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- How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Flagellin Immunization
- COVID-19 in the Context of Aging
- The Existence of Senolytics May Trigger the Shift Towards Targeting Root Causes of Disease
- Notes on Self-Experimentation with Sex Steroid Ablation for Regrowth of the Thymus
- Turning Navitoclax into a PROTAC Senolytic with a Better Safety Profile
- Autoimmunity in Parkinson’s Disease
- Implicating Glymphatic System Dysfunction in Glaucoma
- The Trials of Running a Public Longevity Industry Company
- The Aging of Muscle Stem Cells
- Direct Reprogramming of Skin Cells into Photoreceptors to Restore Light Sensitivity in Mice
- Complement C5 Protein is a Biomarker of Preclinical Atherosclerosis
- Adjusting Macrophage Polarization for Therapeutic Effects is not Straightforward
- Photobiomodulation to Enhance Mitochondrial Function as a Potential Therapy for Parkinson’s Disease
- Mechanisms of Neurodegeneration Interact to Contribute to Multiple Conditions
- Hearing Loss Impairs Synaptic Plasticity and Memory Function in Mice
How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Flagellin Immunization
This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess whether a ten week course of flagellin immunization will significantly and beneficially affect gut microbe populations. Flagellin is the protein making up a flagellum, the appendage that bacteria use to move themselves around. As it happens, the presence of flagellae correlates decently well with harmful gut microbes, and the absence of flagellae correlates decently well with helpful gut microbes. In principle, provoking the immune system into greater efforts to chase down and destroy anything with a flagellum will better manage the microbial populations of the gut. These populations change with age in ways that promote chronic inflammation and reduce the generation of beneficial metabolites.
Flagellin has been used in human clinical trials as an adjuvant to improve vaccination efficacy: it is recognized by the immune system, and helps produce a greater immune response. It was shown to have only minimal side effects in those trials. There is no human data for effects on the gut microbiome, but a very interesting paper describes the effects in mice. The outcome is a shifting of the relative population sizes of gut microbes in a beneficial direction that will reduce chronic inflammation. It is unknown as to how long such an effect might last – whether rousing the immune system is a short-term process, or whether it will continue to better guard against unwanted microbes.
The purpose in publishing this outline is not to encourage people to immediately set forth to follow it. There are, for example, important caveats in the above mentioned mouse study regarding links between flagellin immunity and human gut diseases. If you come away thinking that you should just jump in, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.
- Why Self-Experiment with Flagellin Immunization?
- Caveats in More Detail
- Summarizing Flagellin Immunization
- Establishing Dosage
- An Introduction to Injections
- Considering Autoinjectors
- Obtaining a Needle-Free Injection System
- Obtaining Vials of the Correct Size
- Preparing Flagellin for Injection
- Obtaining Flagellin
- Storing Flagellin
- Validating the Purchased Flagellin
- Establishing Tests and Measures
- Guesstimated Costs
- Practice Before Working with Flagellin
- Schedule for the Self-Experiment
- Where to Publish?
Why Self-Experiment with Flagellin Immunization?
Gut microbes are an important influence on long-term health and aging. If forced to guess, they might be in the same ballpark as exercise. The first changes occur around age 35, reducing the generation of beneficial metabolites. Later changes are more detrimental. There is at present little that people can do to reliably influence gut microbe populations, beyond improving diet, there is considerable variability between individuals, and only a few services such as Viome that offer assays to assess progress. Any sort of therapy that works for a majority of people without aggressive customization would be a step forward, and responsible self-experimentation can help to determine whether this is a viable path forward. These reasons must be balanced against a sober assessment of the risk involved in vaccinating oneself with a bacterial protein that has been used in only a few human trials, and an acceptance of personal responsibility for consequences should one choose to run those risks.
Caveats in More Detail
There are two areas of personal responsibility to consider here. Firstly, this involves injecting a bacterial protein that has little published data on human use (even when that data shows good safety in the short term). As the mouse study paper points out, human patients with inflammatory bowel disease exhibit greater immune reactivity to flagellin, and it is unclear as to whether this drives disease pathology or is a beneficial adaptation. That is a leap into the unknown. So is crossing the road, or indeed getting out of bed in the morning, but there are definitely different degrees of risk and comfort.
Secondly, obtaining flagellin in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is permitted to proteins that are not defined as a therapy for research use, but not to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.
Choosing to purchase and use flagellin would therefore likely be a matter of civil disobedience, as is the case for anyone obtaining medicines or potential medicines outside the established national system of prescription and regulation. People are rarely prosected for doing so for personal use in the US – consider the legions of those who obtain medicines overseas for reasons of cost, despite the fact that doing so is illegal – but “rarely” is not “never.” If you believe that the law is unjust, then by all means stand up against it, but accept that doing so carries the obvious risks of arrest, conviction, loss of livelihood, and all the other ways in which the cogs of modern society crush those who disagree with the powers that be.
Summarizing Flagellin Immunization
Flagellin is classified as a pathogen-associated molecular pattern, something that both innate and adaptive immune systems recognize and react to. The use of flagellin as an adjuvant to spur greater immune response to another, attached protein or protein fragment is an established field of development. This is largely because of the favorable characteristics of flagellin: it doesn’t appear to be associated with mechanisms that might cause severe reactions. As noted earlier, human trials have taken place, and the safety data is good – at least over the short time frame in which safety is assessed.
Immunization with flagellin versus its use as an adjuvant is a matter of injecting the protein on its own at an appropriate dose, to rouse the immune system to greater activity against anything connected to flagellin. Given that we mammals, mice and humans, are already sensitized to flagellin, it is interesting that boosting that reaction has a noticable affect on gut microbes. That was only recently demonstrated in mice. A larger literature on this topic has yet to be established.
For small molecule drugs, it is possible to produce a starting point for human dosage given a mouse dose via standard equations, as shown in “A simple practice guide for dose conversion between animals and human”. Unfortunately this doesn’t apply to vaccines. In fact, it is fair to say that there is no rigorous way to determine how vaccines scale from mice to people, as the systems involved are enormously complex. Outcomes depend on immune system behavior, not on animal size, and modeling the immune system is a speculative activity at best. Many approved vaccines are probably using wildly suboptimal doses.
Insofar as there is any common wisdom, it is that similar doses in mice and humans appear to be a good starting point, based on those cases in which rigorous dose-finding has been carried out. In the case of flagellin, the mouse study employed a 10 μg dose injected weekly for 10 weeks. A 2017 human clinical trial used two monthly doses of 1 to 10 μg via intramuscular injection. Studies in non-human primates used used doses of 1 to 10 μg.
Much larger doses have been given to animals without apparent toxicity, but the failure mode of immunization at too high a dose is intense inflammation or, worse, a cytokine storm that has the potential to be very harmful or even fatal. Whether such adverse consequences are triggered, and how likely they are, is species-dependent. There is no human data for higher doses than those mentioned above, so at the end of the day, the best starting point appears to be to stick with the 10 μg dose repeated weekly over 10 weeks via intramuscular injection, on the basis that this has been trialed in humans.
An Introduction to Injections
The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.
The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1 ml, and intramuscular is limited to 2-3 ml depending on location. These are all very fuzzy numbers, but these upper limits don’t really matter for the purposes of injecting 10 μg of a protein: it can be dissolved in a very small amount of liquid, 0.5 ml or less.
Different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you’ll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.
It seems sensible to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections. In the case of flagellin, it makes sense to stick with the human trial approach of intramuscular injections.
