We are constantly pushing the envelope to come up with methods to improve our results clinically. We think that our new cytokine formula may be such a game changer. Our new formula makes use of Velvet Deer Antler. But the formula is much different from those formulas out there both in strength and the formula itself. It is a propriety mix.   The above illustration represents the growing portion of the antler. Deer Antler Velvet has been used in traditional Chinese medicine for thousands of years but has recently gained popularity in Western medicine. Deer antler can enhance immune system function, improving athletic performance, increasing muscle recovery, enhance sexual function, improve disease recovery, enhance cardiovascular function, and a host of other conditions. Deer Antler Velvet covers the growing bone and cartilage that develops into deer antlers. The growing antler contains a number of necessary cells, including fibroblasts, chondroblasts, chondrocytes and osteocytes. The tips of the antlers begin as undifferentiated mesenchymal stem cells which are transformed into cartilage. Later, the cartilage is turned to bone, due to the effects of testosterone. Deer antler velvet is antler that is still in its cartilaginous stage.  One of the problems with Velvet Deer Antler is the purity and concentration of the product. Our antler product is sourced from a very reliable source. Typically, there is a concentration of 1500 mg of velvet extract per bottle. Honestly, that will have some effects but it is not exactly what we are looking for. We have sourced a concentration of 4500 mg of Velvet Deer Antler per bottle. This concentration would not be legal for any professional athlete and thus we would not use it on them. Typically, these higher concentrations will help balance hormones and promote tissue repair. In addition to this we have actually added certain supplements to this very potent formula. These supplements will increase stem cell efficiency and output from the bone marrow. This combination is totally unique to our practice and network and it is proprietary in nature.  WHAT ARE THE MAIN COMPONENTS OF VELVET ANTLER? The above diagram gives some idea of the many benefits of deer extract. An important concept that is a common theme of many research papers is that the combination of all components of velvet antler provides a synergistic effect that is greater than the total effect that would be achieved by the separate use of each of its individual constituents. That means that if velvet antler is broken down into its constituents that are used separately, their combined effect is significantly less than the effect realized when the nutrients are provided in the naturally combined form of velvet antler. In summary, the effect of the complete product is greater than the summed effect of all components. Let us take a better look at exactly what is found in the antler products. Most of the antler product consists of protein. The active ingredients include collagen, lipids, glycosaminoglycans, minerals, and various growth factors.  We will take a look at the major subgroups of components. The first component to take a look at are the glycosaminoglycans              GLYCOSAMINOGLYCANS Glycosaminoglycans are complex carbohydrates. Glycosaminoglycans (GAGs), have widespread functions within the body. GAGs play a critical role in Regenerative Medicine. They play a crucial role in the cell signaling process, including regulation of cell growth, proliferation, promotion of cell adhesion, anticoagulation, and wound repair. They are an integral component of what is called the extracellular matrix. The extracellular matrix (ECM) is the non-cellular component present within all tissues and organs, and provides not only essential physical scaffolding for the cellular constituents but also initiates crucial biochemical and biomechanical cues that are required for tissue morphogenesis, differentiation and homeostasis. Research has shown GAG’s exist in velvet antler in several forms including: Chondroitin Sulphate – a carbohydrate that helps protect and rebuild degenerating cartilage and is regarded as a potent anti-inflammatory agent Glycosphingolipids – are compounds involved with growth and metabolism of cells and with memory and learning Glucosamine Sulphate – is a component of Chondroitin Sulphate and is a major component of cartilage and synovial fluid Hyaluronic acid – a substance that binds cartilage cells together and lubricates joints. It also acts as a signaling molecule in many biological processes. Phospholipids – the major structural lipid of most cell membranes  Here is a good illustration of the Extra Cellular Matrix. It is the substance between the cells.     Antlers grow by endochondral ossification, the same way that long bones do. A major non-collagenous protein, proteoglycan, a protein substituted with glycosaminoglycan chains, occurs in the cartilaginous tissue of antler. While its use in the antler is not understood, it has been shown that proteoglycan in cartilage, also called aggrecan, regulates differentiation of chondrocytes and may control calcium concentration in the growth plates where endochondral ossification occurs. This may have important implications when we are treating joints with regenerative medicine.   More importantly, the next two illustration shows the ramifications of the Extra Cellular Matrix and repair of various tissues. We can see why enhancing the Extra Cellular Matrix is so important in Regenerative Medicine. We can see that these represent the pillars of regenerative cell therapy. There are few if any other products which have these effects on the extracellular matrix. This is certainly a huge benefit that comes with the Velvet Deer Antler product.   The Glycosaminoglycans have a hand in all of the above repair processes. Most products are only addressing the cellular portions of repair while the antler products are more comprehensive in their approach. The next illustration shows how the Glycosaminoglycans are involved in the new field of tissue engineering. In the case of tissue engineering we call the synthetic ECM a scaffold.   GROWTH FACTORS: THE CLONES OF WHAT STEM CELLS PRODUCE When we look velvet antler growth factors we see a list of who’s who in the growth factor universe. Growth factors, which are generally considered as a subset of cytokines, refer to the diffusible signaling proteins that stimulate cell growth, differentiation, survival, inflammation, and tissue repair. The major growth factors which are found in deer antler include Insulin Like growth factors (IGF-1), Bone morphogenetic growth factors (BMPs), Transforming growth factor family (TGF), Fibroblast growth factor (FGF), Platelet derived growth factor (PDGF), Vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF), Interleukins, and a variety of other factors. When looking at the various growth factors we realize that this appears to be similar to what is found in a Platelet Rich Plasma (PRP) product, and for that matter stem cells themselves release. The following shows some of the major growth factors found in the antler product.             Another aspect of deer antler deals with its Amino acid contents. Remember that amino acids are natures building blocks. Amino acids, often referred to as the building blocks of proteins, are compounds that play many critical roles in your body. They’re needed for vital processes like the building of proteins and synthesis of hormones and neurotransmitters. These are broken down to essential and non-essential amino acids. Another type of amino acid is the free form amino acid. These amino acids aren't joined together with any other amino acids in a protein 'string'. This allows the individual amino acids to be instantly absorbed and used by the body without digestion. Of all the growth factors, the most consequential might be IGF-1. IGF-1 is a banned substance in the world of professional sports. The precursor of IGF-1 is Human Growth Hormone (HGH). IGF-1 is actually the active form of HGH. It is considered performance enhancing. In the smaller doses this is typically not an issue but in the higher doses we are using this is a problem and across the board we will not give this formula to any athlete in the high school, college, or the professional sports arena. Putting this aside, why do we like IGF-1? We can see some of the benefits below. Perhaps more importantly, we need to look at IGF-1 on the basis of cell biology. IGF-1 has been shown to enhance the migratory response of stem cells. We must realize that the IGF-1 supplied by deer antler is a natural form. It seems to be safer than taking the synthetic anabolic agents which can have disastrous consequences to one’s health. The following diagram is somewhat complicated but we see the importance of IGF-1. It will interact with the stem cell and cause the cell to go on to repair tissue or differentiate into that tissue.       IGF-1 and the other growth factors can also result in a number of other health benefits. These extra health benefits may include preservation of a person’s muscle mass, improving the functioning of the immune system, increasing bone density thus helping to improve Osteoporosis, a valid treatment for fibromyalgia conditions, and lastly it may help in weight loss. The bottom line is that these growth factors are all very important in treating damaged tissue. This tissue could be a tendon, a joint, or muscle. Remember, with the antler we are complimenting the growth factors that are supplied by the stem cells and the Platelet Rich Plasma. Also, by taking these supplements on a daily basis we continue the repair process. The one small caveat that we follow is that in those patients who have a history of certain cancers we will typically recommend a lower dose of the antler product. We should also keep in mind that antler products also contain small amounts of the sex hormones testosterone and estrogen. In the right doses these are also important for regeneration.   Another related factor found in the antler is Prostaglandins. They are substances with varying physiologic effects, acting as a vasodepressor, smooth muscle contraction or relaxation, inflammation and uterine stimulation. As components of deer antler velvet, prostaglandins may assist in the capacity of the extract to reduce the swelling associated with arthritis and injury. They also have physiological responses in lipid metabolism, as seen in the cholesterol-lowering effects of deer antler velvet on laboratory animals.   AMINO ACIDS Velvet antler contains many different types of amino acids. Amino acids, often referred to as the building blocks of proteins, are compounds that play many critical roles in your body. Amino acids are organic compounds composed of nitrogen, carbon, hydrogen and oxygen, along with a variable side chain group. Your body needs 20 different amino acids to grow and function properly. Though all 20 of these are important for your health, only nine amino acids are classified as essential. These are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Unlike nonessential amino acids, essential amino acids are those that can’t be made by your body and must be obtained through your diet. Another type of amino acid is a Free-form amino acid. This refers to single amino acid that is already in a pre-digested form and ready to be used by your body. Some nutritional products, especially amino acid blends, contain whole proteins and large peptides (chains of amino acids), which the body must first break down into smaller peptides and individual amino acids before use. For faster utilization and better bioavailability, look for free-form amino acids. We can see that all the various different type of amino acids works synergistically. They all have their purpose.   WHAT SUPPLEMENTS ARE ADDED TO THE FORMULA TO ENHANCE THE EFFECTS?   In addition to the Velvet Deer Antler which provides all the aforementioned compounds. There are a host of other compounds which enhance the efficacy of the product.  These include a natural matrix of herbs bounded by research and science to help one’s stem cells become more active and body supportive. The following compounds highlight these ingredients.   FUCOIDAN – found in the cell walls of certain seaweed species that is has been used medicinally for a wide variety of health purposes. Okinawa inhabitants have a diet rich in Wakame seaweed, which contains the highest concentrations of Fucoidan. Okinawa is also known for its high concentration of centenarians (people who are at least a century old), which researchers believe is linked to their fucoidan-rich diet. The anti-aging effects are associated with Fucoidan's remarkable ability to facilitate tissue regeneration, immune function as well as improving cell-to- cell communication. Not only is fucoidan known for its anti-aging effects, it is also believed to combat cancer, metabolic syndrome and other degenerative disorders. With stem cell therapy, there is always the risk that the adult stem cells could migrate to other areas of the body unintentionally. However, the daily use of fucoidan has been proven to increase mobilization of stem cells to the appropriate area/ site of injury. Not only can fucoidan point the stem cells in the proper direction, it has also shown to improve the stem cells' survival during the differentiation process.   PTEROSTILBENE is a stilbene molecule and demethylated derivative of resveratrol ( it is more bioavailable than resveratrol) that is found in antioxidant-rich foods like blueberries, cranberries and grapes. These all help to slow down the aging process. It is an antioxidant that helps fight free radical damage. It is known to stimulate a series of pathways in the body called Sirtuin gene pathways. The sirtuin genes have effects on a variety of other pathways in the body. Ultimately, the Sirtuins are involved in the regulation of the mitochondria and subsequent ATP production. Research has shown Pterostilbene protects against memory loss, high cholesterol, high blood pressure levels and even certain types of cancer. CARNOSINE is an important nonessential amino acid that helps support brain, heart, and eye health. It offers antioxidant protection from free radicals and oxidative stress, boosts endurance, aids in the recovery process, and offers electrolyte support. BLACK RASPBERRY EXTRACT, one of the least known, but yet strongest anti-oxidants that insures chromosomes retain their health and rebuild themselves to optimum health. RHODIOLA – an adaptogenic herb that can elevate your mood and mental stamina, reduce the stress hormone, cortisol and fight depression due to its protective effects on key mood neurotransmitters. Rhodiola rosea extracts have recently demonstrated its anti-aging, anti-inflammation, immuno-stimulating, DNA repair and anti-cancer effects in different model systems. An adaptogenic herb helps the body adapt to and resist physical, chemical, and environmental stress.   ASTRAGIN – a 100% natural compound which is patented and promotes a healthy gut lining reducing inflammation in the intestinal lining and increasing absorption of nutrients. AstraGin has the ability to increase the assimilation of important amino acids which increase nitric oxide levels in the human body, making it the perfect performance enhancer when taken pre-workout.  Higher levels of nitric oxide result in enhanced blood flow to the muscle which leads to better pumps, muscular contraction, and improved nutrient transportation. AstraGin also aids in glucose absorption so having this pre-workout ensures that you have sufficient energy levels whilst you train.    THE FINAL QUESTION HOW DOES THE ANTLER PRODUCT GET TO WHERE IT IS NEEDED? One could have the absolute best formula on paper but the question is will it work? When we evaluate medications and supplements a very important aspect to consider is what we call the pharmacokinetics of the product. This concerns the science of how the drug moves around in the body. Unfortunately, on paper many compounds seem very promising but in real life are a failure. The main reason for this is that they cannot be absorbed by the body. The mode of absorption of the antler product is what is referred to as sublingual. This means it is absorbed under the tongue. This is many times a very effective mode of absorption. It by-passes the gut and goes directly into the blood stream where it is needed. The components are not broken down. Also, in the saliva are very small particles called exosomes which can be considered a rising star in drug delivery. Saliva is a very rich source of exosomes. Thus, we have a very efficient method of delivering the velvet components in a safe and reliable manner.  If we were to undertake the task of trying to design a complete supplement to utilize in Regenerative Medicine and Stem Cell therapy, Velvet Deer Antler would be at the top of our list. It has most of the ingredients needed for success. Furthermore, there is a very efficient way to deliver the goods to the cell where they are needed. Deer antler will not be the only modality we will use, but it certainly has an important place on our mantle. We are constantly working to push the envelope and I suspect more advances will come.   Thanks, JP

