures (i.e., systolic, diastolic, and mean arterial blood pressure and pulse pressure index [PPi]) on WMH burden in cognitively unimpaired (CU) older adults and those with mild cognitive impairment (MCI).

559 older adults (mean age: 72.4 years) from the Alzheimer's Disease Neuroimaging Initiative were categorized into those with diabetes (DM+; CU = 43, MCI = 34) or without diabetes (DM-; CU = 279; MCI = 203). Participants underwent BP assessment, from which all vascular hemodynamic measures were derived. The presence of DM, but not PPi values, was independently associated with greater WMH burden overall after adjusting for covariates. Higher PPi values predicted greater WMH burden in the DM group only. No significant interactions were observed in the CU group.

Results indicate that higher PPi values are positively associated with WMH burden in diabetic older adults with MCI, but not their non-diabetic or CU counterparts. Our findings suggest that arterial stiffening and reduced vascular compliance may have a role in development of cerebrovascular pathology within the context of DM in individuals at risk for future cognitive decline. Given the specificity of these findings to MCI, future exploration of the sensitivity of earlier brain markers of vascular insufficiency (i.e., prior to macrostructural white matter changes) to the effects of DM and arterial stiffness/reduced vascular compliance in CU individuals is warranted.

Link: https://doi.org/10.3389/fnagi.2021.716638

Research of the past decade makes it clear that it is plausible and possible to alter the aged gut microbiome in ways that will reduce chronic inflammation and improve long-term health. This goal has been achieved in animal models via a range of different means, including flagellin immunization and fecal microbiota transplantation. In short-lived species, healthy life spans can be extended by restoring a more youthful gut microbiome, and in our own species, the detrimental changes that take place in gut microbiome populations are increasingly well catalogued. The next step has yet to be taken in earnest, however: to roll out human trials of the known ways to beneficially alter the gut microbiome.

Changes in the intestinal microflora with aging are related to the pathogenesis of age-related chronic diseases. Dietary intervention, exercise, and drug therapy are currently the most studied anti-aging measures and can improve the intestinal microbial imbalance caused by aging and promote a healthier intestinal environment to achieve anti-aging effects. In addition, gut microbiota modification represents a promising intervention for anti-aging and aging-related diseases, such as the use of probiotics, prebiotics, and synbiotics.

Studies suggest that modifying the gut microbiota of the elderly population by the intake of functional food as probiotics, prebiotics, or synbiotics may be an effective strategy to counteract natural aging. At the same time, these functional products may be suitable, affordable, and economical to most elderly people. However, their effects on health are complex, depending on individual populations and the duration of treatments. Evidence of probiotic, prebiotic and synbiotic use in elderly people is in its infancy compared with other measures.

Despite much research on these interventions, there are no firm conclusions about the benefits for human health. The reasons may be as follows: (1) Most of the relevant studies have been conducted in laboratory and animal models. These findings do not necessarily apply to humans directly. (2) Most clinical trials with humans are short-term and insufficient to understand long-term health effects. (3) Humans are quite different from each other in terms of sex, size, age, genetics, environment, lifestyle, and other factors. An anti-aging intervention that was found to help one person might not have the same effect on another. (4) Although many probiotics have proven strong safety profiles, we should still be careful to monitor their potential risks in different populations in the development of new probiotics.

Therefore, future research needs to focus on addressing these issues to better understand the safety and efficacy of these anti-aging measures in humans. In addition, although much hope and investment are currently focused on drug development, the application of anti-aging drugs in humans still has a long way to go. It is important to note that sensible habits may be more effective at extending healthspan than taking a medication. This means eating healthy foods, exercising, drinking alcohol in moderation or not at all, not smoking, getting adequate sleep, and maintaining an active lifestyle.

Link: https://doi.org/10.1080/19490976.2021.1994835

The portion of the medical research and development community that is focused on aging spends most of its time and funding on classes of treatment that cannot outperform good lifestyle choices when it comes to improving health and slowing degenerative aging. Why is this? If billions and decades are to be expended on building a pipeline from fundamental research through to clinical trials, why is the goal only an incremental benefit to health, smaller than that produced by regular exercise, intermittent fasting, or the practice of calorie restriction? Why such a lack of ambition, given the many possible projects that could achieve far more?

The small patient advocacy community focused on the treatment of aging as a medical condition spent long years convincing scientific and industry groups that it is both possible and desirable to extend the healthy human life span. The result of that work is, it appears, largely the initiation of projects that simply don't matter in the bigger picture, that won't meaningfully change the shape of later life, that won't greatly extend healthy human life spans.

Today's research materials are a reminder that the lion's share of effort and investment in the longevity industry is devoted to treatments and potential treatments that upregulate cellular stress responses, such as autophagy, to recreate a thin fraction of the natural metabolic outcomes of exercise, fasting, hypoxia, or calorie restriction. It remains the case that far too little attention is given to work that can in principle produce rejuvenation, by repairing the molecular damage that is the underlying cause of aging. Yes, senolytic therapies to clear senescent cells have made the leap, but senolytics see a fraction of the interest given to calorie restriction mimetics.

This is an important topic for continued patient advocacy. It is clearly not enough to convince the institutions of the world to work towards the treatment of aging; that is an important and ongoing battle, but it is only a first step. We must also advocate for a focus on the right sort of research programs, those that are in principle capable of producing sizable gains in health and life span, versus those that are not. If another two decades slip away with nothing to show for it but the clinical approval of varieties of mTOR inhibitor and other calorie restriction mimetic small molecule drugs, then a great opportunity to improve the human condition and save countless lives will have been squandered.

Diet trumps drugs for anti ageing and good metabolic health

A study comparing the impact of diet versus drugs on the inner workings of our cells has found nutrition has a much stronger impact. The pre-clinical study suggests the makeup of our diet could be more powerful than drugs in keeping conditions like diabetes, stroke, and heart disease at bay. Conducted in mice, the research showed nutrition (including overall calories and macronutrient balance) had a greater impact on ageing and metabolic health than three drugs commonly used to treat diabetes and slow down ageing: metformin, resveratrol, and rapamycin.

The research builds on the team's pioneering work in mice and humans demonstrating the protective role of diet and specific combinations of proteins, fats, and carbohydrates against ageing, obesity, heart disease, immune dysfunction, and risk of metabolic diseases, such as type 2 diabetes. Drugs can also target the same biochemical pathways as nutrients. There has been a huge effort to discover drugs aimed at improving metabolic health and ageing without requiring a change in diet. "We discovered dietary composition had a far more powerful effect than drugs, which largely dampened responses to diet rather than reshaped them."

Nutritional reprogramming of mouse liver proteome is dampened by metformin, resveratrol, and rapamycin

Nutrient sensing pathways influence metabolic health and aging, offering the possibility that diet might be used therapeutically, alone or with drugs targeting these pathways. We used the Geometric Framework for Nutrition to study interactive and comparative effects of diet and drugs on the hepatic proteome in mice across 40 dietary treatments differing in macronutrient ratios, energy density, and drug treatment (metformin, rapamycin, resveratrol). There was a strong negative correlation between dietary energy and the spliceosome and a strong positive correlation between dietary protein and mitochondria, generating oxidative stress at high protein intake. Metformin, rapamycin, and resveratrol had lesser effects than and dampened responses to diet. Rapamycin and metformin reduced mitochondrial responses to dietary protein while the effects of carbohydrates and fat were downregulated by resveratrol. Dietary composition has a powerful impact on the hepatic proteome, not just on metabolic pathways but fundamental processes such as mitochondrial function and RNA splicing.

There is mixed evidence for exercise to improve neurogenesis and cognitive function in very old mice. Researchers here suggest that this is because the duration of an exercise program matters greatly, and there is a comparatively narrow window of time in which the result is a net gain in function. This may be an example of the frailty of old age: mild stresses such as exercise that are robustly beneficial in younger individuals become more of a balancing act between cost and benefit in age-damaged individuals. The results are interesting, and will likely guide further explorations of the effects of exercise in very old human patients. That said, it isn't clear that the findings are in any way informative as to the dose-response curve for the effects of exercise on neurogenesis and cognitive function in old humans.

Hippocampal function is critical for spatial and contextual learning, and its decline with age contributes to cognitive impairment. Exercise can improve hippocampal function, however, the amount of exercise and mechanisms mediating improvement remain largely unknown. Here, we show exercise reverses learning deficits in aged (24 months) female mice but only when it occurs for a specific duration, 5 weeks, with longer or shorter periods proving ineffective.

While others have reported that a 5-week period of daily voluntary exercise results in the improvement of hippocampal cognitive function in aged mice, surprisingly, our results reveal that if we extended this period of exercise, there was abrogation of cognitive improvement. The discovery of a "sweet spot" of exercise duration critical for both neurogenic activation and improved spatial learning may help explain previous conflicting reports of the efficacy of exercise in improving cognitive improvement in aged mice.

A spike in the levels of growth hormone (GH) and a corresponding increase in neurogenesis during this sweet spot mediate this effect because blocking GH receptor with a competitive antagonist or depleting newborn neurons abrogates the exercise-induced cognitive improvement. Moreover, raising GH levels with GH-releasing hormone agonist improved cognition in nonrunners. We show that GH stimulates neural precursors directly, indicating the link between raised GH and neurogenesis is the basis for the substantially improved learning in aged animals.

Link: https://doi.org/10.1016/j.isci.2021.103275

Heterochronic parabiosis is the surgical linking of the circulatory systems of an old individual and young individual, usually mice. The older mouse shows signs of rejuvenation, while the younger mouse shows signs of accelerated aging. There is a robust ongoing process of debate and discovery regarding the mechanisms by which these effects are mediated. At present it appears that a dilution of harmful factors in old blood is likely to account for most of the outcome, but there is evidence for beneficial factors in young blood to be involved. In the study here, researchers show that epigenetic and transcriptomic measures of age are reduced in old mice following heterochronic parabiosis, and that this effect persists for at least a few months following the end of the intervention.

Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on the biological age of organisms and their long-term health remains unknown. Here, we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the biological age of blood and liver based on epigenetic analyses across several clock models on two independent platforms; remarkably, this rejuvenation effect persisted even after 2 months of detachment.

Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a comprehensive multi-omic rejuvenation effect. In addition, old HPB mice showed transcriptome changes opposite to aging, but akin to several lifespan-extending interventions. Altogether, we reveal that long-term HPB can decrease the biological age of mice, in part through long-lasting epigenetic and transcriptome remodeling, culminating in the extension of lifespan and healthspan.

Link: https://doi.org/10.1101/2021.11.11.468258

With advancing age, regulatory pathways involved in bone remodeling are activated inappropriately in smooth muscle cells of blood vessel walls and cardiac tissue. The result is calcification of tissue, making it inflexible, and disrupting the normal elasticity. That leads to hypertension and other, worse cardiovascular issues. Inflammatory signaling and the presence of senescent cells appear to be involved in causing this process, but firmly proven chains of cause and effect are yet to be established, as is the case for all too much of the panoply of dysfunctions that arise in the progression of degenerative aging.

Separately, the mechanisms of bone remodeling are disrupted in bone tissue itself, giving rise to an imbalance between deposition on the part of osteoblast cells and resorption on the part of osteoclast cells. The density and resilience of bone decreases over time, leading to osteoporosis. In today's review materials, researchers discuss the regulatory mechanisms involved in both osteoporosis and vascular calcification, noting that while incidence of these conditions are correlated with one another to some degree, it remains unclear as to whether one condition is driving the other, or whether they develop independently from shared root causes, such as the age-related increase in chronic inflammation.

Are vascular calcification and bone loss linked disorders of aging?

A recent review paper elucidates the numerous pathophysiological mechanisms shared by vascular calcification and bone loss, identifying the following key associations. Firstly, vascular calcification is an active process of calcium and phosphate precipitation that involves the transition of the vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Secondly, among the molecules involved in this process, parathyroid hormone (PTH) plays a key role, acting through several mechanisms which include the regulation of the RANK/RANKL/OPG system and the Wnt/ß-catenin pathway, the main pathways for bone resorption and bone formation, respectively. Thirdly, some microRNAs have been implicated as common regulators of bone metabolism, vascular calcification, left ventricle hypertrophy and myocardial fibrosis.

The increase or decrease in tissue and/or serum levels of PTH, the RANK/RANKL/OPG system and the Wnt/bcatenin pathways, calcium, phosphate, FGF23, among the most studied factors, may play a pathogenic role but can also be used as markers of bone and cardiovascular diseases. However, levels of some serum markers should be interpreted with caution, as the correlation between hormone levels and tissue levels needs to be better investigated.

Pathophysiology of Vascular Calcification and Bone Loss: Linked Disorders of Ageing?

This review shows that vascular calcification and bone loss that often coexist in ageing individuals, share numerous pathophysiological mechanisms. In this context, PTH, the RANK/RANKL/OPG system and the Wnt/ß-catenin pathway are the most studied factors. High PTH thus increases bone resorption and bone loss, but also triggers mechanisms that favour vascular calcification involving the RANK/RANKL/OPG and Wnt/ß-catenin pathways. Furthermore, other closely related factors such as calcium, phosphate, FGF23, Klotho, vitamin D and other regulatory factors that regulate PTH render these interactions extremely complex. The presence of low or high PTH levels, and consequently of low or high bone turnover, facilitate the process of deposition of hydroxyapatite in the wall of the vessels, leading to progression of vascular calcification when present for prolonged periods. The process eventually becomes severe, potentially increasing vascular molecular signals in order to reduce "bone deposition in the vessels", which in turn could favour the reduction of normal bone formation. Thus, in the presence of severe vascular calcification, a vicious circle may be established, further reducing bone mass.

The increase or decrease in tissue and/or serum levels of any these factors may play a pathogenic role in both bone loss and vascular calcification, and may be potentially promisingly used as a marker of bone and cardiovascular disease. However, caution should be exerted in the interpretation of these markers. For instance, whereas higher serum levels of sclerostin have been associated with vascular calcification and poor outcomes, this may not necessarily be due to a cause and effect relationship, but to a potential overproduction of sclerostin as a protective factor against vascular calcification. Similarly, serum sclerostin levels have been positively, and not negatively, associated with higher bone mass.

Although the pathogenesis and progression of vascular calcification and bone loss shares several common factors and pathways, it remains a "chicken-and-egg" situation, where it difficult to stablish cause and effect as to whether bone loss is driving vascular calcification or vice-versa, or whether there is a higher level of dysregulation generated by the ageing process that impacts on both tissues simultaneously, using common mechanisms.

Exercise delays the aging of the immune system, particularly changes in the distribution of T cell populations, but what are the underlying mechanisms? Researchers here note the existing focus in the scientific community on mitochondrial metabolism in T cells; exercise beneficially affects mitochondrial function, and energy metabolism is an important determinant of T cell behavior. The researchers suggest, however, that the existing evidence is more supportive of effects based on differences in metabolite production and consumption. It is an interesting viewpoint.

The effect of exercise on immune health has been widely studied in various population cohorts, however, exercise-research targeting CD4+ T cells is less represented. Nevertheless, exercise has been shown to modulate both the number and activity of CD4+ T cells, with an increased number of CD4+ T cells observed in the blood of athletes after training. Furthermore, physical fitness can modulate the concentration of immune cell subsets (VO2max exhibiting a large correlation with regulatory T cells (Treg) populations in the blood outside of training). Moreover, exercise can alter the balance of Th17/Tregs (increased Th17 and decreased Treg populations) improving chronic heart failure outcomes in a murine model. In reports that have identified CD4+ T cell subsets, the mechanisms driving these observations are not characterized, however, it is likely that metabolism plays an integral part.

Metabolic programs engaged by T cells directly affect their identity and function. Substrate utilization during exercise will vary depending on the exercise type, intensity, and duration. During low to moderate intensity exercise, the main substrates are glucose, glutamine, and fatty acids; with glucose becoming a more prominent fuel source as intensity increases. Considering the importance of mitochondria in cell metabolism, the effects of exercise in mitochondria have been studied extensively. Although it is expected that exercise-related adaptations mainly affect muscle mitochondria, exercise can influence surrounding cells via the availability of metabolites.

In summary, CD4+ T cells include a diverse population with highly varied function. Plasticity is exhibited between CD4+ T cell subsets and linked to numerous maladies such as development and exacerbation of autoimmune disorders and tumorigenesis. Exercise has been shown to effect CD4+ T cells, however, our understanding of the mechanisms surrounding these changes is limited. We propose that exercise could alter CD4+ T cell identity through metabolic responses to exercise, which in turn, affect the availability of metabolites and induce epigenetic remodeling events. To substantiate these claims, more research is needed to profile the epigenetic landscape of CD4+ T cells in response to exercise at the single cell level, identifying intermediate cell subsets, and deciphering the role of exercise on immune cell plasticity.

Link: https://doi.org/10.3389/fimmu.2021.729366

The yearly Longevity Week events in London are organized by Jim Mellon's network of allies, to promote the longevity industry and the concept of working towards therapies to treat aging as a medical condition. Sadly I could not attend this year, a combination of conflicting events in the US and it being too soon after conferences started up again post-COVID-19 to commit to international travel. Fortunately, one can still find a few notes on the proceedings online, and video of presentations will usually follow.

This week was "Longevity Week" with numerous events organised in the UK on what we now call "geroscience". Geroscience is predicated not only on the idea that human lifespan can be extended well beyond the biblical standard of three score years and ten, but also that we can avoid the degenerative diseases of old age which afflict so many seniors. So "healthspan" - the length of time that we can live without chronic and debilitating conditions such as diabetes, osteoarthritis, rheumatoid arthritis, macular degeneration, Parkinson's, or dementia - is just as important in this discussion as lifespan. That is an important starting point since, although life expectancy has increased significantly across the world within my own lifetime (from about 50 to 70), for many people those additional years are marred by ill health.

This November, a Master Investor event in central London brought together some of the leading scientists and entrepreneurs in this rapidly growing field. "Investing in the Age of Longevity" was the third event of this kind organised by Master Investor, inspired by our chairman Jim Mellon's passionate advocacy for geroscience. Indeed, it all started about five years ago when Jim took a road trip across the USA in a Honda Odyssey with the aim of meeting all the key players - academic scientists, biotech entrepreneurs, and visionary thinkers - who were formulating a body of ideas around the theme of longevity.

There is, as ever, a demographic and economic backdrop to these nascent medical technologies. In terms of the raw demographics, for the first time in the history of humanity there will be more old people (that's over-65s) than youngsters (under-16s) on the planet by 2050 - just 28 years away. The dependency ratio - that's the ratio of the number of people in work and paying tax to the number of people who are retired and receiving pensions - is in free fall in the developed world and is beginning to decline too in developing countries on account of declining fertility rates. These over-65s, to the extent that they get sick, are proving to be an increasing burden on state-funded and insurance-backed healthcare systems. The USA spends just over 17 percent of its GDP on healthcare today; but by 2040, based on current trends, the healthcare burden will double to 34 percent of GDP.

Just as the focus of medical science in the 19th and much of the 20th century was on the need to improve infant mortality and to cure the diseases of childhood, so the primary focus of medical science in the 21st century will be to prevent and cure the diseases of old age. Three leading healthcare economists have calculated that increasing the healthspan of humanity by just one year would be worth $38 trillion a year to the global economy. By increasing healthspans, lifespans will lengthen too.

Link: https://masterinvestor.co.uk/latest/longevity-science-is-alive-and-well/

Today's open access paper makes for an interesting companion piece to the recently proposed adaptive-hitchhike model for the evolution of longevity. Here also, the length of species life spans and the degree to which age-related degeneration (senescence) takes place over time is suggested to be a side-effect of adaptations to specific ecological niches. The authors of this paper observe that a greater proportion of marine clades have a long life span and lesser degrees of senescence than is the case for land-dwelling clades. Further, a greater fraction of marine species continually grow throughout life, a capability that has implications for the processes of regeneration and tissue maintenance. In addition, species that evolved a return to marine life, such as aquatic mammals, tend to be longer lived than their near relative species that remained land-dwelling.

The paper provides a great deal of data and sorting of that data, but is light on detailed conclusions. It is interesting to see the clear advantage in life span enjoyed, on average, by marine species. It is already the case that many in the research community look at the existence of multiple unusually long-lived species in specific niches, such as naked mole-rats and near relative species in their oxygen-poor underground environment, and hypothesize that longevity arises as a side-effect of the evolutionary adaptations required to thrive in that environment. The work here provides much more food for thought on this topic, to be taken up by molecular biologists in search of compelling mechanisms to explain the observed data on life span, senescence, and ecological niche.

Senescence as a trade-off between successful land colonisation and longevity: critical review and analysis of a hypothesis

Our analysis of the maximum life expectancy of species belonging to the groups of animals revealed distinct groups. The first group was insects, with a 99% share of short-lived species. The second group consisted of amphibians and terrestrial reptiles, with an average maximum life expectancy of about 10 years, though some animals, such as olm (Proteus anguinus), show extremely high life expectancy. The third group consisted of land mammals and some birds. The average life expectancy of animals in these groups is approximately 15 years. Only humans (Homo sapiens) and few other species can be classified as long-lived. The next group comprised of about 20% long-lived species that live for over 35 years. This group included fish with short-lived species, but also a large number of very long-lived species, such as Greenland shark (Somniosus microcephalus). High average maximum life expectancy was also observed in soaring birds. However, aquatic mammals were completely different from the other groups, with over 40% of them being long-lived.

