03. April 2017 · Comments Off on How to Validate New Cryonics Technologies · Categories: Cryonics, Science

Evidence in cryonics is a complicated concept. For starters, it is not possible to “prove” cryonics will work, here and now, because the fundamental idea of cryonics is to stabilize critically ill patients (people considered “dead” by less rigorous criteria) in anticipation of more advanced future medical technologies. What we can do is validate cryonics technologies with reversible cryopreservation (“suspended animation”) as a benchmark. As a general rule, we can state that we make progress in cryonics when stabilization, cryopreservation, and maintenance (“storage”) technologies cause less damage than the technologies that preceded them. But how do we know if this is the case?

The most rigorous form of validation, human clinical trials, is usually not available in cryonics. There are often new (approved) emergency medical technologies, however, that can be modified to be used in cryonics procedures. A major advantage of adopting such technologies is that the validation has already been done by other organizations or companies. Examples of such technologies will often fall under the rubric of emergency medicine. For example, an FDA-approved technology that improves blood flow during cardiopulmonary resuscitation can be added to Alcor’s stabilization equipment to improve stabilization procedures.

One step down from rigorously designed human clinical trials are animal studies. In cryonics we often make a distinction between small animal studies (e.g., mice, rats) and large animal studies (e.g., pigs, dogs) etc. It seems common sense to think that large mammals provide stronger evidence for a technology than smaller animals but the real issue at stake here is not how large an animal is but how closely an animal model tracks what happens in humans. For example, if cat brains have an uncharacteristically high tolerance for cerebral ischemia, the (smaller) rat may actually be a more realistic model for validating neuroprotective strategies in humans.

One area where choosing the correct animal model has proven itself to be of crucial importance concerns the effect of cryoprotectants on the brain. Most mammalian species experience dehydration of the brain after equilibration with a vitrification agent. Because it is reasonable to assume that severe dehydration adversely affects brain viability it is tempting to select an animal model that experiences little cryoprotectant-induced dehydration. But one thing that we have learned from burhole measurements and CT scans in human cryonics patients is that under optimal conditions cryoprotective perfusion with both glycerol and the modern vitrification agents produces severe shrinkage of the brain. So if we want to validate strategies to eliminate this dehydration the most important consideration is not how “large” the animal is but how well the animal tracks the effects of cryoprotectants on the human brain.

Most technologies in cryonics need to be evaluated with ultrastructure and/or viability as an endpoint. But there are also new developments in cryonics where such a benchmark would not make a lot of sense. For example, if we build a new patient enclosure to keep the patient cold during cryoprotective perfusion we can just measure the core temperature of the patient to see if we have done a satisfactory engineering job. Another example is the design of new dewars where we can look at variables like the boiloff rate and long-term durability of the design.

In conclusion, there are a number of ways to validate new technologies in cryonics. If a new technology has undergone human clinical trials we often can just adapt that technology for cryonics without designing new experiments. In the case of more cryonics-specific technologies animal studies can be conducted and the choice of animal model will be dictated by how close a model tracks what we know to occur in humans (among other considerations like ethics and cost). Finally, when a new development in cryonics is mostly an engineering challenge, validating its efficacy is often just an issue of doing basic physiological measurements or practical tests.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine, August, 2014

20. November 2016 · Comments Off on Who Decides What We Can Do With Our Body (and Brain)? · Categories: Cryonics, Science, Society

Statement on the High Court ruling concerning 14 year-old cancer victim’s right to cryonics

Click here for PDF

Our hearts go out to the young British woman whose battle with cancer ended sadly earlier this month at age 14, as well as to her parents as they cope with this very difficult time. And we commend the British High Court Judge for his important ruling enabling the girl to obtain her wish to be cryogenically preserved. While we have no comment on the specifics of this case, and do not ourselves offer services of this nature, we hope we can shed some light on the project of experimental medical biostasis / cryonics more generally.

Over the past decade, scientists have made significant advances in low-temperature biology, and scientists developing molecular machines will receive this year’s Nobel Prize on December 10. Many, including scientists at places like Cambridge, Oxford, MIT, NASA and Harvard, now openly support cryonics as a legitimate scientific endeavor. Of course there is no guarantee that any cryonics patients will be revived in the future, but as discussed by four tenured professors in this recent MIT Technology Review piece, the best evidence suggests that cryonics deserves open-minded consideration.

Coordinator of the UK Cryonics and Cryopreservation Research Network, Dr João Pedro de Magalhães, when asked for his thoughts, observed that “no matter the probability you assign to the procedure, we think it’s important to give people the choice, just as we give dying patients the opportunity to try other experimental medical therapies to save their lives”.

Cryonics is a similar experimental treatment, albeit one with different legal and ethical implications, and whose probability of success is unknown. Many parts of the world are now taking progressive stances towards the idea of death with dignity. It seems incongruous with these beliefs to stigmatize a procedure for what is at worst an over-optimistic belief about the state of the future.

Despite the many intermediate successes in low-temperature biology over the past few decades, no cryonics organization can currently revive a patient. Nobody has claimed otherwise, and arguments based on this premise are missing the point.

Cryonicists look at how medicine has progressed over the past hundred years, at the millions of people whose lives would have been cut short if not for advances in technology, and it fills them with hope about what might be possible for the future. The goal of cryonics is not to be able to revive someone with contemporary technology, rather the goal is to preserve a person and her brain well enough that future technologies may be able to (repair and) revive the person. One can think of this as transporting the body forward through time or as medical time travel. This depends on technologies that will be developed in the next decades or centuries, not on the world’s capabilities today. All the major cryonics organizations in the western world are non-profits with the goal of surviving for centuries.

As Aschwin de Wolf, President of The Institute for Evidence-Based Cryonics, explained, “Cryonics is based on the premise that the neuro-anatomical basis of identity is more robust than folk wisdom suggests, and we envision future technologies that can infer the healthy state of the brain from the injured state – and even repair any damage that occurs during the cryopreservation process itself. As such, cryonics is not an act of faith, but an act of reason.”

We will cure cancer one day, and it is reasonable for this girl, born too early through no fault of her own, to choose for herself the best chance to make it to that world where more is possible.

Contact / interviews:

Dr João Pedro de Magalhães

Coordinator, UK Cryonics and Cryopreservation Research Network

+44 151 7954517 / aging@liverpool.ac.uk

www.cryonics-research.org.uk

Aschwin de Wolf

President, Institute for Evidence-Based Cryonics

contact@evidencebasedcryonics.org

www.evidencebasedcryonics.org

Appendix of key supporting materials

  • “The patient should participate responsibly in the care, including giving informed consent or refusal to care as the case might be…The patient’s right is based on the philosophical concept of respect for autonomy, the common-law right of self-determinationAmerican College of Physicians Ethics Manual, 2016
16. September 2016 · Comments Off on The Multi-Headed Hydra · Categories: Science, Society

This article explores some of the regulatory challenges facing those who would bring rejuvenation biotechnologies, like those pursued by Dr. Aubrey de Grey and the SENS Foundation, to market. It does not presume familiarity with Dr. de Grey and his work; I’ve tried to make it informative to all alike.