Sticking a needle into one’s own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately largely not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.
Option 1: Subcutaneous Autoinjection with Needle and Syringe
If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.
Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection
Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I’ll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.
Obtaining a Needle-Free Injection System
There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. So far as I can tell, PharmaJet is the only available needle-free system that is capable of intramuscular rather than subcutaneous injection.
PharmaJet is the better engineered and more expensive of these two systems, and its specialized 0.5 ml syringes are built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.
The instructions for both of these systems are extensive, and include videos. They are fairly easy to use. One caveat is that needle-free systems produce a puncture that more readily leaks injected material back out again than is the case for needles. It is a good idea to have a less absorbent plaster ready to apply immediately after injection, such as one of the hydrocolloid dressings now widely available in stores.
Obtaining Vials of the Correct Size
If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.
There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place – this requires a crimping tool, and removing it requires the use of another tool.
There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.
It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.
Preparing Flagellin for Injection
If using a needle-free injection system, you will likely be limited to injecting 0.5ml amounts. Thus the objective here is to obtain 10 μg of flagellin dissolved in 0.5 ml of phosphate buffered saline in each of ten sealed vials, ready to be used with the injection system, with as little contamination as possible from the environment, and stored a freezer until it is ready to use. Depending on the size of the vial, it might contain doses for multiple injections, but stick to one dose per vial. It is not a good idea to carry out repeated freeze-thaw cycles on protein solutions, which is what you’ll have to do if all the doses are in 5ml of solution in one vial. Multiple freeze-thaw cycles degrade the protein.
When ordering flagellin, it will typically arrive in 10 μg to 100 μg vials. Empty 100 μg of flagellin into a single vial holding 5 ml of phosphate buffered saline. Mix well. Place that vial and 10 empty vials into a vial rack. Then use a pipette to transfer 0.5 ml into each empty vial. Seal and crimp each vial as you go. Then put the whole set into the freezer.
Keeping Things Sterile is Very Important
Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. Laboratories use autoclaves, which sterilize with steam. These are largely expensive devices, but a range of smaller, cheaper options exists. There are many best practices guides and summaries available online. This extends to the injection itself. Even with needle-free systems, an injection site should still be wiped down with alcohol first. It is all too easy to infect an injection site if skipping the precautions, and this can have severe consequences.
Not all flagellin is exactly the same molecule, nor is it created in the same way. The mouse study used a standard option of flagellin protein from Salmonella typhimurium (there are numerous strains, but strain differences are probably irrelevant). This is typically produced using recombinant protein manufacture techniques. There are many established companies that manufacture and sell proteins, including flagellin, to high standards of quality and safety, but as a general rule they will not ship to any customer other than an established and validated lab business. The usual way for everyone else to obtain cost-effective supplies of this sort is to search Alibaba for suppliers who offer that compound in their catalog, but unfortunately this isn’t an option for flagellin. Instead one must search Alibaba for protein synthesis companies, pick a smaller one, and negotiate a price for flagellin synthesis.
As noted at the outset of this post, all of these efforts to obtain, ship, and use any random protein for self-experimentation are to some degree illegal – it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary protein isn’t the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.
Open a Business Mailbox
A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.
Use Alibaba to Find Manufacturers
Alibaba is the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers. The company has done a lot of work to incorporate automatic translation, to reduce risk, to garden a competitive bazaar, and to make the reputation of companies visible, but it is by now quite a complicated site to use. It is a culture in and of itself, with its own terms and shorthand. There are a lot of guides to Alibaba out there that certainly help, even if primarily aimed at retailers in search of a manufacturer, but many of the specific details become obsolete quickly. The Alibaba international payment systems in particular are a moving target at all times: this year’s names, user interfaces, and restrictions will not be the same as next year’s names, user interfaces, and restrictions.
Start by searching Alibaba for protein synthesis companies. There are scores of biotech companies in China for any given specialty. Filter the list for small companies, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a protein, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices – they may vary widely, and it isn’t necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.
Many manufacturers will state that they require a large (often ridiculously large) minimum order; that can be ignored. Only communicate with gold badge, trade assurance suppliers with several years or more of reputation and a decent response rate. Make sure the companies exist outside Alibaba, though for many entirely reliable Chinese businesses there are often sizable differences between storefronts on Alibaba, real world presence, and the names of owners and bank accounts. Use your best judgement; it will become easier with practice.
Arrange Purchase and Shipping via Alibaba
Given the names of a few suppliers, reach out via the Alibaba messaging system and ask for a quote for a given amount of flagellin; you will have to provide the sequence and a reference to the paper in which it is described. Buy more than you’ll think you need, and make sure it is packaged into multiple vials, as one vial will be used to validate the quality of the batch. Payment will most likely have to be carried out via a wire transfer, which in Alibaba is called telegraphic transfer (TT). Alibaba offers a series of quite slick internal payment options that can be hooked up to a credit card or bank account, but it is hit and miss whether or not those methods will be permitted for any given transaction. Asking the seller for a pro-forma invoice (PI), then heading to the bank to send a wire, and trusting to their honesty should work just fine when dealing with companies that have a long-standing gold badge.
To enable shipping with tracking via carriers such as DHL, the preferred method of delivery for Chinese suppliers shipping to the US or Europe, you will need to provide a shipping address, email address, and phone number. Those details will find their way into spam databases if you are dealing with more than a few companies, and will be, of course, sold on by Alibaba itself as well. Expect to see an uptick of spam after dealing with suppliers via Alibaba, so consider using throwaway credentials where possible.
Chinese manufacturers active on Alibaba are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized proteins. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier. Note that different carriers tend to have different processes and rates at which shipments are checked for validity.
Proteins are shipped in a solid freeze-dried (lyophilised) form. While in this form they are easily stored in a refrigerator for the short-term or in a freezer for the long term. It has a much shorter life span once it has been mixed with liquid for injection, however, and should be kept frozen, and used within a matter of a few months at the most.
Validating the Purchased Flagellin
A protein may have been ordered, but that doesn’t mean that what turns up at the door is either the right one or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a protein is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.
Obtain an Extra Vial from the Same Batch as the Others
Since it is extra work to attempt to split out microgram amounts of protein or mail protein in solution rather than lyophilised protein, just order an extra vial from the same batch and send it off to be tested.
Use Science Exchange to Find Lab Companies
Science Exchange is a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Here again, pick a small lab company to work with after searching for LC-MS (liquid chromatography and mass spectrometry) services. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Companies registered with Science Exchange largely don’t provide their rates without some discussion, but a little over $100 per sample is a fair price for LC-MS to check the purity of the compound.
Work with the Company to Arrange the Service
The process of request, bid, acceptance, and payment is managed through the Science Exchange website, with questions and answers posted to a discussion board for the task. Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by NovoPro or other sources.
Ship the Samples
Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.
Examine the Results
Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor or other sources.
Establishing Tests and Measures
There are a few options for testing before and after, the most compelling of which is the gut microbiome. Changes there, given a stable diet, should be indicative that something happened. But to assess outcomes beyond that, inflammatory markers should be tested at the very least. It might also be interesting to look at DNA methylation assessments of biological age, though it is an open question as to what exactly these tests are measuring, and whether they are all that useful to an individual.
The most direct measure of effectiveness is to assess the gut microbe populations directly. There are lab services that do this, but few commercial direct to consumer services. One US based service is Viome, though it doesn’t present results in a useful tabular form, and does not provide the raw data. Nonetheless, it is a measurable endpoint. Gut microbe populations are fairly slow to change, given a consistent diet, so large differences over a ten week course of flagellin injections would be interesting to observe.
