Immuno-Senescence: What Clinicians Need to Know with Dr. Joseph Raffaele

Folks, if you’ve been following me for any time, you know I am fascinated with reversing epigenetic aging. With such research rapidly evolving, there’s always something new to talk about and my guest today has become a friend and a regular on the New Frontiers podcast – Dr. Joseph Raffaele: a renowned expert on telomere biology and age management medicine. Dr. Raffaele, who is board-certified in internal medicine, diplomat at the American Board of Anti-Aging Medicine, AND co-founder of PhysioAge Systems, joins me for the third time to talk about the latest in immunosenescence and aging. (You can find links to our previous conversations and related content in the shownotes.)

I’m excited to continue the conversation with Dr. Raffaele, as we dive into the connection between viral infections, including COVID-19 and CMV, and cell senescence and what we can do clinically to reduce immune risk phenotypes and rejuvenate not only our immune system but our entire bodies. We also touch upon various lab markers that can help us assess immune cell health and epigenetic age, and the anti-aging properties of telomerase activator TA-65 derived from the Astragalus membranaceus plant. There are too many great take-aways to list here, so tune in and get ready to take some notes! Thanks for listening, and please leave a review if you can. ~DrKF

Are chronic viral infections accelerating our aging? Could they also be contributing to neurodegeneration? And is immune-senescence at the root of aging? These are some of the questions addressed by our guest Dr. Joseph Raffaele MD, a clinician and educator in age management medicine and the co-founder of PhysioAge Medical Group, specializing in physiologic age assessment. Dr. Raffaele is also a clinical researcher with a particular focus on telomere biology and in this episode of New Frontiers, he talks to us about the fascinating connection between immunosenescence and telomere length. Together we explore the virome and how infections can lead to accelerated aging, what lab assessments of immune cells can tell us about longevity, and the latest research on reversing epigenetic age with plant-derived telomerase activator molecule TA-65. Tune in to hear Dr. Raffaele’s clinical pearls on assessing immunosenescence and dosing TA-65 for immune rejuvenation.

In this episode of New Frontiers, learn about:

Dr. Joseph Raffaele received his BA in philosophy from Princeton, his MD from Drexel. He trained at the New York Hospital Cornell University Medical Center, and was formerly a clinical assistant professor of medicine at Dartmouth Medical School while in practice at Hitchcock Clinic. He’s a member of the Endocrine Society, is board-certified in internal medicine, and is a diplomat at the American Board of Anti-Aging Medicine.

In 1997, he co-founded PhysioAge Medical Group, where he exclusively practiced age-management medicine with a focus on personalized hormone optimization, and physiologic age assessment. And we’re going to be focusing on this today, in 2007, he co-founded PhysioAge Systems, a web-based biomarker data collection and reporting system now used by age management practices around the world to assess, monitor, and communicate to patients the effectiveness of their treatments.

Since 2009, he’s been involved in clinical telomere biology research, and he’s published four studies of the effect of oral telomerase activators on normal aging adults. He’s lectured nationally and internationally on the clinical application of telomere biology. In 2015, he founded the Raffaele Medical Group and blogs regularly about telomere biology, hormone optimization, and biomarkers of aging on raffaelemedical.com, and physioage.com.

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Dr. Kara Fitzgerald: Hi, everybody, welcome to a New Frontiers in Functional Medicine where we are interviewing the best minds in functional medicine. Of course, today is no exception. I’m thrilled to be here again with Dr. Joseph Raffaele. You’ve actually become really kind of a friend over these years, we’ve been chatting about things that interest us for a long time. Let me give you his background and then, we’ll jump right into some cool topics today.

He received his BA in philosophy from Princeton, his MD from Drexel, he trained at the New York Hospital, Cornell University Medical Center, and was formerly a clinical assistant professor of medicine at Dartmouth Medical School while in practice at Hitchcock Clinic.

He’s a member of the Endocrine Society. He’s board certified in internal medicine, and is a diplomat at the American Board of Anti-Aging Medicine. In 1997, he co-founded PhysioAge Medical Group, where he exclusively practiced age management medicine with a focus on personalized hormone optimization and physiologic age assessment. We’re going to be focusing on that today. In 2007, he co-founded PhysioAge Systems, and this is a web-based biomarker data collection and reporting system now used by age management practices around the world. In fact, he was just telling me about some cool epigenetic testing that will be incorporated into this system that he’ll tell us about.

Since 2009, he’s been involved in clinical telomere biology research, and he’s published four studies of the effect of oral telomerase activators on normal adult aging. He’s lectured nationally and internationally on the clinical application of telomere biology. In 2015, he founded Raffaele Medical Group, and blogs regularly about telomere biology, hormone optimization, and biomarkers of aging at raffaelemedical.com and physioage.com. You can find him now at Raffaele, MD on Instagram and stay tuned because he and I will jump in and do IG Live in the near future. Joe, again, welcome back to New Frontiers.

Dr. Joseph Raffaele: Thank you very much, Kara, it’s always a pleasure to be talking to you about this really fascinating line of work that we’re both in.

Dr. Kara Fitzgerald: Right, and it’s just evolving so fast. Folks, we’re going to link in the shownotes to the previous conversations that I’ve had with Dr. Raffaele and any of the content that we’ve dropped onto those show notes, we’ll just harvest and bring over to this. So just consider your show notes for this particular podcast to be your go-to for all of the previous conversations I’ve had with Joe. Two years ago, we actually did… We talked again, about testing and treating telomeres. We talked about some of the interesting studies happening in recent publications at that time, including telomeres and COVID-19. We also talked about the ancient and the new – is there interaction between cytomegalovirus and SARS-CoV-2 infection.