The Conquest of Aging

Biomedical gerontologist Aubrey de Grey predicts that the first human being to live to 1,000 years old is alive today. Who exactly that person might be – or rather, how old they are today – is a detail that Dr. de Grey has wavered on, but he has remained firm in his commitment to that prediction, and is certainly one of the most prominent figures working towards realization of the technologies required to make his prophecy reality. In his book, Ending Aging, Dr. de Grey describes his proposed approach to the “problem” of aging, and how it differs from those presently in practice.[1]

In Dr. de Grey’s opinion, the current paradigm devotes a vast majority of resources to geriatric care, which attempts to cure or manage age-associated diseases only after they emerge as recognizable groupings of symptoms. To quote an apt metaphor from another longevity advocate:

“[T]he challenge of treating illnesses in the elderly must at times seem like Heracles’ trials of combating the multi-headed Hydra. Each time one head was severed, two more would sprout in its place. Likewise, a patient might survive a serious cardiac episode with help of antihypertensive drugs only to succumb to cancer and dementia.”[2] [emphasis in original]

Meanwhile, the (significantly smaller) remaining portion of research dollars goes towards biogerontology, which studies the upstream causes of aging, any benefit of which is probably unrealizable for several human generations. However, Dr. de Grey argues that without necessarily knowing much more about the upstream causes of aging than is currently understood, it is already possible to categorize the different forms of aging “damage,” and devise therapies that will repair the damage at a sufficient rate to achieve what he terms “longevity escape velocity.”

Dr. de Grey calls his theory “Strategies for Engineered Negligible Senescence” (SENS). There are seven strategies, each related to one of the seven major categories of aging damage thus far discovered. Those categories (and proposed therapies) are: (1) cancer-causing nuclear mutations (removal of telomere-lengthening machinery, aka OncoSENS); (2) mitochondrial mutations (allotopic expression of 13 proteins, aka MitoSENS); (3) intracellular junk (novel lysosomal hydrolases, aka LysoSENS); (4) extracellular junk (immunotherapeutic clearance, aka AmyloSENS); (5) cell loss & tissue atrophy (stem cells and tissue engineering, aka RepleniSENS); (6) cell senescence (targeted ablation, aka ApoptoSENS); and (7) extracellular crosslinks (AGE-breaking molecules and tissue engineering, aka GlycoSENS). The SENS Foundation was established in 2009, helped in part through seed funding provided by Peter Thiel, co-founder of PayPal and a managing partner of The Founders Fund. The SENS Foundation’s stated purpose is “to research, develop and promote comprehensive regenerative medicine solutions for the diseases and disabilities of aging.”[3]

Delving into the details of each of Dr. de Grey’s proposed strategies is beyond the scope of this article, but it is worth taking a closer look at one of the seven. LysoSENS aims at “junk” molecules which cannot be broken down by human lysosomal enzymes. Over time, these molecules accumulate within cells, contributing to conditions like macular degeneration, atherosclerosis, and Alzheimer’s disease (AD)[4]. Dr. de Grey’s proposition is to search for novel lysosomal enzymes (novel to humans, that is) in bacteria, molds, and other microbes that are involved in “recycling” dead animal bodies, since the “junk” inside our cells is — along with the  rest of us — food to them. SENS research being carried out at Rice University has already identified one such enzyme that, when targeted to the lysosome, decreases cytotoxicity of 7-ketocholesterol (7KC), an oxysterol associated with atherosclerosis and Alzheimer’s disease.[5] Enzyme replacement therapy is already used for the treatment of lysosomal storage diseases not associated with aging, like Gaucher’s disease. Insofar as it could be used to treat a condition (or conditions) remedially, a therapy targeting 7KC with a novel lysosomal enzyme might otherwise resemble traditional approaches to disease treatment, but it could also be used preventively. Other SENS pose even greater challenges to the traditional distinctions between cure, prevention and enhancement. The objective of MitoSENS, for instance, is to render the recipient immune to the fallout consequences of mitochondrial DNA mutations by placing backup copies of the thirteen mitochondrial genes — which naturally reside only inside the mitochondria — into the cell nuclei. Significant research progress is being made into this strategy as well.[6]

The problem of normative definitions of aging

Dowsing for fountains of youth is well and good, but won’t get us very far unless they can be tapped and piped to the marketplace, and while there are many scientific obstacles to overcome before rejuvenation biotechnologies are realized, there are also social, political and legal ones. Many of these problems are definitional. For one, what exactly distinguishes age-associated diseases and conditions from “normal” features of aging? In the words of one scholar: “[F]rom the perspective of modern biogerontology, there is little to distinguish biological ageing from a disease state…. To argue that ageing is not a disease by virtue of its universality is as misleading as it is to argue that the Basenji is not a dog because it does not bark.”[7] But to dismiss this debate as purely semantic or philosophical would be to misunderstand the true difficulty the definitional problem poses.

The U.S. Food, Drug and Cosmetic Act defines “drug” as, inter alia, “articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals” and “articles (other than food) intended to affect the structure or any function of the body of man or other animals.” [8] So far so good, because even if the U.S. Food and Drug Administration (“FDA”) did not agree that a particular undesired physical state was a “disease” for the purposes of the first definition, it would be difficult to deny that a proposed therapeutic (whether a chemical entity or a biological product[9]) was not intended to affect the structure or functioning of the body, at some level. However, present regulatory approval pathways indirectly require that a drug be “indicated for the treatment, prevention, mitigation, cure, or diagnosis of a recognized disease or condition or of a manifestation of a recognized disease or condition, or for the relief of symptoms associated with a recognized disease or condition.”[10] [emphasis mine]. The phrase “recognized disease or condition” is not defined in this context[11], and the FDA is not itself the recognizer, but rather looks for consensus within the clinical and/or scientific communities regarding the existence of a particular disease or condition, and of clear criteria for clinical diagnosis thereof.[12] To quote one author: “To the extent that many problems of ageing have not been formally recognized by any of these processes, the FDA has no clear guidance on how to determine if a proposed indication would be acceptable.” [13]

For many age-associated conditions, the question of “recognition” is a valueladen debate. While some commentators will no doubt accuse longevity advocates of “disease-mongering”[14], Dr. de Grey would probably argue that that sort of reaction is a symptom of what he terms the “pro-aging trance”[15] — a terror management strategy that accepts and embraces the apparently unavoidable progressive wasting of one’s body (and mind), instead of rejecting and resisting it. But the cognitively dissonant distinction between normal, “healthy” aging on the one hand, and “diseases” of aging on the other is not impermeable. For some historical perspective, it is worth considering the example of Alzheimer’s disease. When it was first described in 1910, AD only included what is now referred to as “earlyonset Alzheimer’s disease,” i.e., when the symptoms of “senile dementia” appeared in someone under 65.[16] Due to its vastly less frequent incidence, this “presenile dementia” was assumed to be distinct from the normal variety. However this normal/ abnormal categorization broke down in 1977, due to professional recognition of their near identical symptomologies, making the early-onset subtype by far the minority of AD incidence.[17]