    NQO1 is a gene which controls the production of the enzyme NAD(P)H dehydrogenase, quinone 1. The above diagram shows some of the many functions of the NQO1 pathway. We can see that there are three major pillars of health that NQO1 directly influences. These pillars include detoxification which entails the ratio of NAD to NADH, its ability as an antioxidant, and lastly how it helps to stabilize the P-53 gene. Detoxification and antioxidant activity go hand and hand. They are intertwined with each other. Each of these pillars have extreme importance for our health and well-being. The detoxification pillar is a very important aspect of NQO1 functions. Much of the detoxification deals with compounds called Quinones. Quinonoid compounds generate reactive oxygen species (ROS). Quinones are ubiquitous in nature and constitute an important class of naturally occurring compounds found in plants, fungi and bacteria. Human exposure to quinones therefore occurs via the diet, but also clinically or via airborne pollutants. For example, the quinones of hydrocarbons are prevalent as environmental contaminants and provide a major source of current human exposure to quinones. The inevitable human exposure to quinones, and the inherent reactivity of quinones, has stimulated substantial research on the chemistry and toxicology of these compounds. NQO1 is employed in the removal of a quinone from biological systems as a detoxification reaction:  NAD(P)H + a quinone → NAD(P)+ + a hydroquinone. The hydroquinone is excreted. This reaction ensures complete oxidation of the substrate without the formation of semiquinones and reactive oxygen radicals that are deleterious to cells. The localization of NQO1 in epithelial and endothelial tissues of mice, rats and humans indicates their importance as detoxifying agents, since their location facilitates exposure to compounds entering the body. In addition to the detoxification, NQO1 helps produce NAD+ which in its own right is very important.   The ratio of NAD+/NADH is of extreme importance. We are well aware of the importance of NAD+ for our body. NAD+ is instrumental in the production of ATP which is the body’s energy currency. However, NAD+ is also used in a variety of biological processes in the body. Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD+depletion may occur in response to excessive DNA damage due to free radicals. This damage results in significant poly (ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD+. PARP is instrumental in DNA repair. Also, chronic immune activation and inflammatory cytokine production results in accelerated CD38 activity and subsequent decline in NAD+ levels.  We now think that the NAD+/NADH ratio may be as important if not more important than the levels of NAD+. One of the insights arising from the scientific studies of calorie restriction is that the ratio of NAD+ to NADH (NAD+/NADH ratio) might be important for the lifespan extension benefits. This ratio has been reported to decline with age, with NAD+ being decreased and NADH increased in older individuals. While boosting the amount of NAD+ has been getting a lot of attention, improving the ratio between NAD+ and NADH might be more significant than the amount of cellular NAD+ in isolation. In yeast experiments, calorie restriction decreases NADH much more dramatically than it affects NAD+. This decrease in NADH is important for enhancing lifespan, because, on its own, it increases activity of the NAD+ consuming enzymes that boost longevity processes (e.g., Sirtuins) and DNA repair (e.g. PARPs) in yeast. This is thought to occur because NADH is an inhibitor of these enzymes, so lowering it releases the inhibition. As an example, inducing the enzyme NQO1—an enzyme that uses NADH as an electron donor increases intracellular NAD+ levels because it shifts the NAD+/NADH redox ratio in favor of oxidation (NAD+). A side effect of this reaction is that intracellular NAD+ levels increase. Upregulation of the pathway that induces NQO1 occurs in calorie restriction and appears to be an important component of producing the benefits. We must remember that cellular levels of NAD+ are more important than the serum levels.  In review, what NQO1 does is convert NADH to NAD+ while at the same time it maintains a very delicate ratio of NAD/NADH. This ratio is not affected by dietary or IV intake. One important fact is that NQO1 will oxidize NADH to NAD+ and thus it increases NAD+ in the cell. HOW IS THE NQO1 PATHWAY REGULATED?  Another name for the NQO1 gene is the longevity gene.  NQO1, regulates the NAD+/NADH ratio in cells. NQO1 does this by oxidizing NADH to NAD+. During aging the ratio of NAD+ to NADH changes in part to a reduced level of the expression of NQO1. As we age the cells accumulate a type of protein called BET proteins. The BET proteins are Bromodomain and Extraterminal Proteins. They are referred to as epigenetic readers. The following diagram shows the various components involved in epigenetics, namely the writers, erasers, and readers all of which effect gene behavior.  In this case, the BET proteins will suppress the induction of the NQO1 gene. There is now much research looking for inhibitors of BET proteins for a variety of conditions including cancer.       Lowered levels of NQO1 will affect the amounts of a compound called Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha). PGC-1a is a member of a family of transcription coactivators that plays a central role in the regulation of cellular energy metabolism. It is strongly induced by cold exposure, linking this environmental stimulus to adaptive thermogenesis. PGC-1alpha stimulates mitochondrial biogenesis and promotes the remodeling of muscle tissue to a fiber-type composition that is metabolically more oxidative and less glycolytic in nature, and it participates in the regulation of both carbohydrate and lipid metabolism. Oxidative metabolism produces far more ATP than the glycolytic type. It is highly likely that PGC-1alpha is intimately involved in disorders such as obesity, diabetes, and cardiomyopathy. In particular, its regulatory function in lipid metabolism makes it an inviting target for pharmacological intervention in the treatment of obesity and Type 2 diabetes. PGC-1a is regulated by the oxidative state of the cell. NQO1 will regulate the PGC-1a levels by controlling the rate of PGC-1a degradation not its synthesis. Like many regulatory factors, PGC-1a has an extremely short half-life.  All of these extremely short-lived proteins are regulated by degradation rates, not synthesis rates. Higher levels of NQO1 shift the ratio of NAD+/NADH and protect proteins from being oxidized. PGC-1 plays an important role in regulating mitochondrial function. Higher levels of PGC-1a help prevent age related mitochondrial dysfunction. Thus, it appears that under conditions of oxidative stress, such as with aging, NQO1 may be a major factor that controls the concentration of PGC-1a in the cell. PGC-1a is not some esoteric co factor, it is extremely important in many different functions as can be seen from the following diagram. PGC-1α is a transcriptional coactivator that is a central inducer of mitochondrial biogenesis in cells.      Thus, it appears that under conditions of oxidative stress, such as with aging, NQO1 may be a major factor that controls the concentration of PGC-1a in the cell.  WHAT ELSE DOES NQO1 STIMULATE?  NQO1 seems to have a significant effect on the P-53 gene. It helps to stabilize the P-53 gene. P-53 is many times referred to an the “Tumor Suppressor Gene”. It is a potent sentinel in the body looking for and destroying cells which may go on to tumor lines. It has the ability to fix DNA damage if it is not too severe or if too severe it will destroy the cell. The following diagram shows P-53 in action:   It is now thought that many cancers arise from a defect in the P-53 gene. It appears that the NQO1-dependent (ubiquitin-independent) pathway is the most important pathway for regulating p53 levels within the cell. Ubiquitin is a small protein that is found in almost all cellular tissues in humans and other organisms. It helps to regulate the processes of other proteins in the body. Through a process known as ubiquitination or ubiquitylation, a ubiquitin molecule can bind to a substrate protein, changing the way it functions. This can lead to a number of different outcomes. It is most widely recognized for its role in apoptosis of proteins, earning it the title of the molecular “kiss of death” for proteins, although it also plays a major part in several other cellular processes related to the regulation of proteins. If P-53 is working properly hopefully the chances of a cancer arising are significantly diminished. Treatment with curcumin augments the levels of P53 in tumor cell lines through incrementing its half-life in a NQO1 dependent manner. Curcumin treatment promotes the interaction between NQO1-p53.   HOW CAN WE INCREASE THE PRESENCE OF NQO1?  One statement that can be made across the board is that anything which increases the NRf2 pathway will increase the NQO1 gene action. The NRf2 pathway has a profound effect on the NQO1 gene. One can read my previous blogs concerning the NRf2 pathway. I call this pathway the thermostat of anti-inflammation. One of the important stimulators of the NRf2 pathway are ozone messengers which are produced by intravenous ozone such as is delivered by the EBO2 protocol. These messengers will allow the NRf2 to enter the nucleus and activate certain genes. Another offshoot of the EBO2 protocol is the use of photodynamic therapy which stimulated the NQO1 gene. Phototherapy also includes the stimulation of heat shock proteins which are encouraged by the exposure of UVA light.          Again, for those people unaware of the EBO2 protocol, it is a protocol which uses a dialysis filter, intravenous Ozone gas, and photo modulation. The following is a picture of the set up used in the EBO2 protocol:   Other compounds which seem to have stimulating influences on NQO1 include resveratrol, Pterostilbene, Taxifolin (also called dihydroquercetin), sulforaphane (broccoli), curcumin, and Fumaric acid derivatives. Another important supplement perhaps the most important, to stimulate the NQO1 gene is Beta-lapachone, a compound found in the bark of the South American Lapacho tree. It is a potent activator of the NQO1 gene and produces ROS in cancer cells, but reduces ROS in non-cancer cells. Beta-Lapachone is a NQO1 activator. In addition to stimulating the NQO1 gene it stimulates the NRf2 pathway which helps to lower inflammation. Beta-lapacho was very popular a number of years ago. It then seemed to lose it way. Now there is a resurgence in the use of Beta-lapachone on multiple fronts including clinical studies in a variety of universities. A few final thoughts, if a clinic is utilizing NAD+ but not stimulating the NQO1 pathway then they are behind the times. There are a number of clinics which like to dabble in utilizing NAD on their patients. Unfortunately, they are not aware of the basic science of NAD, its effect on senescent cells, methods allowing the body to handle NAD better, and the importance of the NQO1 gene. If you encounter a clinic which is “just” utilizing NAD without addressing these related matters, your best bet is to seek treatment elsewhere!! This will ensure you the best chance of success. It is all a matter of knowing the basic science of the various pathways and how these can be manipulated to the benefit of the patient.  The following illustration gives all the salient points about the NQO1 gene. There is a reason why this is called the Longevity Gene. The answers lie in the illustration. When all is said and done this seems to represent the essence of aging.   Thanks,   Dr. P    

  There now seems to be intense interest in EBO2 techniques. Copious volumes of research have provided evidence that Ozone’s dynamic resonance structures facilitate physiological interactions useful in treating a myriad of pathologies. The comment of Ozone opponents is that ozone therapy looks like a panacea for all diseases. Indeed, it seems so, but in reality, this is due to the multitude of compounds originated at first from the reaction of ozone with body fluids, and eventually able to display pleiotropic effects delivered by different organs. Ozone can be considered a “PRODRUG”. What exactly is a Prodrug? A prodrug is a medication or compound that, after administration, is metabolized into a pharmacologically active drug. Instead of administering a drug directly. The following illustration shows how a prodrug works: One of the issues raised by the scientific community is: how does Ozone really acts on Humans? Ozone is quite different from a drug and its action is not a consequence of a binding reaction between one molecule (drug) and one receptor (cellular membrane protein). For this reason, we cannot look at Ozone in the classical terms of pharmacology. Ozone like other agents, and unlike the common drugs that act on a specific receptor, induces small stress to the whole cell when used at adequate doses. This, in turn, triggers a series of intracellular metabolic processes and promotes a myriad of intracellular activities. Because of these reactions, the defense mechanisms of the cell are alarmed and pushed to improve cell activity, explaining in part the surprising therapeutic actions of Ozone. By the same token Ozone is not truly a prodrug since it can have direct effects on phospholipids, lipoproteins, the cell membrane envelopes of bacteria and viruses. These bioactivities can eradicate bacteria and viruses. In order to fully understand the biochemical basis underlying the pharmacological effects of ozone, it is important to illustrate its effects on various coenzymes. These coenzymes are responsible for ozone cell metabolism regulation. These effects on metabolism have profound effects on many aspects in the body. One of the significant effects of ozone is the acceleration of glycolysis. Glycolysis results in breaking glucose into pyruvate and getting a very valuable H+ hydrogen ion. The free energy released in this process is used to form the high-energy molecules ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). ATP is cellular energy that all cells depend upon. Ozone will allow the ratio of NAD/NADH to be about 700/1. A fundamental condition for guaranteeing the continuity of this process is the reoxidation of NADH as it occurs following ozone exposure. The NADH is converted to NAD+. The above diagram shows some important relationships. The ratio of NAD/NADH should be about 700/1. Ozone will stimulate the NQO1 gene which has profound effects on this ratio. NQO1 also stimulates the Sirtuin genes which are very significant in anti-aging and general well-being. As far as protein metabolism, ozone intervenes mainly due to its remarkable affinity towards sulfhydryl groups. A sulfhydryl is a functional group consisting of a sulfur bonded to a hydrogen atom. The sulfhydryl group is one of the most reactive and ubiquitous binding molecules in biological systems. It is found in most proteins and also in a few low molecular-weight substances such as glutathione, CoA (Coenzyme A, notable for its role in the synthesis and oxidation of fatty acids), lipoate, thioglycolate, and free cysteine. It is the most studied of chemical groups, particularly in relation to its role in enzymatic activity and properties of proteins. Similarly, ozone reacts with essential amino acids such as methionine, tryptophan, and other amino acids containing sulfur (i.e., cysteine). In this case, the amino acids are protected from ozone inactivation by two reactions that prevent their degradation: first the oxidation of glutathione and then the oxidation of the coenzymes NADH and NADPH, which are key reactions in the biochemical mechanism of ozone. Nicotinamide adenine dinucleotide, or NAD, is in all living cells, where it functions as a coenzyme. It exists in either an oxidized form, NAD+, which can accept a hydrogen atom (i.e., a proton), or a reduced form, NADH, which can donate a hydrogen atom. Note that "donate a proton" and "accept a pair of electrons" translates to the same thing in biochemistry. Nicotinamide adenine dinucleotide phosphate, or NADP+, is a similar molecule with a similar function, differing from NAD+ in that it contains an additional phosphate group. The oxidized form is NADP+, while the reduced form is NADPH. Finally, ozone reacts directly with unsaturated fatty acids, which have a double carbon bond and are therefore available for an oxidative reaction, leading to the formation of peroxides following cleavage of the lipid chains. In addition to the direct contributions to cellular metabolism described above, both NADH and NADPH may take part in other important physiological processes, including mitochondrial functions, calcium regulation, antioxidation and its counterpart (the generation of oxidative stress), gene expression, immune functions, the aging process and cell death. As a result, some biochemistry researchers have proposed that further investigation Ozone dissolved in the plasma reacts immediately with a number of biomolecules producing two compounds. There are two compounds Reactive Oxygen Species (ROS) and Lipid Oxidative Products (LOPS). They represent the “ozone messengers” and are responsible for many of the biological and therapeutic effects attributed to ozone. ROS are produced immediately in the early phase (mainly Hydrogen peroxide or H2O2) and are responsible for the early biological effects on blood (erythrocytes, leucocytes, platelets). Hydrogen peroxide, now universally recognized as one of the main intracellular signaling molecules, acts on the different blood cells. Hydrogen peroxide is one of the most significant cytokine inducers in white blood cells. The mass of erythrocytes mops up the bulk of hydrogen peroxide. H2O2 diffuses easily from the plasma into the cells and its sudden appearance in the cytoplasm represents a triggering stimulus. This stimulus depends upon the cell types. Different biochemical pathways can be concurrently activated in erythrocytes, leukocytes and platelets resulting in numerous biological effects. On the other hand, Lipid Oxidative Products (LOPS), which are simultaneously produced at the same time as ROS have a far longer half-life. They reach the vascular system and interact with several organs, where they trigger late effects. Some of these real targets are liver, vascular system, while other organs are probably involved in restoring normal homeostasis including the central nervous system, gastrointestinal tract, mucosal associated lymphoid tissue. The LOPS molecules can elicit the upregulation of antioxidant enzymes such as superoxide dismutase (SOD), GSH-peroxidases (GSH-Px), GSH-reductase (GSH-Rd) and catalase (CAT). Moreover, LOPS exert a neuroimmunomodulatory effect highlighted by a feeling of well-being reported by patients during ozone therapy.   