As time goes on, we’re learning more and more about these correlations, floodgates of scientific data are providing us significant clues to the underpinnings of this ongoing pandemic. What’s new in the scientific world as it relates to viral interaction, immunosenescent changes. Before you go into that and catch us up on current science, just give me a little bit of definition on… Actually talk a little bit about telomeres and why we’re concerned about that relating to COVID-19 and immunosenescent cells and why we’re really thinking about those as well.

Dr. Joseph Raffaele: Sure, the stuff that’s coming out now in literature, a lot of it is focused on, for obvious reasons, the COVID-19 pandemic and trying to figure out ways to approach it, ways to stratify risk, ways to treat people that have gotten infected with SARS-CoV-2. Very interestingly, it’s turning out that because we knew from the beginning that the major risk factor for getting severe disease and dying is age; I think 20% of the elderly get infected, but they account for 80% of the deaths; people have been starting to look at things other than age as a way to stratify and then also to think about ways in which the biology behind that might help us treat or prevent severe COVID disease. That all really revolves around the concept of immunosenescence. Elderly are getting higher rates of hospitalization and death, excuse me, because they have succumbed to at least a degree of immunosenescence. What is that? Immunosenescence is the process in which your immune system becomes less able to fight off as broad an array of antigens, either from tumors or viruses or other kinds of pathogens and not being able to respond to vaccinations as well.

But in addition to that, this whole concept of inflammaging occurs when your immune system becomes immunosenescent. The cells start to produce inflammatory cytokines like TNF-alpha, IL-6, the components that people are becoming more aware of, because it’s part of the cytokine storm that causes all the damage when one gets infected with severe COVID disease. Looking at an immunosenescent cell, it not only doesn’t do its job, which I sort of liken to a watchdog trying to keep the intruders out, but an old watchdog not only doesn’t keep the intruders out, but actually turns around and bites its owner, bites its friends, doesn’t really know what it’s doing. It’s just kind of, what’s the term? A hot mess. It’s always that of an immune system.

This is what happens with the immune system and we really know at this point that it’s not so much the virus itself, as often is the case with viral infections, chronic viral infections in particular, it’s the immune system’s overreaction to it that causes a lot of the damage. What causes your immune system to become senescent? Well, there’s a couple of mechanisms but one of the major mechanisms is really through shortening of telomeres. After repeated stimulation of T cells in vitro, it’s been shown by Rita Effros at UCLA and others in subsequent studies, that after three rounds of strong stimulation, the cells stop dividing, their telomeres get really short, the telomerase is no longer expressed, which is the enzyme that makes telomeres longer and they don’t express this very important molecule called CD-28. And that helps to mount a brisk response when the T cell links up with the antigen presenting cell through the MHC, you need that CD-28 molecule to mount a really brisk proliferative response to the antigen is trying to knock out.

If this occurs regularly from chronic viral infections, from many other things, then your telomeres get short, and you have an accumulation of these senescent T cells. That’s one way, that’s called replicative senescence. There are other ways in which cells can become senescent, meaning that they don’t divide, but then also produce this, what is called the SASP, senescence-associated secretory phenotype, which is all those inflammatory cytokines I mentioned.

If there’s DNA damage, sometimes cells will go into that, recent paper showed that a viral infection can cause what’s called an acute senescence response. There’s a number of different ways to get there, but the bottom line is you have an ineffective cell that also causes damage. Telomeres, just to review real quickly, are the caps on the ends of your chromosomes that are non-coding DNA of the sequence TTAGGG that repeats between eight and 12,000 base pairs or eight to 12 kilobases at around age 25 is what you have then.

Then every time cells divide, they get a little bit shorter because the enzyme telomerase which works to lengthen telomeres, is really suppressed after birth, for the most part, except with some activity in highly proliferative tissues like the bone marrow and skin cells and gut, white blood cells and in stem cells, of course. That’s not enough expression to completely stop that slow attrition of telomere length. Telomeres are known to decline about 50 base pairs or 0.05 kilobases per year between ages 20 and 80, so that by the time you get to 80, sort of the average life expectancy, your telomeres are under five kilobases, sometimes even around between four and five. A lot of bad things start to happen at that point. That’s sort of a brief introduction to what immunosenescence is, and the relationship of telomeres are to it.

Dr. Kara Fitzgerald: Let me just throw a couple of thoughts. Basically, the aging journey. I guess it really a normal aging process and then of course, you could have sort of the standard American version of aging where you’re turning the volume up on inflammaging. You’ve got the aging journey, just increasing vulnerability to getting infections, chronic infections, or something acute, like COVID. We’re putting a lot of attention in looking at SARS-CoV-2 these days. But, it’s a snapshot into aging as well and I would imagine that if we’re able to improve response to COVID, or if we’re able to just address immunosenescence, we’re actually probably slowing down or even reversing biological age. It’s that related.

Dr. Joseph Raffaele: Well, yeah, and it’s funny that you mentioned that there actually was a paper published this year, showing that immunosenescence actually has effects on non-lymphoid tissues such as heart, lung, skin, brain, et cetera. In a model of aging, Yousefzadeh took mice and deleted a DNA repair protein called ERCC1, but did it selectively in hematopoietic cells to see whether or not senescence of the immune system alone caused problems other than just immunosenescence, it caused aging of other tissues. What they found in these mice that they were kind of normal up until about age five months, at which point they were sort of more resembled two years old mice, which is like an eighty-year-old mouse.

They were sort of healthy into young adulthood basically, but then with the increase in senescent cell that they measured in them and they had an increase in SASP, that’s cytokine production, that sort of smoldering inflammation that is what SASP is, they also had decreased response to delayed hypersensitivity testing. Their immune system was definitely senescent. But they also found that in non-immune cells, there was senescence and damage in liver, spleen, kidney, intervertebral discs, there were fewer glycosaminoglycans. There’s brain degeneration. There are markers for senescence, also, the P-16 and P-21 were increased in SA beta-gal (senescence-associated beta-galactosidase) staining and their lifespan was decreased.