There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Since inflammatory markers are the topic of interest, a service that offers more individual tests rather than packages might be a better choice. The Life Extension Foundation offers a wide range of tests, for example, including a selection of inflammatory markers.
DNA methylation tests can be ordered from either vendors such as Epimorphy / Zymo Research – note that it takes a fair few weeks for delivery. From talking to people at the two companis, the normal level of variability for repeat tests from the same sample is something like 1.7 years for the Zymo Research test. The level of day to day or intraday variation between different samples from the same individual remains more of a question mark at this point in time, though I am told they are very consistent over measures separated by months. Nonetheless, it is wise to try to make everything as similar as possible when taking the test before and after a treatment: time of day, recency of eating or exercise, recent diet, and so forth.
The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.
- Business mailbox, such as from UPS: $250 / year
- Cytokine blood panel test from LEF: $300 / test
- MyDNAage kits: $310 / kit
- Viome kits: $150 / kit
- Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: $60
- Small pack of 13mm sterile serum vials: $35
- PharmaJet Needle-free Injection Kit: $1020
- Comfort-in Needle-free Injection Kit: $470
- Bulk 13mm serum vial parts and capping tools: $750
- 100 μg of flagellin via Alibaba: $1000
- Shipping and LC-MS analysis of a sample: $200
Practice Before Working with Flagellin
Do you think you can reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with saline solution rather than finding out that your manual dexterity and methods are lacking while handling an expensive protein. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case.
Schedule for the Self-Experiment
One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of flagellin to take a couple of months. Once all of the decisions are made and the materials are in hand, pick a start date. The schedule for the self-experiment is as follows:
- Day 0: Perform the various tests: bloodwork, gut microbiome assessment, etc.
- Day 1: Intramuscular injection of 10 μg of flagellin.
- Day 8: Intramuscular injection of 10 μg of flagellin.
- Day 15: Intramuscular injection of 10 μg of flagellin.
- Day 22: Intramuscular injection of 10 μg of flagellin.
- Day 29: Intramuscular injection of 10 μg of flagellin.
- Day 36: Intramuscular injection of 10 μg of flagellin.
- Day 43: Intramuscular injection of 10 μg of flagellin.
- Day 50: Intramuscular injection of 10 μg of flagellin.
- Day 57: Intramuscular injection of 10 μg of flagellin.
- Day 64: Intramuscular injection of 10 μg of flagellin.
- Day 65: Repeat the tests.
Where to Publish?
If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial. Equally if you run your own website or blog, why not there?
When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results! Publish whatever the outcome.
COVID-19 in the Context of Aging
It is widely appreciated that old people have a poor time of it when it comes to infectious disease. Seasonal influenza kills tens of thousands of older people every year in the US alone. The aged immune system functions poorly, and vaccinations for many conditions have low success rates in older people. Thus the vast majority of COVID-19 deaths are old people exhibiting immunosenescence. Given that the world at large seems to be entirely accepting of the yearly toll of influenza, while COVID-19 is classed as an apocalypse of some sort, one has to wonder how much of the hysteria surrounding COVID-19 stems from the rare – but highly publicized – deaths of younger individuals. Or perhaps if the rising toll of every influenza season was reported in the same way as deaths from COVID-19, more might be done? Human psychology is a strange thing.
An enormous amount of government and other funding will be directed towards fundamental infectious disease research in the years ahead, once things have settled down somewhat and COVID-19 has faded into the backdrop. That is one consequence of a pandemic that captures the attention of the world to the degree that this one has, deservedly or otherwise. This was perhaps the perfect storm, as ominous rumblings and awareness initiatives have been ongoing for some time regarding the threat of SARS-like viruses making the leap from animals to humans. A critical mass was finally reached. That COVID-19 has so far turned out to be less terrible than suspected at the outset is beside the point. The organizations of the world were primed to react in the way they are now to the first SARS-like virus that appeared remotely threatening.
The prospect of a large increase in funding for infectious disease and immunology research means that scientists in every relevant field of study are racing to position themselves to try to capture a portion of those funds. We outsiders don’t see the ferocious pace of grant writing, but published papers are a visible sign of this energetic process. A few recent examples are noted below. Researchers involved in immunology and aging are taking this moment in history to remind the world that, yes, the immune system decays with age, old people bear the brunt of infectious disease as a result, and perhaps we should do something about this, now that we can target the mechanisms of aging – the cause of immunosenescence.
Covid-19 and Immunity in Aging Populations – A New Research Agenda
As we age, health conditions associated with aging, particularly noncommunicable diseases such as heart disease, cancers, and metabolic and autoimmune diseases, combined with treatments for these diseases and with immune senescence, substantially affect responses to vaccines and infectious diseases. Angiotensin-converting enzyme 2 (ACE2) has been identified as the receptor for SARS-CoV-2, the virus that causes Covid-19, and it has been suggested that differential levels of ACE2 in the cardiac and pulmonary tissues of younger versus older adults may be at least partially responsible for the spectrum of disease virulence observed among patients with Covid-19.
Even as the brunt of severe illness from Covid-19 is being borne by aging adults, we are navigating partially blind in efforts to develop vaccines and therapies to stop this and future pandemics, since we lack knowledge of the mechanisms of immunity to protect this population. If we can delineate principles of effective immunity in the elderly, we might also be able to develop new strategies for broader disease prevention and control in older populations.
COVID-19 is an emergent disease of aging
Here, we found that the case fatality rate for COVID-19 grows exponentially with age in Italy, Spain, South Korea, and China, with the doubling time approaching that of all-cause human mortality. In addition, men and those with multiple age-related diseases are characterized by increased mortality. Moreover, similar mortality patterns were found for all-cause pneumonia. We further report that the gene expression of ACE2, the SARS-CoV-2 receptor, grows in the lung with age, except for subjects on a ventilator. Together, our findings establish COVID-19 as an emergent disease of aging, and age and age-related diseases as its major risk factors. In turn, this suggests that COVID-19, and deadly respiratory diseases in general, may be targeted, in addition to therapeutic approaches that affect specific pathways, by approaches that target the aging process.
Inflamm-Aging: Why Older Men Are the Most Susceptible to SARS-Cov-2 Complicated Outcomes
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by a high mortality of elderly men with age-related comorbidities. In most of these patients, uncontrolled local and systemic hyperinflammation induces severe and often lethal outcomes. The aging process is characterized by the gradual development of a chronic subclinical systemic inflammation (inflamm-aging) and by acquired immune system impairment (immune senescence).
Here, we advance the hypothesis that some key features of aging contribute to the disproportionate SARS-CoV-2 mortality suffered by elderly men. At least four well-recognized aging-related characteristics that are strongly expressed in older men go some way towards explaining why these patients account for the vast majority of fatalities: (i) the presence of subclinical systemic inflammation without overt disease, (ii) a blunted acquired immune system and type I interferon response due to the chronic inflammation; (iii) the downregulation of ACE2 (SARS-CoV-2 receptor), which triggers inflammation, particularly in patients with age-related comorbid diseases such as type II diabetes; and (iv) accelerated biological aging, as measured by epigenetic and senescence markers (e.g. telomere shortening) associated to the chronic inflammatory state.
The Existence of Senolytics May Trigger the Shift Towards Targeting Root Causes of Disease
It is sadly true that little medical research and development involves targeting the root causes of the treated condition. This is exactly why therapies largely fail in trials, and are largely only marginally effective when approved. In the case of age-related disease, efforts to target root causes are a tiny faction of the field. This is slowly changing for the better, but the state of affairs today versus a decade ago is still only a matter of making that tiny fraction a little bit larger.