OZONE THE WONDER DRUG Ozone is a wonder drug because it can produce four extraordinary phenomena: 1) the induction of Oxidative Shock Proteins (OSP) 2) the upregulation of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) 3) the reduction and/or normalization of oxidative stress; and 4) the release of bone marrow stem cells. Ozone mobilizes bone marrow stem cells (BMSC) and produces LOPS, which induce Nitric Oxide synthase (NOs). This produces inhibition of platelet-leukocyte aggregation.NO is responsible for producing neovascularization and neoangiogenisis. Also, NO activates MMP-9 (matrix metalloproteinase 9) which is indispensable for stem cell mobilization. MMP-9 actually releases the bond that holds stem cells in the bone marrow and releases them to the circulation. The production of Nitric Oxide is one of the main mechanisms by which hyperbaric oxygen will increase stem cell output from the bone marrow. These numbers will include a wide variety of the stem cells that are released from the marrow including Hematopoietic and Mesenchymal Stem cells. Ozone forms lipid oxidative products which in turn produce nitric oxide which makes proteases which release stem cells from the bone marrow into the circulation. The release of stem cells from the marrow is a multi-step process dependent on many different factors. We can see from the following diagram the very process of how Ozone affects stem cell release: The above diagram shows release of stem cells (HSC) from the bone marrow to the circulation. This is very dependent upon the production of MMP-9. This stimulation releases the stem cells from the bone marrow and allows them to go into circulation. The fact that Ozone releases stem cells from the bone marrow is very important. It should be mentioned that this concept is similar to the mechanism of hyperbaric oxygen. In the above diagram it is demonstrates that LOPS, throughout the treatments, act as acute oxidative stressors in the bone marrow microenvironments activate the release of metalloproteinases, of which MP-9 particularly may favor the detachment of stem cells. These cells, once in the blood circulation, may be attracted and home at sites where a previous injury (a trauma or an ischemic-degenerative event) has taken place. The potential relevance of such an event would have a huge practical importance and will avoid the unnatural, costly and scarcely effective practice of the bone marrow collection with the need of the successive and uncertain re-infusion.   OZONE AND OXIDATIVE STRESS One of the most important aspects of Ozone therapy is activation the Nuclear Factor Erythroid 2 Related Factor 2 commonly known as NrF2. NrF2 is like the body’s “missile defense system”. It helps keep oxidative stress under control. Oxidative stress arises from free radicals. Free radicals are compounds which have free electrons. These rogue molecules can cause havoc by “stealing” electrons from other molecules. Some free radicals are capable of damaging the DNA or crippling the proteins and lipids that make up various tissues. As a result, these crucial molecules become damaged, weak, dysfunctional, or otherwise incapable of fulfilling their roles. Produced deep within cells as a byproduct of ordinary energy-producing processes, free radicals are thought to be responsible for aging and disease, including even cancer. How does NrF2 protect us from free radicals? NrF2 is basically like a thermostat. But instead of regulating temperature, it regulates stress levels known as oxidative stress. It responds by binding with the DNA, signaling the cells to make thousands of molecules to shield the cells. They will later activate a new response to form a new barrier. This “barrier” will protect the cells from future stress. Furthermore, Nrf2 will remove the toxins that cause cell damage and boost the normal function. Both of these functions will return the stress levels to normal, minimizing the negative effects. We must realize that we do not want to constantly have NrF2 activated. Ozone is a selective NrF2 activator. How does this work? Ozone produces Antioxidant Responsive Elements (ARE). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system and ultimately the NrF2 pathway. Under normal conditions, Nrf2 is expressed at very low levels, and is mainly sequestered in the cytoplasm by its specific inhibitor called Kelch-like ECH associated protein-1 (Keap-1) that also promotes its rapid degradation. The effectiveness of this mechanism allows a rapid turnover of Nrf2, which displays a half-life of a few minutes. Under specific stimuli, Nrf2 dissociates from Keap1 and translocates into the nucleus and transactivates the ARE-driven genes. These genes encode for proteins involved in a multitude of vital biological functions which include protein homeostasis, oxidative stress response, detoxication, DNA repair, proliferation, autophagy (body's way of cleaning out damaged cells), mitochondrial biogenesis and function, inflammation, and the metabolism of lipids, carbohydrates and amino acids. When you stimulate NrF2 you have a healthier patient. The following illustration shows these concepts: NrF2 goes on to control a wide variety of processes in the body including proper formation of proteins, control of inflammation, healthy mitochondrial function, and healthy adipose tissue. WHAT ARE SOME OF THE OTHER EFFECTS OF OZONE THERAPY The oxidation chemistry of ozone is known to produce Hydrogen Peroxide (H2O2) that enters cells where it has various effects. In red blood cells (RBCs), it shifts the hemoglobin dissociation curve to the right and facilitates release of oxygen, while in leukocytes (WBCs) and endothelial cells it can stimulate the production of interleukins, interferons, growth factors and nitric oxide production. In platelets it favors release of growth factors. As a further aspect of the therapeutic action of ozone, there is the capacity to regulate the cell antioxidant network positively. This aspect is of key relevance in all those conditions in which an imbalance between production and neutralization of ROS (Reactive Oxygen Species) may develop, resulting in oxidative stress. Again, this relates to the implications of the NrF2 pathway and its activation or inactivation. These events may turn into a self-feeding cycle in which oxidative stress is sustained by micro- and macro-inflammatory reactions that lead to cell and tissue degeneration and necrosis. This scenario features the pathogenetic role of oxidative stress in several chronic, degenerative disease states such as chronic viral infections, atherosclerosis, tumor growth, neurodegenerative diseases and accelerated aging. Ozone can help stop these problems in their tracks.   HEAT SHOCK PROTEINS Another aspect of Ozone therapy is the production of HEAT SHOCK PROTEINS (HSPS). Shock proteins are a family of proteins that are produced by cells in response to exposure to stressful conditions. They were first described in relation to actual heat shock, but are now known to also be expressed during other stresses including exposure to cold, UV light (this is one of the reasons we are utilizing a UVA light to stress the blood cells) and during wound healing or tissue remodeling. We can also stimulate Heat Shock Proteins by using a sauna or by subjecting our body to very cold temperatures. Many members of the Heat Shock Protein group perform chaperone functions by stabilizing new proteins to ensure correct folding or by helping to refold proteins that were damaged by the cell stress. This increase in expression is genetically regulated. In the packed, busy confines of a living cell, hundreds of chaperone proteins vigilantly monitor and control protein folding. From the moment proteins are generated in and then exit the ribosome until their demise by degradation, chaperones act like helicopter parents, jumping in at the first signs of bad behavior to nip misfolding in the bud or to sequester problematically folded proteins before their aggregation causes disease. People often mistakenly think that proteins are free to live out their lives in a cell. As it becomes increasingly clear that folding is not a once-in-a-lifetime event for proteins but instead a part of day-to-day life in the cell. Scientists are discovering that problems in this sophisticated system of protein folding are implicated in diseases as diverse as cancer, diabetes, and Alzheimer’s. As time goes on we are finding more and more diseases associated with misfolded proteins. This will open up a new realm in medicine in which Ozone therapy and Photo modulation may be integral players on taking the science of Heat Shock Proteins to the next level. Not only is Heat Shock Proteins important in disease control but it seems to have a place in improving athletic performance. The dramatic upregulation of the heat shock proteins is a key part of the heat shock response and is induced primarily by heat shock factor (HSF). The following illustrations demonstrate the very important work of the Heat Shock proteins: HSPs are found in virtually all living organisms, from bacteria to humans. HSPs are known to modulate the effects of inflammation cascades leading to the generation of ROS and intrinsic apoptosis through inhibition of pro-inflammatory factors, thereby playing crucial roles in the pathogenesis of human inflammatory diseases and cancer. There is a scientific push for studying the HSPs for the treatment of various inflammatory diseases and cancer. Also, it appears that a safe and effective method of stimulating HSPs is by the use of Ozone in blood. Heat shock proteins may play a critical role in reducing recovery time and boosting muscle development. The following illustration shows the mechanism of HSPs:     We can see that the HSPs can have profound implications for a person’s well-being. This is a field where more research will certainly be needed. We can see that when we have an altered expression of Heat Shock proteins many bad things happen. It appears that many autoimmune diseases are related to altered Heat Shock proteins. The following diagram is a good illustration of this. Chronic inflammatory conditions are accompanied by a partial or sometimes large resetting of the immune system to a pro-inflammatory and pro-oxidant state. This response has gross implications also in the integrity of vascular components and may represent a sensitive therapeutic target of chronic, degenerative conditions. On the basis of these theoretical foundations, reported therapeutic applications for ozone therapy were the activation of the immune system in infectious diseases and cancer, and an improved oxygen utilization and release of growth factors that can reduce the extent of ischemic lesions in vascular diseases.   - Dr. P

  The above diagram represents the FOXO gene pathways. FOX proteins are named for a gene found in fruit flies that cause the insects to have forked structures on their heads (supplying the “F”) and a particular group, known as “box”, of specialized genes (supplying the “OX”). They’re named alphabetically, from FOXA to FOXS. There are over 100 subclasses of FOX proteins in humans, such as FOXA, FOXR, FOXE, etc. and they have many functions. An important group of FOX proteins is the class “O”. This class is regulated by the insulin/Akt/mTOR signaling pathway. Invertebrates have a single FOXO gene, whereas mammals have four: FOXO1, FOXO3, FOXO4, and FOXO6. FOXO proteins regulate stress resistance, cellular turnover, apoptosis, glucose and lipid metabolism, and inflammation. FOXO factors are evolutionarily conserved mediators of insulin and growth factor signaling. FOXO proteins act as transcription factors by binding to specific regions on DNA, thereby controlling the transmission of genetic information and influencing the chemical "blueprint" for proteins. Of all the different groups the FOXO group may be the most important. The following is a summary of these ideas: As stated, there is accumulating evidence that FOXO factors play an important role in stem cell biology and tissue homeostasis. There is also a great deal of research on the FOXO pathway and its relationship with osteoarthritis and osteoporosis both of which consume a large portion of our health care dollars. During aging, the balance of removal and regeneration of cells in tissues becomes disturbed mainly due to a decrease in the regenerative potential of adult stem cells. The FOXO family of transcription factors (proteins that can bind to DNA and “switch on” other genes) regulate the expression of genes in cellular physiological events including apoptosis (cellular programmed death), cell-cycle control, glucose metabolism, oxidative stress resistance, and longevity. These six pillars can be the blueprint for significant anti-aging strategies in addition to allowing for greater stem cell success in both the lab and the real world. Many transcription factors play a key role in cellular differentiation and the delineation of cell phenotype (the physical appearance from the expression of one or more genes). Transcription factors are regulated by phosphorylation, ubiquitination, acetylation/deacetylation and interactions between two or more proteins controlling multiple signaling pathways. The regulation of these various processes typically involves the addition or removal of certain chemical compounds to a protein. These pathways regulate different physiological processes and pathological events, such as cancer and other diseases. The forkhead transcription factors have four members: FOXO1, FOXO3, FOXO4, and FOXO6. FOXO1 and FOXO3 are expressed in nearly all tissues. FOXO4 is highly expressed in muscle, kidney, and colorectal tissue while FOXO6 is primarily expressed in the brain and liver. The following illustration shows the various Forkhead transcription factors. It shows the far-ranging influences that these transcription factors have: Over the last decade, studies have demonstrated that FOXOs play critical roles in a wide variety of cellular processes. FOXOs transcriptionally activate or inhibit downstream target genes, thereby playing an important role in proliferation, apoptosis, autophagy, metabolism, inflammation, differentiation, and stress resistance. Remember when we are dealing with anti-aging we want to influence downstream events from an upstream process. Deletion of FOXOs has given insight into their function. For instance, deletion of FOXO1 is lethal; it causes embryonic cell death due to incomplete vascular development. Deletion of FOXO3 is not lethal but affects lymph proliferation, widespread organ inflammation, age-dependent infertility, and decline in the neural stem cell pool. Deletion of FOXO4 exacerbates colitis in response to inflammatory stimuli. Deletion of FOXO6 displays normal learning but impaired memory consolidation. The process of aging is accompanied by a decline in physiological function and cellular maintenance. It is known that aging dramatically alters gene expression and transcription factor activity. FOXO functions downstream of insulin/insulin-like growth factor (insulin/IGF). Studies have found that lifespan extension effects of insulin/IGF deficiency depend on FOXO activity, probably through the transcriptional regulation of key longevity assurance pathways. However, how FOXO elicits this response remains to be fully elucidated. FOXO proteins are tightly regulated to ensure that transcription (first step in protein synthesis) of specific target genes is responsive to environmental conditions. A major form of regulation is Akt-mediated phosphorylation of FOXO in response to insulin or growth factors. This can be seen on the following diagram: In the absence of insulin or growth factors, FOXO transcription factors are located in the nucleus, where they specify target gene expression. We are able to see the various tasks accomplished by the FOXO genes including DNA repair, Cell Cycle arrest, help eliminate reactive oxygen species, and have some effects on glucose metabolism. When insulin and other growth factors are present they result in phosphorylation which subsequently results in the export of the FOXO proteins from the nucleus to the cytoplasm thereby decreasing expression of FOXO target genes. This is regulated by the Akt-pathway. The opposite happens when the FOXO genes are stimulated. FOXO proteins are phosphorylated by other protein kinases which phosphorylate FOXO under conditions of oxidative stress. This phosphorylation causes the movement of FOXO from the cytoplasm to the nucleus, thus opposing Akt’s action. Once in the nucleus the FOXO genes can do their work.   