This is really only from a deletion of the ability to repair DNA in the cells of the immune system. It spreads exactly your point is if you start to have immunosenescence, then you’re going to have accelerated senescence of the rest of the body. We know from studies that Judith Campisi did at the Buck Institute that it only takes one or two or even 3% of cells and a tissue to be senescent to wreak havoc. Because, they sit in a little niche and then they start to secrete these inflammatory cytokines, which then starts to cause phenotypic changes in the surrounding cells.

But that’s in a local neighborhood. Okay, so as we talked about, ZIP codes. There’s good ZIP codes and bad ZIP codes in neighborhoods. But they’re kind of a little bit more localized with the immune system, they have access to every tissue. A bad senescence cell to secrete inflammatory cytokines is able to affect multiple tissues. In fact, the whole body. This one study and other studies of different types, I think, are proof that rejuvenation of the immune system, and likewise, aging the immune system has profound effects on the rest of the body.

Dr. Kara Fitzgerald: Yeah, that’s awesome. Thank you that is just very elegantly stated. Again, folks, we will make sure that we corral together all of the papers that Joe’s mentioned, and we’ll put them on to the show notes. I think what you’ve said in summary is that the immune system is the system. The immune system is interwoven with every other organ, it’s not a separated entity. And aging, I think Jeff Bland really is linking, well lots of people are linking it but, perhaps Jeff is somebody I’ve been listening to most recently.

If we’re going to be addressing aging, we can’t not put most of our attention on the immune system. I want to ask you to that end, we talked about this last time. We talked about COVID and then we mentioned the cytomegalovirus paper, these smoldering viral infections that really probably most of us have, chronically can be a problem. Maybe you can talk a little bit about that for anybody who didn’t listen to our previous conversation, and we’ll link to those papers.

Dr. Joseph Raffaele: Yeah, smoldering viral infections, particularly what we have the most evidence on is the herpes viruses. Because they are, as the phrase goes, the gift that keeps on giving, and that’s being facetious. Basically, they sit latent most of the time in various tissues depending on which one you’re talking about. They require the immune system to work to keep them at bay and if the immune system has to turn its attention to other stressors or infections, you can have reactivations of any one of the seven herpes viruses. There has significant showing that the more herpes viruses you have, the sort of more information you have. Certainly, I think we mentioned last time, there’s a paper following people for three years, and those with cytomegalovirus, and then any other three herpes viruses. The more herpes virus you have, the greater your telomere attrition is, and not a small amount of greater, I mean like 20-30% more telomere attrition, with four versus one herpes virus.

It certainly increases telomere attrition, which is known to be associated with virtually every disease of aging. That’s evidence for the herpes viruses. It’s associated with increased risk of cardiovascular disease, people who are CMV positive. Anything that stresses the immune system increases senescent T cells is definitely going to do that. There was a paper that came out recently as well, looking at the title was, “Are the Healthy Vulnerable?” They started the study before the pandemic, it’s by Poloni et al at McGill. They looked at CMV serial positivity in healthy aging adults. They found that it was associated with immune dysregulation, but also with accelerated epigenetic age.

You and I have talked about and you’re deeply involved in what these epigenetic clocks and DNA methylation changes due to the aging and various disease processes and the ways in which we can affect them. They looked at it in the group of 90 adults between 60 and 90 average age 72 years of age. In the CMV negative there was 4.6 that were in this what we call immune risk profile, which is where the CD-4 count divided by the CD-8 count, the CD-4 to CD-8 ratio is less than one that’s been shown in a series of studies to correlate with 50% increase mortality over just six years in those who are not versus those who are not in this what we call immune-risk phenotype.

There was 4% in the CMV negative, 16% in the positive. There was 55% CD28- [cells] and 25% CD28 versus 23.5% in the CMV negatives,* [see footnote below] so they had definitely more senescent immune system. But what they found was when they did DNA methylation and used an epigenetic clock that was available at the time they were doing the study, they had 5.1 years epigenetic age acceleration versus the CMV negatives. Yeah, having these chronic viral infections, particularly CMV. I’ve said this before, and I wonder whether I should say it again, but I really think that CMV is a really, really slow HIV. It just requires… but it’s not in the CD-4 compartment, it’s in the CD-8 compartment, but it leads to increase in immune aging, inflammation. There are just hundreds of studies showing these associations from various different organ systems and various different diseases.

I think that they’ve worked really hard and found a vaccination for SARS-CoV-2, I hope that they really turn their attention on finding one for CMV because it is so damaging really, over time. Interestingly, they’re also finding reports in the literature now, I’m finding reports in the literature, where patients that have severe COVID disease or ended up getting opportunistic infections when they’re CMV positive, like CMV proctitis, CMV pneumonitis, other things; because it’s reactivating and it’s really, really bad virus that causes these infections when you don’t have a fully competent immune system.

Dr. Kara Fitzgerald: That’s so interesting. So these smoldering viruses are pro-aging, and they can wreak havoc if the system is vulnerable from another illness such as SARS-CoV-2, or just the aging journey itself. That’s a pretty strong statement about correlating it with HIV. Well, indeed, it begs the question, since so many of our patients have one of the herpes viruses hanging around that may come and go, I mean, what are some of your go-to interventions for dealing with this?

Dr. Joseph Raffaele: Yeah, it’s a good question. Unfortunately, there is not a good antiviral that’s not a little… The ones we currently have are a little bit toxic, nephrotoxic and liver toxic. You use them when someone’s going to succumb to the disease as we did when we were treating, or I did, when I was treating AIDS patients during the AIDS epidemic in the late 80s, early 90s. But, we wouldn’t give that to a relatively healthy person to try to keep CMV at bay. We don’t have a direct CMV one.