Senolytic therapies that selectively destroy senescent cells are the first rejuvenation therapies, treatments that target something close to a root cause of aging and age-related disease. The accumulation of senescent cells with age is highly detrimental to tissue function, even though they are only a tiny fraction of all cells even in late life. Removing senescent cells produces impressive benefits for dozens of age-related conditions in animal models. Going by the present data, senolytics are far and away more effective and useful than any other approach so far undertaken to intervene in the aging process.
Effective outcomes have a way of dragging the field along with them. It is hard to argue against large effect sizes and robust, replicated evidence of efficacy. For the period in which the majority of this data remains associated with animal models, it is only the research community engaged in revising its ideas on how to approach aging. As human data accumulates, we can hope that the broader medical and funding communities will follow, and then the public at large.
Insights from In Vivo Studies of Cellular Senescence
Recent observations using genetically-modified animal models indicate that the elimination of senescent cells attenuates aging and age-related diseases. These findings have opened new avenues to explore pharmacological approaches to induce apoptosis in senescent cells termed senolytics. With the absence of a unique marker for senescence, interventions have been developed to take advantage of some vulnerabilities that senescent cells have.
Senescent cells, like cancer cells, are resistant to apoptosis through the upregulation of BCL-2 anti-apoptotic proteins. Efforts in cancer research have found ABT263 (navitoclax), a potent inhibitor of BCL-2 and BCL-xL anti-apoptotic proteins, can be used to treat lymphomas and other types of cancer. Interestingly, due to the overexpression of BCL-2 in senescent cells, ABT263 exhibits senolytic activity and prolongs healthy lifespan in normally-aged mice. Importantly, using mouse models of age-related chronic diseases, including atherosclerosis and neurodegeneration, in which the accumulation of senescent cells is detrimental, treatment with ABT263 attenuated disease pathology.
Additional pharmacological interventions have been shown to target senescent cells in mice. These include a peptide that disrupts the interaction of FOXO4 and p53 leading to apoptosis, as well as nanoparticles that target senescence-associated β-gal positive cells. Interestingly, using a mouse model for osteoarthritis (OA), it was shown that senescent cells accumulate in articular cartilage and synovium promoting the development of OA. Consistent with the concept that senescent cells drive pathology, local administration of a new molecule named UBX0101, was used to disrupt the interaction of MDM2 and p53 to trigger apoptosis in senescent cells, yielding positive results in attenuating OA, plus validating UBX0101 as a senolytic. Consequently, UBX0101 was initiated in a clinical trial with adult patients diagnosed with femorotibial osteoarthritis to evaluate the safety, tolerability, and pharmacokinetics of the drug. It is currently being evaluated in phase 2 clinical trials for the effectiveness in treating musculoskeletal diseases with an emphasis on patients with OA.
Natural compounds, such as quercetin and fisetin, have also been used in combination with anti-cancer drugs, particularly the pan-tyrosine kinase inhibitor dasatinib to treat naturally aged mice and senescence-related diseases. The combination of dasatinib and quercetin (D + Q) target particular sensitivities of pro-survival pathways found in senescent cells, known as senescent cell anti-apoptotic pathways (SCAPs). These drugs could theoretically influence a broad spectrum of pathways in all cells, which may make it difficult to assess if senescence ablation occurs or there has been some amelioration of key features of senescent cells, such as the SASP. Nevertheless, these findings set a foundation to start clinical trials in adult patients with idiopathic pulmonary fibrosis and diabetic kidney disease. These studies have provided evidence that intermittent doses of senolytics can be systematically used in humans and tolerated.
The current philosophy of the healthcare system is to systematically treat chronic diseases with medications that, for the most part, address the consequence rather than the cause of the malady. Our desire to improve human care has motivated us to investigate how and why senescent cells accumulate with age and whether they may play therapeutically-relevant casual roles in age-related diseases. The objective is to start treating chronic diseases from the root and not the symptoms of the disease. As we are starting to enroll patients in “senolytics-clinical trials,” it will be imperative to assess if senolysis efficiently targets the primary cause of disease or if it works best in combination with other drugs. Additional basic science research is required to address the fundamental role of senescent cells, especially in the established contexts of disease.
Notes on Self-Experimentation with Sex Steroid Ablation for Regrowth of the Thymus
I periodically publish thoughts on self-experiments that seem interesting and relevant to aging. Despite the influence of the quantified self movement, the broader self-experimentation community is largely terrible on matters of research, rigor, reporting, and safety. My motivation is to something to raise the bar on all of these items.
For every discussion I’ve published on a particular self-experiment, there are half a dozen others sitting at some stage of research and interest. Over the past year or so, I’ve been on and off looking into sex steroid ablation as a mechanism for thymus regrowth. Since my company, Repair Biotechnologies, works on thymus regrowth as a way to reverse immune system aging, I have read up on most of the ways in which that has been attempted or achieved in animals and humans. The thymus is responsible for the maturation of T cells, a complicated process in which the cells are educated to attack pathogens and dangerous cells, but not normal tissue. The thymus atrophies with age, and by age 50 there is usually little active tissue left. Lacking a robust supply of new T cells, the immune system slowly declines into immunosenescence.
In humans, only two approaches to thymus regeneration have produced positive results. The small Intervene Immune trial used lengthy treatment with growth hormone alongside a few other substances to try to offset the negative effects of growth hormone. More impressive results, at least in the metrics measured, have been obtained from the use of sex steroid ablation in prostate cancer patients. For this approach, thymic growth has only been assessed in animal models. In human patients, the metrics have been naive T cell and recent thymic emigrant populations.
One has to ask the question: is it plausible for a 50-plus-year-old male to undergo a short period of sex steroid ablation in order to regrow the atrophied thymus, and rebuild the naive T cell population? Well, yes and no. Can it be done, yes. The evidence is pretty good. A fresh naive T cell population will last a decade or more without further reinforcement before it starts to become problematic – the normal course of aging, and people who have had the thymus surgically removed, tells us that. Many animal studies of castration and sex steroid ablation show thymic regrowth and revitalized production of T cells, for at least a short period of time. Based on trials of hematopoietic stem cell transplantation carried out in conjunction with sex steroid ablation, it is reasonable to think that three to six months would be required for a human naive T cell population to be reconstituted. Lesser duration of upregulated production of T cells would produce lesser benefits.
There is an interesting sidebar on what happens to the thymus in castrated mice. It doesn’t grow in the sense of producing new cells. The cortical thymic epithelial cells grow in size. Then a few weeks later they shrink again. But this does produce upregulation of T cell production, and to a certain degree we don’t really care that much about how the upregulation occurs so long as the T cells are of the right types and behavior. In human prostate cancer patients, increased T cell production is observed after months following sex steroid ablation, so the mechanisms are clearly distinct.
So why not conduct this self-experiment? Because the challenges and unknowns are not insignificant. Firstly, all human data and near all mouse data is in males – obviously necessarily the case in the prostate cancer studies. We have no good idea as to how to apply this sort of approach to females of either species. Secondly, while sex steroid ablation can be carried out very effectively using more modern drugs such as degarelix, a majority of the older prostate cancer patients it is used with do not recover the testosterone levels they exhibited prior to treatment. This is true even for the shortest treatment duration of 3-6 months, and appears to become worse the longer the treatment is continuously applied. There is really no established way of producing sex steroid ablation and very low testosterone levels with both rapid onset and rapid recovery, to get the overall duration to be any shorter than this.