WHAT ABOUT THE DIFFERENT CELLULAR PROCESSES? AUTOPHAGY is a key player in the aging process. Autophagy involves the disassembly and recycling of unnecessary or dysfunctional cellular components. It allows the orderly degradation and recycling of cellular components. Premature aging and age-related disorders have been related to defects in autophagy. FOXO proteins regulate many genes responsible for autophagy. Autophagy has important effects that occur both within the cell and outside of the cell. Within the cell, autophagy helps to decrease oxidative stress, increase genomic stability (which aids in the prevention of cancer), increase bioenergetic metabolism, and increase the elimination of waste. Outside of the cell, autophagy helps to decrease  inflammatory responses, increase neuroendocrine homeostasis, increase surveillance of cancer by the immune system, and increase the elimination of aging cells. CELL CYCLE ARREST Cells constantly monitor their cell cycle status at various checkpoints. These checkpoints help ensure the accuracy of DNA replication and division and provide time for DNA repair. In some scenarios, FOXO blocks the cell cycle by either switching on cell cycle inhibitors or by switching off cell cycle activators. But FOXO is highly sensitive to physiological context and needs, and under conditions of cellular stress, it mediates cell cycle arrest to allow time for repair of damaged DNA and cellular detoxification. When dealing with the cell cycle it might appear strange that FOXO could induce both stress resistance and cell death? The regulation of stress-resistance genes and pro-apoptotic genes by FOXO is not necessarily a paradox. FOXO factors may orchestrate different patterns of gene expression based on the intensity of the stimulus, perhaps activating stress-resistance genes under mild conditions but pro-apoptotic genes when the intensity of stress stimuli increases beyond a certain threshold. It is also possible that FOXO factors regulate different genes in different cell types, causing apoptosis in some cells (e.g. neurons, lymphocytes) while promoting survival in others. Importantly, the induction of apoptosis by FOXO may cause the death of damaged or abnormal cells, therefore benefiting the longevity of the entire organism. FOXO PATHWAYS AND ENERGY HOMEOSTASIS The FOXO pathway has been called the Transcriptional Chief of Staff of Energy Metabolism. FoxO1 is highly expressed in insulin-responsive tissues, including pancreas, liver, skeletal muscle and adipose tissue, as well as in the skeleton. In all these tissues FoxO1 orchestrates the transcriptional cascades regulating glucose metabolism. Indeed, FoxO1 is a major target of insulin which inhibits its transcriptional activity via nuclear exclusion. In skeletal muscle FoxO1 maintains energy homeostasis during fasting and provides energy supply through breakdown of carbohydrates, a process that leads to atrophy and underlies glycemic control in insulin resistance. In a dual function, FoxO1 regulates energy and nutrient homeostasis through energy storage in white adipose tissue, but promotes energy expenditure in brown adipose tissue. In its most recently discovered novel role, FoxO1 acts as a transcriptional link between the skeleton and pancreas as well as other insulin target tissues to regulate energy homeostasis. We can see the importance of these concepts in the following: FoxO1 is a unifying regulator of energy metabolism through the skeleton and peripheral organs FOXO PATHWAYS AND OSTEOARTHRITIS FoxO transcription factors protect against cellular and organismal aging, and FoxO expression in cartilage is reduced with aging and in OA. Observations suggest that FoxO transcription factors play a key role in cartilage development, maturation, and homeostasis and protect against OA-associated cartilage damage. FoxO transcription factors control the expression of genes that are essential for maintaining joint health. The following illustration shows what the lack of FOXO protein transcription and subsequent oxidative stress contribute to in the joint: The next illustration shows this more succinctly: FOXO PATHWAYS AND OSTEOPOROSIS Just like in the joint, FOXO pathways have significant effects on osteoporosis. The effects can sometimes be confusing. How the FOXO proteins function in bone metabolism is a bit more complicated than in the joint. The proper stimulation of the FOXO pathways will encourage the formation of new bone. The cells which make new bone, namely the osteoblasts will have increased survival by FOXO stimulation. At the same time the FOXO pathway will diminish activity of cells which cause bone resorption. Aging increases oxidative stress and osteoblast apoptosis and decreases bone mass, whereas FoxO transcription factors defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis. Conditional deletion of FoxO1, 3 and 4 in three-month-old mice resulted in an increase in oxidative stress in bone and osteoblast apoptosis and a decrease in the number of osteoblasts, the rate of bone formation, and bone mass at cancellous and cortical sites. The effect of the deletion on osteoblast apoptosis was cell autonomous and resulted from oxidative stress. Conversely, overexpression of a FoxO3 gene in mature osteoblasts decreased oxidative stress and osteoblast apoptosis, and increased osteoblast number, bone formation rate and vertebral bone mass. FoxO-dependent oxidative defense provides a mechanism to handle the oxygen free radicals constantly generated by the aerobic metabolism of osteoblasts and is thereby indispensable for bone mass homeostasis. In the future, research will become devoted to the study of supplements and medication which stimulate the FOXO pathway which may become a viable alternation for Osteoporosis treatment. The following diagram shows some of the relationships between the FOXO proteins and the various cells in bone metabolism. There is still much we need to learn concerning this topic.   How to Increase FOXO Proteins The enzyme SIRT1 increases FOXO DNA binding by deacetylating FOXO in response to oxidative stress. So, what happens is that the FOXO leaves the cytoplasm and enters the nucleus ultimately affecting the DNA. FOXO proteins get increased in response to cellular stress and increased energy depletion. Taking it one step further we find that many things which stimulate the Sirtuin genes will stimulate the FOXO genes. Calorie restriction increases sirtuins as well as FOXO factors. For instance, fasting for forty-eight hours elevates FOXO1,3, and 4 by 1.5-fold and but when one eats it will drop back to baseline. FOXO1 is also critical for adapting to fasting by activating gluconeogenesis in the liver, which can make the liver produce glucose whether from amino acids or fatty acids. This can be important in someone who is following a Keto diet. Another method of increasing FOXO is high intensity exercise. FOXO factors are important for regulating muscle energy homeostasis. In response to heat stress, FOXO contributes to increased heat shock protein levels. Heat shock proteins will protect DNA from damage and maintains cellular resistance. One way they do this is to make sure that proteins fold properly in the cell. Taking this to a more practical level, taking a sauna or exercising and sweating can promote FOXO activation and subsequent heat shock protein. Exposure to cold stress production. Hypoxia will also activate FOXO3. The general trend for increasing FOXO follows the same pattern as the other longevity pathways such as AMPK and Sirtuins. Energy deprivation and adaptation to stress can lead to more resilient and longer life. It forces the body to continue producing energy and survive in situations of low nutrients and thus become really efficient at its own metabolic processes. FOXO3 is activated by dietary components, such as EGCG, which is found in green tea, and by quercetin, which is found in onions and apples. WHY STIMULATE THE FOXO PROTEINS? Why would you want to activate FOXO proteins? FOXO proteins activate genes that maintain healthy joints and bone structure. People with osteoarthritis have significantly lower FOXO proteins. FOXO transcription factors modulate autophagy, which promotes cellular turnover and maintenance. Defects in autophagy are associated with age-related diseases. FOXO factors are important for stem cell production and DNA repair. FOXO1 and FOXO3 promote mitophagy which is mitochondrial autophagy FOXO proteins suppress tumorigenesis in cancer. FOXO factors increase the antioxidant capacity of cells, which influence aging and promote longevity. Reactive oxygen species and oxidative stress activate FOXO pathway to adapt to the stress. Inactivity of FOXO factors accelerates atherosclerosis and compromises stem cell proliferation. HOW ABOUT THE FUTURE OF FOXO PROTEINS? Is there a connection between FOXO and cancer? FOXO proteins were originally identified in human tumors. They play an important role in cell-cycle arrest, DNA repair, and apoptosis cell functions that go awry in cancer the FOXO family is thought to coordinate the balance between longevity and tumor suppression. An example of this is found in certain breast cancers. In these cancers, FOXO3 is sequestered in the cytoplasm and inactivated. Expression of active forms of FOXO in tumor cells prevents tumor growth in vivo. Additionally, protein partners of FOXO, such as p53 and SMAD transcription factors, are tumor suppressors. Investigating the ensemble of FOXO protein partners will provide insight into the connection between aging and cancer. The following illustration best defines this relationship: The above entities show the far-reaching hands of the FOXO proteins. These hands all have a direct effect on aging and disease prevention. Thanks, Dr. P  

The above diagram shows just a portion of the symptoms that some patients who are recovering from Covid-19 infection are manifesting. They present with these symptoms long after they have tested negative for the covid-19 virus. These are a group of patients that are now given the name Covid Long Haulers. I have written a few articles on some methods that may help prevent patients from becoming infected with the Covid-19 virus. These recommendations include a number of supplements such as Zinc, vitamins C, D, and melatonin . Also, lifestyle changes such as weight loss and exercise may also help. These recommendations are based on science. BUT WE MUST REALIZE THAT THESE ARE JUST RECOMMENDATIONS AND NOT TO BE TAKEN AS THE GOSPEL TRUTH. THEY HAVE NOT BEEN BASED ON RIGOROUS SCEINTIFIC STUDIES. They are based on clinical hunches and observations. The new question is what might we offer those patients who have had a Covid infection and they continue to be symptomatic. We see some of the manifestations below. These seem to be the most common post Covid symptoms. Patients having long lasting symptoms after a viral infection are not new. This has been seen in the past with Ebola, and the first SARS virus in the early 2000s. Both viruses gave rise to long-lasting symptoms after some people recovered. A 2009 study in Hong Kong found that psychiatric problems and chronic fatigue still plagued SARS-1 survivors up to four years later. People who completely recover from an Ebola infection can still suffer from fatigue, headaches, muscle, joint and stomach pain, eye problems, memory and hearing loss, and mental health issues. The Ebola virus can persist in their bodies, including in the eyes and the central nervous system, even after being cleared from the rest of the body. Covid Long Haulers, recovering patients whose symptoms persist after their coronavirus infections disappear, are a mix of younger people who never needed hospital care and older people with chronic conditions that predate Covid. Their symptoms trail the infection’s path through their lungs, hearts, muscles, nerves, and brains. Deadening fatigue can dog them for weeks or months. Sometimes their problems wane, then resurface in a stuttering pattern that leaves them wondering if they’ll ever get over the condition. Long-haulers include two groups of people affected by the virus. Those who experience some permanent damage to their lungs, heart, kidneys, or brain that may affect their ability to function. While the second group continue to experience debilitating symptoms despite no detectable damage to these organs. Dr. Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases at the National Institutes of Health, has speculated that many in the second group will develop a condition called myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). ME/CFS can be triggered by other infectious illnesses such as mononucleosis, Lyme disease, and severe acute respiratory syndrome (SARS), another coronavirus disease.   WHAT MAY BE THE ETIOLOGY OF THE LONG HAULER SET OF SYMPTOMS? This may be due to an immune-inflammatory response gone amok, or perhaps to ongoing viral activity that might not be clinically detectable. The etiologies are almost certainly multifactorial, but may involve overzealous immune responses, cardiopulmonary or systemic inflammation, vascular inflammation or clotting disorders, and direct damage from viral replication during acute illness. COVID-19 often strikes the lungs first, but it is not simply a respiratory disease, and in many people, the lungs are not the worst-affected organ. In part, that’s because cells in many different locations harbor the ACE2 receptor which is the virus’s major target. However, the infection can harm the immune system, which pervades the whole body. ACE2 is a protein on the surface of many cell types. It is an enzyme that generates small proteins by cutting up the larger protein angiotensinogen that then go on to regulate functions in the cell. The following diagram gives an idea of the ACE 2 receptors and their relationship to Covid 19. Some people who have recovered from COVID-19 could be left with a weakened immune system. Many other viruses are thought to do this. It has been suggested that people who have been infected with measles are immunosuppressed for an extended period and are vulnerable to other infections. This may or may not be case for COVID-19. SARS, for instance, is known to decrease immune-system activity by reducing the production of signaling molecules called interferons. The real problem most long haulers are facing is what is stated earlier by the concept from Dr. Fauci. Many post Covid patients may develop a chronic fatigue syndrome (CFS). CFS, is considered an immune-mediated disorder. It has long been considered a "mystery illness," but that viewpoint is becoming dated. Now it is becoming evident that in CFS there are roles of both of inflammation and autoimmunity. Inflammation is part of a healthy response to problems in the body. When inflammation becomes chronic due to ongoing damage or a misfiring immune system, then you've got a problem. Autoimmunity is when the immune system mistakenly identifies a part of your body as a foreign invader, treating it much like a virus, it needs to get rid of. Your own body triggers its inflammatory process and sends specialized cells to destroy the target and begin the healing process. Only with autoimmunity, the healing process creates more of whatever body part your immune system doesn't like, so it continues to attack and heal and attack in a vicious fashion. The process continues indefinitely. Autoimmunity is a specific type of immune-system dysfunction, but it's important to note that not all immune-system dysfunction is autoimmunity. If we look at the diagram below we see that the symptoms of the Covid Long Haulers and those patients with CFS very much overlap I could substitute the name Covid Long Haulers for CFS. Thus, one big clue in treating patients with Post Covid syndrome is treat them as we would treat Chronic Fatigue Patients. It seems that patients with the Post Covid syndrome and those who have CFS have many similarities. Both conditions seem to have increased levels of cytokine growth factors such as Interleukin 1 and Tumor Necrosis Factor which cause inflammation. This diagram gives a better picture of the relationship to inflammatory growth factors and the Covid Long Haulers. Thus, it seems that the evidence is overwhelming as far as inflammatory growth factors being associated with post Covid symptoms. It would seem logical if we diminish inflammation we can diminish many of the symptoms of the Post Covid Long Haulers. WHAT IS THE ANECDOTAL EVIDENCE WE LOOKED AT FOR COVID LONG HAULERS?   