A present-day example of this process of recognizing “normal” features of aging as diseases or conditions of aging, is the case of sarcopenia. Sarcopenia (literally “poverty of the flesh”) describes the degeneration of skeletal muscle mass and strength that occurs with aging that contributes (in part) to disability, frailty, and morbidity in older persons.[18] Until fairly recently, sarcopenia and related conditions like sarcopenic obesity were considered “normal” aspects of aging, much like senile dementia prior to 1977. To be fair, both sarcopenia and senile dementia are normal, insofar as they are common conditions in older persons — but that does not mean they are untreatable, nor that they should be left untreated. A number of potential drug targets have been identified that may be of use in treating sarcopenia[19], but if consensus recognition of the condition is lacking there may not yet be a regulatory pathway for licensing therapeutics to treat it.[20]

Thus, as it stands, forging a regulatory pathway for treatments of a common, disabling (and in some cases indirectly lethal) feature of aging involves two distinct steps: first, persuade the scientific and clinical communities that a particular symptomology of aging can and should be treated, and second, persuade the FDA that everyone else is persuaded. But this is not insurmountable. The European Working Group on Sarcopenia in Older People published a “practical clinical definition and consensus diagnostic criteria for agerelated sarcopenia” in 2010[21], which was followed by a consensus definition from The International Working Group on Sarcopenia in 2011[22]. In the U.S., the Foundation for the National Institutes of Health, the National Institute on Aging, and the FDA held a Sarcopenia Consensus Summit on May 8-11, 2012.[23] A number of clinically meaningful end points have been proposed for assessing treatment efficacy[24], including patient-reported outcomes.[25] Under appropriate regulatory supervision, medicalization of sarcopenia would help older persons maintain or even regain functional independence and quality of life — and perhaps boost lifespan, via a reduction in comorbidity with diseases like osteoporosis.

The problem of causally interrelated disease states

There is another definitional problem: What distinguishes one age-associated disease from another? This may seem like a facetious question, given the obvious symptomatic differences between atherosclerosis and AD. However, as mentioned above, the oxysterol 7KC has been implicated in the pathogenesis of both those disease states. If 7KC is indeed a metabolic byproduct that is causally related to both atherosclerosis and AD then, in addition to being a promising drug target itself, it could conceivably qualify as a surrogate endpoint for clinical trials of new drugs indicated for those diseases. FDA has issued a draft guidance regarding qualification of biomarkers as drug development tools[26], but surrogate endpoints may only be used in lieu of direct measures of clinical benefit under the FDA’s “Fast-Track Program,” which is only available for new drugs intended for the treatment of a serious or lifethreatening condition and that demonstrate the potential to address unmet medical needs for such a condition.[27] However, it would not be necessary to qualify 7KC reduction as a surrogate endpoint for both AD and atherosclerosis. Doing so for one or the other based on which is thought to be the more serious condition and/or the greater unmet need would allow its use in a fast-tracked New Drug Application for the one indication, and then if safety and efficacy in humans is established and the therapeutic is approved, data from (likely compulsory) post-marketing studies could be used in a new indication claim for the other condition.

Surrogate endpoints need only be “reasonably likely to predict clinical benefit”[28], and some commentators have pointed out that applying this lower standard to the screening of surrogate endpoints may result in drugs approved on the basis of evidence of an effect on a biomarker which, while related to the disease, is not actually causally related to any clinical benefit.[29] Of course, given its ambitious objective, the SENS Foundation has a strong vested interest in tying 7KC to clinical benefit, and the fact that FDA-qualified biomarkers are released into the public domain also fits within the Foundation’s public interest mandate, and could enhance perceptions of the legitimacy of its research goals. But more importantly, it could shorten clinical trials, an oft-criticized source of delay and drug costs. While its work to date has primarily been proof-of-concept research, in the future the SENS Foundation might devote some of its resources to running forms of aging damage like 7KC through the biomarker qualification process. Although publishing both the proof-of-concept and such valuable drug development tools might cut out some of the traditional patenting opportunities[30], it also offsets costs ordinarily borne by pharmaceutical companies. A little low-hanging fruit might stir up some productive competition in an industry sometimes criticized for chasing after minor therapeutic improvements and patent trolling.

Another option that is very in line with the social agenda of longevity advocates would be to promote the rebranding of multiple disease states with significantly overlapping low-level chemistry as single unified conditions that present varied symptom groupings based on exposure to particular environmental factors (including the endogenous “environment,” like certain genes or epigenetic variations, along with more traditional exogenous contributors like diet, exercise, etc). Admittedly, this would be the more difficult path, because it relies on the two-step process of disease recognition, discussed above, and considering how long it took AD and senile dementia to be folded into AD with an early-onset subtype, trying to replicate this process with diseases that present as differently as atherosclerosis and AD may be a Sisyphean task. On the other hand, academic pressure of this kind could have trickle-out effects on the public, re-situating the discourse of age-associated diseases further upstream, pressing on towards greater recognition of aging as disease.

Finally, slight augmentations to the SENS branding could be in order. Dr. de Grey gave unique names to his proposed strategies (LysoSENS, MitoSENS, etc.), but not to the categories of damage which are the targets of those strategies. Devising and promoting novel medical names for these categories of damage, like idiocytotoxicosis[31] for the “intracellular junk” targeted by LysoSENS, might prompt frame-shifting in the academic and clinical communities that could consequently (albeit indirectly, and thus probably slowly) broaden the scope of “recognized disease or condition”. Sadly for the planet, ‘junk’ doesn’t seem to be something humans take terribly seriously — idiocytotoxicosis, on the other hand, well that’s clearly a monster. Perhaps this suggestion borders on “disease-mongering” — but isn’t that term itself equally agenda-driven, given the not-so-subtle association with war-mongering? Dr. de Grey and other longevity advocates consider themselves to be on moral high ground, so these kinds of accusations are only of consequence if they provoke counter-productive public response, and reframing well-recognized diseases like AD and atherosclerosis as symptoms of underlying “metabolic pathology” can hardly be characterized as “questionable new diagnoses — like [premenstrual dysphoric dysfunction] and social anxiety disorder — which are hard to distinguish from normal life,” the likes of which give at least one critic concern. [32] And perhaps it is the very idea that “normal” is the ultimate objective — as opposed to simply “better” — that is the problem.

What’s the alternative?

If the perceived burden is too high, and the cost of doing nothing too great, stakeholders may look to circumvent the FDA. The SENS Foundation characterizes the assault on aging as the next space race.If the U.S. doesn’t take action to foster local development of what will assuredly be highly sought-after therapies, the movement may simply move underground (i.e. further in the vein of DIYbio), and overseas (medical tourism, and seasteads), which will only hamper the FDA’s mandate to protect Americans from harm.

Endnotes

[1]: Aubrey de Grey & Michael Rae, Ending Aging: The Rejuvenation Breakthroughs That Could Reverse Human Aging in Our Lifetime, (New York: St Martin’s Press, 2007).

[2]: David Gems, “Tragedy and delight: the ethics of decelerated aging” (2011) 366 Philosophical Transactions of the Royal Society B [Phil Trans R Soc B] 108 at 110.

[3]: SENS Foundation, SENS Foundation, online: <http://www.sens.org/about-thefoundation>.

[4]: Jacques M Mathieu et al, “7-Ketocholesterol Catabolism by Rhodococcus jostii RHA1” (2010) 76:1 Applied and Environmental Microbiology 352.

5]: Jacques M Mathieu et al, “Increased resistance to oxysterol cytotoxicity in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase” (2012) Biotechnology and Bioengineering, online:
<http://www.wileyonlinelibrary.com> DOI 10.1002/bit.24506.