Terrestrial habitats have been dominated mainly by animals that show a clear senescence phenotype. Since the emergence of insects, mammals, and birds occurred at different points of time, senescence must have evolved independently. Consequently, senescence in these groups of animals is likely to be the result of convergent evolution. Thus, we considered the original questions regarding why these three diverse terrestrial clades, show symptoms of senescence and whether senescence could be adaptive. The arguments presented herein suggest that the appearance of senescence in the three major groups of terrestrial animals was a consequence of the evolution of their life histories and as a side effect of the cessation of growth in sexually mature adults. The main mechanisms leading to the loss of this ability for growth were different across clades and occurred at distinct periods of time. The appearance of senescence in various unrelated clades during various periods of animal evolution suggests convergent evolution of senescence, and hence, a lack of homology.

Evidence that the loss of ability of indeterminate growth in terrestrial mammals results in senescence, is provided by secondarily aquatic mammals. In addition to lower rate of senescence, tetrapods that have undergone secondary aquatic adaptation include the longest-living mammals, such as the bowhead whale (Balaena mysticetus), all of which live for over a hundred years. Researchers have examined reproductive materials from mature female bowheads but did not see positive evidence of senescence. Similarly, the maximum and average lifespan of aquatic and semi-aquatic reptiles, which also secondarily returned to the water, exceed those of their terrestrial relatives.

Our analysis suggests that senescence may have emerged as a side effect of the evolution of adaptive features that allowed the colonisation of land. Perhaps specialisation and adaptation of animals to life on land was accompanied by senescent phenotypes, as a side effect of evolution. Thus, senescence in mammals (including humans) may be a trade-off compromise between land colonisation and longevity. In our relatively short synthesis, we presented adaptations that involve animals best suited to life in terrestrial environments. We emphasised that senescence occurred in parallel with highly adaptive traits. Examples of secondary aquatic mammals indicate that it is evolutionarily possible to delay the onset of senescence symptoms. The aquatic environment, which offers conditions that allow animals to grow larger, due to greater density of fluid medium (water) and facilitates the maintenance of appropriate body temperatures, seems to favour negligible senescent phenotypes or an extremely delayed appearance of the signs of senescence, shortly before the death of the individual (as seen in fish, octopus, and whales). Similarly, the functioning of soaring birds seems to be less costly in terms of energy compared to their relatives with passerine-type flights. Hence, we may conclude that an energy-efficient life in a stable environment can delay the symptoms of senescence and promote a longer life.

A common factor in calorie restriction, intermittent fasting, and time-restricted feeding is time spent in a state of hunger. More time spent hungry may be better, up to a point. The signaling associated with hunger upregulates cellular maintenance processes such as autophagy, known to improve health over the long term. While the biochemistry is under ever greater exploration, there is still a lot of work to be accomplished in order to map the dose-response curve for calorie intake versus time spent hungry, as well as how that dose-response curve may differ in different mammalian species. One might look at recent discussions regarding the structure of studies in mice to see that assessments are not as straightforward a matter as might be imagined: mice in studies of calorie restriction are fed once a day. How much of the resulting health benefits are due to this time-restricted feeding versus reduced overall calorie intake?

A variety of diets have been studied for possible anti-aging effects. In particular, studies of isocaloric time-restricted feeding in laboratory rodents have found evidence of beneficial health outcomes. Companion dogs represent a unique opportunity to study diet in a large mammal that shares human environments. The Dog Aging Project has been collecting data on thousands of companion dogs of all different ages, sizes, and breeds since 2019.

We leveraged this diverse cross-sectional dataset to investigate associations between feeding frequency and cognitive function (n = 10,474) as well as nine broad categories of health outcomes (n = 24,238). Controlling for sex, age, breed, and other potential confounders, we found that dogs fed once daily rather than more frequently had lower mean scores on a cognitive dysfunction scale, and lower odds of having gastrointestinal, dental, orthopedic, kidney/urinary, and liver/pancreas disorders. Therefore, our findings suggest that once-a-day feeding in dogs is associated with improved health across multiple body systems.

Link: https://doi.org/10.1101/2021.11.08.467616

Live Longer World's Aastha Jain maintains an interesting selection of podcasts and video interviews with various people in and around the longevity industry and the related scientific community focused on interventions in the aging process. That includes an interview with me, from a couple of months ago, should you find yourself interested in a random walk through some of my views on the state of the field.

Live Longer World provides you information on the science and practical mechanisms on how we can slow and reverse aging. For those baffled by me calling aging a disease, the easiest way to think of aging is damage accumulation happening in our bodies. Over time, this damage accumulates, wears us down, leads to onset of diseases, aging, and death. But what if this damage could be reversed? And science is showing that it is indeed possible. We can slow the accumulation of damage and also reverse it, and Live Longer World provides you information on how both are possible through different communication platforms and methods.

The newsletter is focused mostly on science-backed lifestyle methods to slow aging, optimize health, biohack, improve healthspan, and feel more energetic. Simple lifestyle practices can go a long way in maintaining optimal health and a robust immune system. If you care to know best science-backed tools to optimize health, sign up for the newsletter.

The science of aging is moving rapidly and making progress at defeating age-related diseases and aging itself. Live Longer World Podcast takes you right where the action is happening! Through conversations with scientists, entrepreneurs, investors and other advocates revolutionizing the field of longevity science, we tell you how you can be disease-free, reverse aging, and maximize longevity in the future. Further, the YouTube channel covers the video versions of the podcast - discussions with scientists, entrepreneurs, investors and other advocates moving the science and technology of longevity forward.

Link: https://www.livelongerworld.com/

Today's open access editorial discusses one quite specific consequence of the age-related disruption of energy metabolism in cells. Mitochondria, the power plants of the cell, falter with age throughout the body, for complex reasons still under exploration. The proximate causes involve too little production of molecular machinery needed for the correct operation of the electron transport chain, or mitochondrial dynamics, in the first case leading to inefficient production of ATP and raised levels of reactive oxygen species, and in the second case leading to failure of quality control mechanisms intended to remove worn and damaged mitochondria.

How these changes connect to the root causes of aging is yet to be established. It is possible that the recently discovered connection between characteristic age-related epigenetic changes, leading to changes in protein production, and cycles of double strand break repair will turn out to be important. Equally, most of the other deeper causes of aging are suspects. It is very challenging to pick through the complex web of interactions to reliably point blame at any one specific mechanism of aging.

Dysfunctional energy metabolism has many consequences, however. It is particularly implicated in age-related diseases of the brain, an energy-hungry tissue. The research materials here are interesting for the demonstration that myeloid cells of the innate immune system are an important part of the problem. A weight of evidence is growing over time for the importance of the immune system and inflammatory signaling in the development of neurodegenerative conditions. Connecting that to the known importance of energy metabolism in neurodegeneration is somewhat novel, however.

Myeloid Metabolism as a New Target for Rejuvenation?

While cognitive decline is clearly underlined by synaptic and neuronal dysfunction, other players are emerging as critical elements in the equation of brain ageing. Inflammation seems to be one of those, with pro-inflammatory factors being associated with poor cognitive performance, pointing to immune cells as important regulators in this process. The immune system is drastically affected with ageing. While the adaptive immune response comprising B-cells and T-cells is diminished, the innate immune system (i.e., cells of the myeloid lineage) shows an increase in the pro-inflammatory state, also known as "inflammaging". This chronic low-inflammatory state is mainly driven by macrophages and pro-inflammatory cytokines.

Cellular metabolism has emerged as a key player in the regulation of immune function, starting already at the level of myeloid versus lymphoid lineage decision and greatly affecting cellular behaviour in the mature immune cells. Several recent studies have suggested that an altered cellular metabolism in aged macrophages might directly contribute to the pro-inflammatory signature. However, the detailed mechanisms initiating this increased inflammation with aging remain unclear.

In a recent publication, researchers have elucidated this cascade using an impressive set of in vitro and in vivo experiments in mice and in human myeloid cells. They found that aged myeloid cells have a decrease in cellular respiration and a decrease in glycolysis, suggesting that aged myeloid cells undergo a general bioenergetic failure. The proposed driving cause is the increased prostaglandin E2 (PGE2) signaling in the ageing myeloid compartment, mediated by the age-dependent upregulation of EP2, one of the four PGE2 receptors.

Conditional knockout of EP2, specifically in the myeloid cells of aged mice proves to be indeed an effective strategy at multiple levels. First, it rescues the expression of some of the immune factors upregulated with age, both in the plasma and in the hippocampus. Second, the loss of EP2 also reduces glycogen levels, normalizing the metabolic state and the associated mitochondrial defects observed in old macrophages. Surprisingly, restoring the PGE2 signaling in myeloid cells to a youthful state is enough to prevent age-dependent cognitive decline.

Overall, this data supports an upstream role of peripheral myeloid cells in orchestrating the process of brain ageing, underscoring the important cross-talk between the immune and the central nervous systems. This study nicely illustrates the importance of the cellular metabolic state of myeloid cells: it highlights that not only the availability of glucose, but also its channeling into different pathways (glycolysis versus glycogen synthesis) contributes to maintaining proper myeloid function.

At first glance, with only a few exceptions in the initial membership, the newly launched Longevity Biotech Association is an advocacy group for the small molecule, stress response upregulation faction within the development community focused on the treatment of aging as a medical condition. More advocacy for the concept and feasibility of treating aging is certainly a good thing, and it is welcome to see the arrival of new high-profile initiatives. A cautionary thought is that the only real merit of the small molecule stress response upregulation approach, mimicking thin slices of the beneficial metabolic response to exercise, calorie restriction, heat, cold, or hypoxia, is that it will be easier to achieve regulatory approval. The outcomes are unlikely to prove better for long-term health than actually undertaking more exercise or adopting some form of restricted calorie intake. If spending decades and billions on medical development, shouldn't be we be aiming higher? Implementing other approaches that are capable in principle of rejuvenation and sizable extension of life span, such as those laid out in the SENS research programs?

Leaders from across the longevity sector came together in London today to announce the formation of the Longevity Biotechnology Association (LBA). The non-profit organisation says it aims to represent those behind the development of "new medicines and therapies to prevent and cure, rather than merely manage, the health conditions of late life." Three of the LBA's founding members announced the launch at today's Investing in the Age of Longevity event at Longevity Week, including Juvenescence co-founder and chairman Jim Mellon, Cambrian Biopharma founder James Peyer, and Mehmood Khan, CEO of Hevolution Foundation.

On the academic side, LBA founding members include Harvard's David Sinclair, Nir Barzilai from the Albert Einstein College of Medicine, and Brian Kennedy from the National University of Singapore. In addition to Peyer, biotech company CEOs include BioAge's Kristen Fortney, Joe Betts-Lacroix of Retro Biosciences, and Matthias Steger of Rejuveron Life Sciences. Beyond Mellon and Kahn, investors are also represented by The Longevity Fund's Laura Deming, Nils Regge of Apollo Health Ventures, Michael Greve of the Forever Healthy Foundation, and Longevity Vision Fund's Sergey Young.

"I think the LBA will be a powerful and necessary force for the coming years… The ambition of the industry over the next 10 years is to completely upend the conception of aging that most people have… and the bottom line is, the industry has come of age." The LBA has several main objectives, the first of which is to "educate governments, the media, the public and the medical field about the promise of emerging therapies with the potential to treat or prevent multiple age-related conditions at once." Other goals include supporting newcomers to the industry and to help foster the creation of industrywide best practices.

Link: https://www.longevity.technology/a-gathering-of-the-longevity-clans/

Rockfish species vary widely in lifespan. Some even exhibit negligible senescence, showing few signs of aging across the majority of their long life spans. When closely related species have divergent life spans, there is perhaps the opportunity to learn something of how metabolism determines longevity. Accordingly, researchers here report on their study of varied rockfish species, in search of the differences in the molecular biology of cells that lead to differences in life span and pace of aging.

In a new study, researchers compare the genomes of nearly two-thirds of the known species of rockfish that inhabit coastal waters around the Pacific Ocean and uncover some of the genetic differences that underlie their widely varying lifespans. Some rockfish, like the colorful calico rockfish (Sebastes dallii), live for little more than a decade, while the most long-lived of the genus Sebastes - the rougheye rockfish (Sebastes aleutianus), which can be found from Japan to the Aleutian Islands - can hang out on the seabed in cold, deep coastal waters for more than 200 years.

Their wide range of lifespans, not to mention differences in size, lifestyle, and ecological niche evolved over a mere 10 million years - one of the most rapid radiations among all fishes. To uncover the genetic determinants of lifespan in rockfish, the researchers obtained tissue samples from 88 species and sequenced their complete genomes. The researchers looked for DNA variations that were more common in fish with longer lives and found 137 longevity-associated gene variations. Not all of these have a direct effect on lifespan, however. The researchers took care to separate out the genetic variations that allowed rockfish to adapt to deeper depths and grow to larger size, since those adaptations themselves have the side-effect of increasing lifespan. Deeper, cooler waters slow metabolism, for example, which is associated with a longer lifespan in many animals.

"We can explain 60% of the variation in lifespan just by looking at the size at maturity and the depth at which a fish lives. So, you can predict lifespan with pretty high accuracy just from these factors. This allowed us to identify the genes that allow them to do those things." The remainder of the longevity-associated variation primarily involved three types of genes: an enrichment in the number of genes for repairing DNA; variations in many genes that regulate insulin, which has long been known to influence lifespan; and an enrichment for genes that modulate the immune system. More DNA repair genes could help protect against cancer, while more immune genes could help ward off infections, as well as cancer.

Link: https://news.berkeley.edu/2021/11/11/pacific-rockfish-and-the-trade-offs-of-a-longer-life/

First generation stem cell therapies have been demonstrated to reduce chronic inflammation, but are unreliable when it comes to producing other benefits. Since very few transplanted cells survive, it was thought that benefits are produced via signaling that changes the behavior of native cells. This has led to work on therapies that deliver extracellular vesicles released by stem cells rather than the cells themselves. That such therapies produce benefits in similar ways to stem cell therapies in animal models suggests that signaling does play an important role.

In some cases, however, transplanted cells die rapidly, too rapidly for signaling to be a plausible mechanism for the resulting suppression of inflammation. Here, researchers provide evidence to show that the death and later clearance of the remains of these transplanted cells is the process by which inflammation is reduced. More research is needed to better understand how exactly this works, perhaps leading to a way to directly manipulate native cells to reproduce the changes in regulation of inflammation that result from the death of transplanted cells.

Mesenchymal stromal cell apoptosis is required for their therapeutic function

Multipotent mesenchymal stromal cells (MSCs) are a heterogeneous population of cells isolated from bone marrow and other tissue stroma that have immunosuppressive and anti-inflammatory properties. In many animal models of disease, MSCs have demonstrated therapeutic efficacy regardless of major histocompatibility complex or species barriers. It remains unclear how MSCs isolated from different tissues or species could exert therapeutic effects on such a wide range of unrelated diseases.

The current consensus is that therapeutic applications of MSCs are based on their secretion of a wide array of cytokines, chemokines, and subcellular particles5. However, MSCs do not engraft and there is little evidence that these cells even survive infusion or injection. Studies tracking MSCs after intravenous administration reported lung entrapment, upregulation of apoptosis-associated genes and presence of apoptotic bodies in the lungs. Only dead MSCs were detected in the lungs and liver 24 hours after administration. In a graft versus host disease study, it was demonstrated that only patients whose immune cells were able to induce apoptosis in MSCs responded to MSC therapy, suggesting that MSC apoptosis may contribute to clinical response.

In the current study, we generated apoptosis-refractory human MSCs to test whether inhibiting cell death in MSCs would abrogate their therapeutic efficacy, thereby establishing that apoptosis of MSCs is necessary for the immunomodulatory effects exerted by their infusion. Our data demonstrated that MSC apoptosis and subsequent efferocytosis are required for their full immunosuppressive effects in vivo, answering the long-standing question of how MSCs mediate therapeutic effects that persist beyond their survival.

Mechanistically, apoptosis of MSCs and their efferocytosis induced changes in metabolic and inflammatory pathways in alveolar macrophages to effect immunosuppression and reduce disease severity. Our data reveal a mode of action whereby the host response to dying MSCs is key to their therapeutic effects; findings that have broad implications for the effective translation of cell-based therapies.

For reasons yet to be determined, the composition of the skin microbiome changes with age. It is also unclear as to whether these changes contribute to skin aging in any meaningful way, or are instead a consequence of skin aging. Researchers here perform a proof of concept study to identify bacterial activities that differ in the microbiome of old versus young skin. There is little to be learned from these initial results, but in principle a much more comprehensive set of data might point the way towards specific experiments that could be conducted, changing the balance of populations in the skin microbiome in measured ways to see if the function of aged skin improves as a result.

The human skin is inhabited by a large number and variety of microorganisms, including bacteria, fungi, and viruses. Although over- and underrepresented bacterial taxa can be readily associated with younger or older skin, the limiting step and challenge in the analysis of these data is the interpretation of biological relevance in context of the research question or hypothesis. Specifically, little insight is currently available in literature on the interplay between microbial functionalities and human cellular processes (referred to as co-metabolism).

To obtain more insight into the connection between the skin microbiome and the human physiological processes involved in skin aging, we performed a systematic study on interconnected pathways of human and bacterial metabolic processes that are known to play a role in skin aging. The bacterial genes in these pathways were subsequently used to create Hidden Markov Models (HMMs), which were applied to screen for presence of defined functionalities in both genomic and metagenomic datasets of skin-associated bacteria. These models were further applied on 16S rRNA gene sequencing data from skin microbiota samples derived from female volunteers of two different age groups: 25-28 years ('young') and 59-68 years ('old').

The results show that the main bacterial pathways associated with aging skin are those involved in the production of pigmentation intermediates, fatty acids, and ceramides. This study furthermore provides evidence for a relation between skin aging and bacterial enzymes involved in protein glycation. Taken together, the results and insights described in this paper provide new leads for intervening with bacterial processes that are associated with aging of human skin.

Link: https://doi.org/10.1371/journal.pone.0258960

UNITY Biotechnology recently reported positive results for their senolytic drug candidate UBX1325, probably derived from navitoclax. Senolytic therapies selectively destroy senescent cells in aged tissues, reducing their inflammatory signaling and contribution to tissue dysfunction. In this case, macular degeneration patients showed improved visual function after treatment, though one should wait for a larger study group before calling this an unqualified success. UNITY Biotechnology's trials are essentially testing the thesis that localized removal of senescent cells can address pathology, and thus low, localized doses of chemotherapeutic-derived senolytic drugs can be used, minimizing side-effects. In this case, the senolytic is injected into the eye.

An earlier trial of localized senolytic administration to arthritic knee joints failed to show meaningful benefits in patients, which led to some discussion in the community over whether or not localized delivery of senolytics could ever work. Is the contribution of inflammatory senescent cell signaling elsewhere in the body sizable enough to continue to cause issues after the local population is removed? UNITY Biotechnology will clearly be continuing to a larger study for macular degeneration, so we shall see if the results continue to look promising.

UNITY Biotechnology, Inc. ("UNITY"), a biotechnology company developing therapeutics to slow, halt, or reverse diseases of aging, today announced 24-week data from its Phase 1 single ascending dose (SAD) safety study of UBX1325 in patients with advanced disease from diabetic macular edema (DME) or wet age-related macular degeneration (AMD). A majority of patients with DME across all doses had rapid improvements in vision, and patients in the higher dose cohorts showed a mean gain of 9.5 ETDRS letters in best-corrected visual acuity (BCVA) at 24 weeks following a single injection of UBX1325. Similarly, a majority of wet AMD patients treated with UBX1325 showed rapid gains in visual acuity, which were maintained through 12 weeks. In most patients, central subfield thickness (CST) remained stable through the study period.

The study enrolled a total of 19 patients with advanced DME (n=8) and wet AMD (n=11) for whom anti-VEGF therapy was no longer considered beneficial. UBX1325 was well-tolerated at all doses tested (through 10 mcg) with no dose-limiting toxicities and no reported incidence of inflammation. The Phase 1 data show rapid improvements in visual acuity as measured by BCVA in patients with DME, with the majority of patients demonstrating sustained responses through 24 weeks.

"A 10 letter gain in DME patients, maintained through six months, is an impressive outcome, and is particularly noteworthy considering that it was achieved with a single injection. Hard-to-treat patients require as many as 10 injections in the first year of treatment to see full benefits from currently available anti-VEGF therapies. A treatment that reduces the frequency of injections while showing meaningful and sustained improvements in BCVA would be of huge value for patients and physicians."

Link: https://ir.unitybiotechnology.com/news-releases/news-release-details/unity-biotechnology-announces-improvement-visual-acuity

Bioviva was at one point developing telomerase gene therapies, work that has transitioned into the medical tourism industry via Integrated Health Systems rather than proceeding towards regulatory approval. The institutional communities of science and funding strongly disapproved of the self-experiment undertaken by the Bioviva founder, and the way that self-experiment was popularized in order to build the company. I think this a pity, given the long history of self-experimentation by noted figures in the scientific community. Nonetheless, we live in an era that frowns upon self-experimentation as a part of the path to progress, and applies very high standards to those who attempt it.

The gene therapies that Bioviva worked upon, and Integrated Health Systems now sells to well-to-do medical tourists, involve localized injection of small amounts of AAV vectors to upregulate expression of telomerase. The alternative option of intravenous injection of large amounts of AAV, in order to reach much of the body in adults, is coming to be looked upon with disfavor in the investment, regulatory, and development communities. This is based on liver toxicity and a few patient deaths at the high doses needed for that mode of administration. Localized injections can use a small fraction of the intravenous injection amount, avoiding issues of toxicity, but are unfortunately still a one-time treatment at present: the immune system will remember the AAV vector used, and clear it next time. Some groups such as Selecta Biosciences are working on ways to enable repeat dosing, but this is still a work in progress.