When we looked at the clinical results of some of our post Covid patients it seemed that when attempts were made at reducing inflammation the patients’ health improved. Again, these are anecdotal observations but they are what we observed nevertheless. When we look at the playbook for treating CFS it seems that it might be adaptable to treating the Post Covid Syndrome Long Haulers. We are trying to reduce inflammation in the body WHAT MIGHT BE OF BENEFIT IN TREATING THESE PATIENTS AND WHAT SEEMED TO WORK? First off, we think intravenous NAD would be a be a great help. NAD will improve the health of the mitochondria. NAD provides the mitochondria with the necessary tools to produce more ATP which is the cells energy currency. NAD has a significant effect on the Sirtuin pathways in the body. These pathways are intimately involved with longevity and health. There are a number of reports that the body is somewhat depleted of NAD levels after dealing with an infection. The diagram below gives good idea of the importance of the Sirtuin pathway. Influencing the Sirtuin pathway in a positive manner is certainly a step in the right direction in improving the health of the Long Haulers. NAD certainly has its value but we must remember one important fact. NAD will make senescent cells flourish which is not something we want in a patient who is trying to recover. Remember senescent cells are those cells that should have died but did not. They unfortunately can cause multiple problems in the body such as secreting the inflammatory growth factors. Senescent cells are one of the reasons why Covid-19 seems to be more unforgiving in the elderly. As we age, we accumulate more and more senescent cells. None the less, in the Post Covid long haulers we want to eliminate a portion of the senescent cells. For our general health we still need some senescent cells but we wish to diminish their numbers. This is accomplished by the use of what are called senolytic agents. One of these agents is Quercetin. There are also some other ones which will be of benefit. INFLAMMATION IN LONG HAULERS We still have the problem of post infection inflammation. There is no simple solution for turning off inflammation. However, by utilizing and taking advantage of the pathways in the body we are aware of methods to reduce inflammation. The body and its cells are like the hardware of a computer and the pathways are the computer software. In this case we are interested in stimulating the NRF2 pathway software. The Nrf2 pathway is the thermostat of anti-inflammation. Get this pathway activated and inflammation is diminished. The NRF2 is the software which stimulates the computer hardware namely the genes in the cells which than produce compounds. The above diagram shows the NRF2 pathway in action. What the NRF2 pathway does is produce reduce inflammation in the body. One very good method of doing this is thru the EBO2 protocol. This is a protocol which uses a dialysis filter and blood ozonation at the same time. By using the blood ozonation certain “Ozone Messengers” are created in the body and they appear to have a direct positive effect on the NRF2 pathway. The NRF2 pathway will help stimulate various compounds which will reduce inflammation. Remember that the NRF2 pathway is the computer software, it causes certain genes to turn on and produce certain compounds which dramatically reduce inflammation. We will also be utilizing a supplement that we specifically designed to stimulate the NRF2 pathway. Another medication that may be of benefit in reducing inflammation in long haulers is a medication called Low Dose Naltrexone (LDN). Naltrexone was approved by the FDA in the USA for the treatment of opioid addiction. For our purposes the dosage we will utilize in long haulers will be much smaller than that used for addiction problems. Naltrexone is an antagonist for the opiate/endorphin receptors. Endorphins are polypeptides made by the pituitary gland and central nervous system to moderate the production of neurotransmitters, such as serotonin and dopamine. Endorphins primarily help to reduce pain and inflammation, promote autophagy, and cellular clean up. For instance, when one gets a high after exercising it is typically from the release of endorphins. In individuals with diagnoses such as depression, fibromyalgia, cognitive degeneration, and autoimmunity we are consistently finding chronically low levels of endorphins. Specifically, low levels of an endorphin called Opioid Growth Factor (OGF). OGF is an endorphin produced in most cells in the body to both influence and regulate cell growth, as well as immunity. When low levels of OGF endorphins exist, it is likely for individuals to develop immune system disorders. Low Dose Naltrexone (LDN) has been shown to increase OGF levels in the body, resulting in positive outcomes for those suffering from a variety of diagnoses. The following diagram gives an idea of how Naltrexone can accomplish its goals. The diagram shows that reducing certain cytokine growth factors will reduce inflammation. We must be cognizant of the fact that these effects of Naltrexone only occur with the low doses. The lower doses are blocking the activation of certain immune cells. This is accomplished by blocking receptors on the cell surface with low doses.   How does LDN work? LDN first binds to opioid receptors which are found on the surface of the cell. In doing so, it helps to displace the body’s naturally produced OGF. As LDN displaces OGF receptors, affected cells become OGF-deficient and, as a result, three vital processes occur. The first is an increased receptor trying to capture more OGF. Secondly, receptor sensitivity is increased to capture more OGF. Lastly, production of OGF is increased to compensate for the perceived shortage of OGF. Since LDN will only block OGF receptors for three to five hours, the body experiences a rebound effect which greatly increases the production and utilization of OGF. Once the LDN has fallen off the OGF receptors and excreted, the increased number of endorphins bind to the now more-sensitive and more-plentiful receptors. As a result, these new and improved receptors assist in regulating cell growth, promoting healing, reducing inflammation, and increasing immunity and autophagy. This is accomplished by utilizing a low dose of Naltrexone per day. This is exactly what we are looking for in treating Chronic Inflammation syndrome and Post Covid Syndrome. This is accomplished by utilizing a low dose of Naltrexone per day. We must realize that the Naltrexone must be used only in a low dose. A higher dose will have will not work and will cause the Naltrexone not to work. One other treatment protocol that seems to have efficacy is the use of a very small embryonic like stem cell, many times referred to as a V cell. V cells are found in each of us. There are some propriety methods of stimulating their numbers and activation. They seem to have some far-ranging effects on various systems in the body. We have utilized these cells for years with great results. I looked at some of the Post Covid patients that we have treated in our clinic and analyzed what worked well for them. The bottom line is what is our approach in treating Covid Long Hauler patients? THE ANSWER IS REDUCING INFLAMMATION!! We feel that NAD will be instrumental in treating these patients. This will initially be done with a loading intravenous dose followed by oral doses. We will employ some propriety methods to increase NAD efficiency. The NAD will stimulate the Sirtuin pathways which have multiple desirous effects. We will also treat the patients with senolytic agents to keep senescent cells at bay. We will also incorporate some other intravenous formulas which will help boost immunity and at the same time help fight inflammation. The EBO2 protocol will help to stimulate the NRF2 pathway and hopefully diminish inflammation. Use of some supplements which will help stimulate the NRF2 pathway, the immune system, and well-being. Utilize low dose Naltrexone. We will utilize a low dose as has been described in the literature. The idea is to again reduce inflammation. Finally, we will utilize some growth factor transdermal patches. These patches have some potent cytokine growth factors. One is called Interleukin 10 and the other is called Interleukin 1 antagonist. I call Interleukin 10 “cortisone with no baggage”. V-cell from the patient which will be beneficial on multiple levels.   RIGHT OFF THE BAT, HOWEVER, LET ME GIVE THE DISCLAIMER THAT THIS ARTICLE DOES NOT CONSTITUTE MEDICAL ADVICE -- ONLY MEDICAL HYPOTHESIS. THESE STATEMENTS HAVE NOT BEEN EVALUATED BY THE FDA. They are based on some of our observations and a review of science that is found in the literature. Before embarking on any treatments, one must consult a physician. Thanks, Dr. P  

The above diagram shows the controversy concerning IGF-1. IGF-1 is both a Dr. Jekyll and Mr. Hyde when it comes to our well-being and longevity. On one hand we see that the IGF-1 axis encourages development, growth, and injury repair. This can give the appearance of vitality and youth. However, appearances can be deceiving. IGF-1 can set the wheels in motion to increase aging by such blocking autophagy and stress resistance and increasing Reactive Oxygen species. These are three of the major hallmarks and causes of aging. WHAT IS AGING? Aging is defined as a physiological decline of biological functions in the body with a progressive decline or loss of adaptation to internal and external damage. In humans aging is extremely heterogeneous and can be described as a complex mosaic resulting from the interaction of several random and environmental events. These include both genetic and epigenetic alterations accumulated throughout our lifetime. Despite its enormous complexity, the molecular basis of aging is limited to few highly evolutionarily conserved biological mechanisms responsible for body maintenance and repair. WHAT IS THE RELATIONSHIP BETWEEN HGH AND IGF-1/INSULIN AXIS? The above diagram shows the intimate relationship between HGH (Human Growth Hormone) and IGF-1. Typically, Growth Hormone is released by the Pituitary gland. One major role of growth hormone in stimulating body growth is to stimulate the liver and other tissues to secrete IGF-I. IGF-I stimulates proliferation of various tissues including chondrocytes (cartilage cells), muscles cells, and bone cells resulting in bone growth. If one takes an external source of HGH it will have to be converted in the liver to IGF-1. The metabolic effects of HGH are, in part, mediated through IGF-1 produced in the liver and in the peripheral tissues influenced by HGH. Change in the GH/IGF-1 can possibly be influenced through amino acid supplementation. Specific amino acids—such as arginine, lysine, and ornithine—can stimulate HGH release when infused intravenously or administered orally. It has also been demonstrated that glycine is also one of the stimulatory agents inducing the pituitary gland to secrete HGH. These are all important amino acids utilized in the growth of tissue cause by HGH. Research shows that one’s metabolism slows down with age. A few reasons for this include less physical activity (exercise), muscle loss (sarcopenia), and the normal aging of the organs. Additionally, loss in lean body mass and muscle tissue can be detrimental when it comes to ill adults. Yet HGH/IGF-1 have major effects on metabolism. It has been shown that HGH’s potential benefits relate to protein metabolism. Many of the functions of HGH are facilitated through IGF-1. Administration of HGH induces a rise in circulating IGF-1 that stimulates glucose and amino acid uptake in muscle, which improves muscle protein synthesis. In catabolic circumstances, the levels of IGF-1 decrease while its binding proteins increase, leading to a lower local IGF-1 activity and contributing to the decreased insulin sensitivity seen in catabolism. Here is another summation of the HGH AND IGF-1 relationship. The bottom line is that IGF-1 will repair and rejuvenate various cells including muscle, bone, and other tissues. The question becomes at what price does IGF-1 accomplish cellular repair and rejuvenation? Also is there a difference between IGF-1 produced naturally and that stimulated by external means? AND NOW FOR THE CONTROVERSY: DOES IGF-1 DECREASE LONGEVITY? This illustration is a great one. It shows one of the great risks of IGF-1. We see the many benefits that can be attributed to the IGF-1 but on the other side of the scale we see one big risk namely an increased cancer risk. But this scale is deceptive because if we were to also add the problem of increased cell growth causing decreased longevity the scales would probably be opposite. Remember that IGF-1 causes cell growth. It can act as a gasoline on a fire when it comes to cell growth. This is where the problem lies. Can IGF-1 increase the growth of a few cancer cells to essentially activate them? No one knows the answer to this question. On the other hand, another school of thought professes that the IGF-1 will strengthen the immune system and prevent cancers. Ultimately it is our immune system which prevents us from developing cancer. So which concept is the correct concept?? The question still comes up what is the relationship between IGF-1 and longevity. What is the mechanism? Why does decreasing growth hormone and IGF-1 signaling increase lifespan when it has such an important role in reviving muscle and brain function? One explanation is the thought that curtailing IGF-1 levels increase the expression of other genes that are involved in stress resistance, particularly oxidative damage. Oxidative damage, which is generated everyday through a variety of mechanisms including toxins in the environment, UV radiation, normal metabolism which puts wear and tear on every tissue in our body and on our DNA. If we can boost the activity of anti-oxidant genes that help stave off this damage, then we should be able to delay the deterioration of our tissues and our DNA, thus extending longevity. The very first illustration in this blog shows that IGF-1 will increase oxidative stress. This increased oxidative stress causes increased aging. The chart below also explains this. We can see the intimate relationship growth hormone signals and longevity. The above chart demonstrates the dichotomy of IGF-1 and longevity. What do we know for sure that will increase longevity? One quick answer is calorie restriction or some form of it. Typically, the faster the cell growth the more various problems will pop up. Calorie restriction will slow down the pace of cell growth. During the last 3 decades one of the most discussed topics in gerontology is the role of the growth hormone (GH)/insulin-like growth factor-1(IGF-1) in the regulation of longevity. Accumulating evidence suggests that this pathway plays an essential role in the pathogenesis of several age-related diseases including cancer, dementia, cardiovascular, and metabolic diseases. More research is needed in this field. In animal models it was shown that down-regulation of the GH/IGF-1/insulin system significantly prolongs the lifespan. However, in humans the data is contradictory. While it is well known that enhanced insulin sensitivity and low insulin levels are associated with an improved survival, there is evidence showing that attenuation of the growth hormone/IGF-1 axis may have beneficial effects in extending lifespan in humans. However, it is still unknown which are the optimal IGF-1 levels during life to live longer and healthier. How much do these levels change with age? In addition, IGF-1 receptor sensitivity and activation of the post receptor pathway were not evaluated in the majority of the study enrolling long-lived subjects. Therefore, it is not possible to define the real activation status of the IGF-1 receptor signaling through the mere dosage of circulating IGF-1 levels. This renders more difficult the identification of pharmacological or environmental strategies targeting this system for extending lifespan and promoting healthy aging which we call healthspan. Nonetheless, striking similarities have been described concerning the endocrine profile between centenarians and subjects after a calorie-restricted diet. The following diagrams shows some of these reasons. The endocrine and metabolic adaptation observed in both models may be a strategy to increase life span through a slower cell growing/metabolism, a slower loss of physiologic reserve capacity, a shift of cellular metabolism from cell proliferation to repair activities and a decrease in accumulation of senescent cells. These mechanisms seem to be, at least in part, mediated through the modulation of the GH/IGF-1/insulin system. The following diagram easily explains this phenomenon. What it boils down to is the fact that IGF-1 will stimulate certain pathways which will stimulate pro-growth and survival but at the same time they will lead to aging due to a variety of effects. IGF-1 activates the Akt pathway, which is a downstream activator of mTOR, both of which are the master regulators of cellular growth and inhibitors of programmed cell death in the body. These are nutrient sensing pathways in the body. These pathways typically oppose longevity. The IGF-1 pathway will lead to further oxidative stress. While decreased amounts of IGF-1 will lead to the expression of stress resistance genes such as superoxide dismutase. Thus, it stands to reason if we can increase the production of various antioxidant response enzymes than these effects of IGF-1 can be ameliorated. One method of doing this is to increase the stimulation of the NRF2 pathway. Remember that the NRF2 pathway is the thermostat of anti-inflammation in the body. In the above diagram we see how IGF-1 stimulates the NFkB pathway. NFkB pathway is the major pathway of inflammation. If you stimulate it you stimulate aging. While the antioxidant compounds will force things to go in the opposite direction. These compounds will help put the brakes on aging. They will help to stimulate the NRF2 pathway. This pathway is a gateway to anti-inflammation and health. WHAT IS THE RIGHT ANSWER? SHOULD WE TAKE ADDITIONAL HGH/IGF-1 AND OTHER PEPTIDES TO LIVE LONGER OR WILL IT SHORTEN OUR LIVES? It’s a trade-off when it comes to growth hormone/ IGF-1 and the effects they have on the body. We know they enhance muscle, neuronal, and bone growth while simultaneously preventing atrophy. At the same time, they will increase oxidative stress in the body leading to a speeding up of aging. They will also increase telomer shortening leading to potential aging. Which do you prefer, having better muscle and cognitive performance or living longer? Or better yet can we have our cake and eat it too? Are we able to get the absolute benefits of IGF-1 while at the same time not cutting short our lives? While it is well known that enhanced insulin sensitivity and low insulin levels are associated with an improved survival, there is evidence showing that attenuation of growth hormone/IGF-1 axis may have beneficial effects in extending lifespan in humans. However, tricky question here to which the answer is unknown is what are the optimal IGF-1 levels during life to live longer and healthier. IGF-1 is double-edged when it comes to our health, with the potential to provide much benefit or harm: too little, and we do not develop properly, we lose muscle mass, bone strength diminishes, and our cognition declines as we age; too much, and our cells can grow out of control, leading to cancer and potentially, premature aging. Balancing IGF-1 is a delicate process which is on a delicate scale. What are some of the methods to balance this scale? Is there a difference in the actions of IGF-1 when it is naturally stimulated in the body? It seems that high intensity exercise will increase IGF-1 but at the same time not necessarily diminish longevity. There appear to be some built in mechanism that gives the benefits of IGF-1 without the usual hit on longevity. The following diagram shows this concept.   