[6]: SENS Foundation, Research Report 2011, online: <http://images.sens.org/reports/ SENS%20Research%20Report%202011.pdf>.

[7]: Supra note 2 at 109.

[8]: 21 USC § 321(g)(1).

[9]: 42 USC § 262(i). The phrase “analogous product” has been used to justify the extension of the FDA’s regulatory authority to human cells, tissues, and cellular and tissue-based products (HCT/Ps). See also Areta L Kupchyk, “Approval of Products for Human Use” in HB Wellons et al, Biotechnology and the Law (ABA, 2007) 591 at 617, note 41

[10]: 21 CFR § 201.57(c)(2) Specifically, this is a labeling requirement, but if a drug cannot be labeled according to the regulation, the New Drug Application cannot be approved. See also 21 CFR § 201.56.

[11]: The term disease is defined in 21 CFR §101.93(g) for the purposes of disease claims relating to dietary supplements, but that is only applicable in that context. See also 21 USC 343(r)(6).

[12]: William J Evans, “Drug discovery and development for ageing: opportunities and challenges” (2011) 366 Phil Trans R Soc B 113 at 114.

[13]: Ibid at 114.

[14]: Barbara Mintzes, “Disease Mongering in Drug Promotion: Do Governments Have a Regulatory Role?” (2006) 3:4 PLoS Medicine e198.

[15]: Aubrey de Grey, “Combating the Tihtonus Error: What Works?” (2008), 11:4 Rejuvenation Research 713.

[16]: GE Berrios, “Alzheimer’s disease: a conceptual history” (1990) 5:6 International Journal of Geriatric Psychiatry 355.

[17]: Robert Katzman et al, Alzheimer’s disease: senile dementia and related disorders (NY: Raven Press, 1978) at 595.

[18]: Eric P Brass & Kathy E Sietsema, “Considerations in the Development of Drugs to Treat Sarcopenia” (2011) 59:3 Journal of the American Geriatrics Society 530.

[19]: Ibid at 531.

[20]: Supra note 12 at 116.

[21]: Alfonso J Cruz-Jentoft et al, “Sarcopenia: European consensus on definition and diagnosis” (2010) 39:4 Age and Ageing 412 (Abstract).

[22]: Roger A Fielding et al, “Sarcopenia: An Undiagnosed Condition in Older Adults. Current Consensus Definition: Prevalence, Etiology, and Consequences” (2011)12:4 Journal of the American Medical Doctors Association [JAMDA] 249 (Abstract).

[23]: See Marco Brotto, “Lessons from the FNIH-NIA-FDA sarcopenia consensus summit” (2012) 9 IBMS BoneKEy 210.

[24]: Supra note 18 at 531-533.

[25]: Ibid at 533. See also Christopher J Evans et al, “Development of a New Patient-Reported Outcome Measure in Sarcopenia” (2011) 12:3 JAMDA 226.

[26]: Center for Drug Evaluation and Research, “Guidance for Industry – Qualification Process for Drug Development Tools,” FDA (October 2010) online: <http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM230597.pdf>.

[27]: 21 USC § 356(a)(1).

[28]: 21 CFR § 314.510.

[29]: Thomas R Fleming, “Surrogate Endpoints And FDA’s Accelerated Approval Process” (2005) 24:1 Health Affairs 67. See also Thomas R Fleming and David L DeMets, “Surrogate end points in clinical trials: are we being misled?” (1996) 125:7 Annals of Internal Medicine 605.

[30]: There may be other intellectual property issues implicated in this shift of paradigm in drug development and regulation, but they are beyond the scope of this article.

[31]: Meaning “self, one’s own” + “cell” + “toxin” + “condition of increase”.

[32]: Supra note 14 at 0463.

[33]: SENS Foundation, Annual Report 2011, online: <http://www.sens.org/sites/ srf.org/files/SENS%20Foundation%20 Annual%20Report%202011.pdf>.

First published as a regular column called In Perpetuity in Cryonics Magazine, March 2013

01. July 2016 · Comments Off on Advances in Cryoprotectant Toxicity Research · Categories: Cryonics, Science

There is little disagreement among cryobiologists that the biggest limiting factor to reversible organ cryopreservation is cryoprotectant toxicity. It is actually not that hard to create vitrification solutions that completely inhibit ice formation at even the slowest cooling rates. The problem is that such highly concentrated vitrification solutions are too toxic to permit recovery of complex tissues. The least toxic vitrification solution for complex mammalian organs as of writing is M22. M22 is the culmination of many years of experimental and theoretical work by cryobiologist Greg Fahy and colleagues using rabbit kidney slices. Studying selected cryoprotectant mixtures on rabbit kidney slices, Fahy and colleagues came to the following conclusions:

1. High concentrations of a cryoprotective agent (or a mixture of different cryoprotective agents) can prevent ice formation during cooldown and warming.

2. The toxicity of some cryoprotectants can be neutralized by combining them with other cryoprotective agents.

3. The non-specific toxicity of a  cryoprotectant solution can be predicted by calculating a quantity (“qv*”) which is intended to measure the average hydrogen-bonding strength of the cryoprotectant polar groups with the water molecules in the solution.

4. Within limits, non-penetrating agents can reduce the exposure of cells to toxic amounts of cryoprotectants without reducing vitrification ability.

5. Synthetic “ice blockers” can be included in a vitrification mixture to reduce the concentration of toxic cryoprotective agents necessary to achieve vitrification.

While M22 is a low toxicity solution, its toxicity profile still necessitates minimizing exposure time and introduction and removal at low (subzero) temperatures. If we had a better understanding of the mechanisms of cryoprotectant toxicity, vitrification solutions with no toxicity at all could be introduced at higher temperatures and exposure times could be increased to optimize complete equilibration of the tissue with the cryoprotectant. It would also allow safer storage at intermediate temperature temperatures (around -130 degrees Celsius) because ultra-stable vitrification solutions could be used that are less prone to de-vitrification upon re-warming. This would be of particular interest for the cryopreservation of large organs or even whole organisms (with applications such as suspended animation and cryonics).

Two major reviews of cryoprotectant toxicity were published in the last 5 years; Gregory Fahy’s “Cryoprotectant Toxicity Neutralization” (Cryobiology, 2010) and Benjamin Best’s “Cryoprotective Toxicity: Facts, Issues, and Questions” (Rejuvenation Research, 2015).

Greg Fahy’s paper is a rigorous exposition of experimental results concerning the phenomenon of cryoprotectant toxicity neutralization. The paper is mostly limited to the discovery that DMSO can block the toxic effects of amides such as formamide. The combination of DMSO and formamide (or other amides such as urea and acetamide) is indeed one of the building blocks of M22 but this combination cannot be used without limit and the paper includes data that indicate the maximum molar concentrations (and ratios) that still permit full viability. In theory, if two (or more) cryoprotectants would completely neutralize each other’s toxicity they could be the sole components of a vitrification solution. But as the formulation of M22 shows, it is still necessary to add weak glass formers such as ethylene glycol, extracellular CPA’s, and “ice blockers” to supplement the toxicity neutralization obtained with formamide and DMSO. An important finding in Fahy’s paper is that n-methylation abolishes toxicity neutralization for amides and combining methylated amides also does not lead to toxicity neutralization between them. In fact, Fahy found that the presence of n-methylated compounds renders even small amounts of DMSO toxic. The remainder of the paper discusses the mechanisms of cryoprotectant toxicity and Fahy now favors protein denaturation as a plausible mechanism of (non-specific) toxicity. While other cases of toxicity neutralization have been reported in the literature, no rigorous studies have been done to produce a body of knowledge that is comparible to what we know about amide-DMSO interactions.