Degarelix is an interesting drug. It causes very rapid sex steroid ablation, within a day or two, unlike other approaches that require a month or more to reach the desired point. When delivered intravenously, it leaves the system within a couple of days. But delivered subcutaneously, it forms a bolus that releases slowly over months. This is great for its intended use, but not so helpful if one wants a rapid recovery from sex steroid ablation. A single injection of degarelix is a 4-6 month commitment from start to (hopefully) final recovery – and the data indicates that the largely older prostate cancer patients do not tend to fully recover prior testosterone levels. Whether this is also true of younger individuals in the 40-50 age range is an interesting question with an unknown answer. Is the incomplete recovery inherent to age, or inherent to the usual secondary effects of sex steroid ablation on the systems of sex steroid regulation?
In principle one could inject degarelix intravenously every few days for a month to obtain a month of sharp onset and sharp recovery of testosterone levels. Then repeat every six months or so for a few years. One might expect that to greatly diminish side-effects, while still causing some degree of thymus regrowth. While the data exists for a single intravenous injection in humans, no-one knows what effects this might have if repeated extensively, or nor the proper calibration of dose and timing. Intravenous injection will produce spikes of much higher levels of the drug than occur for subcutaneous injection, and there is no data on what this does over time. That would all have to be discovered.
So while this might be an interesting project for someone with a high tolerance for personal risk, and an interest in conducting many trial runs with many blood tests along the way, it doesn’t seem all that practical. Meanwhile, the practical approach of using degarelix in the well-established and well-characterized way it is used with prostate cancer patients, for the shortest period possible, produces lasting side-effects that most men might be somewhat unhappy to experience.
Setting aside that conclusion for a moment, if one was going to do this, how to measure outcomes? That should always be the question, regardless of whether or not a potential project is ever undertaken. Inability to come up with good metrics is a very compelling reason, in and of itself, not to carry out a self-experiment. In this case, there are good, well-established measures, however, and they will be applicable to any approach to restoration of thymic activity. Firstly, one can look at the Intervene Immune paper for some simpler approaches. In bloodwork to assess immune populations, the lymphocyte to monocyte ratio, CD4+ and CD8+ naive T cell counts, and recent thymic emigrants are all tests that exist and are to varying degrees easily accessed. One of course has to measure testosterone levels in order to prove that sex steroid ablation is taking place. For the thymus itself, careful CT scans have to be performed. The trick is to ensure that exactly the same location in cross-section is imaged at each assessment. There are papers covering how to do that, such as by taking increments in distance from the sternal notch as an anchor point. Assessing the cellularity of the thymus (it is unlikely to change in size) emerge from an examination of before and after images.
So on the whole, sex steroid ablation as an approach to regrowing the thymus and repopulating the naive T cell pool in older adults has good human evidence for efficacy in males, but is also risky and comparatively costly, and the existing tools are adapted to a use case that doesn’t match well with this goal. Better approaches are needed, but developing them is likely beyond the means and interest of most self-experimenters.
Turning Navitoclax into a PROTAC Senolytic with a Better Safety Profile
It is interesting to compare today’s open access paper on converting the senolytic drug navitoclax into a PROTAC with recent efforts to improve navitoclax by conjugation with galactose. In both cases the objective is to reduce side-effects, but the strategies are quite different. Navitoclax is arguably the worst of the viable first generation senolytic drugs capable of selectively destroying senescent cells in old tissues. Senescent cells accumulate with age and cause great harm via their inflammatory signaling. Removing them has been shown to extend healthy life, reverse aspects of aging, and turn back a wide range of age-related diseases in animal models. Nonetheless, navitoclax is a toxic and unpleasant chemotherapeutic, and in addition to being poorly selective in comparison to more recent senolytic compounds, it also kills platelets to produce thrombocytopenia.
Galactose conjugation is pretty straightforward: link navitoclax with galactose to produce a molecule that is innocuous until the β-galactosidase found in large amounts in senescent cells cleaves away the galactose to reveal navitoclax. Thus the activity of navitoclax is largely restricted to senescent cells, reducing its off-target effects. Making a PROTAC, a proteolysis targeting chimera, is somewhat more complex and needs less of the structure of navitoclax – it is more of an evolution of the compound to produce a new small molecule. In this case, the part of navitoclax capable of binding to Bcl-xl is connected to sequences that cause a cell to degrade the Bcl-xl protein, an activity that will force a senescent cell into apoptosis. This only takes place if the cell has the right molecular machinery to kick off that degradation process, however. Platelets do not have that machinery, and so the PROTAC derived from navitoclax will not activate in platelets. The platelets survive, and thrombocytopenia is evaded.
Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity
ABT263 (also known as navitoclax), a Bcl-2 and Bcl-xl dual inhibitor, is one of the most potent and broad-spectrum senolytic agents identified to date. Bcl-xl inhibition with ABT263 and other small molecular inhibitors induces platelet apoptosis and results in severe thrombocytopenia, which prevents the use of ABT263 and other Bcl-xl specific inhibitors in the clinic – even for cancer patients – because platelets solely depend on Bcl-xl for survival. By contrast, Bcl-2 is dispensable for thrombopoiesis and platelet survival in mice and humans and inhibition of Bcl-2 with ABT199 (also known as venetoclax) does not induce thrombocytopenia. We hypothesize that we can reduce ABT263 on-target toxicity and generate a safer senolytic agent by converting ABT263 into a platelet-sparing Bcl-xl proteolysis-targeting chimera (PROTAC).
PROTACs are bivalent small molecules containing a ligand that recognizes a target protein linked to another ligand that recruits a specific E3 ubiquitin ligase. PROTAC binding induces proximity-induced ubiquitination of the target protein and its subsequent degradation by proteasomes. Importantly, because PROTACs rely on E3 ligases to induce protein degradation, it is possible to achieve cell/tissue selectivity, even when the target proteins are ubiquitously expressed as long as they target the proteins to an E3 ligase that is cell- or tissue-specific.
Here, we report the use of PROTAC technology to reduce ABT263 on-target toxicity by converting ABT263 into PZ15227 (PZ), a Bcl-xl specific PROTAC (Bcl-xl-P), which targets Bcl-xl to the E3 ligase cereblon (CRBN) that is poorly expressed in platelets. We find that PZ is less toxic to platelets but equally or slightly more potent against senescent cells compared with ABT263. These findings provide an approach to reduce on-target toxicity of toxic senolytic agents. With further improvement, Bcl-xl-Ps have the potential to be developed into safer and more effective senolytics than ABT263.
Autoimmunity in Parkinson’s Disease
Is Parkinson’s disease in part an autoimmune condition? Parkinson’s is an age-related neurodegenerative condition in which the primary motor control symptoms result from the death of a specialized population of neurons that generate dopamine. There are also other harms done to neurological function, however. Under the hood, processes such as chronic inflammation, mitochondrial dysfunction, and aggregation of α-synuclein contibute to that cell death. Researchers here speculate on an autoimmune component to the pathology of Parkinson’s disease, in that these mechanisms also drive the immune system into greater inflammatory activity that harms healthy tissue.
A new study adds increasing evidence that Parkinson’s disease is partly an autoimmune disease. In fact, the researchers report that signs of autoimmunity can appear in Parkinson’s disease patients years before their official diagnosis. Scientists have long known that clumps of a damaged protein called alpha-synuclein build up in the dopamine-producing brain cells of patients with Parkinson’s disease. These clumps eventually lead to cell death, causing motor symptoms and cognitive decline.