The question beckons what about those patients who wish to take supplemental peptides of IGF-1 etc to increase its effects? Why does decreasing growth hormone and IGF-1 signaling increase lifespan when it has such an important role in muscle and brain function. As was mentioned earlier, IGF-1 and its cohorts tend to increase oxidative stress. Thus, it seems like a no brainer if we employ methods which will increase the expression of genes that are involved in oxidative stress resistance, particularly oxidative damage, we may get the benefits of IGF-1 without the fall off in longevity. One of the best methods to accomplish this goal is to utilize the EBO2 protocol. This protocol involves blood filtration and direct ozonation of the blood. Blood ozonation will produce intermediate metabolites which will markedly stimulate the NRF2 pathway. This pathway can put a strangle hold on inflammation in the body. The EBO2 protocol will produce potent anti-oxidants enzymes. THIS IS A VERY IMPORTANT TREATMENT FOR THOSE PATIENTS WHO WILL SUPPLEMENT THEIR IGF-1 LEVELS BY TAKING ADDITIONAL PEPTIDES ETC. EBO2 MAY BE THE SAVING GRACE FOR PATIENTS TAKING SUPPLEMENTAL IGF-1. The next illustration shows a portion of the EBO2 protocol. There are some other lifestyle factors that can also boost the expression of stress resistant genes without the downsides to low levels of growth hormone and IGF-1. This is called hormesis. Hormesis refers to the beneficial effects of a treatment that at a higher intensity is harmful. In one form of hormesis, sublethal exposure to stressors induces a response that results in stress resistance. The principle of stress-response hormesis is increasingly finding application in studies of aging, where hormetic increases in life span have been seen in several animal models. The “hermetic effect” is actually the mechanism of action of many catechins and polyphenols that are often mislabeled as antioxidants. Catechins and polyphenols are found in: Green tea, Blueberries and other purple-pigmented fruits/vegetables, Dark Chocolate, Wine, Turmeric. Catechins and polyphenols on their own have no ability to “scavenge “free” radicals like classic anti-oxidants such as vitamins C and E. Rather, they are a little toxic to our cells and thus induce a “hormetic response” by increasing the expression of anti-oxidant genes, and this is why they are put into the category of anti-oxidants. The next illustration shows the benefits of Hormesis. Again, the key here is stress resistance genes. The IGF-1 pathway is one pathway which will in the future be involved in much research. Manipulating this pathway may lead to many dividends. There is no question of the benefits of IGF-1 etc in increasing our healthspan. There is also no question that these same compounds may diminish our lifespan. We may increase life span through a slower cell growing/metabolism, a slower loss of physiologic reserve capacity, a shift of cellular metabolism from cell proliferation to repair activities and a decrease in accumulation of senescent cells. These mechanisms seem to be, at least in part, mediated through the modulation of the growth hormone/IGF-1/insulin system. We feel that if we modulate the stress resistance genes to produce potent anti-oxidant enzymes we are getting very close to achieving that delicate balance between health span and longevity. If you are taking IGF-1 or a variety of its stimulating peptides and not addressing oxidative stress etc. such as we have mentioned, you are gambling with your longevity. Thanks, Dr. P  

 NF- kB is a central regulator in stress response. The NF- kB signaling pathway can be activated by numerous stimuli as listed in the blue boxes: In response to these different stimuli NF- kB transcriptionally regulates hundreds of genes, the generalized categories of which are listed in the red circles. NF-kB is a short name of Nuclear Factor kappa-light-chain-enhancer of activated B cells. It is not a single protein, but a small family of inducible transcription factors that play an important role in almost all mammalian cells. As a common responder to varied stress stimuli, NF-kB is well positioned to play a key role in driving aging. NF-kB has been directly implicated in the aging process. Many biologic pathways implicated in aging, including immune responses, cell senescence, apoptosis, genotoxins (a genotoxin is a chemical or agent that can cause DNA or chromosomal damage), oxidative stresses, cell cycle progression, and inflammation all stimulate the NF-kB family of transcription factors. Transcription factors are proteins that help turn specific genes "on" or "off" by binding to nearby DNA. The function of transcription factors is to regulate or turn on and off genes. It is very important that transcription factors make sure that the genes are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. NF-κB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. NF-kB plays a key role in regulating the immune response to infection. Incorrect regulation of NF-kB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection and improper immune development. For instance, the Covid-19 virus seems to have an affinity for activating the NF-kB causing potentially a cytokine storm and dire consequences. Damage to cellular macromolecules and organelles is thought to be a driving force behind aging and associated degenerative changes. However, stress response pathways activated by this damage may also contribute to aging. The IKK/NF-κB signaling pathway has been proposed to be one of the key mediators of aging. It is activated by a variety of factors as mentioned above. Transcriptional activity of NF-κB is increased in a variety of tissues with aging and is associated with numerous age-related degenerative diseases including Alzheimer’s, diabetes and osteoporosis. Pro-growth survival pathways known to promote aging, specifically Insulin/IGF-1 and mTOR are known to stimulate NF-κB. Insulin/IGF-1 act via two mechanisms, AKT and mTOR signaling, to activate NF-κB. However, through AKT, Insulin/IGF-1 signaling also interacts with known longevity processes by inhibiting FOXO. longevity factors such as SIRT and CR, FOXO inhibits NF-κB signaling. Stress/damage pathways known promote age-associated changes including genotoxic stress, ROS, and inflammation also activate NF-κB. NF-κB then acts to promote aging related changes by contributing to cellular senescence, SASP, apoptotic signals and inflammatory responses. The preceding diagram gives some idea as to the relationship between the NF-kB pathway and aging. One is able to see the relationship between many different pathways in the body and the NF-kB pathway. Interestingly, some of the pathways seem to be opposed to each other. For instance, the pro-growth and survival pathways seem to be opposed to the longevity pathways. The pro-growth pathways are nutrient sensing pathways. They favor cell growth and survival but not necessarily longevity. This is where we start getting some controversy in Anti-Aging medicine. There seems to be a perception that HGH, better known as Human Growth Hormone, is a fountain of youth. This concept is cloaked in controversy. Indeed, HGH can reverse some aspects of aging BUT, AND THIS IS A BIG BUT, this will also stimulate NF-kB which is associated with aging. On the other hand, caloric restriction and the Sirtuin pathways will block NF-kB and thus block aging. For those of you who wish to take a regimen of HGH you may want to think twice about it. However, research shows if HGH is increased naturally (intensive exercise etc.) than NF-kB may not be stimulated in the same manner and aging not encouraged. We also see that stimulation of mTOR will encourage NFkB and possible increased aging. In the above diagram we also see that the AKT pathway (another nutrient sensing pathway) will encourage NFkB activation by disabling the FOXO pathway which is an important longevity pathway. We must remember that not every aspect of the NFkB pathway is negative. It can be important in infections and general stimulation of the immune system.   HOW DOES NF-kB PATHWAY GET ACTIVATED? The above diagram shows what is called the Canonical NF-kB signaling pathway. NF-kB signaling is initiated when a cytokine growth factor receptor (PRR) recognizes its cytokine, starting a signaling cascade (1) that converges on the phosphorylation of the IKK2 complex. IKK2 then phosphorylates IκBα (2), leading to NF-kB activation and (3) subsequent degradation by the proteasome of some of the NF-kB components. This releases NF-κB (the two green colors) from negative regulation while they are in the cytoplasm. (4) allows the NF-kB dimers to translocate to the nucleus to (5) initiate inflammatory gene transcription. (6) De novo synthesis of IκBα acts as a negative regulator of NF-κB-dependent transcription, limiting inflammation in the absence of further signaling events. (7) Primary response genes include those encoding cytokines such as TNF. (8) Release of these proteins leads to autocrine signaling through cytokine receptors. This, or (9) continued PRR ligation, create a positive feedback loop wherein NF-κB is periodically activated until these signals are eliminated. This is a somewhat complicated picture of how NF-kB works. The important thing to remember is that when the NF-kB is in the cytoplasm of the cell it is essentially inactive. Remember it is a transcription factor and thus it must move into the nucleus and stimulate the DNA to start its work. When it gets stimulated to move into the nucleus it starts to turn on certain inflammatory genes to produce biological compounds called cytokines etc. The following diagram is complicated yet it is not as complicated as we might think. What we see here is receptors for two master inflammatory growth factors namely IL-1 and TNF. These two growth factors are responsible for a variety of conditions including osteoarthritis, cancer and a host of other diseases. What we need to know on this diagram is that these inflammatory growth factors ultimately stimulate NF-kB. We must also realize that these growth factors can also help to stimulate an immune response in the face of an infection. Pro-growth survival pathways known to promote aging, specifically Insulin/IGF-1 and mTOR are known to stimulate NF-κB. Insulin/IGF-1 act via two mechanisms, AKT and mTOR signaling, to activate NF-κB. However, through AKT, Insulin/IGF-1 signaling also interacts with known longevity processes by inhibiting FOXO. longevity factors such as SIRT and CR, FOXO inhibits NF-κB signaling.  Stress/damage pathways known promote age-associated changes including genotoxic stress, ROS, and inflammation also activate NF-κB. NF-κB then acts to promote aging related changes by contributing to cellular senescence, SASP, apoptotic signals and inflammatory responses. In the above diagram we are able to see that two main inflammatory cytokines namely IL-1 and TNF are picked up by the cell surface receptors and stimulate the release of NF-kB from the cytoplasm when it goes into the nucleus stimulating the production of inflammatory agents. BESIDES CAUSING AGING WHAT ELSE DOES NF-kB DO? SOME GOOD THINGS The above diagram shows the many faces of NF-kB. Inflammation in cells and tissues has a common pathway: activation of nuclear factor kappa B (NF-kB). “Nuclear” in this case refers to the nucleus of the cell, where chromosomes carry genetic information that influences NF-kB. When NF-kB gene expression signals move into a cell’s nucleus, it activates pro-inflammatory signals called cytokines. These cytokines travel through the circulatory system to trigger inflammatory changes in tissues everywhere in the body. Inflammation promotes diseases through an array of biochemical pathways. Inflammation has even been shown to shorten telomeres (nucleotide sequences at the ends of chromosomes). When telomeres shorten, cells eventually stop functioning, directly contributing to shortened cellular lifespans. What we also see in the above diagram is the fact that NF-kB will also influence the type of macrophages in the “neighborhood”. It is responsible to produce type 1 macrophages. Type 1 macrophages are very important in fighting infections. While a type 2 macrophages are important in tissue regeneration. It also appears that NF-kB may have a similar effect on the polarization of mesenchymal stem cells (MSCs). Like macrophages, there are two types of MSCs namely type 1 and type 2. Type 1 is very efficient in fighting infections while type 2 is important in tissue regeneration. NF-kB will cause the polarization of MSCs to the type 1. What is also very important in this diagram is the fact that NF-kB is intimately involved with various aspects of the immune system. NF-κB is a master regulator of innate immune responses, and vital to many of the roles that macrophages and other innate immune cells play in orchestrating the inflammatory response to pathogens. It can help extend the cell life of various cells of the immune system by inhabiting apoptosis. Considered broadly, immune responses can be divided into innate and adaptive responses. The immune response begins with the host recognizing the presence of foreign pathogens, followed by responses at the cellular, tissue and organismal levels, that ultimately lead to the clearance of the pathogen. As such, immune responses can be broken down into individual signal transduction events through which changes in the extracellular environment elicit altered gene expression at the cellular level. In many instances, NF-κB is the transcription factor that mediates these transcriptional changes. The gene products characteristic of early events in immune responses include cytokines and other soluble factors that propagate and elaborate the initial recognition event. The activation and modulation of NF-kB is also a common target of these factors. Thus, in a surprising number of situations NF-kB mediates the critical changes that are characteristic of innate and adaptive immune responses. This has significant implications in the management of the Covid-19 virus. THE ROLE OF THE NFKB PATHWAY IN MECHANOTRANSDUCTION  Apart from its prominent role in immune response regulation, NF-kB is also identified as a mediator of mechanotransduction in several cell types. Mechanotransduction refers to the processes through which cells sense and respond to mechanical stimuli by converting them to biochemical signals that elicit specific cellular responses. This role is carried out through changes in both its activation, and localization, in response to mechanical signals. Altered cytoskeleton dynamics of a cell, for example, will activate NF-κB. A good example of this is osteoarthritis as is noted above. WE CAN NOT LIVE WITHOUT NF-kB Given the enormous number of genes activated by NF-kB and the diverse modes of signaling that impinge upon these transcription factors, there remains much to learn. As can be seen there are a number of roles for the NF-kB pathway. In the past years NF-κB dynamics emerged as key regulators of cell life and death. This family of transcription factors, which in healthy tissues controls tissue homeostasis, responds to external stimuli and coordinates cell growth and differentiation, is often deregulated in cancer cells. Most of these pathways are bad in that they cause inflammation in the body. However, the effects of the NF-kB on our immune system especially in times for a bacterial or viral invader can be lifesaving. There are a host of supplements that seem to have significant effects of the NF-kB pathway. One which comes to mind is Curcumin. A variety of supplements seem to be the very effective in blocking the stimulation of the NF-kB pathway. -Dr. P    