Benjamin Best’s paper is more general in scope but presents a lot of experimental data and also critically discusses Fahy’s work on cryoprotectant toxicity. As Ben Best points out, different (and seemingly contradictory) results do not necessarily mean that cryoprotectant toxicity is a species or cell-type dependent phenomenon. One could imagine a meta-analysis of cryobiology data in which variables such as concentration, loading- and unloading protocols, exposure temperature, exposure time, and the type of viability assay are matched to ensure methodological consistency. It is also important to compare cryoprotectants at their minimum concentration to vitrify to make meaningful toxicity comparisons. If the work at 21st Century Medicine is an indication, universal low-toxicity cryoprotective solutions should be feasible. Perhaps the most interesting part of the paper is where Best offers a critique of Grag Fahy’s “qv* hypothesis of cryoprotectant toxicity”, which aims to show that non-specfic toxicity concerns the degree to which cryoprotectants leave water available to hydrate macromolecules. This discovery allowed for the substitution of ethylene glycol for propylene glycol in Fahy’s lower toxicity vitrification solutions, despite the resulting higher CPA concentrations. Best observes, “it seems contradictory that water remains available for hydration, but not available for ice formation.” A potential rejoinder to this observation is that so called “bound water” does not participate in ice formation but can be disturbed by strong glass formers. Best also suggests a potential refinement of qv* that allows for more precise calculation of the hydrogen bonding strength of the polar groups that are used to calculate qv*. It is conceivable that such a refinement would eliminate the few remaining outliers in the data that support the qv* hypothesis. The paper also draws attention to the possibility of kosmotropic co-solvents and changes of pH and microenvironment polarity to mitigate cryoprotectant toxicity.

Neither of the papers discusses cryopreservation of the mammalian brain, but there is good reason to believe that in the case of this organ modification of low-toxicity vitrification solutions is required. Conventional cryoprotective agents such as PG, EG, and DMSO have poor blood brain barrier (BBB) penetration and the brain may not tolerate the CPA exposure times that other organs do. For example, while M22 can be used for cryopreservation of the brain, many of its component have poor BBB penetration and PVP and the ice blockers (X-1000 and Z-1000) are assumed not to cross the (non-ischemic) BBB at all. One potential solution is to (reversibly) open the BBB with so- called BBB modifying agents like detergents or perhaps to search for cryoprotective agents that can cross the BBB.

The most fundamental question in the design of vitrification solutions remains whether it is possible at all to introduce high concentrations of cryoprotectants without creating any kind of irreversible molecular and ultrastructural adverse effects. Understanding what specific and non-specific cryoprotectant toxicity exactly is should enable us to answer this question.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine, September-October, 2016

05. February 2015 · Comments Off on An End to the Virus · Categories: Health, Science

Breakthroughs in medicine have increased substantially over the last hundred years, and most would agree that the introduction of antibiotics in 1942 has been one of the largest milestones in the history of medicine thus far. The success in treating bacterial infection has only accentuated the glaring lack of progress in developing effective therapeutics for those other enemies of the immune system, viruses. But Dr. Todd Rider and his team at MIT have dropped a bombshell with their announcement of a new broad spectrum antiviral therapeutic, DRACO, which appears not only to cure the common cold, but to halt or prevent infections by all known viruses.

Before talking specifically about this exciting news, let us first review viral biology and why viral infections have been so difficult to treat.

As you may recall from your early education, a virus particle, or virion, consists of DNA or RNA surrounded only by a protein coat (i.e., naked virus) or, occasionally, a protein coat and a lipid membrane (i.e., enveloped virus). Viruses have no organelles or metabolism and do not reproduce on their own, so they cannot function without using the cellular machinery of a host (bacteria, plant, or animal).

Viruses can be found all throughout our environment and are easily picked up and transferred to areas where they may enter our bodies, usually through the nose, mouth, or breaks in the skin. Once inside the host, the virus particle finds a host cell to infect so it can reproduce.

There are two ways that viruses reproduce. The first way is by attaching to the host cell and entering it or injecting viral DNA/RNA into the cell. This causes the host cell to make copies of the viral DNA and translate that DNA to make viral proteins. The host cell assembles new viruses and releases them when the cells break apart and die, or it buds the new viruses off, which preserves the host cell. This approach is called the lytic cycle.

The second way that viruses reproduce is to use the host cell’s own materials. A viral enzyme called reverse transcriptase makes a segment of DNA from its RNA using host materials. The DNA segment gets incorporated into the host cell’s DNA. There, the viral DNA lies dormant and gets reproduced with the host cell. When some environmental cue happens, the viral DNA takes over, makes viral RNA and proteins, and uses the host cell machinery to assemble new viruses. The new viruses bud off. This approach is called the lysogenic cycle; these viruses are called retroviruses and include herpes viruses and HIV.

Once free from the host cell the new viruses can attack other cells and produce thousands more virus particles, spreading quickly throughout the body. The immune system responds quickly by producing proteins to interfere with viral replication, pyrogenic chemicals to raise body temperature, and the induction of cell death (apoptosis). In some cases simply continuing the natural immune response is enough to eventually halt viral infection. But the virus kills many host cells in the meantime, leading to symptoms ranging from the characteristic runny nose and sore throat of a cold (rhinovirus) to the muscle aches and coughing associated with the flu (influenza virus).

Any virus can be deadly, especially to hosts with a weakened immune system, such as the elderly, small children, and persons with AIDS (though death is actually often due to a secondary bacterial infection). And any viral infection will cause pain and suffering, making treatment a very worthwhile goal. So far, the most successful approach to stopping viral infections has been prevention through the ubiquitous use of vaccines. The vaccine— either a weakened form of a particular virus or a mimic of one—stimulates the immune system to produce antibodies specific to that virus, thereby preventing infection when the virus is encountered in the environment. In another approach, antiviral medications are administered post-infection and work by targeting some of the specific ways that viruses reproduce.

However, viruses are very difficult to defeat. They vary enormously in genetic composition and physical conformation, making it difficult to develop a treatment that works for more than one specific virus. The immense number of viral types in nature makes even their classification a monumental job as there is more enormous structural diversity among viruses. Viruses have been evolving much longer than any cells have even existed and they have evolved methods to avoid detection and to overcome attempts to block replication. So, while we have made some progress in individual battles, those pesky viruses have definitely been winning the war.

Which is why the announcement of a broad spectrum antiviral therapeutic agent is such huge news. In their paper, Rider et al. describe a drug that is able to identify cells infected by any type of virus and which is then able to specifically kill only the infected cells to terminate the infection. The drug, named DRACO (which stands for Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer), was tested against 15 viruses including rhinoviruses, H1N1 influenza, polio virus, and several types of hemorrhagic fever. And it was effective against every virus it was pitted against.