Today's paper is, I think, one of the first more formal reports from the Bioviva / Integrated Health Systems collective. A small number of Alzheimer's patients underwent localized delivery of an AAV gene therapy to the brain, upregulating telomerase and klotho. The paper is light on details regarding specifics of the treatment, and there was no control group. I am told via other channels that trial costs were sponsored by a non-profit, and that the patients are representative of the general Alzheimer's population. Still, all that can really be determined from the outcome is that there were no meaningful side-effects. One can compare the patient's cognitive performance with the age-matched Alzheimer's population as a whole, and see that it is better, but the study size is small, and people who have the will, connections, and support to get into a trial of this nature are likely obtaining significantly better care than the average patient.

It is worth noting that a few other companies are developing treatments based on klotho upregulation or telomerase gene therapies within the established regulatory system. There is evidence from animal studies for both approaches to be beneficial in a range of age-related conditions. These are not unreasonable approaches to compensatory therapies that may help patients with neurodegenerative conditions, albeit without addressing the underlying causes of neurodegeneration, and thus having a necessarily limited upside.

Safety Study of AAV hTert and Klotho Gene Transfer Therapy for Dementia

Five patients participated in the study and were classified as having mild or moderate dementia. All patients performed witnessed informed consent and provided information from their primary doctor including diagnosis, brain images, and bloodwork. All accepted to participate in genetic testing and telomere testing. Pre-treatment Folstein cognitive testing was performed on all patients and repeated post treatment at intervals of approximately once a month following the initial treatment. Pre-treatment medical laboratory blood analysis was performed and repeated post treatment along with doctor office visits at 3, 6-, 9-, and 12-month periods. Pre-treatment brain Magnetic Resonance Imaging (MRI) was performed and repeated 10 months post treatment.

Recent reports of fatalities in the ASPIRO (AT132) trial highlighted the potential dangers of AAV therapy regarding dose limitations and immunological issues. In the AT132 trial, large doses (1E14 vg/kg or higher) of AAV administered via an intravenous route in patients with preexisting hepatic disease resulted in the death of 4 participants. Mechanisms felt to be causal in these deaths are direct hepatic toxicity from the large viral load as well as stimulation of an aggressive immune response.

Regional AAV gene transfer therapy is felt to be less risky due to the lower does utilized or required, minimal viral load to the liver, and that the regional therapy is often delivered to immune privileged areas like the ocular globes or the central nervous system. The patients in this study received the equivalent of 1/10000 of the dose of the patients in the AT132 trial. Additionally, in this case the dosing was regional rather than intravenous. This extremely low dose essentially minimized the risk of hepatoxicity to almost zero. Additionally, the regional delivery to the CNS minimized the risk of a significant humoral response thereby maximizing the potential for cell transduction.

No short term or long term clinical of laboratory complications were observed or identified. Monitored serum chemistry, electrolyte, and hematological values remained stable throughout the follow-up time. No serious adverse effects such as a visible or laboratory detected innate or humoral immune response was observed or reported. Hepatic function as monitored by clinical laboratory values remained within normal parameters without evidence of hepatic insult. The treatment was well tolerated with only brief minimal physical discomfort at the injection site.

Pretreatment brain MRI compared to post treatment Brain MRI demonstrated no significant changes. Our telomere analysis before and after treatment demonstrated evidence of increased telomerase function. The median and the short telomeres were measurably elongated. Reduced biological age was seen in 4 of 5 participants. Cognitive assessment before and serial cognitive testing after treatment demonstrated a rapid improvement in cognitive function during the first 3.5 months after which the improvement slowed until it leveled off at five months. Thereafter there is a slight decrease slope in the average test score with an average drop of 0.07 points per month. Given that Patients with Alzheimer's dementia typically show an annual decline of 3 points on the on the Folstein test, our results indicate that the AAV hTert and Klotho gene transfer therapy was successful, and the effects of that gene therapy reversed some of the dementia pathology.

The accumulation of senescent cells in later life is a contributing cause of aging. While these errant cells never amount to more than a tiny fraction of all cells in a tissue, their inflammatory signaling is harmful. Researchers here describe an approach to diminish that inflammatory signaling, thereby reducing the ability of senescent cells to force nearby cells to also adopt a senescent state. This could shift the balance between the pace of creation and pace of clearance of senescent cells, allowing the burden of senescent cells in aged tissues to fall to more youthful levels. The result is improved function and a reduction in inflammatory markers of aging.

NF-κB is an inducible transcription factor capable of regulating diverse biological processes, including inflammation, immunity, stress responses, cell proliferation, differentiation, and survival. A wide range of external, internal, and environmental inducers can activate the NF-κB signaling including growth factors, viral or pathogenic assaults, tissue injury, genotoxic, oxidative, and inflammatory stresses. Subsequent signaling cascades in most cases converge on the IKK complex formed by two catalytic subunits, IKKα and IKKβ, and a regulatory subunit IKKγ (NEMO). Activated IKK complex phosphorylates IκB proteins, leading to its subsequent degradation. As a result, the NF-κB dimer sequestered in the cytoplasm is liberated and translocated to the nucleus to activate specific transcriptional machinery.

Normal activation of NF-κB is required to maintain many physiological functions, whereas its abnormal or chronic activation have been linked to many inflammatory and age-related diseases. NF-κB also plays key roles in cellular senescence and the aging process. Chronic inflammation, a hallmark of aging, also induces cellular senescence and promotes tissue aging. Bioinformatics studies demonstrated that NF-κB is the transcription factor most associated with mammalian aging. Furthermore, constitutive NF-κB activation drives senescence and mammalian aging, conferring expression of senescence-associated secretory phenotype (SASP) factors including pro-inflammatory cytokines and chemokines.

Acute genetic blockade of NF-κB signaling in epidermis of old mice reduced the expression of age-associated genes and reverted many features of aging to that observed in young mice. We and others also demonstrated that inhibition of NF-κB by genetic depletion of one allele of the p65 subunit of NF-κB delayed the onset of aging-related symptoms and extended healthspan in progeroid mice.

Given its key role in senescence and aging, the NF-κB signaling pathway presents a therapeutic target for extending healthspan. Previously, we developed the small molecule SR12343 capable of inhibiting NF-κB activation by disrupting the association between IKKβ and NEMO. SR12343 was developed to act as a mimetic of the NEMO Binding Domain (NBD) peptide shown previously to improve pathology in many pre-clinical models as well as to reduce senescence and improve healthspan in a mouse model of accelerated aging. Treatment with SR12343 demonstrated positive effects in murine models of acute inflammation. Here, we examined the therapeutic potential of SR12343 in reducing cellular senescence and extending healthspan using both cell-based models and mouse models of accelerated and naturally aging.

SR12343 reduced senescence-associated beta-galactosidase (SA-β-gal) activity in oxidative stress-induced senescent mouse embryonic fibroblasts as well as in etoposide-induced senescent human IMR90 cells. Chronic administration of SR12343 to mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2-year-old wild-type mice. Taken together, these results demonstrate that the IKK/NF-κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan, and treating age-related diseases.

Link: https://doi.org/10.1111/acel.13486

Asking whether an age-associated disease is a part of normal aging is an exercise in boundary drawing. The very definition of an age-related disease as something distinct from aging is the result of past boundary drawing. Many of these boundaries are quite arbitrary. Aging is a complex phenomenon, and people like to lay taxonomies on top of a complex space of many interacting mechanisms in order to try to make some sense of it. The results are sometimes helpful, sometimes not. The discussion in this open access paper is perhaps a good example of where the exercise of drawing boundaries can lead, while trying to separate out categories from the interplay of cancer, precancerous processes, and mechanisms of aging.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders characterized by ineffective hematopoiesis resulting in peripheral blood cytopenias. MDS typically occur at an advanced age with a median age at diagnosis of 68 to 75 years. MDS as clonal disorders may be preceded by a state of clonal hematopoiesis (CHIP) in which MDS defining features cannot (yet) be substantiated. Whereas CHIP-specific somatic mutations are rarely detected in persons younger than 40 years, they reveal an increasing incidence with advanced age.

Considerable progress has been made in deciphering the biology of normal aging, which includes the distinction of normal aging from pathologies associated with aging; additional progress has been made in describing MDS preceding states and elucidating initiation and progression of this disease. Despite these data, the provocative question, if MDS is simply a variant of the aging process, remains challenging.

The earliest answers supporting this hypothesis come from epidemiologic data with a clearly increasing incidence of MDS with age. As always, this observed correlation needs to be supported by establishing a causal relationship. Some similarities between aging and MDS have undoubtedly been defined. Especially changes affecting hematopoiesis are suggestive for an involvement of aging in the development of a hematologic disorder. One example is aging hematopoiesis as a result of clonal selection of hematopoietic stem cells leading to an alteration of the HSC pool. Another is clonal hematopoiesis such as defined in CHIP which is recognized as a potential pre-MDS state with a continuous increase at an advanced age.

CHIP has not only implications for MDS but also for other conditions or diseases associated with aging such as cardiovascular disease which further supports the connection with aging. Finally, many of the biologic features that drive the MDS process can also be observed in processes of aging or are key players in non-hematologic diseases of the elderly. On the other hand, there are clear data demonstrating that MDS is not inevitable with aging: for example, the risk of developing hematologic malignancies, particularly MDS, is higher in patients with clonal hematopoiesis than in persons without, but by far not all of them develop MDS. In addition, as far as we know, not every person contracts MDS, if he or she gets just old enough.

A possible solution for this conundrum is the notion that aging certainly contributes to the development of MDS. One might hypothesize that in many cases aging is the main driver of MDS, whereas in others aging promotes the specific phenotype. MDS might thus be seen as an interplay of clonal disease and normal or premature aging. Probably different subtypes or disease entities of MDS are distinctively affected by aging.

Link: https://doi.org/10.1007/s11912-021-01136-5

Senescent cell accumulation is an important contributing cause of aging. Senescent cells secrete a mix of signals that provokes growth and inflammation, useful in the short term in circumstances such as wound healing and cancer suppression, but damaging to tissue function and health when sustained over the long term. Senolytic drugs that can selectively destroy senescent cells have produced impressive displays of rejuvenation when used in aged mice. Some are in human trials, while many others are in development.

The most studied of present senolytic treatments are navitoclax, the dasatinib and quercetin combination, and fisetin, a mix of small molecule chemotherapeutics and plant extract supplements. Navitoclax has side-effects that make it undesirable. Administration of dasatinib and qercetin is the most proven of senolytic therapies, with data in human patients showing a similar reduction in senescent cells to that observed in mice. Fisetin is undergoing human trials, but results have yet to be reported.

Researchers here report on a study of earlier life intermittent administration of dasatinib and quercetin, and fisetin: monthly administration from 4 months to 13 months of age in mice. Mouse age does not correspond linearly to human age, if going by the usual exterior manifestations of aging. A 3 month old mouse is equivalent to a mid-20s human. A 12 month old mouse is equivalent to a 60 year old human. A 24 month old mouse is equivalent to a 70 year old human. The results here suggest that earlier life administration of current senolytics can have long term downsides that vary by gender and treatment, at least at the frequency used here.

This caution is aimed at those who would like to use senolytic therapies as a preventative approach to aging, starting earlier to stop the senescent cell burden from increasing at all - or at least to slow its progession considerably. To do so, senolytic therapies with better side-effect profiles are needed. Highly targeted small molecule approaches such as the prodrugs that only take effect in cells expressing high levels of β-galactosidase are one possible path towards achieving this goal.

Sexual dimorphic responses of C57BL/6 mice to Fisetin or Dasatinib and Quercetin cocktail oral treatment

Fisetin and quercetin (Q) are plant-derived flavonoids that offer cytoprotection against cellular stress and act as anti-inflammatory, chemopreventive, chemotherapeutic, and senotherapeutic agents. Dasatinib (D) is a tyrosine kinase inhibitor used to treat leukemia and is routinely used in combination with Q to improve the senotherapeutic potency. Fisetin and D+Q selectively clear senescent cells, thereby delaying aging-associated disorders and improving healthspan and lifespan. This has been observed after reducing senescent cell burden in progeroid mice or in 22-24 month old C57BL/6 mice. Moreover, deletion of senescent cells from the brain genetically or pharmacologically with senolytic drugs led to functional improvements in mouse models of neurodegenerative diseases such as Parkinson's and Alzheimer's=. These studies have shown senolytics can reduce senescent cell burden and have positive impacts on animals with accelerated aging, advanced age, or neurodegenerative disorders. Accordingly, senotherapeutics are currently marketed as anti-aging therapies where young, healthy adults can take these products as dietary supplements.

However, less is known regarding their anti-aging effects of these compounds when administered prior to significant senescent cell accumulation. Thus, the experiments were designed to examine the long-term effects of monthly oral treatment with Fisetin or a D+Q cocktail when administered to C57BL/6 mice starting at 4 months age. Since aging alters numerous biological functions, we examined morphological, metabolic, and cognitive components that are known to be affected by senescent cell accumulation. The results presented here indicate that monthly administration of Fisetin or D+Q had sexually dimorphic effects which also depended on treatment type in C57BL/6 mice.

Our study indicates that both age and sex may also determine the therapeutic outcomes of senolytic treatment. When the treatment was started at 4 months of age, before the reported senescent cell accumulation, Fisetin had beneficial effects in male mice while a D+Q cocktail had adverse consequences in female mice. Fisetin treated male mice had reduced senescence-associated secretory phenotype (SASP), enhanced glucose and energy metabolism, improved cognitive performance, and increased hippocampal expression of adiponectin 1 receptor and glucose transporter 4. D+Q treated females had increased SASP expression along with accumulation of white adipose tissue, reduced energy metabolism, and cognitive performance.

These observations provide novel information with translational relevance. First, senolytic drugs can be taken at an age before significant senescent cell burden to reduce or prevent their prevalence later in life. Second, males and females have differential responses to the same senolytic treatment when initiated at younger ages. Third, a particular senolytic treatment may have beneficial, negligible or detrimental effects depending on the age, sex, or disease. These observations should serve as a note of caution in this rapidly evolving and expanding field of investigation.

TLR4 is one of a number of cell surface receptors that mediate innate immune cell reactions to molecules indicative of damage in the body. Some fraction of the chronic inflammation of aging is caused by increases in damage-associated molecular patterns that trigger receptors of this nature, and consequent maladaptive reactions on the part of the innate immune system. Does it help to block this trigger? In mice, yes. Knockout of TLR4 leads to mice that have improved insulin metabolism and cardiovascular health in old age. TLR4 might be a good example of antagonistic pleiotropy. Natural selection has produced mice equipped with a more aggressive innate immune response, helpful in youth, at the cost of a more rapid deterioration in later life.

A growing amount of evidence suggests that inflammation plays a critical role in the physiological aging process. Many studies have shown that the activated chronic inflammatory response is involved in aging-related diseases. Aging-related inflammation is characterized by increased levels of IL-6, IL-1β, TNF-α, and type I interferon. This chronic activation of the innate immune system in the absence of infection during the aging process is called inflammaging. The activated innate immune system in aging causes insulin resistance and oxidative processes, making the cardiovascular system more vulnerable to stress, thereby increasing the risk of cardiovascular diseases. Moreover, some studies have shown that inhibiting inflammation could reduce the occurrence of cardiovascular diseases in aging, suggesting the important role of inflammation in aging-induced cardiovascular injury.

The mechanism responsible for inflammaging is still far from clear. Metabolic disorders, mitochondrial dysfunction, DNA damage, and autophagy deficiency are all involved in inflammaging. The damaged DNA or self-derived molecules released from damaged cells are called damage-associated pattern molecules (DAMPs). Usually, DAMPs are transferred into lysosomes and then degraded. The accumulation of excessive DAMPs will lead to inflammation. TLR4 is an innate immune receptor that specializes in sensing DAMPs. When sensing DAMPs, TLR4 triggers intracellular signaling pathways which subsequently activate downstream inflammatory responses, leading to the release of inflammatory factors.

The effects of TLR4 on the cardiovascular system of aged mice were investigated in TLR4-/- mice. In wild-type mice, TLR4 expression increased in the hearts and aortas of mice in an age-dependent manner. Loss of TLR4 increased insulin sensitivity in aged mice. Moreover, loss of TLR4 improved cardiac performance and endothelium-dependent vascular relaxation in aged mice. Importantly, the increases in serum inflammatory cytokines and oxidative stress in the heart and aorta were also inhibited by TLR4 deficiency. The reduced inflammatory responses and oxidative stress may be the reason for the protective effects of TLR4 deficiency during aging. Our study indicates that targeting TLR4 is a potential therapeutic strategy for preventing aging-related cardiovascular disease.

Link: https://doi.org/10.1186/s12979-021-00251-y

Cellular senescence contributes to degenerative aging. The burden of senescent cells increases throughout the body with age, and these cells disrupt tissue function and health via their inflammatory secretions. Senescence is an end state in nature, irreversible once a cell has become senescent. Researchers have nonetheless found ways to force cells out of senescence, and while this line of work is not very advanced, it has been suggested as the basis for alternatives to senolytic therapies that force the destruction of senescent cells.

The majority of cells entering senescence do so because their telomeres have shortened due to cell division, reaching the Hayflick limit, not because they became damaged and potentially cancerous. But reversing senescence has always come with the accompanying question of what to do about cells that are senescent due to mutational damage to the genome: returning potentially cancerous cells to normal operation does not seem like a good idea. Researchers here expand the importance of that question. It seems plausible that all senescent cells have significant levels of genomic damage, generated on entry to senescence, regardless of the cause of senescence.

Researchers have shown that cellular senescence, which occurs when aging cells stop dividing, is caused by irreversible damage to the genome rather than simply by telomere erosion. This discovery goes against the scientific model most widely adopted in the last 15 years, which is based on one principle: telomeres, caps located at the ends of chromosomes whose purpose is to protect genetic information, erode with each cell division. When they get too short, they tell the cell to stop dividing, thus preventing damage to its DNA. Made dormant, the cell enters senescence.

"What's most surprising is that, before really entering senescence, the cells divide one last time. In fact, the cell division caused by telomere dysfunction is so unstable that it ends up creating genetic defects. Contrary to what was believed, senescent cells have an abnormal genome. That's what we show in our study. Genetically, we were able to reproduce the phenomenon of cellular aging in the laboratory and ensured that all the telomeres of a population of cells became dysfunctional. With our equipment, we then observed in real time what was happening inside each single cell."

With time, senescent cells build up in the body and are responsible for the development of diseases such as cancer. This study, therefore, opens up new research opportunities. For example, could telomeres be repaired prior to the senescence phase, thereby preventing cellular aging and genomic instability? The scientific community has been debating this potential cellular rejuvenation for several years now. Nevertheless, these emerging therapeutic approaches still need fine-tuning.

Link: https://nouvelles.umontreal.ca/en/article/2021/11/09/cellular-aging-a-basic-paradox-elucidated/

The UK is somewhat further ahead than the US when it comes to progress towards an official government policy on medical research and development aimed at increasing healthy longevity. A variety of groups are attempting to influence the political and bureaucratic class into seeing the benefits, and are doing so in a more organized and well supported fashion than is the case for US lobbying efforts. Today's article is an example of this sort of approach from the UK lobbying community.

For my part, I believe that meaningful success in such initiatives requires the first rejuvenation therapies to have been deployed in human trials, with solid evidence for significant benefits to result to patients. The best thing we can do at this stage of the new longevity industry is to robustly prove that approaches such as senolytics actually work in humans, and can reliably reverse age-related disease. Government institutions follow but slowly in the wake of technological progress. All large organizations and sizable sources of funding, particularly those run by governments, are very conservative, and rarely get involved until quite late in the process of developing new approaches to medicine.

Live Longer or Healthier? The Science That Is Making Both Possible

One of humanity's greatest success stories of the past century is the increase in global life expectancy as a result of the social and medical advancements that have dramatically improved basic living conditions and reduced vulnerability to infectious diseases. However, longer lives haven't fully translated into healthier lives, and as we grow older the likelihood that we will live with debilitating chronic illness rises significantly. Beyond unacceptable personal suffering, this places extreme pressure on both health-care systems and the economic models of advanced economies.

However, a new frontier of science is emerging - longevity research, also referred to as geroscience - that is helping us understand the underlying biological mechanisms of how and why we age, with the potential to develop treatments that delay, prevent or even reverse the onset of ageing and multimorbidity. In short, the nature and speed of the ageing process, and the aches and pains that accompany it, may not be inevitable. If we can successfully intervene to ensure ageing is healthy for as long as possible, then the potential gains in personal, public and economic health would be enormous.

In this paper, we examine the public-health and economic imperative to act, look at some of the most promising areas of innovation in longevity research across academia and industry, and propose practical actions that governments should take to invigorate this critical but under-resourced area of science. Given the profound implications, we believe governments should set ambitious targets: the UK and other advanced economies should aim for "30 in 30" - to increase healthy life expectancy by 30 years (to approximately 95 years) by 2050. While our work is international in scope, we also include a case study on how the UK can play a global leadership role in longevity research and development. With its world-leading academic institutions, strong life-sciences industry, access to vast NHS data sets and a global financial hub in London, the UK is already primed to become a global leader in this nascent field.

A range of convincing data shows correlations between the presence of airborne particulates, such as those in wood smoke, and raised late-life mortality due to cardiovascular disease and respiratory disease. The likely mediating mechanism is an increased burden of chronic inflammation, due to the effects of particulates on lung tissue. The challenge in any such study is the associated correlation between wealth and air quality: wealthier people do not tend to use wood stoves for cooking, or live in more heavily polluted areas, but it is also the case that wealthier people tend to live longer for many reasons.