The above diagram shows the locations of the Sirtuin family of proteins as they are found in the cell. Sirtuins are a family of seven proteins that regulate cellular health. The sirtuins are present in the mitochondria (Sirt 3,4,5) and in the cell nucleus (Sirt 1,6,7). The sirtuins represent one of the most important pathways in the body. Remember that the cell and its organelles are analogous to computer hardware while the pathways represent the computer software. As I have said it is much easier to modify the software rather than the computer. When we modify the “software” we potentially modify upstream causes of aging. The Sirtuins are responsible for a host of functions in the body which can make the difference between health and disease. The following diagram shows just some of the functions. Let us briefly talk about each aspect of this illustration. SIRTUINS AND CHROMATIN REGULATION Chromatin is the material that makes up a chromosome that consists of DNA and protein. The major proteins in chromatin are proteins called histones. Their primary function is packaging long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication. We see the names facultative and constitutive heterochromatin. All we need to know about these is that constitutive heterochromatin can affect the genes near itself; while facultative heterochromatin results in genes that are silenced but under specific conditions of developmental or environmental signaling cues, it can lose its condensed structure and become transcriptionally active. Thus, we can see that the facultative heterochromatin might be able to be manipulated. This leads to the field of Epigenetics. We will see down the road this can have profound implications in Anti-Aging medicine and disease control. CELL CYCLE CONTROL The cell cycle represents the essence of life. It deals with how cells reproduce. Typically, problems in the cell cycle result in many degenerative diseases and cancers. As cells move through the cell cycle, do they breeze through from one phase to the next? If they're cancer cells, the answer might be yes. Normal cells, however, move through the cell cycle in a regulated way. They use information about their own internal state and cues from the environment around them to decide whether to proceed with cell division. This regulation makes sure that cells don't divide under unfavorable conditions (for instance, when their DNA is damaged, or when there isn't room for more cells in a tissue or organ). Again, we can see the importance of the Sirtuins and the cell cycle. Our health is very dependent upon the proper functioning of the cell cycle. SIRTUINS AND CELL FATE DECISIONS The cell fate control is one of the Holy Grails of Regenerative Medicine. Sirtuins have a direct effect on what type of cell a stem cell may become. Sirtuins seem to have importance in the process of creating an IPS (induced pluripotential stem) cell. iPS cells are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes. Again, the relationship between sirtuins and stem cells is extremely important. It can have profound implications on Regenerative Medicine procedures. We can see how the sirtuins can control stem cell fate by taking notice of various environmental clues. Sirtuins give clues to the stem cells as to what to do and when to do it. We are leaning more and more about the relationship between the Sirtuin proteins and their effects on stem cells. They maintain stemness which is an essential characteristic of a stem cell that distinguishes it from ordinary cells namely self-renewal.   SIRTUINS AND DNA REPAIR The sirtuin pathways are very important in the repair of DNA damage. The following diagram give us some insight into this process. The ssDNA is damage to a single strand of DNA while DBS repair represents a double strand break in the DNA. The following illustration shows the difference between single (ssDNA) and double Strand (DSB) DNA breaks. DNA safekeeping is one of the most important functions of the cell, allowing both the transfer of unchanged genetic material to the next generation and proper cellular functioning. Therefore, cells have evolved a sophisticated array of mechanisms to counteract daily endogenous and environmental assaults on the genome. These mechanisms rely on the recognition of the damaged DNA and its subsequent signaling. If DNA repair is not accomplished, this can lead to a variety of medical problems from cancer to a variety of degenerative diseases. SIRT1, SIRT3 and SIRT6 are involved in the signaling of different DNA repair pathways through key signaling factors. They help accomplish this with a variety of enzymes. Actually, DNA repair is intimately tied into chromatin regulation. Again, we go back to the histones and their importance. The Sirtuin histone targets are essentially gene targets. A major enzyme the sirtuins work with is PARP. The main role of PARP (found in the cell nucleus) is to detect and initiate an immediate cellular response to metabolic, chemical, or radiation-induced single-strand DNA breaks (SSB) by signaling the enzymatic machinery involved in the SSB repair. One very significant fact is that the PARP enzyme is a very big consumer of NAD+. This is one of a number of reasons that as we age we need to increase the amount of NAD+ or its precursors that is available to our body. More about NAD+ in a bit.   SIRTUINS AND MITOCHONDRIA Mitochondria play a critical role in energy production, cell signaling and cell survival. Defects in mitochondrial function contribute to the aging process and aging-related disorders such as metabolic disease, cancer, and neurodegeneration. As time goes on we are realizing the mitochondrial malfunctions leave their footprints on most diseases. Alterations in the expression/activity of SIRT3, SIRT4, SIRT5 are linked with many different diseases. Overall, mitochondrial sirtuins regulate mitochondrial protein networks, orchestrate mitochondrial function, and allow cells to adapt to metabolic stresses. In addition, emerging evidence indicates that sirtuins regulate yet another important cellular process, autophagy. Autophagy is extremely important in the body’s recycling process. It allows the orderly degradation and recycling of cellular components. The sirtuins and mitochondria are intimately involved in this process. If you encourage autophagy you encourage anti-aging. SIRT3, SIRT4 and SIRT5 (mitochondrial sirtuins) belong to the sirtuin family proteins and are located in the mitochondria. They catalyze NAD+ into a variety of different compounds. They modulate the function of various targets to regulate the metabolic status in cells. Emerging evidence has revealed that mitochondrial sirtuins coordinate the regulation of gene expression and activities of a wide spectrum of enzymes to orchestrate oxidative metabolism and stress responses. Mitochondrial sirtuins act in synergistic or antagonistic manners to promote respiratory function, antioxidant defense, insulin response and adipogenesis to protect individuals from aging and aging-related metabolic abnormalities. The next diagram shows how mitochondria produce energy, namely ATP, the body’s energy currency. ATP keeps the cells alive and it is instrumental in many of the body’s repairs. This is the Krebs cycle and other energy producing cycles. HOW DO THE SIRTUINS ACCOMPLISH THEIR VARIOUS TASKS? The basic role of sirtuins is that they remove acetyl groups from other proteins. An acetyl group is a small molecule made of two carbon, three hydrogen, and one oxygen atoms. When Acetyl groups are added to or removed from other molecules that may affect how the molecules act in the body. In the above diagram the Acetyl group is represented by Ac in the green hexagons. Acetyl groups control specific reactions. Sirtuins work with acetyl groups by doing what’s called deacetylation. This means they recognize there’s an acetyl group on a molecule then remove the acetyl group, which tees up the molecule for its job. One way that sirtuins work is by removing acetyl groups (deacetylating) biological proteins such as histones. For example, sirtuins deacetylate histones, proteins that are part of a condensed form of DNA called chromatin. Here we see the DNA strands wound about the histones. The histone is a large bulky protein that the DNA wraps itself around. Think of it as a Christmas tree, and the DNA strand is the strand of lights. When the histones have an acetyl group, the chromatin is open, or unwound. This unwound chromatin means the DNA is being transcribed, an essential process. But it doesn’t need to remain unwound, as it’s vulnerable to damage in this position, almost like the Christmas lights could get tangled or the bulbs can get damaged when they’re unwieldy or up for too long. When the histones are deacetylated by sirtuins, the chromatin is closed, or tightly and neatly wound, meaning gene expression is stopped, or silenced. Sirtuins deacetylate a multitude of targets including histones, transcription factors, and metabolic enzymes. Taken together, sirtuins have been implicated in numerous cellular processes including stress response, DNA repair, energy metabolism. Sirtuins are a family of proteins that act as metabolic sensors. They deacetylase a coenzyme NAD+ into free nicotinamide. Basically, they break down acetyl from proteins to maintain their functioning for longer. The ratios of NAD+ and NADH determine the nutritional status of the cell and sirtuins are there to respond to it. NAD+ is an essential currency for energy metabolism and DNA repair. Sirtuins are proteins that evolved to respond to the availability of NAD in the body. The preceding diagram spells out the relationship between the Sirtuins and NAD+. We see that the sirtuins in response to NAD+ levels influence a number of other pathways which are responsible to a variety of different diseases. The bottom line is the Sirtuins are a family of proteins that act as metabolic sensors. They deacetylase a coenzyme NAD+ into free nicotinamide. The ratios of NAD+ and NADH determine the nutritional status of the cell and sirtuins are there to respond to it. NAD+ is an essential currency for energy metabolism and DNA repair. Sirtuins are proteins that evolved to respond to the availability of NAD+ in the body. SIRTUINS AND OXIDATIVE STRESS The sirtuins help regulate oxidative stress and inflammation. The above diagram shows this very well. In this diagram we see that the sirtuins have a direct effect on a pathway called the NRf2 pathway. This is the major pathway in the body that helps reduce inflammation. Consider it a thermostat of inflammation. When this pathway is stimulated it produces very powerful antioxidant enzymes which will significantly lower inflammation. The antioxidant properties also tie into the NAD/NADH ratio which ideally, we like to be 700/1. HOW TO INCREASE SIRTUINS There’s a lot of evidence pointing to the longevity benefits of increased sirtuin activity. If not in over-expression, then increasing sirtuins can still be good for your health in most cases. Glucose restriction extends the lifespan of human fibroblasts because of increased NAD+ and sirtuin activity. Inhibiting insulin shuttles SIRT1 out of the cell’s nucleus into the cytoplasm. Cancers use primarily glucose and glutamine for fuel with the exception of some ketones in rare cases. Caloric restriction and fasting increase SIRT3 and deacetylate many mitochondrial proteins. Reduction of calorie intake without causing malnutrition is the only known intervention that increases the lifespan of many species including primates. It’s thought that these effects in longevity require SIRT1. The following illustration demonstrates a variety of methods to help increase sirtuin activity. The chart is broken down into a few different categories. The input and effector categories are the most important to us. We see the sirtuins are very important sensors. Activating AMPK elevates NAD+ levels, leading to increased Ketosis. Ketone bodies like beta-hydroxybutyrate promote sirtuin activity. This is essentially a ketogenic diet. Thus, one simple way of increasing the sirtuins is by following a somewhat ketogenic diet. Natural ways of caloric restriction and fasting are still the best ways of signaling energy deprivation which promotes longevity. In an everyday context, a low carb diet is also pro-sirtuin to a certain extent because of the low levels of insulin and glucose. Exercise has anti-inflammatory effects and it increases SIRT1. The long-term benefits of exercise are even thought to be regulated by SIRT1. Cyclic-AMP (CAMP) pathway activates SIRT1 very rapidly to promote fatty acid oxidation independent of NAD+. CAMP is linked with AMPK (another very important pathway in the body) which gets activated under high energy demands while being energy deprived. This can be accomplished with cold exposure and high-intensity exercise training. Heat exposure and saunas increase NAD+ levels which promote SIRT1 as well. Sweating, cardio, yoga, or infrared saunas will probably have a similar effect on activating heat shock proteins which can increase sirtuins. Chronic oxidative stress and DNA damage depletes NAD+ levels and decreases sirtuin activity. This will then disrupt DNA repair and impair mitochondrial functioning. That’s why you want to keep stressors acute and followed by recovery. Melatonin can activate sirtuins and has anti-aging effects. It’s also the main sleep hormone and a powerful antioxidant that helps the brain get more recovery from deeper stages of sleep. Sirtuins also affect the circadian clocks so keeping a consistent circadian rhythm is incredibly important for longevity. NAD+ is under circadian control and when you you are misaligned you’ll have less energy and lower SIRT1 and SIRT3 activity. The enzyme SIRT1 increases FOXO DNA binding by deacetylating FOXO in response to oxidative stress. FOXO is another very important pathway. FOXO proteins get increased in response to cellular stress and increased energy depletion. PERHAPS NAD IS THE BEST WAY TO INCREASE SIRTUIN FUNCTION Maybe the best way to consider NAD+ is to consider it as the energy currency to purchase increased Sirtuin activity. Of all the methods to increase Sirtuin activity NAD+, especially when given intravenously, may be one of the most effective sirtuin stimulators. In the above diagram, STACs are sirtuin activating compounds. In addition to Resveratrol, there is also Pterostilbene which is a better effector than Resveratrol. THE BOTTOM LINE IS EAT THE ABOVE FOODS, EXERCISE, ESPECIALLY INTERMITTENT HIGH INTENSITY EXECISE, INTERMITENT FASTING, AND CALORIE RESTRICTION WILL INCREASE YOUR SIRTUINS. TAKE PLENTY OF NAD OR ITS SUBSTRATES. WHEN YOUR SIRTUINS ARE INCREASED IT WILL HAVE A WATERSHED EFFECT ON THE OTHER PATHWAYS IN THE BODY. THE DIAGRAM BELOW SAYS IT ALL. Thanks, Dr. P

  In 1972, Easter Island, called Rapa Nui, famous for its Moai statues, offered a new wonder: the discovery of the drug rapamycin named after the island's Polynesian name, Rapa Nui. Over the past three decades, rapamycin, which was isolated from soil bacteria, has been applied as an immuno-suppressor in a multitude of ways, including to coat coronary stents and to reduce the immune responses in people who receive organ transplants. Currently, it’s garnering attention because of its potentials in anti-cancer and neuroprotection as well as anti-aging therapies. Rapamycin has extended the expected lifespan of middle-aged mice by 28 percent to 38 percent. In human terms, this would be greater than the predicted increase in extra years of life if cancer and heart disease were both cured and prevented. Aging researchers currently acknowledge only two life-extending interventions in mammals: calorie restriction and genetic manipulation. Rapamycin appears to partially shut down the same molecular pathway as restricting food intake or reducing growth factors. We can see in this illustration of this concept. What we do notice is that caloric restriction intimately tied in with longevity. Rapamycin works by targeting a master regulator of cell growth in our cells called mTOR pathway. mTOR is a kinase which is a protein that phosphorylates (adds a phosphate group) to an amino acid. This is important because the phosphate group basically acts as a flag telling other proteins to bind on top of the phosphate. A bit more about kinases. As one of the most abundant gene families in humans, protein kinases tightly control cell signaling via pathways and cell function through protein phosphorylation. What is a pathway and how do we put it into the proper prospective? I saw an excellent article that gave a great analogy on how pathways function in the body. Think of the cell and all its different organelles (such as mitochondria) as the computer hardware and the biological pathways act as the software. We might not be able to change our hardware but we can modify the software. This is the essence of Anti-Aging medicine. When we want to truly alter the course of aging we typically need to influence the pathways or the “software”. All too often aging problems are attacked by altering the downstream causes of aging. This is typically not an effective method of treating aging. When rapamycin targets mTOR, it inhibits cell growth. Many of the problems that come with aging arise from uncontrolled growth or aging cells that have accumulated so much internal cellular debris that they lose healthy functionality. This makes rapamycin a potent anti-cancer drug, since cancer is marked by uncontrolled cell growth. The inhibition of mTOR also triggers autophagy, a process by which lysosomes, the so-called recycling centers of cells, clean up misfolded proteins and damaged organelles. During stress conditions, autophagy can lead to the cell survival by degrading dysfunctional components and providing the building blocks of cells, such as amino acids and lipids for reuse by the cell. mTOR is involved in every aspect of cellular life and existence. The following diagram shows some aspects of the mTOR pathway. In the case of mTOR we are actually trying to apply the brakes to cell growth and proliferation. In addition, the mTOR pathway is a direct target of the IGF-1 signaling pathway, which is a major driver of aging. Despite talking about mTOR as if it is just one molecule, it functions, in fact, as two separate complexes called mTORC1 and mTORC2 as we see from the following diagram: mTORC1 and mTORC2 which are composed of discrete protein binding partners to regulate cell growth, motility, and metabolism. These complexes are sensitive to distinct stimuli, as mTORC1 is sensitive to nutrients while mTORC2 is regulated via growth factor signaling. mTOR can affect cell division processes, the response to stress, and general cell and protein tasks. These signaling pathways are very complicated and depend on many feedback loops, energy supply, and a great variety of