If somebody has chronic recurrent, either HSV-2, I’m sorry, HHV-2 genital herpes, I sometimes put patients on chronic Valtrex treatment to keep it at bay, particularly if they have very high titers of CMV. Because, the thinking behind it is, and again, this is not been backed by a study. But the thinking behind it for me is that if you can keep the attention on CMV and not have it diverted if they’re going to have an outbreak of HSV-1 or general herpes, then perhaps you can decrease the amount of damage that CMV can do. At the same time, they have decreased recurrence of the herpes symptoms either labial or general.

There’s also some really interesting data that has come out on the role of herpes HHV-1 and Alzheimer’s disease and as a risk factor for increased risk of Alzheimer’s. Studies showing that people who were either on prophylaxis or were recurrently treated with anti-viral such as Valganciclovir have reduced incidences of Alzheimer’s, which makes sense, again, through the sort of senescence pathway. It’s not the neurons that are becoming senescent because they don’t divide, they’re post mitotic, but the supporting cells, the glia, the microglia are the ones that get senescent and then turn nasty with not doing their job, not energetically supporting them, and also secrete inflammatory cytokines that cause damage to the neurons.

Yeah, so that’s my justification for that, I think it’s a relatively low toxicity. I’m following titers, I haven’t seen a strong signal yet is whether or not I get a decrease in the CMV IgG, as I mentioned, to CMV IgG titers are the way you diagnose somebody with CMV. But also, higher titers are associated with more mortality and morbidity and presumably if you can bring those down you’ve done the patient some good.

Dr. Kara Fitzgerald: Do you see them shift with the diet and lifestyle?

Dr. Joseph Raffaele: I don’t have… We’re still collecting data, you have to get a pretty large data set on that, to make that assessment. I can’t say that I’ve seen that, I mean, most of my patients come in doing pretty well from that standpoint, and we certainly make adjustments as needed in them. But I haven’t looked at that systematically, I know you do a lot more of that than I do. I’ve got to work on my database to add more collection of things like, what’s your calorie intake, et cetera and what’s your phytonutrient intake, but that I don’t have that ability just yet.

Dr. Kara Fitzgerald: Well, it’ll be pretty cool when you can kind of data crunch the massive amount of information you guys are building into your system, it’ll be really exciting. Let’s see, there was a study published just recently, “Virus-induced senescence is driver and therapeutic target in COVID-19.” This was a Lee et al, just kind of underscoring everything you’ve said, anything you want to add? Any comments on this study?

Dr. Joseph Raffaele: Yeah, I think again, it’s interesting, because, I think it shows that it is the immune system’s response to the virus that causes the problem. When it got in, the virus increased the senescence-associated secretory phenotype, the SASP, you had increases in… Let me to see that paper, it just came out, and I read through it pretty quickly. But when I read through it, basically the concept was that viruses can cause this, this is what’s happening with COVID. Then at the end, they also use rapamycin, interestingly, in a model to sort of reverse some of those changes and that’s a whole other area that I think is interesting and evolving, that others are starting to get involved in, and I don’t know if you’ve done anything with that in your practice. It’s, I think, starting to be thought less as an immunosuppressant, which is what it’s approved for in transplant patients, but in more as an immunomodulator through mTOR1, and not even really a senolytic either. It’s more to sort of prevent cells from becoming senescent versus remove senescent cell. I just think that that paper was sort of more evidence that viruses, and I gave a talk last year or early this year, you know how this stuff all runs together.

Dr. Kara Fitzgerald: Especially in the pandemic, time has definitely changed.

Dr. Joseph Raffaele: Where I was talking about COVID, and immunosenescence. I really think that this is going to be the decade of the virome versus the decade of the microbiome, there’s still so much more to learn about the microbiome, obviously, but I think the virome has gotten a little bit of short shrift up until recently. That is, again, a silver lining of this pandemic, if you say there is one, because attention has been turned towards viral mechanisms for inducing senescence, for causing disease, and things to do about it. I think that that’s really going to be interesting to watch the story. As I suppose I mentioned, I hope that we see an increase in efforts, and there are still very strong efforts toward finding a vaccination for CMV. But, having a really good vaccination, the one that works for CMV could be really pretty amazing.

Dr. Kara Fitzgerald: Right, I agree with you actually and maybe as we move towards this RNA technology and see that it appears to be rather extraordinary, where else can we apply that? I know some of my colleagues will disagree on that.

Dr. Joseph Raffaele: I’ve got more than a few colleagues on Instagram get a lot of backlash when we say that but that’s a whole another discussion.

Dr. Kara Fitzgerald: I agree with you and being an ND I’m thinking about diet and lifestyle and botanical and, all of the other interventions, the foundational pieces that need to be in place for somebody to age gracefully. Just putting more attention towards what we do for viruses, I agree that the microbiome, the bacteria have gotten a lot of attention and turning over to look at viruses is long overdue. You published recently, you and a large group of folks in a multi-clinic trial with 500 patients, which is impressive, using TA-65. This is a telomerase activator that you can actually give us a little background on as well and we’ll again link to it in the shownotes. Look, it decreases immunosenescent CD8+CD28- T cells and it was published in May, in OBM Geriatrics Research. We’ll link to this on the show notes and congratulations on your publication and just talk to us about what you guys did and what you found and why it’s important.

Dr. Joseph Raffaele: Yeah, so the impetus for this study was a study that I was involved in back in 2007, just started a cohort of patients in my practice getting TA-65. As well as a supplement pack, and the usual interventions we do for diet and exercise. After a year of TA-65, we found that they had a decrease in senescent T cells, a slight increase in naive T cells and a decrease in percentage of the critically short telomeres. But, it was a cohort, it wasn’t a randomized control trial and there was other interventions that were taking place at the same time. That was published in 2011 I guess, and this study was started in 2016 to try to prove that it was the telomerase activator, which I’ll talk about in just a second, that caused the beneficial effects in the immune system.