Dr. Rider looked closely at living cells’ own defense mechanisms in order to design DRACO. First, he observed that all known viruses make long strings of doublestranded RNA (dsRNA) during replication inside of a host cell, and that dsRNA is not found in human or other cells. As part of the natural immune response, human cells have proteins that latch onto dsRNA and start a biochemical cascade that prevents viral replication. But many viruses have evolved to overcome this response quite easily. So Rider combined dsRNA detection with a more potent weapon: apoptosis, or cell suicide.

Basically, the DRACO consists of two ends. One end identifies dsRNA and the other end induces cells to undergo apoptosis. When the DRACO binds to dsRNA it signals the other end of the DRACO to initiate cell suicide, thus killing the infected cell and terminating the infection. Beautifully, the DRACO also carries a protein that allows it to cross cell membranes and enter any human or animal cell. But if no dsRNA is present, it simply does nothing, leaving the cell unharmed.

An interesting question is whether any viruses are actually beneficial and whether wiping all viruses out of an organismal system may have negative consequences (as happens when antibiotic treatment eradicates both invading pathogenic bacteria and non-pathogenic flora, often leading to symptoms such as digestive upset). After his recent presentation at the 6th Strategies for Engineered Negligible Senescence (SENS) conference in September 2013, Dr. Rider fielded this question and stated quite adamantly that there are no known beneficial, symbiotic, or non-harmful viruses. This point is further emphasized in a recently published interview in which he is asked whether DRACO-triggered cell death could lead to a lesion in a tissue or organ. Rider responds that “Virtually all viruses will kill the host cell on the way out. Of the hand-full that don’t, your own immune system will try to kill those infected cells. So we’re not really killing any more cells with our approach than we already have been. It’s just that we’re killing them at an early enough stage before they infect and ultimately kill more cells. So, if anything, this limits the amount of cell death.”

So far, DRACO has been tested in cellular culture and in mouse models against a variety of very different virus types. Rider hopes to license DRACO to a pharmaceutical company so that it can be assessed in larger animal trials and, ultimately, human trials. Unfortunately, it may take a decade or more to complete this process and make the drug available for human therapeutic purposes, and that’s only if there is enough interest to do so. Amazingly, the DRACO project was started over 11 years ago and has barely survived during that period due to lack of interest and funding. Even now, after the DRACOs have been successfully engineered, produced, and tested, no one has yet reached out to Rider about taking them beyond the basic research stage. Let us hope that those of us who do find this work unbelievably exciting can make enough noise that Rider’s work continues to the benefit of all mankind.

Originally published as an article (in the Cooler Minds Prevail series) in Cryonics magazine, November, 2013

26. January 2015 · Comments Off on HIV, Immunosenescence, and Accelerated Aging · Categories: Health, Neuroscience, Science

After a few articles considering Alzheimer disease from several angles, I would like to switch gears this month and talk more generally about the interaction between the immune system and aging.

In his 2012 paper[1], Caleb E. Finch documents the evolution of life expectancy in the course of human history. The life expectancy at birth of our shared ape ancestor 6 millions years ago is hypothesized to approximate that of a chimpanzee, 15 years. The first Homo species appeared 1-2 million years ago and had a life expectancy of ~20 years, while H. sapiens came onto the scene ~100,000 years ago and could expect about 30 years of life. But starting around 200 years ago, concurrent with industrialization, human life expectancy jumped rapidly, to somewhere between 70 and 80 years today.

As many readers are likely aware, the huge recent increases in life expectancy are commonly attributed to improvements in hygiene, nutrition, and medicine during the nineteenth and twentieth centuries that reduced mortality from infections at all ages. Finch hypothesizes, generally, that early age mortality over the course of human history is primarily due to (acute) infection, while old age mortality is primarily due to (chronic) inflammation. Further analysis of mortality rates over the last several hundred years leads him to further hypothesize that aging has been slowed in proportion to the reduced exposure to infections in early life. These hypotheses are supported by twentieth century examples which strongly demonstrate influences of the early life environment on adult health, such as the effects of prenatal and postnatal developmental influences (e.g., nutrition, exposure to infection) on adult chronic metabolic and vascular disorders as well as physical traits and mental characteristics. This leads Finch to suggest “broadening the concept of ‘developmental origins’ to include three groups of factors: nutritional deficits, chronic stress from socioeconomic factors, and direct and indirect damage from infections.”

Finch also considers the effects of inflammation and diet on human evolution, proposing several environmental and foraging factors that may have been important in the genetic basis for evolving lower basal mortality through interactions with chronic inflammation, in particular: dietary fat and caloric content; infections from pathogens ingested from carrion and from exposure to excreta; and noninfectious inflammagens such as those in aerosols and in cooked foods. He hypothesizes that exposure to these proinflammatory factors, which one would expect to shorten life expectancy, actually resulted in humans evolving lower mortality and longer lifespans in response to highly inflammatory environments.

A means for this, he argues, was the development of the apoE4 genotype. Noting that the apoE4 allele favors advantageous fat accumulation and is also associated with enhanced inflammatory responses, Finch argues that heightened inflammatory response and more efficient fat storage would have been adaptive in a pro-inflammatory environment and during times of uncertain nutrition. As has been discussed in prior articles in Cooler Minds Prevail, the apoE alleles also influence diverse chronic non-infectious degenerative diseases and lifespan. “Thus,” Finch concludes, “the apoE allele system has multiple influences relevant to evolution of brain development, metabolic storage, host defense, and longevity.”

With the general relationship between inflammation and the evolution of human aging and life expectancy in mind, let us now consider immune system involvement in more detail, and the relationship between HIV and immunosenescence more specifically.

Immunosenescence refers to the ageassociated deterioration of the immune system. As an organism ages it gradually becomes deficient in its ability to respond to infections and experiences a decline in long-term immune memory. This is due to a number of specific biological changes such as diminished self-renewal capacity of hematopoietic stem cells, a decline in total number of phagocytes, impairment of Natural Killer (NK) and dendritic cells, and a reduction in B-cell population. There is also a decline in the production of new naïve lymphocytes and the functional competence of memory cell populations. As a result, advanced age is associated with increased frequency and severity of pathological health problems as well as an increase in morbidity due to impaired ability to respond to infections, diseases, and disorders.

It is not hard to imagine that an increased viral load leading to chronic inflammatory response may accelerate aging and immunosenescence. Evidence for this is accumulating rapidly since the advent of antiretroviral therapies for treatment of HIV infection. An unforeseen consequence of these successful therapies is that HIV patients are living longer but a striking number of them appear to be getting older faster, particularly showing early signs of dementia usually seen in the elderly. In one study, slightly more than 10% of older patients (avg = 56.7 years) with wellcontrolled HIV infection had cerebrospinal fluid (CSF) marker profiles consistent with Alzheimer disease[2] – more than 10 times the risk prevalence of the general population at the same age. HIV patients are also registering higher rates of insulin resistance and cholesterol imbalances, suffer elevated rates of melanoma and kidney cancers, and seven times the rate of other non-HIV-related cancers. And ultimately, long-term treated HIV-infected individuals also die at an earlier age than HIV-uninfected individuals[3].