other molecules or signals. During certain diseases and aging, the function of mTOR can become deregulated. What does all of this have to do with aging? Many processes that lead to better longevity outcomes, like calorie restriction, fasting, and protein restriction, stimulate autophagy – a process within cells that cleans up broken proteins, bacteria, and viruses. Autophagy stimulates longevity. Aside from regulating cell growth and metabolism, mTORC1 also controls autophagy, an intracellular process that allows orderly degradation and recycling of cellular components. mTORC1 negatively regulates autophagy. The following illustration demonstrates Autophagy. The lysosomes are essentially recycling centers. mTOR SUPPRESSES AUTOPHAGY, WHICH SUPPRESSES LONGEVITY, THUS SUPPRESS mTOR INCREASE AUTOPHAGY Activating mTOR prevents autophagy from occurring. Autophagy seems to be a crucial component of longevity. Therefore, suppressing mTOR which rapamycin does should have longevity benefits. It is this effect, plus others like its suppression of cell division and growth, that immediately raised interest for its study in the field of aging sciences. But herein lies a cautionary tale for all potential aging therapeutics. Remember, mTOR affects many processes important for biological functioning. We cannot just eliminate mTOR without subsequent problems. We need to walk a fine balance line. mTORC1 and mTORC2 generally promote an anabolic response by stimulating protein synthesis, glycolysis, mitochondrial functions, and lipid synthesis to influence proliferation and survival, effector and memory responses, innate training and tolerance as well as hematopoietic stem cell maintenance and differentiation. Deactivation of mTOR restores cell homeostasis after immune activation and optimizes antigen presentation and memory T‐cell generation. Antigen presentation and memory T-cell generation are the basis of the Covid-19 vaccines. These findings show that the mTOR pathway integrates information of the environmental and cellular energy status by regulating cellular metabolic responses to guide immune cell activation. Elucidation of the metabolic control mechanisms of immune responses will help to generate a systemic understanding of the immune system and may eventually help us in dealing with the Covid-19 virus. Should we be concerned when we utilize rapamycin to block the mTOR pathways? Remember, the mTOR pathway affects many processes to keep our cells functioning. Suppressing it too intensely is clearly not good; we need a lighter touch. Like many things in life we need a fine balance. Not only do we need to establish the right dose for us, but we also need to identify the right treatment regimen. In some clinical studies, it took about two weeks to demonstrate significant suppression of mTOR1C. Sensible usage strategies (if there are any) for rapamycin could, for example, be for those aged 60 years and older to either use a low daily dose or to use it for 30 days at a time every 6 months or so. Low-dose, short-term rapamycin in healthy humans has been found to be safe, with few or no side effects. It’s possible that rapamycin can be given to healthy humans with fewer side effects than occur in transplant recipients. Healthy men given a single dose of rapamycin of roughly 16 mg/kg had no significant difference in the incidence of adverse effects between treatment and placebo. Healthy elderly volunteers given everolimus (a rapalog of rapamycin) at 0.5 mg daily, 5 mg weekly, or 20 mg weekly, for six weeks, had no serious adverse effects associated with the drug. The most common associated side effect was mouths ulcers. The treated groups had higher antibody titers in response to flu vaccines compared to controls, implying better immune function. Note that this is a much lower dose than the doses given in mouse studies of rapamycin. A rapalog is a class of molecules that has rapamycin-like activity but is not confined to chemical derivatives of rapamycin. A rapalog is also known as an analog. Unfortunately, treatment with rapamycin and its analogs/ rapalogs is associated with negative side effects that limit their potential utility as anti-aging therapies. These side effects include immunosuppression, glucose intolerance, an increased risk of type 2 diabetes, and disruption of lipid homeostasis. Although short term, low dose, and/or intermittent rapalog-based regimens may partially limit side effects, and trials of rapamycin in the elderly have begun, the efficacy of such treatment regimens in treating age-related diseases remains to be determined, particularly as the effects of rapamycin on longevity are dose-dependent. All three FDA-approved rapalogs sirolimus, everolimus, and temsirolimus have similar effects on the glucose metabolism and immune cell profile of mice. It is clear that there is an urgent need for new molecules that inhibit mTOR signaling with reduced side effects. There are some studies that claim the Metformin may also target mTOR. Taken together, rapamycin and metformin are promising candidates for life and healthspan extension; however, concerns of adverse side effects have hampered their widescale adoption for this purpose. It appears that the two may cancel out the unwanted side effects of each. Recent studies have demonstrated that a number of natural products, nutraceuticals, isolated from plants (e.g. fruits, vegetables, spices, nuts, legumes, herbs, etc.) also inhibit PI3K/Akt/mTOR pathway, and exhibit potent anticancer activities and probable antiaging activities. As most of the natural products occur in our diet every day, and are very safe, the results suggest that those natural products may be explored for cancer prevention and treatment. This special issue selects apigenin (present in many fruits, vegetables), curcumin, cryptotanshinone (Asian herbal compound), fisetin (compound from various berries), indoles (Cruciferous vegetables), isoflavones (genistein and deguelin), quercetin, resveratrol, and tocotrienol (a form of Vitamin E). Immunologically, mTOR has a fundamental part in controlling and shaping diverse functions of innate and adaptive immune cells, in particular, T-cell subsets differentiation, survival, and metabolic reprogramming to ultimately regulate the fate of diverse immune cell types. We must remember m TOR is a kinase. As one of the most abundant gene families in human, protein kinases tightly control cell signaling and cell function through protein phosphorylation. There are 634 protein kinases encoded in human genome to date, and 85% kinases are observed dysregulated in human disorders. However, only 49 small molecule kinase inhibitors have been approved for treating human diseases, urging for more in-depth investigations on the rest 77% possibly druggable kinome. Among all protein kinases, mTOR (mechanistic target of rapamycin, previously known as mammalian target of rapamycin) has drawn extensive attention. This is due to its indispensable roles in regulating key cell function such as proliferation, metabolism, autophagy, ageing, and others, and its ability to sense environmental changes including nutrients and growth factors to adjust cell physiology. Dysregulation of mTOR signaling is observed in solid tumors, epilepsy, obesity, and diabetes. The bottom line for the mTOR pathway is intermittent fasting, calorie restriction, a number of different supplements, and some intermittent rapamycin in the prescribed dosages and regimens which include Metformin. I suspect, as we refine things, we will see much more of rapamycin use. I suspect mTOR inhibition is just the tip of one of the icebergs. Thanks, Dr. P  

  As a physician and scientist, I always like to put politics aside. I must give accolades to Operation Warp Speed. It is difficult for people outside the biomedical research circuit to grasp the magnitude of creating a working vaccine for any disease, within a year. To put that in perspective, the fastest vaccine development cycle on record was four years, for mumps. To have a vaccine developed, found to be relatively safe, and available for wide distribution in such a short period of time is unheard of. This dedication and mind set reminds me of back in the 1960s when we were involved in the space race for the moon. It took us 10 years to go from dream to reality. For the vaccine race it took us 10 months to go from dream to reality. These two achievements are not that dissimilar. The space race gave our world many different gifts of technology ranging from different types of plastics to computer technology. Our society would be much different without the “technological gifts” from the space race. I suspect we are going to see similar “gifts” from the vaccine race that will forever change our society. First let’s talk about the implications for the here and now about the vaccine.   One big question everyone has if it will still be necessary to wear a mask after receiving the Covid-19 vaccine? The answer for this is yes. For a couple reasons, masks and social distancing will still be recommended for some time after people are vaccinated. First off remember that the protection the vaccine affords will take a few weeks to develop. We will need to get two shots and effective immunity may take a few weeks after the second injection. The still unknown out there is if the vaccine protects us from the infection or does it just prevent symptoms? Potentially, vaccinated people might still be able to get infected and pass the virus on, although it would likely be at a much lower rate. Lastly, the sheer numbers of people that will require vaccination will take several months to complete. So, for now I do not see masks going away until sometime late in the second half of 2021.   WHAT ABOUT THE VACCINE ITSELF? Any vaccine has controversy to it, yet by and large, they have changed the course of history in a positive way. Just read about the struggles with the Polio virus until an effective vaccine was produced. The Polio Virus was a scourge that would cause paralysis in many otherwise healthy people. Also, unlike the Covid-19, the Polio Virus had a predilection for younger patients actually it was also known as “infantile paralysis”   WHAT IS THE VACCINE MADE OF? THE ANSWER IS MESSENGER RNA (mRNA) Every strand of mRNA is made up of four molecular building blocks called nucleosides. But in its altered, synthetic form, one of those building blocks, like a misaligned wheel on a car, can throw everything off by signaling the immune system. Thus, scientists simply subbed it out for a slightly tweaked version, creating a hybrid mRNA that could sneak its way into cells without alerting the body’s defenses, a giant step. That was an aha moment. It changed the course of history. Looking at things further, the above diagram is an excellent representation on how the Covid-19 vaccine works by using mRNA also known as messenger RNA. In molecular biology, messenger RNA is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene. One can consider mRNA as a blueprint. The mRNA is read by a ribosome which subsequently synthesizing a protein in the cell. In step 1 of the above diagram we see the protein spike on the virus outer shell. What the scientists do is make a mRNA sequence that codes (blueprints) for the virus spike protein. Once the spike is produced it is mixed with a lipid coating and is prepared for injection into the patient. Once inside the patient the mRNA prompts the cells to produce millions of spike proteins. The spike proteins than stimulate the immune system to produce antibodies to protect the cell when a virus attacks the cells. Although this is a simplistic view it is gives a good idea on how the vaccine works. What I like about the next diagram is that it shows more specifics about the immune system. We see that the immune system is making use of what are called T-helper cells. The T-helper cells recognize the virus and activate either cytotoxic T cells which kill virus infected cells or B cells which launch antibodies which will block the virus from infecting healthy cells. Although the studies don’t clarify whether people who have had a clear a SARS-CoV-2 infection or receive the vaccine can ward off the virus in the future, both identified strong T cell responses to it. In another words this bodes well for the development of long-term protective immunity. The Antibodies that are produced may not last that long but the T cells should give us long lasting immunity. This may explain why a large portion of the population may be able to deal with the Covid 19 virus due to previous exposure to common cold viruses which indeed have some cross over to Covid-19. I feel comfortable that the immunity we receive from the vaccine will be long lasting. I feel the same about the immunity received from an infection. SOME COMMON QUESTIONS ABOUT THE VACCINE One important question people are asking is how the new vaccine is different from traditional vaccines. The above diagram shows the major differences on how the vaccines are produced. Traditional vaccines use bits of injected proteins to train the immune system to take down future viruses displaying those same proteins. Manufacturing them takes months, a timescale too slow to combat emerging epidemics. mRNA vaccines, on the other hand, simply encode these protein fragments in a single mRNA strand. As mRNA companies optimize and scale up their enzymatic production of mRNA, scientists anticipate these vaccines could be made in a matter of weeks.   A number of questions arise concerning the vaccines. One question is if the mRNA will incorporate into our DNA. The simple answer to this is NO. mRNA is designed to transcribe proteins etc. Its job is not to act as an enzyme and affect the structure of the DNA. mRNA is not able to alter or modify a person’s genetic makeup (DNA). The mRNA from a COVID-19 vaccine NEVER ENTERS THE NUCLEUS OF THE CELL, WHICH IS WHERE OUR DNA IS KEPT. This means the mRNA does not affect or interact with our DNA in any way. The vaccine will not cause you to test positive on the viral tests however if you are tested for antibodies that test may be positive.   Another question is if a person has had the virus should they get the vaccine? There is no simple answer to this question since we really do not know. Some studies show that natural immunity may not last long while others say it conveys the best immunity of all. I suspect that if one was infected with the Covid- 19 virus they would have long lasting immunity. However, we will need to view the landscape in a few months to see where we are on this question. It may very well turn out that both natural immunity and vaccine induced immunity may provide the best protection of all.   When will there probably not be a need for the vaccine?  The goal of the vaccination program is to achieve heard immunity. What percentage of the population that needs to receive the vaccine or have had a previous infection is still not known to achieve herd immunity.   MANY PEOPLE ASK ME IF I WILL TAKE THE VACCINE. THE ANSWER TO THIS QUESTION IS YES. I suspect that a vaccination will be required to travel to many different countries. Also, airlines such as Qantas will probably start requiring passengers to be vaccinated to travel on their planes. As a matter of fact, I reserved the domain name Immunityvisas.com for this very reason. I would suspect that the cruise industry may also require a vaccination to take a cruise. The bottom line is for the world to return to some semblance of normal we will have to achieve herd immunity either by immunization or actually having the disease or both.   ANYTHING ELSE WE WILL BENEFIT FROM WITH THE COVID-19 VACCINE BESIDES IMMUNITY? THE ANSWER IS mRNA THEURAPEUTICS The two preceding illustrations give the essence of mRNA therapy. This is a therapy that already has and will continue to disrupt medicine. Here is a quote that I read in an article that was written in early 2019 before we realized the pandemic was here: “When the first mRNA therapeutic crosses the finish line, we’ll all be winners, suggests CureVac, a biopharma that anticipates an mRNA stampede”. Like the space race of the 1960s, there will be much in the way of spinoffs from the Covid-19 vaccine. The first vaccines against Covid-19 aren’t just a landmark in the fight against the pandemic. They’re also the stepping stone for an unconventional technology that could one day defeat other ailments that have eluded doctors, from cancer to heart disease. What I am talking about here is the new field of mRNA therapeutics. This is possibly at the very heart of Stem Cell and Regenerative Medicine therapies. This is one of the basic mechanisms that can allow stem cell to help regenerate tissue. We can see how the Big Pharma and the Biotech industry has embraced this new technology. Perhaps they can come up with a vaccine for many types of cancer. There are already important achievements in the field of immunology and cancer with some promising new therapies. Remember that the Covid-19 virus was not the first kid on the block. Every year influenza kills at least 650,000 people. Perhaps with mRNA technology we can make one vaccine to essentially eliminate influenzas. This is not as far-fetched as it sounds. There is also a possibility that mRNA technology may be used for reprograming stem and other cells in the future. There are still problems that we will encounter with mRNA therapy but these will be overcome. I think the appropriate way of ending this piece on the vaccine and mRNA therapy is to take some poetic license on the quote from Neil Armstrong as he took his first step on the surface of the moon: That’s one small step contributed by many people, but one giant leap for mankind. Thanks, Dr. P