There was also beneficial effects in bone density, in other markers of metabolic health, that was subsequently in a randomized control trial also shown to be from the TA-65 in metabolic syndrome. But to really prove out the improvements in sort of reduction in senescent T cells, this large randomized control trial was undertaken. TA-65 is a natural molecule derived from the natural medicine called Astragalus membranaceus, it’s been in traditional Chinese medicine for millennia. The company T.A. Sciences acquired the rights of it from Geron Corporation who found it through a natural product screen of over 5000 different molecules to see which ones would turn on telomerase, hoping to sort of use a telomerase activator to slow down the aging process, sort of the brainchild of Calvin Harley, who first came up with the idea that the Hayflick limit might be from telomeres, and a major cause of aging.

It has been shown to be a telomerase activator in the gold standard assay, called the TRAP assay. It’s a moderate telomerase activator, it’s transient, so it turns it on for… It’s in the bloodstream for about eight hours. We have randomized control trial data, as well, on lengthening telomeres in a trial done in Spain. We don’t know the exact mechanism as to how it works, but we think it may derepress the repressor of the gene for telomerase for the catalytic component, which is TERT. There’s the TERT and that’s the sort of catalytic part that then interacts with the RNA template to make more TTAGGG and add length to the telomeres.

They recruited as you mentioned, 900 sorry, 500 subjects, which were followed over nine months between the ages of 45 and 75. I think the average age is about 57. The previous study, the average age was a little older of 62 to 63 and it was more males, I think 75% males. They corrected that and this one was 65% women, and age was about the same. It was really fascinating to me, well, let’s just talk about the trial setup and it was to see what the effect was on senescent T cells and naive T cells. Naive T cells being those that haven’t encountered their antigen that they’re uniquely designed to recognize, the marker CD95. The lack of that on the CD8 cell sort of marks it as a naive T cell.

To look at both senescent T cells and naive T cells because we saw signals in the previous study that there was an increase in naive T cells, decrease in senescent T cells and really a remodeling of the immune system in CMV positive subjects toward a more youthful sort of profile. They did a dose finding 100 units, 250 units, 500 units and also to see whether or not there was a benefit from splitting the dose up into twice a day. They did baseline studies, what I found was really fascinating is that two studies of different populations, both in the US but, separated by whatever seven years or even longer than that, had almost exactly the same amount of senescent T cells in the CMV positive subset.

There was 221 per microliter, in the what’s called the QPS study. This is the study we’re talking about that was just published. The average age of 57 and in the other study, which we’ll call the Patton Protocol study, because we use the Patton Protocol for that, there were 272. A little bit more because they were a little bit older. The CD95 negatives were about the same as well. Then what was also reconfirmed, but it’s been also shown in other studies published in the literature over the past decade, that the CMV positive subjects versus the CMV negative subjects had two times the number of senescent cells. Even more than two times, two times the percentage of them, what was it? 22 versus 47%, in CMV negative versus CMV positive.

If you’re CMV positive you have double the percentage, but actually three to four times the number of them. Almost exact replication of the sort of profile of a person who is CMV positive versus CMV negative. Then what we did find, which was very gratifying, was that taking TA-65, in any of the doses actually 100, 200, 250 reduces senescent T cells quite markedly. About 20, excuse me, 21% in the CMV positive subjects, and about 13% in the CMV negative subjects. Also, even if you weren’t CMV positive, you benefited from it because you had a reduction in the small number of CD28- cells you had, but also an increase of the same magnitude in the CD95- cells. Improvement in both compartments a decrease in the senescence cells about 50 cells, highly significant result, the p value is less than 0.0007.

Dr. Kara Fitzgerald: That’s awesome.

Dr. Joseph Raffaele: Then also what you want to see which is an increase in T cells that are naive and can fight off new infections and respond to vaccination. Again, that remodeling of the immune system towards a more youthful profile in more of an effect than those that needed it, the CMV positive ones, but also an effect in CMV negative ones. It’s just sort of goes along with the idea that when we have a lot of senescent T cells, you take up what’s called immunological space and when you remove them, there’s space for the immune system to produce and circulate more naïve or younger T cells that can fight off new infections. It was very gratifying to see these two studies done and have virtually the same results and it really proves that while the other stuff that was being done was beneficial, probably in terms of supplement packs, et cetera. But the main driver was this molecule TA-65 that caused that.

Dr. Kara Fitzgerald: So listen, I just want to say a few things about it. First of all, congratulations I know a clinical trial, especially one as large as that is just a huge undertaking and it’s really cool what you found. I just want to go back earlier I was asking you about what kind of intervention you might do thinking when you’ve likened CMV to HIV, how we think about caring for these patients and obviously you know the answer is right here.

Dr. Joseph Raffaele: Obviously, duh, I should have said that.

Dr. Kara Fitzgerald: Yeah. Well, I know.

Dr. Joseph Raffaele: All my patients are on TA-65 so forgot about that…

Dr. Kara Fitzgerald: You’ve already got that that’s a foundational piece. Okay, so then those people who might have like breakthrough are very aggressive.

Dr. Joseph Raffaele: Right, exactly.

Dr. Kara Fitzgerald: Then you’re thinking about that. Okay, so let’s just clarify that. We know traditionally astragalus is an amazing botanical and it has been used as an antiviral and likely it’s this particular component or at least this component is doing the heavy lifting as you guys are demonstrating. I find that very exciting and then if you connect the dots, it’s rejuvenating the immune system. Okay, so yeah, it’s influencing telomerase, but it’s rejuvenating the immune system. It’s keeping cytomegalovirus at bay. You can jump to the fact that it’s anti-aging.