Recent research is beginning to explore and unravel the interplay between HIV infection and other environmental factors (such as co-infection with other viruses) in the acceleration of the aging process of the immune system, leading to immunosenescence. In the setting of HIV infection, the immune response is associated with abnormally high levels of activation, leading to a cascade of continued viral spread and cell death, and accelerating the physiologic steps associated with immunosenescence. Despite clear improvements associated with effective antiretroviral therapy, some subjects show persistent alterations in T cell homeostasis, especially constraints on T cell recovery, which are further exacerbated in the setting of co-infection and increasing age.

Unsurprisingly, it has been observed that markers of immunosenescence might predict morbidity and mortality in HIV-infected adults as well as the general population. In both HIV infection and aging, immunosenescence is marked by an increased proportion of CD28- to CD57+, and memory CD8+ T cells with reduced capacity to produce interleukin 2 (IL-2), increased production of interleukin 6 (IL-6), resistance to apoptosis, and shortened telomeres. Levels of markers of inflammation are elevated in HIV infected patients, and elevations in markers such as high-sensitivity C-reactive protein, D-dimer, and interleukin 6 (IL-6) have been associated with increased risk for cardiovascular disease, opportunistic conditions, or all-cause mortality[4].

But even as we are beginning to identify markers that appear to be associated with risk of poor outcome in HIV infection, it is still unclear how patients should be treated on the basis of this information. To that end, several trials are underway to evaluate the effects of modulation of immune activation and inflammation in HIV infection. At the same time, clinicians at the forefront of advancing knowledge and clinical care are performing research aimed at optimizing care for aging HIV patients.

The implications for such research may be far-reaching. In fact, many HIV clinicians and researchers think that HIV may be key to understanding aging in general. Dr. Eric Verdin states, “I think in treated, HIV-infected patients the primary driver of disease is immunological. The study of individuals who are HIV-positive is likely to teach us things that are really new and important, not only about HIV infection, but also about normal aging.”

Dr. Steven Deeks stresses the collaborative efforts of experts across fields. “I think there is a high potential for tremendous progress in understanding HIV if we can assemble a team of experts from the world of HIV immunology and the world of gerontology,” he says. “Each field can dramatically inform the other. I believe HIV is a well described, well studied, distinct disease that can be used as
a model by the larger community to look at issues of aging.”

References

[1] Finch, C (2012). Evolution of the Human Lifespan, Past, Present, and Future: Phases in the Evolution of Human Life Expectancy in Relation to the Inflammatory Load. Proceedings of the American Philosophical Society, 156:1, 9-44.

[2] Mascolini, M (2013). Over 10% in Older HIV Group Fit Alzheimer’s Biomarker Risk Profile. Conference Reports for NATAP: 20th Conference on Retroviruses and Opportunistic Infections, March 3-6, 2013.

[3] Aberg, X (2012). Aging, Inflammation, and HIV Infection. Topics in Antiviral Medicine, 20:3, 101-105.

[4] Deeks, S, Verdin, S. and McCune, JM (2012). Immunosenescence and HIV. Current Opinion in Immunology, 24: 1-6.

Originally published as an article (in the Cooler Minds Prevail series) in Cryonics magazine, June, 2013

23. January 2015 · Comments Off on Apolipoprotein E Genotype and Viral Infections · Categories: Neuroscience, Science

Last month this column considered current and future progress in Alzheimer Disease (AD) diagnosis, management, and treatment. Because AD is a terrible brain disease with an increasing rate of prevalence with age, and because it represents one of – if not the – worst conditions that can afflict a person with cryopreservation arrangements, I would like to continue our consideration of this well-known and widely-feared neurodegenerative disease. Specifically, our focus will be on apolipoprotein E (apoE) and research regarding its role in the modulation of physiological responses to certain viral infections.

ApoE protein is primarily synthesized peripherally in the liver and mediates cholesterol metabolism systemically, but it is also made in the central nervous system by astroglia and microglia (non-neuronal cell types) where it transports cholesterol to neurons. In the CNS, neurons express receptors for apoE that are part of the low density lipoprotein receptor gene family. Historically, apoE has been recognized for its role in lipoprotein metabolism and its importance in cardiovascular disease. Of course, apoE carrier status is also widely known as the major factor determining one’s risk of developing late-onset Alzheimer disease (AD). But more recent research has indicated that the various isoforms of apoE may also have significant immunological impact by conferring different susceptibilities to other diseases, as well.

The human apoE gene is located on chromosome 19 and is composed of 79 individual single nucleotide polymorphisms (SNPs). The three major alleles of apoE, named Epsilon-2 (Ɛ2), Epsilon-3 (Ɛ3), and Epsilon-4 (Ɛ4), are determined by differences in SNPs s429358 and rs7412. The products of these alleles are the protein isoforms apoE2, apoE3, and apoE4, which differ only by a single amino acid at two residues (amino acid 112 and amino acid 158). These amino acid substitutions affect noncovalent “salt bridge” formation within the proteins, which ultimately impacts on lipoprotein preference, stability of the protein, and receptor binding activities of the isoforms (see Table 1).

Isoform Amino acid 112 Amino acid 158 Relative charge Lipoprotein preference LDL receptor binding ability

apoE2

cysteine

cysteine

0

HDL

low

apoE3

cysteine

arginine

+1

HDL

high

apoE4

arginine

arginine

+2

VLDL, chylomicrons

high

Table 1. ApoE isoform amino acid differences and resulting chemical and physiological changes.

There are also two minor alleles, Epsilon-1 (Ɛ1) and Epsilon-5 (Ɛ5), which are present in less than 0.1% of the population. The three major alleles are responsible for three homozygous (Ɛ2/Ɛ2, Ɛ3/Ɛ3, Ɛ4/Ɛ4) and three heterozygous (Ɛ2/Ɛ3, Ɛ2/Ɛ4, Ɛ3/Ɛ4) genotypes. [I will pause to mention here that it is now quite easy to determine one’s genotype through services such as 23andme.com.]

An interesting document in the field is the literature review by Inga Kuhlman, et al. (Lipids in Health and Disease 2010, 9:8) which assesses hepatitis C, HIV and herpes simplex disease risk by ApoE genotype. An important finding is that the Ɛ4 allele is found less frequently in populations as they age (e.g., 14% of the general German population vs. 5% in centenarians), indicating that Ɛ4 is a major mortality factor in the elderly. This is assumed to be a result of the Ɛ4 allele’s well-known predisposition to Alzheimer and cardiovascular diseases.

The authors explain that “apoE4 carriers have a tendency for 5-10% higher fasting total cholesterol, LDL-cholesterol and triglyceride levels relative to homozygote Ɛ3/Ɛ3” and that this tendency towards higher lipid levels is probably responsible for the 40-50% greater cardiovascular disease risk in Ɛ4 carriers. They also point out that “although the molecular basis of the pathology is poorly understood, and likely to be in part due to apoE genotype associated differences in brain lipid metabolism, an apoE4 genotype has been highly consistently associated with the risk of an age-related loss of cognitive function, in an allele dose fashion.” This means, of course, that Ɛ4/Ɛ4 carriers are at greatest risk for cognitive dysfunction with increasing age.

In the field of immune regulation, a growing number of studies point to apoE’s interaction with many immunological processes. In their article, Kuhlman, et al., summarize the impact of the Ɛ4 allele on the susceptibility to specific infectious viral disease. The authors review a number of studies of the effects of apoE4 genotype on hepatitis C (HCV), human immunodeficiency virus (HIV), and herpes simplex (HSV) infection and outcome in humans.