An earlier study showed that alpha-synuclein can act as a beacon for certain T cells, causing them to mistakenly attack brain cells and potentially contribute to the progression of Parkinson’s. This was the first direct evidence that autoimmunity could play a role in Parkinson’s disease. The new findings shed light on the timeline of T cell reactivity and disease progression. The researchers looked at blood samples from a large group of Parkinson’s disease patients and compared their T cells to a healthy, age-matched control group. They found that the T cells that react to alpha-synuclein are most abundant when patients are first diagnosed with the disease. These T cells tend to disappear as the disease progresses, and few patients still have them ten years after diagnosis.
The researchers also performed an in-depth analysis of one Parkinson’s disease patient who happened to have blood samples preserved going back long before his diagnosis. This case study showed that the patient had a strong T cell response to alpha-synuclein ten years before he was diagnosed with Parkinson’s disease. Again, these T cells faded away in the years following diagnosis. “One of the most important findings is that the flavor of the T cells changes during the course of the disease, starting with more aggressive cells, moving to less aggressive cells that may inhibit the immune response, and after about 10 years, disappearing altogether. It is almost as if immune responses in Parkinson’s disease are like those that occur during seasonal flu, except that the changes take place over ten years instead of a week.”
Therapies exist to treat inflammation from autoreactive T cells, and these TNF therapies are associated with lower incidence of Parkinson’s disease. Going forward, the researchers are especially interested in using a tool called a T cell-based assay to monitor patients already at risk for Parkinson’s to see if they could benefit from TNF therapies.
Implicating Glymphatic System Dysfunction in Glaucoma
There is growing interest in the systems of drainage that carry away cerebrospinal fluid and molecular waste from the brain. The failure of these systems due to the damage and dysfunction of aging may be an important cause of neurodegenerative conditions, allowing protein aggregates such as amyloid-β to build up to pathological levels in brain tissue. One branch of this work is focused on drainage through the cribriform plate, while another is focused on the comparatively recently discovered glymphatic system. Here, researchers note that a portion of the glymphatic system is implicated in glaucoma, a retinal degeneration that is proximately caused by rising pressure in the eyeball. Underlying causes are thought to include autoimmunity, senescent cells, and chronic inflammation in general.
Instead of a traditional lymphatic system, the brain harbors a so-called glymphatic system, a network of tunnels surrounding arteries and veins through which fluid enters and waste products drain from the brain. In a new study researchers show that the rodent eye also has a glymphatic system that takes out the trash through spaces surrounding the veins within the optic nerve. They also found that this system may be compromised in glaucoma and is capable of clearing amyloid-β, the build up of which has been implicated in the development of Alzheimer’s disease, glaucoma, and age-related macular degeneration.
The majority of the aqueous humor – the fluid that fills the chamber between the cornea and the lens – drains from the eye to the surrounding vasculature through a circular lymph-like vessel called Schlemm’s canal. This helps regulate intraocular pressure. Researchers decided to connect the knowledge about the front of the eye with their questions about the back of the eye. Because a 2012 study that the brain’s glymphatic system was capable of clearing amyloid-β, they used that molecule to investigate the existence of an ocular glymphatic system.
The researchers found that the constriction of the pupil in response to light in both mice and rats increased glymphatic clearance from the eye. They also showed that the pressure in the eye, which is higher than that in the cranium, is necessary to drive the drainage through the ocular glymphatic system. Because of this, they hypothesized that glymphatic clearance might also be disrupted in glaucoma, a disease that typically involves an increase in intraocular pressure.
The research team determined that there was more amyloid-β clearance from the eyes of two different mouse models of glaucoma than from age-matched control mice. But the increased drainage did not seem to be caused by changes in intraocular pressure. Instead, excessive outflow was actually leakage into the spaces between axons in the optic nerve, not into the glymphatic system, that was related to a breakdown in the barrier between the eye and the optic nerve. The researchers hypothesized that this barrier normally diverts fluid, facilitating solute transport along the optic nerve’s veins, a process that may fall apart in a diseased eye.
The Trials of Running a Public Longevity Industry Company
Taking a company public is a deal with the devil. One does it to gain access to capital, and at the behest of early investors who want an exit. Thereafter, however, one has to deal with all sorts of complications and perverse incentives that make the process of developing therapies that much more challenging. A little of this topic is discussed in this article, in the context of longevity industry biotech companies. Only a few of these companies are public at the moment, but that will change as the field advances and broadens.
When we spoke to Aubrey de Grey recently, he highlighted the potential challenges facing public companies in the longevity field. Juvenescence portfolio company AgeX Therapeutics is publicly traded and Greg Bailey of Juvenesence points out that many of the macro challenges are the same as any other biotech business. “You can go months between positive news, and during that period of time, the shorts can play havoc with your stock. And the other thing is, you fundamentally always have to have two years of working capital. If you don’t, the short sellers circle – knowing you’re going to have to do a financing and that they’ll be able to buy in when you do that financing at a 10 to 15% discount to market.”
But the work conducted by companies working on anti-aging and regenerative therapies also adds another level of variability to the challenge. “Now you also need to be concerned about the fact that one of your competitor’s trials fails and everybody says ‘Ah, anti-aging is rubbish – all hype, no reality’ and you get caught in the downdraft. And, unfortunately, there have been a number of downdrafts with public companies where their trials have not lived up to expectations.”
With investors beginning to realise the scale of the Longevity market, Bailey feels that this sort of reaction will become less common, but that hasn’t stopped Juvenescence from taking action to mitigate the risk by moving AgeX Therapeutics to a licencing model. “So now you can’t short because we did a deal with a major Japanese company in January. We did a deal with UC Irvine that’ll see us in clinical trials in 18 months from January. So now as a short investor you’ve got to worry that I sign a license tomorrow, and the stock jumps and you get caught offside.”
Like almost everyone in the world at the moment, dealing with the impact of COVID-19 is a significant concern. However: “If Bank of America is right, and the fact that every government investor is going to be looking at boosting the immune system, which is definitely affected by aging, there’s going to be an enormous amount of money available in this. So it’s just about getting in front of the right people and them understanding there’s going to be a company that is modifying aging that’s going to be worth 100 billion dollars, sooner as opposed to later. You’ve just got to pick which model you think is the right business model to invest in to get your institution or entity correctly positioned.”
The Aging of Muscle Stem Cells
Impaired function of muscle stem cells is presently thought to be the dominant contributing factor in the development of sarcopenia, the loss of muscle mass and strength that affects everyone with advancing age. The mechanisms that cause stem cell decline are less well mapped, both in terms of their relative effect sizes, as well as their positions in the complex web of cause and consequence that links fundamental forms of cell and tissue damage, the root of aging, to downstream manifestations of aging.
Adult skeletal muscle has its own stem cell population, namely muscle satellite (stem) cells (MuSCs). Under sedentary conditions in the adult stage, MuSCs are mitotically quiescent and reside beneath the basal lamina of the myofiber; this position between the myofiber and the surrounding extracellular matrix is crucial for maintaining the stem cell state. After muscle injury, quiescent MuSCs promptly get activated, resulting in proliferation and their differentiation into myoblasts. Hence, myoblast fusion is critical not only for skeletal muscle development but also for regeneration. However, the function of MuSCs gradually declines during physiological and pathological aging. Although loss of the muscular regenerative capacity in aging is partly due to this impairment of MuSC function, the precise mechanism of how stem cell function is maintained and impaired remains unclear.