Some natural experiments allow the correlations between wealth and health and between particulate air pollution and health to be better distinguished. For example, the Puget Sound area has quite varied levels of airborne particulates and there is localized data on pollution and health going back decades. Other examples can be found in Asia, where happenstance has led to populations with similar socioeconomic circumstances exposed to different sources and degrees of particulate air pollution.

Long-term exposure to household air pollution (HAP) is a public health problem worldwide and is among the top 10 hazard factors for disease. Solid fuel is the largest source of HAP worldwide, where nearly a third of the population in low- and middle-income countries (LMICs) rely on it for primary domestic cooking, heating, or lighting energy. Notably, exposure to air pollution from solid fuel burning may directly or indirectly contribute to over 4 million deaths and 110 million disabilities annually.

The association of solid fuel use and common diseases has been proved in many studies on the elderly. Owing to physical limitations and social factors, the elderly are generally most active in and around the house, and they willingly perform housework and spend more time on it as they age. A cohort study indicated that the use of biomass fuel is associated with higher hazard ratios (HRs) of hypertension among the elderly (aged ≥65 years). The Chinese Longitudinal Healthy Longevity Survey (CLHLS) is an open, prospective, and national cohort of community-dwelling Chinese aged 65 years. In this study, we investigated whether the type of fuel used for cooking is associated with subsequent 8-year mortality and whether switching the fuel used for cooking for 4 years is associated with changes in HR with successive 5 years of follow-up.

Among the participants in the 2011-2018 survey, 53% reported using solid fuel. Such group was associated with a 9% increase in mortality risk relative to clean fuel users (HR = 1.09). Among participants in the 2014-2018 survey, 339 reported a switch from solid to clean fuels and they were not at increased mortality risk relative to the 488 people that reported a stable use of clean fuels (HR = 1.14) although the estimated HR was similar to the one for stable solid fuel users (HR = 1.19). Interaction and stratified analyses showed that solid fuel use had an impact on mortality in participants who were non-current smokers, had low dietary diversity scores, and were living in areas with high PM2.5 concentrations (over 50 μg/m3) and city population below 8 million. These findings showed a clear association between solid fuel use and mortality among older Chinese, and an even stronger association between risk of mortality and solid fuel use among individuals exposed to high levels of PM2.5.

Link: https://doi.org/10.3389/fpubh.2021.741637

A few strategies offer the possibility of growing additional redundant blood vessels, though this is far from rigorously proven. Intermittently provoking hematopoietic stem cells to leave the bone marrow via CXCL12 upregulation, for example. Humans are not completely uniform in their major blood vessel networks, there are variant populations with more redundancy. The value of that greater redundancy is illustrated here by a look at a patient possessing a Kugel's artery, a rare additional vessel that can allow survival in cases of obstructed coronary arteries due to the progression of atherosclerosis. Redundancy in blood vessel networks is a poor substitute for addressing the causes of atherosclerosis, such that no-one suffers blocked vessels, but if redundancy can be engineered, then it would be a benefit.

Kugel's artery is defined as a rare anatomical variant of the coronary artery vascular bed consisting of an anastomotic connection between branches of the right coronary artery (RCA) and/or left circumflex artery (LCX). Kugel's artery has been reported to have an incidence of 6% in the general population. The presence of this anastomotic communication may play a pathophysiological role in a patient with a right dominant coronary circulation and an underlying coronary artery disease (CAD) affecting the right coronary system. There exist only a few reported cases of Kugel's artery with the pathophysiological relationship in CAD remaining unclear. Herein, we present a case with multivessel occlusion myocardial infarction found to have anomalous vascular anastomosis between the proximal RCA and distal segment of the same artery.

A 67-year-old African American male with no significant past medical history presented to the emergency room for an out-of-hospital witnessed cardiac arrest. In this case, the Kugel's artery was found to connect the proximal RCA to the branches of the distal RCA. The patient was found to have inferior anterolateral myocardial infarction on EKG and coronary angiogram revealed complete total occlusion of RCA and obtuse marginal. With total occlusion of the RCA one would expect to have a clinically significant myocardial infarction earlier in life, however, because of the patent Kugel's artery, the territory of RCA had viable blood supply and the presentation was most likely due to occlusion of the second obtuse marginal branch of the LCX. This bears out the observation by previous studies that collateral circulation was not seen in angiography until the degree of arterial occlusion is greater than 90%.

A favorable long-term prognosis is associated with good collateralization in patients with angiographically-proven single or double vessel CAD. Based on our patient's EKG and coronary angiogram findings, his acute presentation was most likely due to myocardial infarction in the LCX territory. He denied previous cardiorespiratory symptoms which may indicate myocardial viability in the distribution of RCA maintained by the Kugel's artery. Understanding the existence and significance of Kugel's artery and the anastomotic network cannot be overemphasized. The presence of an anomalous vascular connection bypassing an area of epicardial vessel occlusion may be a lifesaving pathophysiological finding that maintains myocardial perfusion and viability.

Link: https://doi.org/10.7759/cureus.17039

Why are long-lived species long-lived? The prevailing view of the evolution of aging is that degenerative aging is the result of natural selection operating more strongly on early life reproductive success than on later life reproductive success for individuals. Natural selection produces biological systems that are front-loaded for immediate success and fall apart over time (cellular senescence is a cancer suppression and wound healing strategy, but causes tissue dysfunction as senescent cells accumulate), or simply cannot function indefinitely even if they were perfectly maintained (the adaptive immune system devotes ever more cells with passing time to the memory of specific threats, at the expense of cells capable of attacking those threats). At the level of individual biological mechanisms, antagonistic pleiotropy takes place: mechanisms are selected for reproductive success in early life, but those same mechanism cause harm in older individuals.

In today's open access paper, researchers suggest that the exceptional longevity of any given species is largely an accidental byproduct of adaptations to a given evolutionary niche. This view has been discussed in past years in the context of naked mole-rats and related species, which are resilient to the low oxygen environment found underground. The mechanisms needed for that resilience likely also contribute to the exceptionally long life spans exhibited by these species. Similarly for many bat species, the mechanisms required for resistance to viral pathogens and sustaining the high metabolic demands of flight likely contribute to a much greater longevity than is found in similarly sized mammals. A counterargument is the case of our own species. We live longer than other primates, and the grandmother hypothesis suggests that this is because culture and intelligence allows old individuals to contribute to the reproductive success of their descendants - our relative longevity amongst primates is not an accidental byproduct of evolutionary adaptation, in other words, but actually selected.

Lifespan Extension in Long-Lived Vertebrates Rooted in Ecological Adaptation

The ability of an organism to survive in a specific ecosystem as a result of changes to its behavioral, physiological, morphological, and genetic response is called adaptation. Ecological adaptation strongly underlies lifespan extension in lineages and species where longevity has been observed despite differences in species ecosystem, morphology, and complexity. Predictably, all long-lived species have low extrinsic mortality due to the nature of their habitat or have evolved mechanisms to evade predators and imminent dangers. However, as much as this ability is expected to contribute to lowering extrinsic mortality, it neither explains the variation observed in lifespan or mechanisms through which lifespan is regulated.

Long-lived species are now known to exhibit efficient adaptive responses in essential pathways that contribute to lifespan with evidence of enhanced genome maintenance, DNA damage response, and repair attributing to their longevity. Thanks to affordable genome sequencing, the availability of genome data revealed widespread adaptation in the genomes of long-lived species where positive selection, rare sequence variants, and genome duplication contributed to ecological adaptation, the evolution of body size, and disease resistance. Although mechanisms of extended lifespan of these species are currently unclear, emerging evidence from genomic analyses points to the important role of species adaptation in longevity.

Although there are a number of genetic adaptions in the wild that contribute to lifespan extension, population genetics postulates that genetic changes are hardly to be fixed if such genetic changes do not increase fitness during long-term evolution. Nevertheless, we posit that strong selection acts to maintain these changes, leading to long lifespans in living organisms. We propose that extended lifespan is not by itself under selection but rather an epiphenomenon (by-product) of species adaptation, a phenomenon we have termed here the adaptive hitchhike model. First, the model implies that a new pleiotropic mutation, with one of its effects being extended lifespan, could be favored by natural selection due to its advantage to some other trait and therefore becomes fixed. Second, the model also applies to new pro-longevity mutations that occur at sites closely (or functionally) linked with the allelic sites under selection; if a new pro-longevity mutation arises at a site that is linked to an adapted genome region, natural selection may cause an increase in allele frequency and fix this pro-longevity mutation through linkage and allelic associations.

Therefore, the adaptive-hitchhike model suggests that the selective constraint acting on the genomic region associated with adaptation and fitness is largely responsible for non-random beneficial pro-longevity effects. For example, patterns of selective sweep across loci of close proximities were reported for adaptation to altitude among the Tibetan population, and a further association was found between longevity and hypoxia response in this same population. In other cases, natural selection might act on an already existing but neutral mutation through a sweeping selection; therefore, if neutral alleles responsible for lifespan extension are close enough to other alleles under selection, the chances of recombination are slim, and together, they become fixed in the population.

This model could be mainly summarized in the following ways: (1) Some adaptive genetic changes could have dual functions, i.e., adaptive and longevity effects. (2) A pro-longevity mutation could come under selection and become fixed through direct selection or linkage and allelic association. (3) In the same way that a pro-longevity mutation could become fixed, a geronic (pro-aging) mutation could also become fixed and lead to a shorter lifespan. (4) In the case where environmental pressure is relaxed, pro-longevity effects may be lost. Therefore, our adaptive-hitchhike model of longevity of animals could be tested by (a) identifying pro-longevity effects of genetic changes that respond to adaptation and (b) detecting signals of linkage disequilibrium between adaptive and longevity variations. The novelty of this model is that it gives a key role to such nucleotide substitutions and loci with dual functions. Functional evaluation and validation of adaptive nucleotide substitutions with the pro-longevity potential could provide answers to the century-long questions surrounding the evolvability of animal lifespan.

This open access paper provides a good summary of present thought on the contributing causes of hearing loss, in which the various issues of noise, aging, and toxicity cause harm via inducing stress in hair cells of the inner ear and their axonal connections to the brain. Autophagy is a cell maintenance process, the recycling of damaged component parts. More efficient autophagy helps hair cells to resist and survive a stressful environments, but autophagy declines with age. Defects arise in many of the component parts of the autophagic system and its regulation. This is likely why the threshold for loss of hair cells in response to stresses diminishes in later life, leading to the onset of hearing loss in a large fraction of the population.

Hearing loss is not only a physical and financial burden in social life, but also causes psychological problems and psychiatric disorders, including cognitive decline and depression. Genetic alterations, noise, ototoxic drugs, and aging can all contribute to hearing loss. Although the causes vary, the most common causes of deafness are damage or loss of hair cells (HCs) and degeneration of spiral ganglion neurons (SGNs). HCs are responsible for converting external sound signals into electrical signals that are transmitted to the brainstem through SGNs. Recent studies have shown that these sensory cells cannot spontaneously regenerate in adult mammals, so damage or loss of HCs and degeneration of SGNs can result in permanent deafness.

Autophagy is responsible for normal cell survival and homeostasis. A variety of human conditions, such as neurodegenerative diseases, cancer, and inflammation, have been reported to be associated with dysregulated autophagic processes. In the inner ear, many studies have shown that autophagy played an important role in cell development, differentiation, and survival, and recently there has been renewed interest in regulating autophagy to prevent sensorineural hearing loss (SNHL).

Noise and ototoxic drugs increased the levels of oxidative stress in HCs, which contributed to cell death, and in a mouse model that was exposed to noise, the level of autophagy was increased in HCs. It is worth noting that the oxidative stress level in response to noise was dose dependent, and moderate noise induced temporary threshold shifts and increased the level of autophagy in outer hair cells, while severe noise produced excess reactive oxygen species (ROS) that induced permanent threshold shifts. Increasing autophagy with rapamycin can reduce the accumulation of ROS and prevent cell death from noise exposure. In contrast, blocking autophagy via pharmacological or genetic means can increase the accumulation of ROS and promote cell death.

Presbycusis (age-related hearing loss) is a common sensory disorder associated with aging. The level of autophagy decreases with age, and the upregulation of autophagy can promote aging HC survival and slow the degeneration of auditory cells. Though we have known that some proteins and miRNAs participate in the autophagic pathways involved in SNHL making them potential targets for treatment of SNHL, the specific signaling pathways they participate in remain unclear, let alone the known connections between these proteins and miRNAs. The application of autophagy as a treatment for deafness is still a long way off. Current research has been limited to cell lines, explants, and animals, and few clinical trials have examined the role of autophagy. Given the complexity of mechanisms and functions of autophagy, the safest and most effective strategies must be studied in future research.

Link: https://doi.org/10.3389/fncel.2021.760422

Researchers here show that forcing mitochondrial uncoupling in macrophages can change their behavior to resolve inflammation. There are a range of therapeutic approaches that might achieve this goal, as mitochondrial uncoupling has been a target for drug discovery for some time, albeit largely for reasons other than the reduction of inflammation. Uncoupling in mitochondria disconnects their activity from the production of ATP to power cellular operations, leading to heat generation instead. This change influences many other cellular processes, largely in beneficial ways; uncoupling occurs during calorie restriction, and intermittently inducing uncoupling is a calorie restriction mimetic strategy, with accompanying benefits to long-term health. To what degree is a reduction in chronic inflammation in later life driving those benefits? That is an interesting question.

Researchers have recently shown how inflammatory reactions can be resolved by changes to the metabolism of macrophages. Danger signals released by damaged cells during inflammation play a role during this process. 'Rewiring' the mitochondria in the macrophages protects them against overloading and can thus improve the way in which parts of damaged cells are eliminated and resolve the inflammatory reaction. Inflammation is a natural and vital response of our immune system to danger signals and tissue damage. Inflammatory processes help the body to eliminate the triggers, for example bacteria, and to initiate repair mechanisms. Terminating this inflammatory reaction quickly and in a coordinated manner is just as important, however, as otherwise there is a risk of developing chronic inflammatory conditions.

Researchers investigated the function of macrophages at the site where inflammation occurs. These cells are capable of ingesting large quantities of cellular waste and digesting and eliminating the molecular components of this waste in their mitochondria, also referred to as the powerhouse of the cell. The scientists were able to demonstrate that the danger signal interleukin 33, which is released from damaged cells, triggers lasting changes to the metabolism of macrophages, so that their waste disposal capacity significantly increases.

The sheer quantity of waste produced during the inflammatory reaction places the mitochondria under severe strain, and they produce increased quantities of damaging oxygen radicals as a result. Interleukin 33 regulates the function of the mitochondria by initiating a process known as uncoupling in these cell components and protecting them from overloading. "This enables the macrophages to 'let off steam' and carry on ingesting waste without interruption despite the heavy strain placed upon them, resolving the inflammation processes as a result. It may be possible to accelerate and support the resolution of inflammatory processes in the long term by influencing the cell metabolism of the macrophages and deliberately uncoupling their mitochondria."

Link: https://www.fau.eu/2021/11/02/news/research/when-macrophages-let-off-steam/

In recent years more careful consideration of epidemiological data has pointed to there being no health advantage to moderate alcohol consumption. Many past studies were looking at a form of alcohol consumption, such as moderate wine intake, that correlates with higher socioeconomic status. That in turn correlates with health and longevity, and the whole web of interlinked factors such as wealth, intelligence, education, and so forth.

The study here suggests another issue, in that groups who do not drink alcohol may have a higher proportion of people with risk factors for poor health and shorter life span than is the case in the general population. For example, people with a history of substance abuse, who do not drink at all as a necessary part of maintaining control over their lives. If one matches people who do not drink versus people who drink moderately, and ensure that other risk factors are the same on both sides, then the apparent advantage to drinking vanishes.

This is perhaps a similar situation to that arising in the epidemiology of excess fat tissue. Studies were showing a survival advantage to being moderately overweight in late life. That, however, is because the population of people who are thin at a given time in later life includes a sizable number who lost weight due to continuing ill health, and are thus at a higher risk of mortality going forward. People who remain thin throughout life have a survival advantage over their moderately overweight peers. There are many such cautionary tales in epidemiology regarding the interpretation of data.

Alcohol abstinence and mortality in a general population sample of adults in Germany: A cohort study

Evidence suggests that people who abstain from alcohol have a higher mortality rate than those who drink low to moderate amounts. However, little is known about factors that might be causal for this finding. The objective was to analyze former alcohol or drug use disorders, risky drinking, tobacco smoking, and fair to poor health among persons who reported abstinence from alcohol drinking in the last 12 months before baseline in relation to total, cardiovascular, and cancer mortality 20 years later. A sample of residents aged 18 to 64 years had been drawn at random among the general population in northern Germany and a standardized interview conducted in the years 1996 to 1997. The baseline assessment included 4,093 persons (70.2% of those who had been eligible). Vital status and death certificate data were retrieved in the years 2017 and 2018.

We found that among the alcohol-abstinent study participants at baseline (447), there were 405 (90.60%) former alcohol consumers. Of the abstainers, 322 (72.04%) had met one or more criteria for former alcohol or drug dependence or abuse, alcohol risky drinking, or had tried to cut down or to stop drinking, were daily smokers, or self-rated their health as fair to poor. Among the abstainers with one or more of these risk factors, 114 (35.40%) had an alcohol use disorder or risky alcohol consumption in their history. Another 161 (50.00%) did not have such an alcohol-related risk but were daily smokers. The 322 alcohol-abstinent study participants with one or more of the risk factors had a shorter time to death than those with low to moderate alcohol consumption.

The Cox proportional hazard ratio (HR) was 2.44 for persons who had one or more criteria for an alcohol or drug use disorder fulfilled in their history and after adjustment for age and sex. The 125 alcohol-abstinent persons without these risk factors (27.96% of the abstainers) did not show a statistically significant difference from low to moderate alcohol consumers in total, cardiovascular, and cancer mortality. Those who had stayed alcohol abstinent throughout their life before (42; 9.40% of the alcohol-abstinent study participants at baseline) had an HR 1.64 compared to low to moderate alcohol consumers after adjustment for age, sex, and tobacco smoking.

The majority of the alcohol abstainers at baseline were former alcohol consumers and had risk factors that increased the likelihood of early death. Former alcohol use disorders, risky alcohol drinking, ever having smoked tobacco daily, and fair to poor health were associated with early death among alcohol abstainers. Those without an obvious history of these risk factors had a life expectancy similar to that of low to moderate alcohol consumers. The findings speak against recommendations to drink alcohol for health reasons.

Changes in fat tissue behavior in the skin take place with age, such as rising levels of inflammation and inflammatory signaling. These changes have a detrimental effect on the ability of hair follicles to produce hair. The growth of hair is a complicated process that cycles through repeated phases of growth (anagen), transition (catagen), and rest (telogen). Aging leads to progressive dysfunction in this process and loss of hair in late life. A better understanding of the details of this dysfunction may lead to interventions, such as those attempted here in mice, to change the signaling of fat tissue in aged skin and thereby restore greater capacity to regrow hair.

Progressive deterioration in the regenerative potential of stem cells is a hallmark of aging, which results in the failure to maintain proper tissue homeostasis. Hair follicles are independent autonomous stem cell niches and undergo continuous regenerative cycling during their lifespan. With aging, hair follicle have diminished self-renewing capacity, manifesting as cycling defects and poor responsiveness to activating stimuli. hair follicle cycling slows down with aging and gradually turns into senescent alopecia.

Hair follicle stem cells are extensively reprogrammed by the aging process, manifesting as diminished self-renewal and delayed responsiveness to activating cues, orchestrated by both intrinsic microenvironmental and extrinsic macroenvironmental regulators. Dermal white adipose tissue (dWAT) is one of the peripheral tissues directly adjacent to hair follicles and acts as a critical macroenvironmental niche. dWAT directly contributes to hair follicle aging by paracrine signal secretion. However, the altered interrelationship between dWAT and hair follicle with aging has not been thoroughly understood.

Here, through microdissection, we separated dWAT from the skin of aged mice (18 months) and young mice (2 months) in telogen and depilation-induced anagen for transcriptome comparing. Notably, compared with young dWAT, aberrant inflammatory regulators were recapitulated in aging dWAT in telogen, including substantial overexpressed inflammatory cytokines, matrix metalloproteinases, and prostaglandin members. Nonetheless, with anagen initiation, inflammation programs were mostly abolished in aging dWAT, and instead of which, impaired collagen biosynthesis, angiogenesis, and melanin synthesis were identified. Furthermore, we confirmed the inhibitory effect on hair growth of CXCL1, one of the most significantly upregulated inflammation cytokines in aging dWAT.

Finally, we proved that relieving inflammation of aging dWAT by injecting high-level veratric acid stimulated hair follicle regenerative behavior in aged mice. Concomitantly, significantly decreased TNF-a, CCL2, IL-5, CSF2, and increased IL10 in dWAT was identified. Overall, the results elaborated on the complex physiological cycling changes of dWAT during aging, providing a basis for the potential regulatory effect of dWAT on aging hair follicles.

Link: https://doi.org/10.3389/fcell.2021.728188

Some researchers consider the metabolic dysfunction of brain tissue in Alzheimer's disease to be similar to that produced by diabetes, both involving disruption of insulin signaling. It was proposed at one point that Alzheimer's should be classified as type 3 diabetes. Diabetic patients have an increased risk of Alzheimer's disease, but it is possible to argue that this is the outcome of raised inflammation. Here, researchers dig a little deeper to instead suggest that a specific regulatory aspect of the diabetic metabolism leads to an increase in the tau pathology characteristic of Alzheimer's disease, and that this mechanism links the two conditions.