Dr. Joseph Raffaele: Well, yeah. No, I mean, I think in my book, anything that maintains telomere length or lengthens it, is anti-aging because, telomere attrition is one of the top four elements of the hallmarks of aging. People argue whether it’s number one or number four or somewhere in between but I think that it’s very upstream in terms of the aging process. People talk about rapamycin being one of the few things that have lengthened lifespan in virtually every animal model. But that’s true of TA-65 as well. I mean not TA-65, of increasing telomere length in mouse models.

Ron DePinho’s study showed improvement and rejuvenation. Maria Blasco published a study showing in normal aging mice at year one, in year two, if you give them, this is gene delivered, increase in telomerase activity that turns on telomerase and lengthens telomeres, you see a 24% increase in median lifespan in the one-year and a 13% increase in median lifespan in the two-year-olds. I would argue that it’s definitely affecting the aging process when you do that.

Dr. Kara Fitzgerald: It’s got to be influencing that epigenome as well. I think, more than I know, there’s a lot of clocks, we were talking about the various DNA methylation clocks before we hit the record button. I think they’re more than just surrogate markers of the aging process, I think that some of them are influencing the aging journey as well. I just anticipate that when attention is placed on TA-65 and the epigenome we’re going to see some interesting stuff. Do you have any comments there?

Dr. Joseph Raffaele: Yeah, well, I’m starting to place that attention on it. These clocks have been available for a little while commercially and I’ve started now probably in the last year, using them (pause) in as many patients as there is an opportunity too, but I think we’re going to see that, and I think that’s right, that we’re going to see a signal. There’s a phenomenon it’s called telomere position effect, whereby you see… You still hearing me because I’m getting this sign?

Dr. Kara Fitzgerald: Yes.

Dr. Joseph Raffaele: Okay, yeah. Whereby the telomere within the loop can loop back to as far as proximal as a mega base, back onto the chromosome and influence gene expression, (pause) The thinking is that, when the telomeres get just a little bit shorter, the area of the proximal chromosome that they’re affecting the gene expression of is going to change and that could be deleterious. This is the telomere position effect. It’s not just the telomeres are important when they get critically short and the cell can no longer divide, they may be, and there’s emerging data but not as much as there needs to be, regulators of the epigenome as well.

I think you’re right, and I think we’re going to pick that up depending on which genes. We were talking earlier about the clocks and looking at CPGs and their degree of methylation. It’s a big, sort of complex systems biology thing. We look at these clocks in a big sort of systems biology problem to sort of… These clocks are trained on large data sets and you can train them on looking to predict chronological age. Which are the first ones they were doing, they did that very, very well. But in the end, the methylation sites that we’re looking at are genes that are either being turned on or turned off, relatively more or less and knowing which ones they are, I think is really going to be key. I think that once they look closely, they’re going to find that their genes that are potentially affected by this telomere position effect and that is one way in which shortening telomeres, way before they become critically short, can affect aging and health. That makes me think that the real thing that we ought to shoot for is keeping everybody at their optimal telomere length, which is probably their telomere length at around age 25.

Dr. Kara Fitzgerald: It wouldn’t be that difficult of a study, as I’m just thinking off the top of my head, you could look at just global methylation change. I wonder, I mean, you’d have to, I’m sure drill down. But just a correlation between global methylation status and telomere length. I guess I’m wondering my hypothesis would be the shorter the telomere, if your idea bears out, then the more hypomethylation, one would see and we know hypomethylation is a fundamental player in the aging journey, I guess. Anyway, there’s my simplistic dot connection. But, it wouldn’t be that difficult and then you could drill down and look in more specifically to see whether there are the sort of the aberrant patterns, the differential. I know, hypermethylation also occurs. But less so, there’s a global trend toward hypomethylation. So anyway, I just think that would be fascinating.

Dr. Joseph Raffaele: The folks that we both worked with at True Diagnostic are looking at that, they are looking at using DNA methylation to predict telomere length.

Dr. Kara Fitzgerald: Right, they could answer that question.

Dr. Joseph Raffaele: Learning particular CPGs and see how well they can predict telomere length. Then the interesting thing is, what else do those CPGs predict? What are the genes that are involved in that? Is it hypomethylation of TERT? The gene involved for TERT? Who knows what’s going on? But yeah, that question does need to be addressed and that’s why we’re collaborating with them, because we have a lot of over 2000 telomere length measurements in our database. Hopefully, we’ll continue to add what we have telomere length, epigenetic data. Now, of course, there’s the senescent T cells, the UCLA lymphocyte subset panel that we do here, and then, glycan age, any marker that’s been shown to be an important predictor of outcomes and health status. I think the more biomarkers, as we talked about in our last podcast, the more information we have.

Dr. Kara Fitzgerald: Yeah, right, absolutely. Okay, so to that end, just, you used the UCLA lab test, you just referred to it, and we’ll link to the test in our show notes. You used it in your study, and you use it in practice all the time. Just, briefly what is it and when do you use that?

Dr. Joseph Raffaele: It’s a flow cytometry test that looks at PBMCs, and then looks for certain markers on them, these CD markers, cluster of differentiation markers, that can tell you sort of what kind of cell it is, what its behavior is going to be. We’ve all heard of the T cell and that’s marked by CD3. Within the T cells there’s the CD4s, which are the helper cells, CD8 which are the cytotoxic suppressor cells. Then, there’s the CD19s which are B cells, CD56 which are natural killer cells. We look at those cells, we get then a further breakdown of the CD8 cells into two populations. The CD28 marker tells us whether they’re senescent, if they lack that marker, whether they’re a healthy suppressor cell, if they have that marker.