Aged MuSCs are also more prone to undergo senescence or apoptosis than young MuSCs. In terms of the ability of MuSCs to differentiate, the adipogenic differentiation program is enhanced in cultured, aged MuSCs. In the context of acute injury, symmetric and asymmetric cell division promote the expansion of MuSCs and maintain homeostasis of the stem cell compartment. Impairment of this process in aged muscle leads to an impaired propensity to proliferate and produce myoblasts necessary for muscle regeneration. While there are reports demonstrating decreases in the number of MuSCs during aging, conflicting reports show no significant differences in the number of MuSCs between young and aged mice. Additionally, since MuSCs are very rare and the number of MuSCs differs in the type and location of skeletal muscles, it is difficult to reach conclusions on the frequencies of MuSCs within young and aged mice.
The decline of MuSC regenerative capacity is due to age-associated extrinsic/environmental changes as well as cell-intrinsic/autonomous changes. As extrinsic factors, inflammatory responses, extracellular components, and changes in interacting cell types definitely affect the function in MuSCs. MuSC function is also impaired by cell-intrinsic damages including oxidative stress, DNA damage, modified signaling pathways, damage to proteins, and altered metabolism. An accumulation of cell intrinsic damages leads to a “point of no return” in aged MuSCs as they go into a pre-senescent state or they undergo apoptosis. Alterations in several intracellular signaling pathways in aged MuSCs affect their self-renewability. The functional decline of MuSCs is partly due to the activation of FGF2, TGF-β, WNT pathways, JAK/STAT3, p16INK4a, and p38. Those pathway modulations could be a therapeutic target for muscle regenerative therapy in elderly.
Direct Reprogramming of Skin Cells into Photoreceptors to Restore Light Sensitivity in Mice
Researchers here demonstrate a direct form of cellular reprogramming, converting skin cells directly into another cell type without going through intermediary stages of induced pluripotency and differentiation. In this case the goal is to produce patient-matched photoreceptor cells to treat retinal degeneration. Proof of concept is demonstrated in blind mice that exhibit restored light sensitivity following treatment. The degree to which vision can be restored via this approach is an open question – that is much harder to assess in mice. In principle this type of reprogramming should be much more efficient, but it remains to be seen as to which approaches to building sources of patient-matched cells will emerge first into widespread clinical practice.
Up until now, researchers have replaced dying photoreceptors in animal models by creating stem cells from skin or blood cells, programming those stem cells to become photoreceptors, which are then transplanted into the back of the eye. In the new study, scientists show that it is possible to skip the stem-cell intermediary step and directly reprogram skins cells into photoreceptors for transplantation into the retina.
Scientists have studied induced pluripotent stem (iPS) cells with intense interest over the past decade. IPSCs are developed in a lab from adult cells and can be used to make nearly any type of replacement cell or tissue. But iPS cell reprogramming protocols can take six months before cells or tissues are ready for transplantation. By contrast, the direct reprogramming described in the current study coaxed skin cells into functional photoreceptors ready for transplantation in only 10 days. The researchers demonstrated their technique in mouse eyes, using both mouse- and human-derived skin cells.
Direct reprogramming involves bathing the skin cells in a cocktail of five small molecule compounds that together chemically mediate the molecular pathways relevant for rod photoreceptor cell fate. The result are rod photoreceptors that mimic native rods in appearance and function. The researchers performed gene expression profiling, which showed that the genes expressed by the new cells were similar to those expressed by real rod photoreceptors. At the same time, genes relevant to skin cell function had been downregulated.
The researchers transplanted the cells into mice with retinal degeneration and then tested their pupillary reflexes. Within a month of transplantation, six of 14 (43%) animals showed robust pupil constriction under low light compared to none of the untreated controls. Moreover, treated mice with pupil constriction were significantly more likely to seek out and spend time in dark spaces compared with treated mice with no pupil response and untreated controls. Preference for dark spaces is a behavior that requires vision and reflects the mouse’s natural tendency to seek out safe, dark locations as opposed to light ones.
Complement C5 Protein is a Biomarker of Preclinical Atherosclerosis
At some point in the years ahead, the research community will develop effective means of reversing atherosclerotic lesions, the fatty, inflammatory deposits that build up in blood vessel walls to ultimately cause stroke or heart attack. Those therapies will be best applied in a preventative manner, rather than in later stages of the condition when lesions are large, complex, and greatly distort and weaken blood vessels. That in turn means that a reliable, low cost test to assess progression of preclinical atherosclerosis is required. Researchers here propose complement C5 protein levels in a blood sample as such a test, based on recent human data.
The purpose of this study was to analyze the temporal and topologically resolved protein changes taking place in human aortas with early atherosclerosis to find new potential diagnostic and/or therapeutic targets. The protein composition of healthy aortas (media layer) or with early atheroma (fatty streak and fibrolipidic, media, and intima layers) was analyzed by deep quantitative multiplexed proteomics. Plasma levels of complement C5 were analyzed in relation to the presence of generalized (more than 2 plaques) or incipient (0 to 2 plaques) subclinical atherosclerosis in 2 independent clinical cohorts, PESA (Progression of Early Subclinical Atherosclerosis) and NEFRONA (National Observatory of Atherosclerosis in Nephrology).
Proteins involved in lipid transport, complement system, immunoglobulin superfamily, and hemostasis are increased in early plaques. Components from the complement activation pathway were predominantly increased in the intima of fibrolipidic plaques. Among them, increased C5 protein levels were further confirmed by Western blot, enzyme-linked immunosorbent assay, and immunohistochemistry, and associated with in situ complement activation. Plasma C5 was significantly increased in individuals with generalized subclinical atherosclerosis in both PESA and NEFRONA cohorts, independently of risk factors. Moreover, in the PESA study, C5 plasma levels positively correlated with global plaque volume and coronary calcification.
In conclusion, activation of the complement system is a major alteration in early atherosclerotic plaques and is reflected by increased C5 plasma levels, which have promising value as a novel circulating biomarker of subclinical atherosclerosis.
Adjusting Macrophage Polarization for Therapeutic Effects is not Straightforward
The innate immune cells known as macrophages can adopt different packages of behaviors, known as polarizations, under different circumstances. The underlying reality is more a continuum than two clear categories, but researchers classify macrophages as being either M1, aggressive and inflammatory, or M2, pro-regenerative and anti-inflammatory. In theory, a number of issues that manifest with age could be slowed or reversed by forcing M1 macrophages to adopt M2 behaviors instead. The open access paper here is chiefly interesting for the discussion on why this is far from simple: a blunt approach to reprogramming macrophages is certainly feasible, but will probably do as much harm as good.
Compared to the classical phagocytotic “M1” macrophages, the alternatively polarized macrophages, called “M2” macrophages, function as modulators of cellular and humoral immunity and as mediators of tissue repair and remodeling. Transforming growth factor beta 1 (TGFβ1) is the most important growth factor enhancing tissue repair and fibrosis, and is believed to be produced and released by a subpopulation of M2 macrophages (M2c) in response to IL-10, in contrast to M2a macrophages which are primarily anti-inflammatory.
Prior work has shown that macrophages are the only inflammatory cells that infiltrate into the closed nucleus pulposus, and the number of macrophages is positively correlated with the severity of intervertebral disc degeneration. Moreover, there is evidence to suggest that macrophages may either directly play a role in phagocytosis, or synergistically regulate lumbar disc metabolism through a neuro-immune mechanism. Likewise, macrophage dysfunction can cause the aggregation, chemotaxis, and diffusion of inflammatory factors, leading to degradation of the extracellular matrix in the intervertebral disc, which in turn leads to lumbar disc degeneration. However, whether macrophage polarization is critical for the development of lumbar disc degeneration (LDD) and by what mechanism it may affect LDD, remains to be experimentally tested. This question was addressed in the current study.