Diabetes mellitus is characterized by hyperglycemia caused by a lack of insulin, insulin resistance, or both. It is associated with the development of secondary complications resulting in several comorbidities. Recent studies have revealed an increased risk of developing cognitive dysfunction or dementia in diabetes patients. Diabetes mellitus is considered a risk factor for many neurodegenerative diseases, including Alzheimer's disease (AD). There is increasing evidence to support a link between diabetes mellitus and AD. Studies have shown the dysfunction of insulin signaling in the brain, resulting in increased tau protein phosphorylation (hyperphosphorylation), a hallmark and biomarker of AD pathology, leading to accumulation of neurofibrillary tangles.

In diabetes mellitus, the insulin dysfunction in the brain is reported to alter the glycogen synthase kinase-3β (GSK-3β) activity showing to enhance tau phosphorylation. In diabetes mellitus and AD, GSK-3β signaling has been involved in the physiological and pathological processes, respectively. This potentially explains why diabetes mellitus patients have an increased risk of developing AD with disease progression and aging.

Interestingly, several in vivo studies with oral antidiabetic drugs and insulin treatment in diabetes mellitus have improved cognitive function and decreased tau hyperphosphorylation. This article will review the relationship between diabetes mellitus and AD as it relates to tau pathology. More understanding of the link between diabetes mellitus and AD could change the approach researchers and clinicians take toward both diseases, potentially leading to new treatments and preventative strategies in the future.

Link: https://doi.org/10.7759/cureus.18362

Spiny mice are one of the very few mammalian species in which adults can repeatedly regenerate at least some tissues without scarring and loss of tissue function. This has been observed in their ability to slough off skin as a way to confound predators, then regrow that skin. They can also regrow nerves and cartilage. Researchers pursuing an understanding of the molecular biology of proficient regeneration have found that the behavior of macrophages and senescent cells is meaningfully different in species capable of regeneration, such as the spiny mice, salamanders, and zebrafish. Salamanders, for example, possess macrophages that are far better at clearing senescent cells than is the case in our species. The hope is that the relevant mechanisms to allow scarless regeneration from injury remain present in most mammals, but are repressed or dormant, silenced in a way that will be comparatively easy to undo.

Regeneration following injury, and tissue maintenance in general, is a complex dance between stem cells, somatic cells, senescent cells, and supporting immune cells such as macrophages. Senescent cells form to help provoke tissue growth and contribute to the inflammatory signaling that draws in immune cells to play their parts. Those immune cells destroy senescent cells after their task is complete. Something in the interplay between macrophages and senescent cells appears critical to scarless regeneration, based on the evidence to date. This may be a complex set of differences in signaling, or it may turn out to hinge on just a few regulatory genes. Whether there is a basis for therapy here that can be exploited in the near future to allow humans to fully regenerate from severe injuries remains an open question.

Spiny mice regenerate damaged kidneys without scarring

Earlier studies of wound healing in spiny mice suggested that the animals had - over the course of their evolution - solved the problem of tissue fibrosis (scarring) after injury. But could they also heal damaged internal organs the same way? To find out, they exposed spiny mice to conditions that are known to cause serious kidney injury. Their studies showed that, although spiny mice suffered the same degree of tissue injury initially, they were nevertheless able to completely heal: they regenerated an apparently healthy kidney with no signs of fibrosis. As expected, other mice treated in the same way progressed to organ failure.

"The dramatic and complete recovery of kidney function over a two-week time course in spiny mice was quite surprising to us. The types of severe injuries we used were chosen because they produce a decisive and rapid loss of kidney function in mice and led to complete organ failure over the same two-week period." To find out how the spiny mice do it, the researchers took a comprehensive look at the genes they express. Their studies suggest that the spiny mouse genome is poised at the time of injury to launch a rapid, scarless regenerative response in surviving kidney cells. The analysis uncovered differences in the activity of 843 genes in six unique clusters. Researchers also saw a delayed response by immune cells called macrophages, which are known to play a role in fibrosis. Unlike in other mice, macrophages didn't appear on the scene for about a week.

Spiny mice activate unique transcriptional programs after severe kidney injury regenerating organ function without fibrosis

Fibrosis-driven solid organ failure is an enormous burden on global health. Spiny mice are terrestrial mammals that can regenerate severe skin wounds without scars to avoid predation. Whether spiny mice also regenerate internal organ injuries is unknown. Here, we show that despite equivalent acute obstructive or ischemic kidney injury, spiny mice fully regenerate nephron structure and organ function without fibrosis, whereas C57Bl/6 or CD1 mice progress to complete organ failure with extensive renal fibrosis. Two mechanisms for vertebrate regeneration have been proposed that emphasize either extrinsic (pro-regenerative macrophages) or intrinsic (surviving cells of the organ itself) controls. Comparative transcriptome analysis revealed that the spiny mouse genome appears poised at the time of injury to initiate regeneration by surviving kidney cells, whereas macrophage accumulation was not detected until about day 7. Thus, we provide evidence for rapid activation of a gene expression signature for regenerative wound healing in the spiny mouse kidney.

One of the interesting findings from research into fasting mimicking diets was that parts of the immune system contract, cell counts diminishing, after about three days. Those cell counts return to normal after the fasting ends. This winnowing and replacement seems beneficial, shedding damaged and problematic cells. In this paper, researchers look at other aspects of the immune system, finding differing beneficial outcomes after a three day fast.

Previous studies have shown that long-term light or moderate fasting such as intermittent fasting can improve health and prolong lifespan. However, in humans short-term intensive fasting, a complete water-only fasting has little been studied. Here, we used multi-omics tools to evaluate the impact of short-term intensive fasting on immune function by comparison of the CD45+ leukocytes from the fasting subjects before and after 72 hour fasting. Transcriptomic and proteomic profiling of CD45+ leukocytes revealed extensive expression changes, marked by higher gene upregulation than downregulation after fasting. Functional enrichment of differentially expressed genes and proteins exposed several pathways critical to metabolic and immune cell functions.

Specifically, short-term intensive fasting enhanced autophagy levels through upregulation of key members involved in the upstream signals and within the autophagy machinery, whereas apoptosis was reduced by down-turning of apoptotic gene expression, thereby increasing the leukocyte viability. When focusing on specific leukocyte populations, peripheral neutrophils are noticeably increased by short-term intensive fasting. Finally, proteomic analysis of leukocytes showed that short-term intensive fasting not only increased neutrophil degranulation, but also increased cytokine secretion. Our results suggest that short-term intensive fasting boost immune function, in particular innate immune function, at least in part by remodeling leukocytes expression profile.

Link: https://doi.org/10.1111/acel.13507

As we age, the vascular system becomes ever more dysfunctional in a number of ways. The density of capillaries declines, lowering the rate at which nutrients are delivered to energy-hungry tissues. The blood-brain barrier begins to leak, allowing inappropriate molecules and cells into the brain to provoke inflammation. Hypertension produces pressure damage to delicate tissues, and the ongoing rupture of tiny blood vessels, destroying small volumes of tissue. As researchers attempt to understand the relative importance of the many possible contributing causes of neurodegenerative conditions such as Alzheimer's disease, in the wake of the failure of protein aggregate clearance to improve patient outcomes, it is a reasonable argument to suggest that vascular dysfunction provides a meaningful contribution to the age-related decline of the brain.

Alzheimer's disease (AD) is the most common form of neurodegenerative disease in elder population worldwide. AD is clinically characterized as cognitive decline and psychiatric manifestations. The pathological hallmarks of AD brain are the accumulation of extracellular β-amyloid (Aβ) (senile plaques) and the intracellular twisted strands of the hyper-phosphorylated tau protein (neurofibrillary tangles). These changes in the brain are accompanied by the neuronal damage.

AD is a progressive neurodegenerative disorder that can start decades before the appearance of clinical symptoms. Although several pathological mechanisms of AD have been identified, no satisfactorily effective therapeutics has been developed. Recently, cerebrovascular dysfunctions, as a possible cause in the development and progression of sporadic AD, have gained increasing attention. Increasing evidence has indicated the involvement of various alterations in cerebrovascular structure or functions, such as the cerebrovascular microstructure, blood-brain barrier (BBB) integrity, composition of neurovascular unit, cerebrovascular reactivity and cerebral blood flow, in AD pathophysiology and cognitive defects.

Recent findings further highlighted the prevalence of cerebrovascular disorders in Down syndrome patients and added to a growing body of evidence implicating cerebrovascular abnormalities as a core feature of AD rather than a simple comorbidity. Moreover, adrenergic system, including adrenergic receptors and their downstream molecular signaling process, might serve as the key approach to modulate these cerebrovascular abnormalities and progressive neurodegeneration.

Link: https://doi.org/10.18632/aging.203482

In today's open access paper, the authors report on a literature search for efforts to reduce cellular senescence in stem cell populations. The majority of the work they list, involving the assessment of pharmacological agents that can influence the onset of cellular senescence, has taken place in cell cultures, an environment that has very little relevance to what happens in stem cell niches in a living organism. Stem cells in a petri dish undergo very different rates of replication, have different stresses and signals, are not subject to interactions with supporting cells of the niche, and so forth.

Thus I'd be inclined to give little attention to the in vitro work, at least in the context of developing drugs to be used to reduce cellular senescence in vivo by altering cellular metabolism in ways that lower the rate at which cells become senescent. The environments are so very different, a petri dish versus living tissue. This in vitro work is of interest in the context of manufacturing stem cell therapies, however.

Recent evidence suggests that the degree to which stem cells become senescent when expanded in culture influences whether or not the therapeutic use of those cells produces benefits. Since small differences in manufacture can lead to sizable differences in the proportion of cells that become senescent, this could explain the wide variability of efficacy from clinic to clinic and patent to patient that is observed in the use of first generation stem cell therapies. Senescent cell accumulation is an important contributing cause of aging, and if even a modest percentage of injected cells are senescent, that may be enough to offset the benefits of such a therapy. Stem cells produce benefits via their signaling, and senescent cells cause harm via their signaling.

In search of elixir: Pharmacological agents against stem cell senescence

Stem cell senescence has been studied in aging, diseases, adverse drug effects, and as a challenging phenomenon in cell therapy. The most investigated types of these cells are endothelial progenitor cells (EPCs), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). Other investigated kinds include cardiac progenitor cells (CPCs), myeloblasts, and induced pluripotent stem cells (iPSCs). EPCs are involved in vascular homeostasis and new blood vessel regeneration. The decrease in their functional cell number is associated with aging and atherosclerotic processes. HSCs are involved in blood coagulation, oxygen transportation, and immune system function, so their senescence leads to blood dysfunction. MSCs exist in many tissues, including bone marrow, adipose tissue, the bloodstream, and cord blood. MSCs have high self-renewal capacity and the ability to differentiate into other kinds of cells, such as adipocytes, chondrocytes, and osteoblasts, depending on their host organ. Although adult stem cells appear to be valuable sources for regeneration, they have limited sources, differentiation, and expansion potential. However, differentiated cells can be reprogrammed to iPSCs and then differentiated to desired cell types.

This manuscript examines protective medicines and supplements which are capable of hindering stem cell senescence. As reviewed in this paper, most of these protective agents increased telomerase activity or decreased oxidative damage via various anti-oxidant mechanisms, which ultimately inhibited cellular senescence. Senescence prevention in the body results in health and longevity. Various medicines inhibit senescence through different mechanisms. As mentioned in this review, 17β-estradiol, melatonin, metformin, rapamycin, coenzyme Q10, N-acetyl cysteine, and vitamin C were the most studied agents in different kinds of stem cells. Although most of these studies were in vitro, we can consider these agents in cell therapy to increase the shelf life and the functional cell number of donated stem cells before transplantation to achieve more clinical success. Moreover, in vitro studies are the first step towards clinical studies. Although more studies are necessary for clinical application, these reviewed agents have been used in the clinical setting for different purposes for a long time; therefore, we only need to evaluate their systemic anti-senescence effects and effective anti-senescence dosages.

We conclude that off-label use of approved medicines and supplements is a convenient, safe, and economical approach to prevent stem cell senescence both in vitro and in vivo. These agents provide a wide range of options based on targeted cells. Since all of them have passed substantial safety trials, we only need to determine their effective dosage to prevent stem cell senescence. Maybe it seems that heterogeneity of administration, patients, and diseases can make repurposing inefficient and time-consuming. Still, in comparison with discovering new anti-senescence agents, this approach is much more economical and accessible. Moreover, performing retrospective studies for each medicine can address these issues.

One logical outcome of a growing ability to treat aging as a medical condition, with therapies that target the underlying causes of aging such as senescent cell accumulation, is that aging will be staged. A patient will be assessed and declared to have stage I aging, or stage III aging, as determined by some combination of factors. The medical community assigns stages to many chronic conditions, an assessment of severity and progression that is used to decide upon treatment strategies. Like those chronic conditions, aging as a whole is the consequence of underlying processes of damage, and has a clear progression. In the paper here, the authors argue that the time has come to set up a staging system for degenerative aging.

In the coming decades, the proportion of older adults in the world will nearly double. Older adults are a heterogeneous population, with many people over the age of 80 continuing to work and travel, while others might be weak, chronically ill, or disabled. A traditional framework for describing different populations of older adults is "young-old," "old," "old-old," and/or "oldest old". Fried's frailty phenotype is a similar three-stage framework in which people are classified as non-frail, prefrail, or frail. Proteomic analysis finds large changes in gene expression at about the age of 40, 60, and 80. However, these frameworks are not adequate to describe the different stages of aging and subpopulations of older adults. Older age is a risk factor not only for normative physiological changes with aging but also for cancer, heart disease, diabetes, dementia, and many other chronic conditions. Genes, disease, and behaviors can pull on the chronological age of a person and make the person appear younger or older with the risk profile ("biological age") of someone younger or older.

Aging can be viewed as a very slow step-wise decline from wellness and independence toward disability, reduced quality of life, and ultimately death. The rhythm of decline of an individual is very personal and depends on the genes, lifestyle, diseases, and geriatric syndromes such as dementia and falls. The biological causes for physiological changes with aging and for the prevalence of age-associated diseases continue to be explored and debated. Dividing the process of aging into phases, and characterization of these phases from various aspects can raise awareness and recognition that old age is not homogeneous or stereotypical as it is often considered. Healthy, active people in their 70's should not be treated like disabled people in their 90's, and active people in their 90's should not be treated like sick, disabled people in their 90's.

Adults in stages one and two of aging (age 60-79) typically remain in the first stage of the four-phase functional scale. The rate of decline to the next phases depends on vascular risk factors, genetics (family history), and social/environmental factors (such as education, career, physical activity, and social engagement or isolation). The goal of medicine is to compress morbidity, allowing old and old-old adults to spend more time in these earlier functional aging phases and less time in the disabled or burdened phase with bothersome symptoms.

Chronological age stratifications may not correlate with medical, functional, emotional, and social changes that an individual may be experiencing for multiple reasons. The chronological age strata used in this study are obviously inexact, but useful. As noted, older adults are heterogeneous and may develop problems earlier than average or later or never. The final chronological age strata in this proposed staging system are older than 85. In fact, there may be substantial differences between 87-year-olds and centenarians. However, at this time, there is insufficient data to describe organ system changes and disease epidemiology in subpopulations like centenarians. With the advancement of modern medicine and with improved preventive care and lower rates of infectious disease, we more often witness an expanding gap between chronological age and biological age. Some people with advanced age have functional and cognitive abilities that are greater than those of younger people.

Link: https://doi.org/10.3389/fpubh.2021.513557

Age-related frailty has a strong inflammatory component. The chronic inflammation of aging is disruptive of tissue function and over time contributes to the loss of muscle mass and strength characteristic of frailty. Some fraction of that chronic inflammation is the consequence of changes in the gut microbiome, the loss of helpful populations and the expansion of harmful, inflammatory populations of microbes. The relationship between age-related changes in the gut microbiome and the age-related decline of the immune system into a state of chronic inflammation is likely bidirectional, but it has been shown that fecal microbiota transplantation from young to old animals improves the situation. This approach should be tested in humans.

As the population ages, frailty syndrome will bring a huge medical burden to society. Previous studies have suggested that gut microbiota imbalance may be a cause of frailty. Animal models and a few human studies have demonstrated that individuals with frailty tend to have increased levels of inflammatory factors (e.g. IL-6, C-reactive protein, and TNF-α) and a chronic inflammatory status. Inflammatory factors have been demonstrated to directly or indirectly reduce key indicators of frailty, such as muscle mass and grip. In addition, gut microbiota imbalance has been demonstrated to be associated with higher expression of inflammatory factors.

Studies have suggested that gut microbiota imbalance leads to enhanced intestinal permeability. This in turn triggers the entry of pathogen-related antibodies like PAMP and DAMP to the circulatory system to subsequently trigger an inflammatory reaction. As a result, investigators believe that the chronic inflammatory status due to gut microbiota imbalance could directly or indirectly give rise to the typical symptoms of frailty (by causing cardiovascular diseases or damaging the musculoskeletal system). In addition, higher levels of inflammatory factors due to gut microbiota may further influence the nervous system of the host via the gut-brain axis to induce neuroinflammation leading to neurodegenerative diseases, i.e., dyskinesia and/or cognitive disorders in patients with frailty.

However, the above assumptions have not been validated in large cohort-based studies. The relationship of gut microbiota imbalance, chronic inflammation, and frailty is not unilateral but complicated and interrelated. Several studies have suggested that chronic inflammation due to gut microbiota imbalance may not be the only cause of frailty. It is worth noting that individuals with frailty are on long-term medication due to preexisting chronic diseases. It has been demonstrated that medications could alter gut microbiome composition. Hence, future studies are necessary to determine whether gut microbiota is a cause of frailty or a result of long-term medication in people with frailty. In addition, factors that may affect the gut microbiota, such as lifestyle, diet, and other health complications, need to be considered comprehensively.

Link: https://doi.org/10.1515/med-2021-0364

The authors of today's open access paper use the provocative question "how young can you die of old age?" as a framing device, a way to consider what is known of the way in which type 2 diabetes and obesity harm people over the course of years and decades. These are, of course, very well studied conditions. A great deal of time has been spent and a great deal of ink spilled on the topic of exactly what excess visceral fat and the pathologies of diabetes do to an individual, at the level of cells, at the level of organs, and, most visibly, to overall health and mortality. What is new, as of recent years, is the understanding that a sizable degree of the pathology of these conditions is mediated by senescent cells.

Factions within the research community have long seen diabetes as a form of accelerated aging, and evidently so given what it does to mice and people. Now, however, one can more literally argue that both obesity and diabetes produce accelerated aging. This is the case because they produce, through raised inflammation, metabolic stresses, and other means, a faster increase in the numbers of senescent cells present in the body. Senescent cells actively cause tissue dysfunction via their pro-inflammatory secretions, and their accumulation is an important contributing cause of degenerative aging. Removing senescent cells produces a narrow form of rejuvenation in mice, and the first senolytic therapies capable of a targeted destruction of senescent cells are undergoing human trials and, in some cases, readily available to self-experimenters.

How young can one die of old age? Our present societies of comfort and calories, with sizable populations of patients who are both obese and diabetic at ever younger ages, seem set on chasing an answer to that question.

Senescence and Type 2 Diabetic Cardiomyopathy: How Young Can You Die of Old Age?

Type 2 diabetes (T2D) is an enormous global medical and economic burden and its prevalence is on the rise with an ever ageing, increasingly obese population. It is a debilitating, chronic disease affecting almost half a billion people worldwide. T2D is often presented as a multimorbid disease cluster as it is a major risk factor for the premature onset of multiple age-related conditions such as chronic kidney disease, stroke, impaired wound healing, infection, depression, cognitive decline, and inflammation. T2D is especially a high-risk factor for cardiovascular mortality and cardiac remodelling, with coronary vessel disease and atherosclerosis being primary reasons for the increased incidence of cardiovascular dysfunction.

T2D is more common in an ageing host and "accelerated ageing" has been proposed as a pathogenic mechanism, including cell ageing leading to a complex phenotype termed senescence. Over the past 30 years, cellular senescence has been identified as a possible trigger of general tissue dysfunction and ageing phenotypes. Senescent cell load is low in young individuals but increases with ageing. When senescent cells accumulate they contribute to tissue dysfunction in the context of ageing and related pathologies. Several studies have shown that senescence accumulates in multiple cardiovascular cell populations and is linked with cardiovascular diseases (CVD) including heart failure (HF).

In T2D, comorbidities including obesity, hypertension, and atherosclerosis all have the ability to increase the number of senescent cells. However, the relationship between T2D and myocardial senescence may be both complex and complementary. The microenvironment of systemic metabolic stress in T2D could be permissive to the development and accumulation of senescent cells. On the other hand, senescent cells may contribute to the cardiac parenchyma dysfunction and comorbidities observed in T2D. Overall, it is likely that these complex interactions might lead to a malicious positive feedback in which systemic metabolic dysfunction in the early stages of T2D leads to immune cell senescence that in turn contributes to the worsening of cardiac function and tissue metabolism, which further increases the formation of senescent cells while decreasing their removal.