Then as I mentioned earlier, this sort of naive T cell which we look at, they don’t express CD95, which is a receptor for the Fas death ligand to sort of kill the cell after it’s done its job fighting off whatever virus or tumor is trying to fight off. It is all of those markers, and then it also gives you that C4 to CD8 ratio, which is that immune risk phenotype. There’s relatively robust literature showing what are the good ranges for those. Certainly, starting with the CD4 to CD8, you want that to be above one, because below that in 80- and 90 year-olds, and there’s early data that perhaps in even younger cohorts, their populations there, there’s an increase in morbidity & mortality. Sort of really good place to be for that is between 1.5 and 2.5, meaning you have 1.5 to 2.5, more CD4 cells than CD8s.

The reason for that is that we all have a really good number like 300 to 350 of the CD28+ cells. That number decreases of the healthy ones. But if you’re accumulating senescent T cells, then that’s going to be CD8+ CD28-, which is not a good thing. What happens is that, that denominator goes up while the helper cells stay about the same and that’s what causes the ratio to go under one. That’s a marker for an increased burden of senescent T cells and likely inflammaging. You want to have that marker be in that sort of, ideally, between 1.5 and 2.5 and certainly not below one. If you get below 0.7 then you’re sort of in this sort of more critical area where you have a lot of senescent cell and you don’t even have that many CD4 or helper cells.

Interestingly, when you get really high in that ratio, it means that you’re not seeing the positive, you don’t have a lot of senescent T cells, but you also don’t have a very good ability to respond to new infections either. That’s actually associated with the frailty and increased mortality as well. As with many things, there’s a sweet spot and I almost always see like, my most healthy patients right there in that sweet spot. That’s kind of how you interpret I mean, I look at the CD95, you’d like to see them be CD95 negatives, I see them be a little higher than. When you’re youthful there somewhere around 35% or more, any sort of healthy aging is between 20 and 35%. Then, I have a lot of patients in 60 and above their 10 to 20%.

When you get below 5%, it’s kind of you don’t have a great, diversity of cells to fight off new infections. I have patients that… The interesting thing about senescent T cells is that, we used to think that it really was an age phenomenon, but I have patients in their 70s and 80s, who have almost no senescent T cells, because they’re not infected with these viruses, or they have some genetic gift that allows them to deal with it, and they don’t have that number. Likewise, I have some patients that have, 700, 800, 900, cells per microliter. They’re still walking around, but they are at risk, I believe. When you’re born you have virtually no senescent T cells, they’re all healthy expressing CD28+.

That’s sort of a quick way in our software that we have, we sort of give you a grade and sort of optimal, you get an A, healthy aging you get a B, aging you get a C, immune risk you get a D and if you’re critical, you get an F and we try to move up your grade point with our therapies.

Dr. Kara Fitzgerald: I just want to remind folks physioage.com is the site that you can go to to learn about the interpretive tools that Dr. Raffaele has developed. Then we will link to the UCLA test that he just spoke to. I have a couple questions. I just wanted to ask you circling back to our TA-65 discussion. Different doses have been studied. What do you dose in your practice? Are you consistent with everyone or do you change based on circumstance?

Dr. Joseph Raffaele: No, I mean, I do change. I tell you I was a little surprised with the results of the, plesantly surprised I guess, but they didn’t necessarily comport with what I see, in that the lowest dose seemed to be effective, and not any less effective than the higher doses. But that, I didn’t look at each individual subjects, which I do in my practice. The way in which I dosed TA-65 is I measure telomere length in everybody but I think as we talked about before, telomere length has a coefficient of variation about 2-3%. But each year you lose a lot less than that. You need a couple of years’ worth of telomere length before you really get an idea about what direction it’s going in and the response isn’t as quick. For the senescent T cells, you’ll see a response within three months, a reduction in senescent T cells in virtually everybody I see that’s on TA-65 and it’s a bigger response if you have more senescent T cells.

But I don’t see that with everybody always in the starting dose, which for me is 250 IUs. If I don’t see that, then I’ll go to 500. If I don’t see that there, then I’ll go to 500 in the morning and 250 at night, the thinking being that if you have more exposure in 24 hours to the telemorase activator, that you can see a signal. I usually will see a signal at that point and the reason for that variability I believe is, there was a pharmacokinetic study done and absorption is quite variable individual to individual.

Dr. Kara Fitzgerald: Make sense.

Dr. Joseph Raffaele: Blood levels can be almost nil for one person, whereas very high and good levels, that would sort of turn onto telomeres in the TRAP assay with the same dose. I think people absorb it differently and so I dose it differently. The reason I do that is because, if you’re not getting that signal, then you’re wasting your money. If you’re, I mean, say wasting your money, but I think that typically, you might need a higher dose, in which case you should take the higher dose. But you shouldn’t take a higher dose if you don’t need it. Because, it’s not crazy expensive, but it’s not inexpensive.

Dr. Kara Fitzgerald: Right, right. Right, so you start them on 250 and then you just bump up based on their response using the UCLA essay?

Dr. Joseph Raffaele: But also when this came out, I started to think about some people that don’t want to spend the money for it, because it showed that the 100 IUs works. Let’s start thinking about dosing it at 100 and seeing what happens, but I haven’t started doing it yet.

Dr. Kara Fitzgerald: Okay, all right. Well, geez, talk about it, I’m sure next time, I’d be curious what you learn, yeah. Because in the context of an overall program, it’s something that you don’t want to overdose on. Well, you don’t want to prescribe excessive amounts, if you don’t need to, you want to be right where you need to be. Alright, let me see. I’ve got a couple other questions for you just sort of interesting things happening in the landscape of some of the gerontology world. Buck Institute researchers recently developed a novel, non-invasive lipid metabolite biomarker that can actually be used to track senolytics. You earlier, I mean, you don’t look at TA-65 as a senolytic exactly, but sort of in the…

Dr. Joseph Raffaele: Well, that’s the thing, not to interrupt you, but…

Dr. Kara Fitzgerald: Yeah, no, no, interrupt me. Go ahead.