The delicate phenotypic control of tissue macrophages is critical for proper tissue repair after injury and is very time-sensitive. Too much M1 polarization results in severe inflammatory responses, severe tissue damage and poor recovery. However, too much M2 polarization may result in insufficient inflammatory responses and incomplete pathogen- and cell debris removal. Importantly, M2-like polarization induces fibrosis, mainly mediated by TGFβ signaling.
In the current study, we co-blocked DNMT1 and TGFβ1 in macrophages. While DNMT1 suppression induced a general M2-like polarization in macrophages, specific inhibition of TGFβ1 may affect IL-10 production and secretion, which in turn reduces the generation of fibrotic M2c macrophages. Our results showed that co-blocking TGFβ1 did not attenuate the effects of DNMT1 inhibition on induction of M2-like macrophage polarization, but did reduce cell apoptosis and pain-associated MMP1, thereby promoting a favorable therapeutic outcome.
Photobiomodulation to Enhance Mitochondrial Function as a Potential Therapy for Parkinson’s Disease
There is evidence for near-infrared light to improve mitochondrial function, although exactly how it works, and how reliably it works, is far from settled. This wavelength of light penetrates tissue deeply enough to be considered as a treatment for neurodegenerative conditions, at least those in which mitochondrial dysfunction is strongly implicated, such as Parkinson’s disease.
As the main driver of energy production in eukaryotes, mitochondria are invariably implicated in disorders of cellular bioenergetics. Given that dopaminergic neurons affected in Parkinson’s disease (PD) are particularly susceptible to energy fluctuations by their high basal energy demand, it is not surprising to note that mitochondrial dysfunction has emerged as a compelling candidate underlying PD.
A recent approach towards forestalling dopaminergic neurodegeneration in PD involves near-infrared (NIR) photobiomodulation (PBM), which is thought to enhance mitochondrial function of stimulated cells through augmenting the activity of cytochrome C oxidase. Notwithstanding this, our understanding of the neuroprotective mechanism of PBM remains far from complete. For example, studies focusing on the effects of PBM on gene transcription are limited, and the mechanism through which PBM exerts its effects on distant sites remains unclear.
Also, the clinical application of NIR in PD proves to be challenging. Efficacious delivery of NIR light to the substantia nigra pars compacta , the primary site of disease pathology in PD, is fraught with technical challenges. Concerted efforts focused on understanding the biological effects of PBM and improving the efficiency of intracranial NIR delivery are therefore essential for its successful clinical translation. Nonetheless, PBM represents a potential novel therapy for PD.
Mechanisms of Neurodegeneration Interact to Contribute to Multiple Conditions
The common neurodegenerative conditions are characterized by the aggregation of a few types of misfolded or otherwise altered proteins, harmful to cell and tissue function. Each form of protein aggregate is not a process occurring in isolation; their presence and their consequences interact with one another. Neurodegeneration is a holistic process, and the scientific and medical communities carve pieces out of that whole and call them diseases or disease mechanisms. It doesn’t do to lose sight of the fact that these neatly boxed classifications are to some degree arbitrary lines in the sand.
Many people with Lewy body diseases (LBDs) such as Parkinson’s disease (PD) ultimately develop dementia, and many have amyloid-β (Aβ) plaques and tau tangles. Do they have two diseases at the same time, or is this combination of scourges a unique entity unto itself? Researchers reported that people with PD who carry genetic risk variants for Alzheimer’s disease (AD) were more likely to become cognitively impaired. Similarly, AD variants predicted which PD patients harbored Aβ and tau proteopathies in their brains. In people with dementia with Lewy bodies (DLB), Aβ plaques seemed to worsen cognitive decline more than tau tangles did, suggesting an etiology distinct from AD. Although the interactions between α-synuclein, Aβ, and tau still need more clarification, some suggested that therapies targeting Aβ might benefit people with Lewy body diseases.
Studies suggest that Aβ could exacerbate both tau and α-synuclein aggregation in LBD. This may be why Aβ-targeted therapies could benefit people with synucleinopathies. However, anti-Aβ therapies have failed to benefit cognition in people with AD, so why would they work for LBD? In LBDs, Aβ appears to exacerbate both tau tangles and α-synuclein aggregation, either of which could lead to dementia. Targeting Aβ might dismantle this toxic triplet. Second, while Aβ plays the role of instigator in AD, it appears to drive progression throughout the disease process in LBD. Therefore, while ridding the brain of Aβ may be too little, too late, for people with symptomatic AD, it could slow the disease in people with LBD.
Hearing Loss Impairs Synaptic Plasticity and Memory Function in Mice
The brain makes use of sensory information in order to form memories. Loss of hearing has an impact on the aging brain, as suggested by the correlation between onset of age-related deafness and onset of dementia. While it is possible that this reflects common processes of neurodegeneration, as age-related deafness appears to result from loss of neural connections between sensory hair cells and the brain, studies such as this one provide evidence for deafness to cause greater loss of function in areas of the brain associated with memory formation.
Brain structures that are essential for the acquisition and encoding of complex associative memories, such as the hippocampus, use spatial sensory information both to generate metric representation of navigable space and to create robust and long-lasting records of spatial experience. The latter is enabled by hippocampal synaptic plasticity, and it has been shown that visuospatial, olfactospatial, and audiospatial experience can be used by the hippocampus to create spatial memories.
Studies of the consequences of loss of visual input and blindness have shown that adaptation occurs as a consequence of extensive reorganization of the cortex that reflects both changes in the affected primary sensory cortex and in other primary and associative sensory areas. One aspect of this that has received little attention is how the cortex and hippocampus functionally adjust to initial loss of input from a specific sensory modality. Recently, we reported that hereditary blindness that becomes manifest in mice within weeks after birth results in massive and progressive reorganization of neurotransmitter receptor expression in the cortex and hippocampus that persists for months after the onset of blindness. This reorganization is accompanied by a profound impairment of hippocampal long-term potentiation (LTP) and debilitation of hippocampus-dependent spatial learning.
Ultimately, both humans and animals recover from this transitional phase. Recent studies in human individuals have suggested, however, that the consequences for cognition of gradual sensory loss are insidious. Age-dependent sensorineural hearing loss (presbycusis) comprises a gradual and cumulative loss of hearing sensitivity. It is closely associated with cognitive decline and is considered a risk factor for dementia.
A causal link between cumulative hearing loss and cognitive decline is currently lacking. In the present study, our goal, therefore, was to explore to what extent a gradual loss of hearing sensitivity can result in cortical reorganization and changes in hippocampal function. The C57BL/6 mouse strain develops cumulative deafness that first becomes manifest at the age of 4 weeks. We show here that widespread changes in plasticity-related neurotransmitter expression become manifest as early as at 2 months of age in C57BL/6 mice. At 4 months of age, neurotransmitter receptor changes occur in both primary sensory and association cortices and also extend to the hippocampus. At this time-point, potent impairments in hippocampal LTP and spatial memory become evident.
The data indicate that gradual hearing loss is accompanied by extensive adaptive changes in the cortex and hippocampus that hinder effective hippocampal information processing and suggest that progressive hearing loss may be causally linked to cognitive decline.