The aim of this review is to spark discussion and help generate hypotheses that may link senescence to cardiometabolic complications in T2D. We hypothesize that clearing senescent pro-inflammatory immune cells or targeting the SASP (Senescence-Associated Secretory Phenotype) may present opportunities for the development of revolutionary therapies for diabetic cardiomyopathy and its complications, leading to advances in its treatment and prevention. Furthermore, we review our current understanding of the metabolic remodelling of both heart tissue and senescent immune cells in T2D, and we discuss potentially fundamental mechanisms by which these metabolic responses influence and intersect each other to ultimately determine the prognosis of the myocardial inflammation.

This open access paper reviews the relationship between DNA damage and inflammation in the specialized environments that support stem cell populations. Aging produces many changes that lead to reduced stem cell function. Changes in the niche, the supporting cells that help to ensure stem cells retain their function, are of increasing interest to the research community. The chronic inflammation of aging is also an area of growing study. The inflammatory response to rising levels of DNA damage with age in stem cells and stem cell niches is an interesting overlap between these two parts of the field.

DNA damage profoundly affects the inflammatory microenvironment where stem cells reside, which can have detrimental consequences for their maintenance and long-term function. Indeed, it has been shown that DNA damage-induced immunostimulatory events can lead to tissue-specific stem cell exhaustion leading to degenerative conditions. Conversely, the release of specific cytokines can also positively impact tissue-specific stem cell plasticity and regeneration of damaged tissues in addition to enhance cancer stem cell activity leading to tumor progression.

This review provides an overview of the main biological mechanisms linked to changes in the stem cell microenvironment and activation of immune processes upon DNA damage induction. Although recent findings have brought to light new insights into these DNA damage-related inflammatory events, some questions remain unanswered. For instance, it is still not clear how to exploit the production of inflammatory cytokines in order to promote on one side immunostimulatory responses against the tumor and on the other side immunosuppressive responses against aging-related degenerative conditions. Especially since the activation of DNA and RNA sensors might change depending on the specific stimulus and cell type.

DNA damage-induced senescence plays a pivotal role in cell cycle arrest and can be used as a barrier against tumor expansion; however, due to the accompanying senescence-associated secretory phenotype (SASP), it is also responsible for loss of tissue function, aging-related diseases and tumor progression. Therefore, further studies are required to understand how to properly modulate the exposure to SASP factors toward the promotion of a regenerative state and against detrimental effects, such as paracrine senescence of neighboring cells and chronic inflammation. Furthermore, it would be interesting to explore how different types of DNA damage can influence senescence and its SASP phenotype in different adult stem cells.

Further understanding of DNA damage immunomodulatory mechanisms, cell- and stimulus-specific variability might unravel novel strategies to regulate the stem cell microenvironment. As mentioned in this review, genotoxic stress can affect the stem cell microenvironment leading to stem cell exhaustion, likely through a combination of a decline in cell number and functional capacity, with the emergence of aging-related pathologies. On the other hand, due to their self-renewal properties, cancer stem cells are also affected by DNA damage and the associated inflammatory microenvironment, which can worsen tumor control and treatment efficacy. Understanding the mechanistic links between stem cell properties and microenvironmental changes initiated upon DNA damage will be critical to counteract the functional decline of adult stem cells in aging-related diseases and effectively diminish cancer stem cell activity and expansion.

Link: https://doi.org/10.3389/fcell.2021.729136

Restricting calorie intake stresses and kills cancer cells, for a variety of reasons both direct (cancer cells have high metabolic requirements) and indirect (calorie restriction improves immune function). Calorie restriction, a flat reduction in calorie intake, and intermittent application of fasting or fasting mimicking diets have been shown to improve outcomes in animal studies and human trials. Which of these options are better, however? Researchers here provide evidence to suggest that calorie restriction is better than a fasting mimicking diet when it comes to slowing cancer.

Calorie restriction (CR) is without a doubt the most robust non-pharmacological intervention against induced and spontaneous cancers. Numerous studies have shown the ability of daily CR to delay neoplasia in multiple tissues and inhibit the growth of chemically induced and spontaneous tumors, including breast cancer. Despite the far-reaching improvements in health and reduction in cancer incidence associated with daily CR, its long-term implementation is not a feasible approach for most humans.

Implementation of protocols that involve intermittent fasting (IF) as clinically viable alternatives to daily CR has been shown to promote similar improvements in metabolic markers and provide protection against cancer growth. However, it is unclear whether optimization of diet composition in these less stringent feeding regimens would provide equal or better protection against tumor growth as daily CR. In this work, we begin to address this knowledge gap by directly assessing the effects of cycles of fasting-mimicking diet (FMD) vs. daily CR.

Using a breast cancer model in mice, we compare the potency of daily CR to that of periodic caloric cycling on FMD or an isocaloric standard laboratory chow against primary tumor growth and metastatic burden. Here, we report that daily CR provides greater protection against tumor growth and metastasis to the lung, which may be in part due to the unique immune signature observed with daily CR. Earlier work has suggested that upregulation of immune-fighting T-cells, specifically CD8+ and CD4+, leads to slower tumor growth and is a good indicator of survival outcome. Conversely, depletion of CD8+ partially eliminates CR-mediated delay in tumor growth. Here, our findings show that daily CR led to an increase in CD8+ and CD4+ cells in peripheral tissues. This is a key observation because T cell exhaustion is one of the major drivers of tumor progression and is indicative of poor prognosis in breast cancer patients.

Link: https://doi.org/10.1038/s41467-021-26431-4

How to crowdfund the development of products, with variants such as crowdfunding to purchase equity in a company rather than a product, is a solved problem. Enabled by the internet, crowdfunding clearly works well when those who provide the funds will obtain something of concrete value in the near term as a result, be it a product or equity. Unfortunately, the established approaches to crowdfunding, exemplified by platforms such as Kickstarter, fail to work at scale when the goal is to fund scientific research. People have tried, numerous times, to make a Kickstarter for scientific research, with Experiment being perhaps the best of the resulting failures. The fundamental issue is that without the quid pro quo of a product or equity, some concrete value obtained, the incentives and feedback loops that make Kickstarter and its ilk work are absent.

The only type of crowdfunding that works to fund scientific research is small in scale, a matter of enabling passionate special interest communities to fund favored projects in ways that are not appreciably different from pre-Internet non-profit pledge drives. I'd like to think that our own, still comparatively small longevity-focused community has proven to be fairly adept at this sort of thing, with groups such as Lifespan.io enabling a number of crowdfunded projects to proceed over the years.

Still, much more is needed. Enter VitaDAO, which is structured in a way made possible by blockchain technology, and which probably could not have arisen absent the present fervor for blockchain implementations such as cryptocurrencies. The core ideas on how to make crowdfunding of research actually work, implemented by VitaDAO, could just as well be put into practice by an ordinary non-profit organization with centralized administrative software, absent a blockchain implementation, however. One could argue that the primary use for blockchains here was to enable VitaDAO to start out with a very large warchest of funds via a token launch, without the need to convince the traditional, highly skeptical sources of non-profit funding to back such a project. That is important!

The core ideas appear good, and VitaDAO does indeed have deep pockets with which to fund research. Stripped down, the concept is to build a self-sustaining long term research fund in which: (a) people buy into the organization, putting in funds to obtain voting rights on which research projects to fund; (b) deals are struck with universities and startups to provide funding for specific projects in exchange for a cut of later patents, royalties, equity, or similar flows of funds; (c) successfully commercialized research will lead to funds flowing back into VitaDAO for investment into research. There are numerous ways in which VitaDAO could perhaps help to make such commercialization easier in the future, such as by building a marketplace akin to, say, BioNeex or similar.

The additions to this core idea made possible by the use of blockchain technologies revolve around decentralization of governance, keeping track of ownership, and perhaps later allowing trading on interests in research programs. But the current administrators have structured the organization and its work, quite carefully, in order to avoid any appearance of conducting a stock offering or producing a trading platform. Regulatory compliance is very important in this part of the field. The core of it is the virtuous feedback loop of funds, governance, and income from successful commercialization - a slow loop, given the timelines for moving from research to clinical, but there nonetheless.

VitaDAO is focused on the longevity industry and aging research aimed at lengthening the healthy human life span, an endeavor with strong support in the broader cryptocurrency community, as illustrated by large donations to organizations such as the SENS Research Foundation in recent years. But there is no reason as to why this model couldn't work for any arbitrary field of research with a strongly interested community of laypeople cheering it on. Environmental science, for example, or many others.

VitaDAO is in its early stages, but they have passed the first few most difficult hurdles. Firstly, they have obtained significant funds to deploy to research programs via a token offering. Secondly they have structured the first deals with a university, laying the groundwork for future similar agreements. After that, the rest is largely a matter of attracting attention and keeping the wheels turning. Any researcher or entrepreneur who is looking for a $250,000 grant in exchange for a modest interest in later patents or royalties can step up today and pitch the VitaDAO community and leadership on their project. We live in interesting times!

VitaDAO

VitaDAO is a new cooperative vehicle for community-governed and decentralized drug development. Our core mission is the acceleration of R&D in the longevity space and the extension of human life and healthspan. To achieve this, VitaDAO utilizes a combination of novel governance (distributed autonomous organizations - DAOs), digital assets (non-fungible tokens - NFTs), and financial market frameworks (automated market makers - AMMs).

Value creation in biopharma centers heavily around intellectual property assets and patents as core drivers for funding and innovation. Yet intellectual property ownership as a business model has barely evolved in the past century. Current biopharma business models carry severe limitations and R&D inefficiencies that cost those who should be the core stakeholders: patients and researchers.

We believe the future of biopharma is transparent, collaborative, and open source. VitaDAO is an open cooperative that anyone can join, with the goal to acquire, support and finance new therapeutics and research data in the longevity space. The VitaDAO collective will directly hold legal IP rights to these projects and may develop a growing portfolio of assets represented as NFTs. Members of the public can join VitaDAO and become owners of its IP by purchasing VITA tokens through contributing funds, work, or valuable research data or IP assets. Ownership and governance of VitaDAO requires VITA tokens. VITA tokens enable their holder to engage in decision-making and governance of VitaDAO's research, signal support for specific initiatives, and govern its data repositories and IP portfolio.

VitaDAO will acquire and commission research, as well as own, develop and monetize the resulting intellectual property assets. VitaDAOs portfolio consists of: 1) NFTs representing intellectual property, patents and licenses to therapeutic research projects; 2) Data assets generated by funding R&D around its research projects and NFTs. Vetted longevity research projects will request funds from VitaDAO, and members will vote to grant or raise those funds in exchange for ownership in the resulting IP. To fund more research and to provide long term funding for the DAO operations, there are several options to monetize owned data and IP. VitaDAO can enter co-development deals with private companies or other DAOs. in single or multi-license agreements, VitaDAO could license data and IP to 3rd parties, or could sell to the highest suitable bidder.

That the INDY gene can influence life span was one of the earlier discoveries made once researchers begin to manipulate the life span of short-lived species, spurred by the study of slowed aging via calorie restriction, and searching in earnest for the mechanisms by which metabolism determines the pace of aging. Progress is very slow in this part of the scientific community. Reviews of what is known of INDY are not that different today then they were a decade ago, and it remains an open question as to how relevant this is to humans. That INDY has effects related to preserved intestinal function in flies may just be a reflection of the great importance of the intestine in fly aging, and not an indication of what to expect in mammals.

Reduced gene expression of fly Indy and its worm homologues extends their life span by altering metabolism in a manner similar to calorie restriction (CR). Fly INDY and homologues in worms and mammals share a preference for transporting citrate. By regulating cytoplasmic citrate levels, INDY acts as a metabolic regulator in modulating glucose and lipid levels, and energy production in mitochondria. Metabolic changes associated with Indy reduction in the fly midgut results in dramatic changes in midgut physiology that lead to preserved intestinal stem cell (ISC) homeostasis. This is vital for replacement of damaged cells and the maintenance of midgut function illustrated by preserved intestinal integrity.

Indy reduction extends lifespan in male and female flies, but the effects of Indy reduction on ISC homeostasis have only been studied in female flies. Male and female flies have different gut pathologies and respond differently to stress and CR, with males having a delay in age-related gut pathology and lower ISC proliferation, while females respond better to stress and CR. Considering these differences, it would be of interest to determine the effects of Indy reduction on the midguts of male flies.

ISC homeostasis is regulated by multiple signaling pathways including IIS, Notch, EGF, Wnt/wingless, BMP/Dpp, JNK, and JAK/STAT, among others. It would be important to assess the status of different signaling pathways in flies with reduced Indy expression, as metabolic changes might delay age-associated activation of these pathways and could contribute to preservation of ISC homeostasis and longevity.

The data reviewed here support the role of INDY as a metabolic regulator: Indy expression changes in response to nutrient availability and requirements of the organism, which, by regulating citrate levels, controls energetic status of the organism to maintain tissue-specific metabolic requirements leading to preserved organismal health and homeostasis. Reduced INDY levels in the midgut could then prevent age-related ISC hyperproliferation by reducing the available energy for proliferation.

Link: https://doi.org/10.3390/metabo11100705

Telomerase gene therapies usual deliver telomerase reverse transcriptase (TERT), which might be thought of as the most important part of the full telomerase complex. Most research has focused on the ability of telomerase to lengthen telomeres, and where overexpression of telomerase is seen to extend life span in animal models, lengthening of telomeres is the mechanism most explored by the research community. However, TERT also acts on mitochondria. Here, researchers advance the understanding of how mitochondrially localized TERT can improve mitochondrial function. Given the importance of mitochondria in aging, it is an interesting question as to the degree to which telomere lengthening versus improved mitochondrial function produce the improved health, lower cancer incidence, and extension of life span observed in mice as a result of telomerase gene therapies.

The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) has protective functions in the cardiovascular system. TERT is not only present in the nucleus, but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection and appropriate tools are missing to dissect this. We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart as well as cellular functions of cardiomyocytes, fibroblasts, and endothelial cells were determined.

All mice were phenotypically normal. While respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wildtype mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after one, two and four weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning.

In conclusion, mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. We conclude that mitochondrial TERT is responsible for cardioprotection and its increase could serve as a therapeutic strategy.

Link: https://doi.org/10.1161/CIRCULATIONAHA.120.051923

There is, in general, a growing appreciation of the relevance of previously dismissed mechanisms of damage in the pathology of common age-related disease, such as cardiovascular conditions. The biggest challenge in aging and age-related disease is perhaps less the identification of relevant mechanisms, but rather understanding the relative importance of those mechanisms. Take transthyretin amyloidosis for example, the accumulation of misfolded transthyretin aggregates that occurs over the course of aging. It is only comparatively recently that the research community has established that this universal process of aging contributes meaningfully to a sizable fraction of heart failure.

Work on treatments for the inherited, accelerated version of transthyretin amyloidosis caused by mutation has produced a drug, tafamidis, that can at least slow the condition, and can also be applied to the vast majority of individuals lacking that mutation. Joining the dots will lead to this treatment being used in heart failure patients with notable amyloidosis. But again, this is all very recent. The lesson to take away from all of this is that improvements in the understanding of even well studied, common age-related conditions are ongoing. Slowly, the mechanisms of aging are being linked to conditions, and that leads to progress towards the development of therapies.

Cardiovascular Diseases That Have Emerged From the Darkness

It is important for both the patient and physician communities to have timely access to information recognizing rapid progress in the diagnosis and treatment of familiar but relatively uncommon cardiovascular diseases. Patients with three cardiovascular diseases, i.e. hypertrophic cardiomyopathy, pulmonary arterial hypertension, and transthyretin (TTR) cardiac amyloidosis (ATTR), once considered rare without effective management options and associated with malignant prognosis, have now benefited substantially from the development of a variety of innovative therapeutic strategies. In addition, in each case, enhanced diagnostic testing has expanded the patient population and allowed for more widespread administration of contemporary treatments.

In hypertrophic cardiomyopathy, introduction of implantable defibrillators to prevent sudden death as well as high-benefit:low-risk septal reduction therapies to reverse heart failure have substantially reduced morbidity and disease-related mortality (to 0.5% per year). For pulmonary arterial hypertension, a disease once characterized by a particularly grim prognosis, prospective randomized drug trials with aggressive single (or combined) pharmacotherapy have measurably improved survival and quality of life for many patients. In cardiac amyloidosis, development of disease-specific drugs can for the first time reduce morbidity and mortality, prominently with breakthrough ATTR-protein-stabilizing tafamidis.

In conclusion, in less common and visible cardiovascular diseases, it is crucial to recognize substantial progress and achievement, given that penetration of such information into clinical practice and the patient community can be inconsistent. Diseases such as hypertrophic cardiomyopathy, pulmonary arterial hypertension, and ATTR cardiac amyloidosis, once linked to a uniformly adverse prognosis, are now associated with the opportunity for patients to experience satisfactory quality of life and extended longevity.

While they cannot explain aging as a whole, single cause theories of aging can be useful tools to frame discussion and investigation aimed at better understanding aging and its evolution. The theory presented here is aging viewed through the lens of cancer, two entwined processes. Aging is viewed as largely a consequence of tumor suppression mechanisms that evolved to keep cancer at a low enough incidence for successful selection and continuation of the species. Cancer and evolution are themselves in a dynamic, competing equilibrium. Evolution requires a certain minimal rate of spontaneous mutation, while cancer thrives on those mutations; the higher the rate, the higher the risk of cancer. An ever more complex arms race results, eventually resulting in the varied pace of mutation and aging, types of tumor suppression mechanisms, and incidence of cancer found across diverse species today.

Single cause theories of aging remain important in aging research. One obvious reason for this is the need for simplification. Another reason is the necessity to at least break up the aging process into potentially treatable parts, even if no single treatment can be expected to do much. Somatic mutations have long been proposed as a cause of aging and genomic instability is one of the four primary hallmarks of aging. The tumor suppression theory of aging outlined here differs from previous theories in that clonal expansion and malignancy is proposed as the relevant consequence of somatic mutation and that impairment, loss of cellular function, or cell death as a consequence of somatic mutation is largely irrelevant. To counter the tumorigenic potential of clonally expanding cells, we have evolved tumor suppression mechanisms that remove or limit proliferation of stem cells. Accumulating senescent cells and loss of capacity for self-renewal and repair eventually cause the phenotypes we experience in very old age.

Obesity and caloric restriction accelerate and decelerate aging due to their effect on cell proliferation, during which most mutations arise. Most phenotypes of aging are merely tumor-suppressive mechanisms that evolved to limit malignant growth, the dominant age-related cause of death in early and middle life.

Cancer limits life span for most long-lived mammals, a phenomenon known as Peto's paradox. Its conservation across species demonstrates that mutation is a fundamental but hard limit on mammalian longevity. Cell senescence and apoptosis and differentiation induced by oncogenes, telomere shortening, or DNA damage evolved as a second line of defense to limit the tumorigenic potential of clonally expanding cells, but accumulating senescent cells, senescence-associated secretory phenotypes and stem cell exhaustion eventually cause tissue dysfunction and the majority, if not most, phenotypes of aging.

If the tumor suppression theory of aging would be correct, the only way to retard human aging would be a reduction of somatic mutation. Preventing aging would be the same as preventing cancer. Unfortunately, reduction or prevention of somatic mutation is something that remains thoroughly out of reach of current medical technology. The problem of aging will probably defy the assault of human ingenuity for some time to come.

In the meantime, removal of senescent cells seems to offer a reasonable chance of alleviating many phenotypes of very old age. Cell senescence is antagonistically pleiotropic, and accumulation of senescent cells probably an evolutionary accident brought about by the unforeseen increase in average human life expectancy. Together with a second antagonistically pleiotropic phenomenon, the age-related emergence of systemic and excessive chronic sterile inflammation, these two phenomena might be mutually reinforcing evolutionary accidents responsible for many of the pathogenic processes promoting the irreversible functional decline of very old age. Even though they do not prevent aging per se, in terms of looking at realistic strategies for increasing human health span, these two processes are, compared to the primary prevention of mutation, probably lower hanging fruit and offer plentiful possibilities in postponing the dreaded symptoms of old age.

Link: https://doi.org/10.1016/j.mad.2021.111583

In recent years, researchers have established that machine learning approaches can be used to produce any number of clocks from biological data that shifts with age, finding patterns that match chronological or biological age to a great enough accuracy to suggest that they can be useful assays for the assessment of potential age-slowing and rejuvenating therapies. It remains an open question as to whether and how exactly the assessed patterns correlate to the specific forms of molecular damage that cause aging, or to any of the specific downstream consequences of that damage. Scientists here raise the possibility that much of the epigenetic change of aging may not in fact be as useful as a basis for measurement as thought, and suggest that more fundamental research is required in order to robustly connect clocks with specific processes of aging.

Our meta-analysis of the largest available age-annotated methylation dataset to date found: 1) as much as one fifth of the measured cytosines contains age-predictive methylation patterns; 2) tissues show largely similar aging patterns despite having methylated regions that define their identity; 3) epigenetic clock sites are enriched in intergenic regions, gene enhancers, and sites near expression quantitative trait loci (eQTLs) and 4) are depleted in the regions generally thought to have the largest direct impact upon gene expression (e.g., CpG Islands and gene promoters); 5) patients with age-correlated diseases did not appear significantly age-accelerated according to the chronological epigenetic clock.

The fact that many different sites can be used to create an epigenetic clock with minimal impact on predictive performance argues against the idea that methylation changes are either programmed or individually important. Yet, because the clock is robustly predictive and age-related methylation changes are mostly similar between tissues, this argues against entropy as a driving force. This could be reconciled by hypothesizing some genomic regions and/or features receive less methylation maintenance than others.