Dr. Joseph Raffaele: We don’t know how it works. We know that it turns on telomerase and presumably, that is what’s happening, but the telomere length was not measured in this study. We don’t know exactly how TA-65 works. I talked about derepressing the repressor. But I don’t think that it’s known at this point. To the extent that it reduces a cell type, which we from many studies, believe is a senescent cell then yeah, I mean, it’s senolytic.

Dr. Kara Fitzgerald: It’s senolytic, it’s not destroying the senescent cells, its upstream

Dr. Joseph Raffaele: Right, but that’s the thing. We don’t really know what’s happening to it. If it is a senolytic, I would call it a sort of a good senolytic, because, I think senolytics are great, but I do worry that if you get rid of a lot of senescent cell, the tissue that you’re getting rid of them has to replace them with new cells, and the cells around them are going to divide, the stem cells in those niches are going to divide, and then their telomeres are going to get shorter. Then you’re going to have more senescent cells.

I think, while you have a short-term benefit, in the long run, what’s going to happen? Most of the studies haven’t gotten that far out. Michael Fossel, who’s a telomere biologists has sort of written a book on it, one of the books on it anyhow, has hypothesized that this phenomenon could occur and you want to maybe go more upstream by turning on telomerase and making sure cells don’t become senescent. But why we’re seeing this reduction in CD28, we just don’t know yet. But I believe it’s certainly a beneficial effect.

It’s a senolytic almost like, maybe the way rapamycin is, it’s not really a senolytic but works in a different way. I think talking about the Buck Institute Study, having other markers that can be easily obtained to see whether or not your senolytic is working, which they developed this oxylipin that is produced by senescent cells as part of their SASP, but not the sort of protein SASP, like IL-6 and TNF-alpha. But instead it’s a modified lipid that I think is great to be able to… Because, this is what we need in the field. You markers that tell you whether what you’re doing is doing good things or not good things.

Dr. Kara Fitzgerald: Have you had any access to that biomarker yet?

Dr. Joseph Raffaele: No, as far as I know, nobody does other than [mid].. perhaps might be collaborating with other researchers. But, I don’t have access to it. There’s an assay for SASP that this company GenFinity does. But I’m not as aware of it as one clinically available and have you worked with them at all?

Dr. Kara Fitzgerald: No, I haven’t.

Dr. Joseph Raffaele: But yeah, that would be a really interesting thing to do with TA-65 is to stand in control trial to see whether we’re getting that oxylipin signal and if we are then we don’t necessarily know what tissue it’s coming from but at least you know it is killing senescent cells. In this case, is it decreasing the production of new senescent cell and therefore, that’s why you see a decrease in CD28- cells? Or is it actually getting rid of CD28- cells faster than they normally would be gotten rid of? I don’t know, nobody knows.

Dr. Kara Fitzgerald: You would need the UCLA to answer that? You’d need them in conjunction to actually really be able to test that out. Well, let me ask you this. We have to wrap up. I know I can continue to pick your brain for a long time. But we’ve been yammering for a while now. What, if anything, is jumping out to you that’s I mean, we’ve talked about a lot of things that we’re excited about in this field, and in the future of medicine in general, but any additional things you’d like to add, any big picture thoughts?

Dr. Joseph Raffaele: Well, I think what’s exciting to me, at least in my practice, is that now I have the tools to really, I think, be able to tell patients that I am beneficially affecting their aging process. We have glycan age, we have epigenetic age, we have what we call TelomerAge, immune subset panels. If patients come to me all the time and say, should I try this new supplement? Should I go on quercetin? Should I go on and fisetin? Should I take alpha ketoglutarate?

Well, I can’t tease out necessarily its effect on us, but if it’s the only thing we change at one point I can certainly say, whether since you started that, these markers have gone in a beneficial direction, and this one hasn’t. I think that’s exciting for me as a clinician in this field, that it’s more and more evidence driven. There’s that whole biohacker field where we can sort of do it in with feedback from the person’s physiology. That part is exciting to me.

I think there’s also just incredible work being done in every area, the whole work in rapamycin and rapalogs is being done. Pharma’s starting to really focus on aging as being the next big frontier, because of the bang for buck you get when you slow the aging process down not only in decreased diseases, but also in functionality. Obviously, Pharma’s going to be chafing at the bit for something that can make people feel better, and look younger, before they get disease, because that’s everybody. Everybody is going to want that. I think that that impetus is there now because we’re changing people in general and certainly, the research community is changing the view of aging. That’s really the bedrock for everything else that we’re trying to prevent.

Dr. Kara Fitzgerald: Yeah, yeah. Amen to that. Good, that’s a great place for us to end. Dr. Raffaele, as always, thanks so much for joining me today on New Frontiers. It was great talking to you.

Dr. Joseph Raffaele: Oh, it’s always a pleasure talking to you, Kara. I’m sure we’ll be chatting again real soon.

Dr. Kara Fitzgerald: Indeed, we will.

*clarification: (actual study finding) All subjects were healthy and asymptomatic. Those CMV seropositive had more lymphocytes, CD8 T cells, CD28 negative T cells, decreased CD4/CD8 cell ratios, and had higher average EpiAge (65.34 years) than those CMV seronegative (59.53 years). Decreased % CD4 (p=0.003) and numbers of CD4 T cells (p=0.0199) correlated to increased EpiAge.

Actual study finding:

All subjects were healthy and asymptomatic. Those CMV seropositive had more lymphocytes, CD8 T cells, CD28 negative T cells, decreased CD4/CD8 cell ratios, and had higher average EpiAge (65.34 years) than those CMV seronegative (59.53 years). Decreased % CD4 (p=0.003) and numbers of CD4 T cells (p=0.0199) correlated to increased EpiAge.