Perhaps the changes occur in regions of the genome where they have no consequence, and instead, vary with absolute time such as in determining speciation time using pseudogene mutation rates. This "pseudomethylation" would be problematic for modeling aging biology, as they would likely not respond to aging intervention. Methylation maintenance mechanisms (e.g., DNMT1) serve as a counterbalance against entropy. However, if some genomic regions are less maintained than others, then we would expect the probability of a methylation state change with age to be correlated with the degree to which it is subject to methylation surveillance and maintenance. Because maintenance costs energy, it is reasonable to hypothesize the degree of maintenance correlates with the adverse impact an unregulated change in methylation would cause. If so, the probability a site's methylation will vary with age would inversely correlate with its impact on an organism's survival.

Given that methylation changes with age are robust across tissues, yet small in magnitude, leads the field to question whether the "ticking" that drives them is due to changes in cell population composition, such as a reduction of pluripotent stem cells or an increase in senescent cells within every tissue, or possibly high magnitude effects in rare cell populations (e.g., immune cells in the central nervous system compared to astrocytes or neurons). In either case, it is not clear whether the phenomenon driving ticking clock sites is due to healthy compensatory changes or deleterious drift toward age-related fragility.

In summary, the predictive power of the epigenetic clock is robust, but such a large fraction of the genome can be used to predict, the magnitude of the changes is small, and these regions tend to be depleted near genes. This leads us to hypothesize that the pan-tissue predictive loci are more likely to be molecularly "silent" methylation changes that accrue outside of strong regulatory regions due to entropy in methylation maintenance, which must be explored in the future studies. Furthermore, if current models inconsistently annotate patients with age-related diseases as "age-accelerated" and the confidence by which one can declare a sample age-accelerated is small, this argues against the idea that epigenetic clocks can disentangle biological age from chronological age.

Link: https://doi.org/10.1111/acel.13492

Cambrian Biopharma started as a venture fund, but is a business development company now. The most important difference between those two business models is that a fund waits for startup companies to form and be ready for investment, while a development company sets out to create startups. The longevity industry is still comparatively small, and the arrival of new investment opportunities is thought by many, including the Cambrian Biopharma principals, to be too sparse to sustain larger funds. The solution, in an environment rich with promising scientific projects, is to create those opportunities: bring together scientists and entrepreneurs, license the relevant technologies from the universities, and fund the resulting startup company.

As noted in today's publicity materials, Cambrian has assembled a sizable fund, joining other large funds and business development companies such as the Longevity Vision Fund, Life Biosciences, Juvenescence, and Kizoo Technology Ventures. As Juvenescence and the Longevity Vision Fund illustrate, much of the investment in the early stages of growing the longevity industry has gone to less promising projects, or biotech and medical opportunities unrelated to the treatment of aging. Running a fund has a timeline built in, and investments must be made on a schedule. This, coupled with the small size of the longevity industry, is why we see a sizable fraction of the larger investment ventures in this space formed as business development companies rather than pure venture funds. In general, one should expect a business development company run by knowledgeable people to be more likely than a standard venture fund to invest in projects that are relevant to treating aging as a medical condition.

Cambrian Biopharma's $100 million Series C will advance healthspan-boosting therapeutics with financing co-led by Anthos Capital and SALT Fund

Longevity biotech Cambrian Biopharma has announced the close of an oversubscribed Series C financing, which raised $100 million. The financing was co-led by Anthos Capital and SALT Fund, with participation from existing investors Apeiron Investment Group, Future Ventures, Moore Capital and others, to develop therapeutics to combat the biological drivers of aging, treat and prevent age-related diseases and lengthen healthspan. With this financing, Cambrian has raised approximately $160 million since the company was founded in 2019.

Age-related diseases account for more than two-thirds of all deaths worldwide, taking 41 million lives every year, or nearly one death every second. Existing approaches to these diseases are almost exclusively reactive - waiting for people to get sick and only then using the rapidly expanding knowledge of biology to try to treat very sick patients. Cambrian believes the future of medicine lies in approaching these diseases proactively, removing damage at a cellular level before a person becomes a patient. Each therapeutic in Cambrian's pipeline targets a different type of damage that builds up with age and will be tested for clinical safety and efficacy in an acute indication before using running multi-disease prevention trials.

Cambrian operates as a Distributed Development Company designed to bridge the gap that exists today between academic discovery and drug development. This unique drug discovery model combines the advantages of a venture capital firm and a big pharmaceutical company, with the nimbleness of a biotech startup. Cambrian's hypothesis-driven approach and industry-leading pipeline of drug candidates provides for reduced risk and multiple "shots on goal". To date, Cambrian has 14 novel therapeutics in development across its majority-held pipeline companies. Proceeds from the financing will support the advancement and expansion of a diversified pipeline of novel therapies, each with the potential to both treat and prevent age-related diseases, with the goal of extending human healthspan. Cambrian expects to initiate clinical trials for three programmes in the next 18 months.

Glutathione is a mitochondrial antioxidant, and additional antioxidant capacity in mitochondria appears to be beneficial to long-term health, improving mitochondrial function and overall health. Mitochondria conduct the energetic process of producing ATP, used to power the cell, with a flux of oxidative molecules as a side-effect. With age, mitochondria tend to become less efficient and produce more oxidizing molecules, harmful to the cell. Glutathione levels decline with age, which may contribute to this age-related mitochondrial dysfunction.

Oral supplementation with gluthatione doesn't have any effect, unfortunately, but researchers recently published a small study of supplementation with large amounts of glutathione precursors. This caused increased gluthatione manufacture, increased glutathione in blood samples, and measurable benefits to health in old individuals. Here, researchers provide evidence for an iontophoresis approach for the delivery of glutathione through the skin via an electrical field, an intriguing option to be compared against intravenous administration when considering relative costs and benefits.

Glutathione (GSH) is the most abundant antioxidant in human cells. Reactive oxygen species (ROS) produced in the body can promote oxidative damage to cells and may cause genomic instability and mitochondrial dysfunction, two hallmarks of aging. The concentration of GSH has been shown to decrease with aging, resulting in reduced antioxidant activity in cells. Consequently, lower GSH levels have been associated with an increased risk of aging-associated diseases. Relatively higher blood levels of GSH, on the other hand, are associated with improved physical and mental health in older individuals. Supplementation of GSH may, therefore, protect against age-related morbidity and mortality.

In recent years, intravenous supplementation has become a popular method to restore GSH levels. It is an effective method but has its limitations as it is only accessible in a specialty clinic setting and is expensive and inconvenient for patients. Two aging patients with low serum GSH levels were supplemented with GSH in our clinic using a non-invasive drug delivery device, the IontoPatch, to deliver GSH through the skin. The IontoPatch technology uses bipolar electric fields, iontophoresis, to deliver molecules across the skin into the underlying tissue. Iontophoresis is widely used in physical therapy for localized treatment of pain and inflammation.

A 1 mL dose of a 200 mg/mL saline solution of GSH was added to the patch's negative electrode for each treatment. The patch was applied on the upper arm's skin and was worn for six consecutive days for at least four hours each day. Serum levels of GSH were assessed at baseline and days 7 and 23 after treatment was initiated. In both cases, serum GSH levels increased after seven days of treatment (64.4% and 21.8%). Serum GSH levels then decreased between days 7 and 23 to 44.5% and 17.2% above baseline. There were no adverse events reported in either case. More extensive studies should be conducted to determine the pharmacokinetics, safety of long-term supplementation, and supplementation health benefits.

Link: https://doi.org/10.7759/cureus.17803

Researchers here put forward an interesting view of the progression of Alzheimer's disease, in which abnormal modes of brain activity are part of a feedback loop that between inflammation and pathological protein aggregation. Evidence strongly suggests that chronic inflammation in brain tissue is an important component of neurodegenerative conditions, and the aggregation of altered proteins such as tau is both caused by inflammation and contributes to it. It is interesting to see that view expanded out to encompass the neural activity of the brain as a part of the downward spiral of interacting dysfunctions.

Scientists have known for a while that Alzheimer's disease is associated with chronic inflammation in the brain. A driver of this inflammation appears to be the accumulation of amyloid proteins in the form of "plaques," a neuropathological hallmark of the illness. In a new study, researchers identified non-convulsive epileptic activity as another critical driver of chronic brain inflammation in an Alzheimer's-related mouse model. This subtle type of epileptic activity also occurs in a substantial proportion of people with Alzheimer's disease and can be a predictor of faster cognitive decline in the patients. "One way this subclinical epileptic activity may accelerate cognitive decline is by promoting brain inflammation."

Researchers discovered that, when they reduced epileptic activity in the mouse brain, one of the inflammatory factors most affected was TREM2, which is produced by microglia, the brain's resident immune cells. People with genetic variants of TREM2 are two to four times more likely to develop Alzheimer's disease than people with normal TREM2, but scientists are still trying to decipher the precise roles this molecule plays in health and disease.

The scientists first showed that TREM2 was increased in brains of mice with amyloid plaques, but reduced after suppression of their epileptic activity. To find out why, they examined whether TREM2 affects the susceptibility of mice to low doses of a drug that can cause epileptic activity. Mice with reduced levels of TREM2 showed more epileptic activity in response to this drug than mice with normal TREM2 levels, suggesting that TREM2 helps microglia suppress abnormal neuronal activities.

"TREM2 has been primarily studied in relation to pathological hallmarks of Alzheimer's disease such as plaques and tangles. Here, we found that this molecule also has a role in regulating neural network functions. The genetic variants of TREM2 that increase the risk for Alzheimer's disease appear to impair its function. If TREM2 doesn't work properly, it could be harder for immune cells to suppress neuronal hyperexcitability, which in turn might contribute to the development of Alzheimer's disease and accelerate cognitive decline."

Link: https://gladstone.org/news/alzheimers-disease-may-cause-vicious-circle-between-brain-network-and-immune-cell-dysfunctions

As you may know, I co-founded Repair Biotechnologies, a company presently focused on developing an approach to rapidly reverse the cholesterol content of atherosclerotic lesions, a goal that is impossible to achieve using the existing panoply of treatments for atherosclerosis. We use gene therapy techniques to provide cells with the ability to safely break down excess cholesterol, enabling the removal of pathological levels of intracellular cholesterol and localized deposits of extracellular cholesterol that characterize conditions such as atherosclerosis (in blood vessel walls) and NASH (in the liver). Atherosclerosis is an important consequence of aging: the structural weakness and narrowing in blood vessels caused by the growth of cholesterol-laden lesions is the cause of death for a quarter of humanity. Closer to half of humanity were cancer somehow removed from the human condition.

The Foresight Institute runs salons these days, and publishes a range of interesting presentations from people in the biotechnology, molecular nanotechnology, and artificial general intelligence fields. Earlier this year, the Foresight Institute staff were kind enough to invite me to present on the work taking place at Repair Biotechnologies, and the understanding of atherosclerosis that informs that work.

I titled the presentation "Atherosclerosis, the As Yet Undefeated Monster" in part as a reaction to a certain type of conversation that recurs when talking to venture capitalists. Many biotech investors, and others too, seem to think that atherosclerosis is a solved problem, and therefore a poor choice of field in which to see a return by supporting the development of new approaches. Many of the best-selling small molecule drugs are statins that have been deployed for decades to treat atherosclerosis by lowering LDL cholesterol in the bloodstream. Physicians reflexively prescribe statins to older individuals. Everyday people obsess about their blood cholesterol levels. New LDL cholesterol lowering drugs that employ modern technologies such as monoclonal antibodies and siRNA are being approved for use on an ongoing basis.

Yet in this environment of decades of ever more attention given to the reduction of LDL cholesterol levels in the bloodstream, atherosclerosis still kills a quarter of humanity. Atherosclerosis is very far removed from being a solved problem! There is an enormous unmet need and ongoing mortality, on a par with the global burden of cancer.

LDL cholesterol reduction as a basis for therapy can lower late life mortality by 20% at most, with many large, robust clinical trials failing to obtain even that degree of benefit. It just isn't the right mechanism if the goal to produce a cure. It doesn't matter how efficiently a therapy reduces LDL cholesterol in the bloodstream, that 20% mortality reduction appears to be a ceiling. PCSK9 inhibitors do no better than statins when it comes to lowered mortality following control of LDL cholesterol, despite being a much more modern and capable technology. I feel that perhaps the research and development community has been encouraged in its near monomaniacal fixation on lowering LDL cholesterol by the discovery of human gene variants (in PCSK9, ANGPTL3, and so forth) that result in lower LDL cholesterol and up to 50% lower cardiovascular mortality. But that result is obtained due to a full lifetime of lowered LDL cholesterol. Therapies built on those discoveries cannot match that outcome.

Atherosclerotic lesions grow over time at a pace that is influenced by LDL cholesterol levels, by the pace at which cholesterol arrives from the bloodstream to a lesion. Reducing that pace via LDL cholesterol lowering therapy cannot reverse established lesions, or even stop them from further growth and eventual rupture, however. Once a lesion is established, the more important mechanism is the dysfunction of the macrophage cells responsible for clearing cholesterol from blood vessel walls. Those cells are overwhelmed, become inflammatory, attract more macrophages, and die, adding their mass to the lesion. The lesion becomes a macrophage graveyard.

To actually reverse atherosclerotic lesions, to actually produce a treatment that could legitimately be called a cure for atherosclerosis, one needs to protect the function of macrophages in the hostile, inflammatory, cholesterol-laden atherosclerotic plaques. If macrophages can be made invulnerable to excess cholesterol, and other harms such as oxidized LDL particles, then given enough time they will do their jobs and repair the blood vessel wall. That is our goal at Repair Biotechnologies, to make a real and meaningful inroad towards that goal.

Atherosclerosis: The As Yet Undefeated Monster

So why are macrophages not able to do their job later in life? We need to understand cholesterol transport first. Cholesterol isn't created or destroyed in near all cells, it is rather ingested and excreted. Cells don't break down or get rid of the cholesterol they don't want locally, they hand it off to other cells and parts of the system when they no longer need it. Cholesterol is created in the liver, gets stuck onto LDL particles and goes into the bloodstream, gets stuck in a blood vessel wall, macrophages eat it and then throw it back into the bloodstream to attach to the HDL particles that flow back into the liver. LDL and HDL particles do pretty much the same work when you're young and old, it's the macrophages that stop doing their job. So why exactly do they stop doing their job? Due to a variety of issues - namely systemic inflammation, systemic oxidative stress, and too much cholesterol, although the last is probably not the worst of those three. What this leads to is a feedback loop. Your plaque is a macrophage graveyard, and the signaling of that draws in even more macrophages trying to fix the problem. That is the underlying cause of atherosclerosis.

As you are aware, there's an entire research community and pharmaceutical industry focused only on lowering LDL cholesterol - taking that part of cholesterol transport from the liver to the rest of your body and turning it down. This probably helps a little, since you're reducing oxidized LDL and altered cholesterols, you end up with less altered cholesterol in the plaques, so you're giving macrophages a little bit more breathing room. But it doesn't work to a great enough degree, even if you reduce LDL cholesterol to 10-20% of what is normal in humans, you won't get rid of the plaques, you won't reverse it.

What are the alternatives? Let's start with those that don't work. I mentioned that systemic inflammation is one of the problems leading to atherosclerosis. But if you reduce inflammation systemically, studies suggest you get about the same benefit to mortality as you would get from lowering LDL cholesterol. Which doesn't mean that somebody cannot come up with a way that could do this in a better and more targeted fashion, but the tools available for control of systemic inflammation are really blunt right now.

The second alternative sounds much better, if limited to mice. Reverse cholesterol transport is the pathway wherein a macrophage sucks up cholesterol and hands it off to HDL particles in the bloodstream. There are a number of genes involved in this - macrophages use ABCA1 to hand off cholesterol to the HDL particle initially and then ABCG1 helps add more cholesterol to the particle. Then the particle heads to the liver and is excreted and ejected from the body. Anything you do in mice to make one or more parts of this system work better, it all works great - up to 50% reversal of plaque lipid content in some cases. But every time it was tried in humans, it failed - there's a whole list of clinical trials over the last 20 years that tried and failed. That tells us that we don't understand something very important about the way in which cholesterol transport is rate-limited in its different steps in humans vs in mice.

So our approach is to make macrophages resilient to the environment in old tissues. There have been a number of people trying this, some of it hasn't made it very far, some of it is interesting, and sometimes there is overlap between those two. There is a recent paper with a hypothesis of effect they showed is that if you target lysosomes in macrophages with antioxidants, it prevents the oxidized LDL particles from messing things up, and therefore more macrophages are doing their job - reversing the plaque by 50% in a mouse model. It's entirely possible their hypothesis is wrong and delivering antioxidants is improving something else in the picture, but it's certainly a result that self-experimenters should pay attention to because these antioxidants are easily available.

Secondly there is the Underdog Pharmaceuticals approach - sequestration of 7-ketocholesterol, which is a highly toxic altered cholesterol, thought to play a big role in atherosclerosis. Lastly there is our approach - engineering macrophages to give the ability to degrade excess cholesterol, whether or not it is altered. The company is named Repair, since I believe that if you're going to address aging and you can't point to something you are actually repairing - a form of damage or dysfunction, where you can clearly say that you are fixing this - then you might not be doing the right thing.

In summary, what we're doing is allowing macrophages to degrade cholesterol and then stepwise approaching the various atherosclerotic conditions in order of number of patients. So starting with an orphan condition - homozygous familial hypercholesterolemia, then you go into larger patient groups as you gain experience doing this. Unlike most therapies, we can actually apply ourselves to any form of atherosclerosis, whether or not it has a genetic cause, we don't care how you got plaques, we just break them down. We've demonstrated AAV delivery of our cholesterol degrading protein - has a very large effect of 48% reversal of plaque lipids in a month, which is large in the scheme of things as compared to other approaches.

Our goal is to produce a universal macrophage cell therapy. As I said, atherosclerosis is basically the encounter of an aged macrophage with cholesterol, at which point you get a lot of cell death and cholesterol-based plaque. If you overwhelm the existing systems of normal macrophages with excess cholesterol, they can't do anything with it and bbecome pathological foam cells - they don't have an way to deal with that level of cholesterol. So with that picture in mind, the whole spectrum of LDL lowering cholesterol drugs really only lowers that input to the problem. And they can't lower it more than a little, because the macrophage is in the plaque, not in the bloodstream, and the plaque is packed full of cholesterol and toxic horrible nastiness, so you're not really getting a lot of boost from lowering the input from bloodstream. The problem is the plaque that's sitting there. You can't reverse it by undertaking this LDL lowering approach, you still have macrophages exposed to excess cholesterol and becoming pathological foam cells as a result.

And the pathological foam cells leading to your plaque brings us to this point, one that has to be made to a lot of people, unfortunately. Your risk of death is not due to LDL cholesterol, it's due how much plaque you have. It's exactly how much plaque you have and how much high risk plaque - the soft plaque laden with cholesterol. That determines your mortality. LDL cholesterol, while widely accepted as a surrogate marker, is not the cause of your death. That's why different people can have different levels of cholesterol in their bloodstream and have quite divergent mortality rates.

The point of the exercise is to figure out what we should do differently - and that is making macrophages invulnerable to the plaque-based environment as best as we can. Our idea of "as best we can" is to give a macrophage the capability to break down cholesterol safely in situ. I should say that this is not a trivial thing to do, because a cell is basically an enormous lump of cholesterol - our body uses cholesterols everywhere in the cell membrane. The reason why we never evolved to break down cholesterol when it's harming us is probably because our cells have cholesterol everywhere. So you couldn't evolve something that just chews cholesterol whenever it sees it. And that's why delivering things like the known cyclodextrins that bind to cholesterol is not quite simple either, because the first thing that will happen if you dump a bunch of cyclodextrins into somebody is that their blood turns to mush, because it will consume all your blood cells by hooking all the cholesterol out of cell walls.

So the objective is a safe way of breaking down cholesterol, but only the excess cholesterol, which is what we achieve by putting in these specific mechanisms into the cells we're working with. We can demonstrate that by putting these mechanisms into any old cell, and the output is exactly the same - we get a catabolite that is safe and more soluble, and quickly leaves the cell, departing into the bloodstream where it is gotten rid of. What this means is that we can take macrophages and give them the ability to express our cholesterol degrading proteins, and then if you dump cholesterol on those cells they remain competent and able to ingest cholesterol and dispose of it - that's what you want in your plaque.

So going forward, we take induced pluripotent stem cells (iPSCs) from mice or humans, the lines are then disrupted in certain ways to make them universal (you get rid of the surface markers that make them recognizable - a very important technology that leads to off the shelf lines of universal cells, you can look at recent reports from Sana Biotechnology, of the delivery of universal iPSCs to primates, for example). We then differentiate iPSCs into macrophages that express cholesterol degrading proteins, and this is the way we produce a cost effective cell therapy. We've injected mice with the first of these cells over the last month or so and we should have initial data by the end of the year.

And then what we do with this is a stepwise approach through the orphan indication of homozygous familial hypercholesterolemia with very few patients and a much easier FDA process, then to the heterozygous familial hypercholesterolemia indication with more patients, and then to the large high risk subpopulations of atherosclerosis, possibly tens of millions of patients at the end of the day. These are all people who will have medical imaging carried out to show the presence of high-risk, cholesterol-laden plaques. Ultimately we think you can take the lion's share of death - of that 27% by atherosclerotic diseases - and use technology such as ours to remove that cause of death from the human condition. How long is it going to take? Who knows, but the most high-risk population is where we start.

Tauopathies like Alzheimer's disease are characterized by the spread of tau aggregates through the brain. Tau is one of the few molecules in the body that can become altered in a way that encourages other copies of the same molecule to also alter, causing aggregates to form. These aggregates and their surrounding biochemistry are disruptive to cell function and toxic to cells. A number of neurodegnerative conditions are associated with protein aggregates of amyloid-β, α-synuclein, and tau. The mechanisms by which this spread occurs are debated, but researchers strongly suspect a role for glial cells in this process.

Dementia is one of the leading causes of death worldwide, with tauopathies, a class of diseases defined by pathology associated with the microtubule-enriched protein, tau, as the major contributor. Although tauopathies, such as Alzheimer's disease and frontotemporal dementia, are common amongst the ageing population, current effective treatment options are scarce, primarily due to the incomplete understanding of disease pathogenesis. The mechanisms via which aggregated forms of tau are able to propagate from one anatomical area to another to cause disease spread and progression is yet unknown.

The prion-like hypothesis of tau propagation proposes that tau can propagate along neighbouring anatomical areas in a similar manner to prion proteins in prion diseases, such as Creutzfeldt-Jacob disease. This hypothesis has been supported by a plethora of studies that note the ability of tau to be actively secreted by neurons, propagated and internalised by neighbouring neuronal cells, causing disease spread. Surfacing research suggests a role of reactive astrocytes and microglia in early pre-clinical stages of tauopathy through their inflammatory actions. Furthermore, both glial types are able to internalise and secrete tau from the extracellular space, suggesting a potential role in tau propagation; although understanding the physiological mechanisms by which this can occur remains poorly understood.

Link: https://doi.org/10.1016/j.bbih.2021.100242

Exercise is beneficial at every age, but most people do not undertake enough physical activity. In a sedentary world, structured exercise programs look like a decent therapy, because that exercise corrects a harmful deficiency in the operation of metabolism. Thus the studies showing a reduction in mortality resulting from exercise as an intervention in older individuals. Exercise improves mitochondrial function, amongst other changes, and these changes should be expected to modestly slow the progression of many age-related diseases.

Neurons are highly specialized post-mitotic cells that are inherently dependent on mitochondria due to their higher bioenergetic demand. Mitochondrial dysfunction is closely associated with a variety of aging-related neurological disorders, such as Alzheimer's disease (AD), and the accumulation of dysfunctional and superfluous mitochondria has been reported as an early stage that significantly facilitates the progression of AD. Mitochondrial damage causes bioenergetic deficiency, intracellular calcium imbalance, and oxidative stress, thereby aggravating β-amyloid (Aβ) accumulation and Tau hyperphosphorylation, and further leading to cognitive decline and memory loss.

Although there is an intricate parallel relationship between mitochondrial dysfunction and AD, their triggering factors, such as Aβ aggregation and hyperphosphorylated Tau protein, are still unclear. Moreover, many studies have confirmed abnormal mitochondrial biosynthesis, dynamics, and functions will present once the mitochondrial quality control is impaired, thus leading to aggravated AD pathological changes. Accumulating evidence shows beneficial effects of appropriate exercise on improved mitophagy and mitochondrial function to promote mitochondrial plasticity, reduce oxidative stress, enhance cognitive capacity and reduce the risks of cognitive impairment and dementia in later life. Therefore, stimulating mitophagy and optimizing mitochondrial function through exercise may forestall the neurodegenerative process of AD.

Link: https://doi.org/10.3389/fnagi.2021.755665

This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess whether a nine month course of injected peptides will significantly and beneficially affect thymus size and cellularity. The thymus atrophies steadily with age. This organ is where maturation of T cells takes place, a complex process of selection to enable T cells to recognize foreign molecule without attacking the body's own component parts. Regrowth of active thymus tissue should improve immune function for the long term by increasing the supply of new T cells, slowing or reversing some of the age-related decline of the immune system.

The peptides in question are three of the Khavinson peptides, the subject of a long-running line of research and development originating in the Russian biomedical community. The original Khavinson peptides were sourced from animal organs, and then over the years synthetic forms were manufactured instead. Some of these natural and synthetic peptides have undergone human trials and have been approved for use as therapies in Russia. The peptides epitalon, thymogen, and vilon have been used to generate animal data to suggest that they improve immune function, at least partially via some degree of regrowth of the aging thymus. But this latter point of thymic regrowth has not been assessed in humans.

The purpose in publishing this outline is not to encourage people to immediately set forth to follow it, though the existing human data for the Khavinson peptides suggests a minimal side-effect profile. If you come away thinking that you should just jump in, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.

Contents

• Why Self-Experiment with Methods of Thymic Regrowth?
• Caveats in More Detail
• Summarizing the Use of Thymus-Relevant Khavinson Peptides
• Establishing Dosage
• An Introduction to Injections
• Considering Autoinjectors
• Obtaining a Needle-Free Injection System
• Obtaining Vials of the Correct Size
• Preparing Khavinson Peptides for Injection
• Obtaining Khavinson Peptides
• Storing Khavinson Peptides
• Validating the Purchased Peptides
• Establishing Tests and Measures
• Guesstimated Costs
• Practice Before Working with Peptides
• Schedule for the Self-Experiment
• Where to Publish?

Why Self-Experiment with Methods of Thymic Regrowth?

Thymic involution with age, the process of atrophy that replaces active thymic tissue with fat, is an important contributing cause of immune system aging. The thymus plays a vital role: thymocytes created in the bone marrow migrate to the thymus, where they mature through a complex process of selection in order to become T cells of the adaptive immune system. As active thymic tissue atrophies, the supply of new T cells diminishes. This lack of continued reinforcements leads to an aged adaptive immune system that is ever more populated by exhausted, senescent, and malfunctioning T cells.

Notably, the decline of the immune system is slow. The thymus loses 1% of its active tissue with each passing year of adult life, and is near all fat by the time most individuals are in their 50s. The adaptive immune system takes another decade after that to become notably problematic. Given a way to wholly replenish the T cell complement of the adaptive immune system, one could likely gain an additional decade of function. Equally, regrowing 10% of the original youthful thymic tissue would also likely gain about a decade of additional immune function. Given the great importance of the immune system to health, a restored thymus seems a goal worthy of pursuit.

Caveats in More Detail

There are two areas of personal responsibility to consider here. Firstly, this self-experiment involves injecting peptides that, while having a fair amount of use outside the US and in self-experimenter communities, come with comparatively little easily accessible primary sources of published human data. Much of that data is actually quite challenging to access, being both dated and from the Russian medical community, so the only English language references are in the form of reviews that describe only the outline of an earlier study. So while the materials that can be reviewed suggest a good safety profile, it is more of a leap into the unknown than is experimenting with compounds that have gone through clinical trials in the English-language world and where the primary sources are easily accessible.

Secondly, obtaining peptides in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is legal to use peptides that are not defined as a therapy for research use, but illegal to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.

Summarizing the Use of Thymus-Relevant Khavinson Peptides

Most of the primary research materials relating to Khavinson peptides are largely in Russian, and largely not available online. The book Peptides in the Epigenetic Control of Aging provides a decent summary of the peptides and what has been done with them, but lacks many of the specifics that would be provided in the original clinical trial materials and study papers. There are 16 Khavinson peptides, derived from different organ tissues, and claimed to have numerous beneficial effects in aged individuals, but only three are of interest for animal data suggesting an effect on the thymus, or an increase in life span, or both. These are epitalon (derived from pineal gland tissue peptide extracts called epithalamin), thymogen (derived from thymus tissue peptide extracts called thylamin), and vilon (also derived from thylamin). While the details are not well understood, evidence tentatively suggests cross-talk between the pineal gland and thymus, and an involvement for pineal signaling in thymic involution.

Epitalon and vilon been claimed to extend life in rodents, while thymogen has been claimed to be geroprotective in rodents. Researchers have reported lower cancer incidence in treated mice. Further evidence suggests benefits to immune function, but the claimed benefits to health are very broad, including reduced incidence of many different diseases. Similar reports exist for the animal organ extracts epithalamin and thylamin from which the more modern Khavinson peptides are derived. Near all of this is from Russian scientific literature, or from old literature that is not fully available online, or from second-hand reports of the same, so should be taken as less robust than would otherwise be the case. A great deal is claimed, and these peptides have been used in human trials, but such claims are by no means as rigorously proven as would be the case if the English-language regulators had been involved, or the work carried out more recently.

Establishing Dosage

The Khavinson peptides have been used orally and via subcutaneous or intramuscular injection. The latter path should provide a greater bioavailability of the peptide. In the reported animal studies, doses vary widely. Where human trials are discussed, there are more useful examples. For example, "Peptides of pineal gland and thymus prolong human life", published in 2003 and hard to find online, reports a trial in which patients underwent intermittent 10 day courses of injections of 10mg per day of thymlamin or epithalamin. The intervals between courses were 6-12 months, with a trial duration of up to 3 years, with 5 years of following observation. Vilon, thymogen, and other later synthetic Khavinson peptides have similarly been reported to have undergone a variety of human trials.

After looking through the unfortunately sparse literature, the dose selected for this self-experiment is 10mg epitalon, 10mg thymogen, and 10mg vilon daily. That dose is to be split between two intramuscular or intravenous injections, carried out 12 hours apart (morning and evening). That dose schedule is repeated for 10 consecutive days, once every 3 months, for 9 months. This choice is aimed to (a) not go too far beyond what has been done in terms of a single injected dose at any one time to minimize the risk of side-effects, while (b) providing a sufficiently high dose and intensive schedule to argue, should no increase in thymus cellularity be observed, that this failure was not due to using too small a dose or too sparse a dose schedule.

An Introduction to Injections

The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.

The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1 ml, and intramuscular is limited to 2-3 ml depending on location. These are all very fuzzy numbers, but these upper limits don't really matter for the purposes of injecting 10 μg of a protein: it can be dissolved in a very small amount of liquid, 0.5 ml or less.

Different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you'll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.

It seems sensible to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections. In the case of flagellin, it makes sense to stick with the human trial approach of intramuscular injections.

Considering Autoinjectors

Sticking a needle into one's own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately largely not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.

Option 1: Subcutaneous Autoinjection with Needle and Syringe

If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.

Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection

Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I'll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.

Obtaining a Needle-Free Injection System

There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. When initially looking at the market a few years ago, PharmaJet was the only available needle-free system capable of intramuscular rather than subcutaneous injection.

PharmaJet is the better engineered and more expensive of these two systems, and its specialized 0.5 ml syringes are built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.

The instructions for both of these systems are extensive, and include videos. They are fairly easy to use. One caveat is that needle-free systems produce a puncture that more readily leaks injected material back out again than is the case for needles. It is a good idea to have a less absorbent plaster ready to apply immediately after injection, such as one of the hydrocolloid dressings now widely available in stores.

Obtaining Vials of the Correct Size

If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.

There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place - this requires a crimping tool, and removing it requires the use of another tool.

There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.

It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.

Preparing Khavinson Peptides for Injection

If using a needle-free injection system, you will likely be limited to injecting 0.5ml amounts. The objective here is to produce doses of the peptides dissolved in 0.5 ml of phosphate buffered saline in sealed vials, ready to be used with the injection system, with as little contamination as possible from the environment, and stored a freezer until it is ready to use. Depending on the size of the vial, it might be able to contain doses for multiple injections, but it is better to stick to one dose per vial. Peptides are sensitive to free-thaw cycles, so you want as few of those as possible.

When ordering epitalon, thymogen, and vilon, they will arrive as lyophilized (freeze-dried) crystals or powder. Thymogen must be shipped on ice, as it is not very stable at room temperature. (a) Divide the lyophized peptides into 100mg amounts, and place into a freezer. (b) Every month pull out 100mg of each peptide, and dissolve all three 100mg amounts into the same 10ml of phosphate buffered saline, with the addition of five drops of DMSO to aid in solubility. Thymogen is a very light, fine powder, and 10ml is right on the edge of what is required to dissolve 100mg. You may see a few flecks remaining. (c) Split the 10ml solution into 20 vials of 0.5ml each. This is a lot of precision pipetting, so practice first! (d) Seal the vials and place them into the freezer, for use in injections that month.

Keeping Things Sterile is Very Important

Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. Laboratories use autoclaves, which sterilize with steam. These are largely expensive devices, but a range of smaller, cheaper options exists. There are many best practices guides and summaries available online. This extends to the injection itself. Even with needle-free systems, an injection site should still be wiped down with alcohol first. It is all too easy to infect an injection site if skipping the precautions, and this can have unfortunate consequences.

Obtaining Khavinson Peptides

A number of peptide vendors worldwide sell epitalon, the most popular of the Khavinson peptides. A much smaller number keep a stock of thymogen and vilon. Most reputable peptide manufacturers could also run up a custom order, but that is much more expensive. An advantage of the companies that advertise epitalon, thymogen, and vilon in their catalog is that they will have established mass spectra and other data sheets for the compound that can be compared with one another, or used as a basis for evaluating the quality of the product. Prices are often outrageous, however. Chinese companies, on the other hand, have a comparatively low price for their offerings, significant lower than that of even the most aggressively competitive companies in the US and Europe. Among European and US synthesis companies out there, Pepscan and Genscript are options, with Genscript recommended by a number of sources.

Without focusing on any of the specific vendors mentioned above, the easiest way to obtain cost-effective synthesis of peptides is to find and connect with smaller-sized suppliers in China. There are a good many reputable peptide synthesis concerns in that part of the world. Sadly, finding such companies has become great deal more challenging, since Alibaba removed all such commerce from their platform in 2021.

As noted at the outset of this post, all of these efforts to obtain, ship, and use any random protein for self-experimentation are to some degree illegal - it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary protein isn't the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.

Open a Business Mailbox

A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.

Finding Overseas Manufacturers

It used to be the case that Alibaba was the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers of peptides and other compounds. Unfortunately, the company banned all such manufacturers from their marketplace as of mid-2021, for reasons that have not yet become clear. Reputable manufacturers exist in China and other countries, but finding them is now more of an exercise without an initial set of connections to work from. Smaller companies are desired, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a protein, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices - they may vary widely, and it isn't necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.

Chinese manufacturers are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized proteins. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier. Note that different carriers tend to have different processes and rates at which shipments are checked for validity.

Storing Khavinson Peptides

Peptides are commonly shipped in a solid freeze-dried (lyophilised) form. While in this form they are easily stored in a refrigerator for the short-term or in a freezer for the long term. Some are fairly stable at room temperature in this state, and some are not - it varies widely from peptide to peptide. A peptide has a much shorter life span once it has been mixed with liquid for injection, and should be kept frozen, used within a matter of a few months at the most, and not subject to repeated freeze-thaw cycles.

Validating the Purchased Peptides

A peptide may have been ordered, but that doesn't mean that what turns up at the door is either the right one or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a protein is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.

At one point, Science Exchange was a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Unfortunately, their service is now expensive and restricted to industry. Thus we must fall back on more laborious approaches to finding small laboratory service companies or university core services to work with. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Rates vary, but $200 per sample is a fair price for LC-MS to check the identity and purity of a compound.

Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by other sources. Finding those sources through PubChem is not hard for more widely used compounds.

Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.

Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor or other sources.

Establishing Tests and Measures

There are a few options for testing before and after effects of an intervention that may provoke the regrowth of active thymus tissue, and thereby increase the production of T cells.

CT Scan

The most direct approach is to undergo a CT scan of the chest, with the aid of a cooperative physician, and obtain a copy of the imaging data to work with. There are a number of freely available viewers for scan data, such as Gingko CADx. The thymus is clearly identifiable in such scans, and one can perform manual or pixel-counting analysis to get some idea as to whether cellularity or size has changed. In humans, the imagery provided in the Intervene Immune trial results suggest that changes in cellularity and thus density are the more likely outcome than any growth in size. It will require some reading around the literature in order to understand the CT images one obtains from a provider. There are a number of useful papers containing sample images from individuals at various stages of thymic involution. See "Normal CT characteristics of the thymus in adults" for example.

Monocyte to Leukocyte Ratio

The monocyte to leukocyte ratio should be altered towards an increased number of leukocytes if T cell production by the thymus is upregulated. This ratio can be obtained from the counts provided in normal bloodwork. There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Though note that some US states require the involvement of a physician!

Naive T Cell and Recent Thymic Emigrant T Cell Counts

Given a cooperative physician who is knowledgable regarding immunology, it should be possible to order tests that count (a) naive T cells and (b) recent thymic emigrants, T cells with characteristics that fade within a few weeks of leaving the thymus. Both populations should be increased by a more active thymus. These are less common, more expensive assays, but are more compelling than a monocyte to leukocyte ratio measure.

Guesstimated Costs

The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.

• Baseline bloodwork for monocyte and leukocyte counts from WellnessFX: $220 / test
• Naive T cell assay via a medical provider: $1500+ / test
• Recent thymic emigrant assay via a medical provider: $1500+ / test
• CT scan: $1000 / scan
• Business mailbox, such as from UPS: $250 / year
• Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: $60
• Phosphate buffered saline and DMSO: $100
• Small pack of 13mm sterile serum vials: $35
• PharmaJet Needle-free Injection Kit: $1020
• Comfort-in Needle-free Injection Kit: $470
• Bulk 13mm serum vial parts and capping tools: $750
• 100mg of each of epitalon, thymogen, and vilon from Chinese peptide manufacturers: $600
• Shipping and LC-MS analysis of samples: $600

Practice Before Working with Peptides

Do you think you can reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with saline solution rather than finding out that your manual dexterity and methods are lacking while handling an expensive protein. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case.

Schedule for the Self-Experiment

One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of peptides to take a couple of months. During that process, also obtain the baseline assessments such as CT scan. Once all of the decisions are made and the materials are in hand, pick a start date. The self-experiment will last for 9 months, with 10 days of twice-daily injections every 3 months. On each injection day, space the injections 12 hours apart. After completion of the self-experiment, rerun the selected assays.

Where to Publish?

If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial. Equally if you run your own website or blog, why not there?

When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results! Publish whatever the outcome.

The NLRP3 inflammasome is a part of the complex regulatory system that controls inflammatory responses, the rousing of the immune system to action. NLRP3 has become an topic of interest to researchers as it seems, potentially, a point of intervention to suppress inflammatory signaling and maladative cell responses to that signaling. While regulatory adjustment is a poor substitute for removal of the causes of chronic inflammation in aging, interfering in NLRP3 activity may diminish the downstream consequences of chronic inflammation, slowing or reversing the progression of inflammatory conditions in later life. Whether the benefit is large enough to merit the effort can only be discovered by making the attempt.

Osteoporosis is a systemic bone metabolism disease that often causes complications, such as fractures, and increases the risk of death. The NLRP3 inflammasome is an intracellular multiprotein complex that regulates the maturation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18, mediates inflammation, and induces pyroptosis. The chronic inflammatory microenvironment induced by aging or estrogen deficiency activates the NLRP3 inflammasome, promotes inflammatory factor production, and enhances the inflammatory response.

In this review, we summarize the related research and demonstrate that the NLRP3 inflammasome plays a vital role in the pathogenesis of osteoporosis by affecting the differentiation of osteoblasts and osteoclasts. IL-1β and IL-18 can accelerate osteoclast differentiation by expanding inflammatory response, and can also inhibit the expression of osteogenic related proteins or transcription factors. In vivo and in vitro experiments showed that the overexpression of NLRP3 protein was closely related to aggravated bone resorption and osteogenesis deficiency. In addition, abnormal activation of NLRP3 inflammasome can not only produce inflammation, but also lead to pyroptosis and dysfunction of osteoblasts by upregulating the expression of Caspase-1 and gasdermin D (GSDMD).

In conclusion, NLRP3 inflammasome overall not only accelerates bone resorption, but also inhibits bone formation, thus increasing the risk of osteoporosis. Thus, this review highlights the recent studies on the function of NLRP3 inflammasome in osteoporosis, provides information on new strategies for managing osteoporosis, and investigates the ideal therapeutic target to treat osteoporosis.

Link: https://doi.org/10.3389/fendo.2021.752546

Exosomes are membrane-wrapped parcels of molecules used by cells to pass signals between one another. Researchers here note that exosomes harvested from stem cells provide a way to induce macrophages to adopt the M2 phenotype that suppresses inflammation and is involved in tissue regeneration. These macrophages can then be injected into mice in order to spur a greater degree of regeneration following spinal cord injury than would otherwise take place. It is far from a complete recovery, but it is certainly better than the alternative.

The spinal cord injury is a site of severe central nervous system (CNS) trauma and disease without an effective treatment strategy. Neurovascular injuries occur spontaneously following spinal cord injury (SCI), leading to irreversible loss of motor and sensory function. Bone marrow mesenchymal stem cell (BMSC)-derived exosome-educated macrophages (EEM) have great characteristics as therapeutic candidates for SCI treatment. It remains unknown whether EEM could promote functional healing after SCI. The effect of EEM on neurovascular regeneration after SCI needs to be further explored.

We generated M2-like macrophages using exosomes isolated from BMSCs, which were known as EEM, and directly used these EEM for SCI treatment. We aimed to investigate the effects of EEM using a spinal cord contusive injury mouse model in vivo combined with an in vitro cell functional assay and compared the results to those of a normal spinal cord without any biological intervention, or PBS treatment or macrophage alone (MQ). Neurological function measurements and histochemical tests were performed to evaluate the effect of EEM on angiogenesis and axon regrowth.

In the current study, we found that treatment with EEM effectively promoted the angiogenic activity of HUVECs and axonal growth in cortical neurons. Furthermore, exogenous administration of EEM directly into the injured spinal cord could promote neurological functional healing by modulating angiogenesis and axon growth. EEM treatment could provide a novel strategy to promote healing after SCI and various other neurovascular injury disorders.

Link: https://doi.org/10.3389/fncel.2021.725573