In general, apoE4 was found to be protective against hepatitis C infection vs. (Ɛ3/Ɛ3) controls. Though the exact mechanisms of apoE genotype-specific effects on HCV life cycle remain uncertain, apoE seems to be involved because “available data indicate that the outcome of chronic HCV infection is better among Ɛ4 carriers due to slower fibrosis progression.”

Concerning the possible influence of apoE genotype on HIV infection and HIV-associated dementia, the authors call attention to the fact that “cholesterol is a crucial component of the HIV envelope and essential for viral entry and assembly.” Given that apoE is essential for cholesterol transport, they hypothesize that apoE genotype influences HIV-induced effects on neurological function. Subsequent review of available research suggests that the 4 allele is associated with higher steady-state viral load and faster disease progression due to accelerated virus entry in 4 carriers, but a correlation between apoE4 and HIV-associated dementia “remains controversial and needs to be clarified by further studies.”

Lastly, a review of the literature regarding the effects of apoE4 genotype on herpes simplex virus (HSV)-1 infection and outcome in humans indicates that apoE4 enhances the susceptibility for HSV-1 “as well as the neuroinvasiveness of HSV-1 compared to other apoE variants” (i.e., HSV-1 is found in more frequently in the CNS of 4 carriers). Importantly, the authors also note that “the combination of apoE4 and HSV-1 may lead to a higher risk of Alzheimer disease (AD) than either factor in isolation.”

Due to its generally being associated with higher risk of cardiovascular disease, dementia, and increased susceptibility to and/or accelerated progression of various viral infections, one may wonder why the 4 allele has not been eliminated by evolutionary selection. This may be explained, in part, by the protective and beneficial effects it exhibits in certain harmful infectious diseases, as demonstrated for hepatitis C.

The exact mechanisms of apoE influence on susceptibility to and course of viral infection remain shrouded. Because the mechanisms of HCV, HIV, and HSV infection are quite similar (i.e., all three viruses compete with apoE for cell attachment and receptor binding), it is interesting to find differences in receptor binding among them.

Involvement or interaction between the immune system, cognition, and brain diseases such as AD is an as-yet widely untouched field of inquiry. Further elucidation of the mechanisms by which apoE may influence the pathogenesis of infectious viral diseases can lead to new developments in the treatment of disease based on an individual’s apoE genotype.

Aside from the role that ApoE plays in susceptibility and progression of infectious disease, there is growing interest in the role that infection or a compromised immune system plays in the development of dementia. For example, despite the successful management of HIV with antiretroviral drugs, some patients are showing signs of memory impairment and dementia at a relatively young age. Interestingly, these people seem to show accelerated aging, too, which raises important questions about the relationship between the immune system, immunosenescence, and aging.

Originally published as an article (in the Cooler Minds Prevail series) in Cryonics magazine, May, 2013

29. November 2014 · Comments Off on Deficiencies in the SENS Approach to Rejuvenation · Categories: Health, Science

This article was originally published in Cryonics Magazine, 2011 Issue #1

I am an ardent supporter of Dr. Aubrey de Grey and his work to advance rejuvenation science. The man is priceless and unique in his concepts, brilliance, dedication, organizational abilities, and networking skill. His impact on anti-aging science has been powerful. I have attended all four of the conferences he has organized at Cambridge University in England. For the February 2006 issue of LIFE EXTENSION magazine I interviewed Dr. de Grey, and for the December 2007 issue of LIFE EXTENSION I wrote a review of ENDING AGING, the book he co-authored with Michael Rae.

Dr. de Grey asserts that aging is the result of seven kinds of damage – and that technologies that repair all seven types of damage will result in rejuvenation. His seven-fold program for damage repair is called SENS: “Strategies for Engineered Negligible Senescence”. Dr. de Grey asserts that repairing aging damage is a more effective approach than attempting to slow or prevent aging, and I agree with him. Being an ardent supporter of SENS has not stopped me from simultaneously being a critic of aspects of his program that I think are flawed or deficient. I will attempt to outline some of my criticisms in simple language, assuming that my readers have some knowledge of basic science.

Two SENS strategies cannot justly be described as damage-repair, in my opinion. To protect mitochondrial DNA from free radical damage he wants to make copies of mitochondrial DNA in the nucleus – and import the resulting proteins back into the mitochondria. I would call this an attempt to slow or prevent aging – it cannot be called repair.

Similarly, SENS aims to eliminate cancer by deletion of genes that contribute to cancer, specifically telomerase and ALT (Alternate Lengthening of Telomeres) genes. I am not convinced that this is the best way to eliminate cancer, and I do not believe that deleting cancer-producing genes can properly be called damage-repair.

My criticisms about a procrustean attempt to force two strategies into a model purporting to only be concerned with damage and repair is minor, however, compared to a more fundamental concern that I have that a significant form of aging damage may be being ignored by SENS. I have written a review expressing my concern entitled “Nuclear DNA Damage as a Direct Cause of Aging” that was published in the June 2009 issue of the peer-reviewed journal Rejuvenation Research, [note 1] a journal of which Dr. de Grey is Editor-in-Chief. A PDF of my review is available in the life extension section of my website BENBEST.COM. Those interested in all the citations for claims I will make in this essay are encouraged to read my review. In this essay, I limit my citations to only a few critical articles.

There are many types of DNA damage, but for the purposes of this essay I will focus on breakage of both DNA strands – resulting in a gap in a chromosome. There are two mechanisms for repairing double-strand DNA breaks: Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ). HR usually results in perfect repair, but HR can only operate when cells are dividing. NHEJ is the more frequent form of double-strand break repair, but it is error-prone. NHEJ is the only DNA repair mechanism available for non-dividing cells. Even in cells that divide, 75% of double-strand breaks are repaired by NHEJ. [note 2]

It is hard to believe that it could be a coincidence that the most notorious “accelerated aging” diseases are due to defective DNA repair. The two most prominent of these diseases are Werner’s syndrome (“adult progeria”) and Hutchinson-Gilford syndrome (“childhood progeria”), both of which are caused by defective nuclear DNA repair, mainly HR. In both diseases the “aging phenotype” is apparently due to high levels of apoptosis and cellular senescence. Apoptosis (“cell suicide”) and cellular senescence (cessation of cell division) are both mechanisms that are induced in cells experiencing nuclear DNA damage that the cell is unable to repair. It is not surprising that victims suffering massive depletion of properly functioning cells should exhibit “accelerated aging”. Mice that are genetically altered to show increased apoptosis and cellular senescence also show an “accelerated aging phenotype”.

Elimination of senescent cells and stem-cell replenishment of cells depleted in tissues by this elimination – as well as depleted by apoptosis – are part of SENS. But these strategies are only applicable to cells that divide – not to non-dividing cells such as neurons. Cryonicists are acutely aware that organs – and even whole bodies – can be replaced, but brains (neurons, axons, dendrites, and synapses, particularly) must be preserved if we are not to lose memory and personal identity. The ability of future medicine to replace all organs and tissues other than the brain would render most of SENS unnecessary – except for the brain.

There is considerable evidence of a significant role for DNA damage in brain aging. There are nearly twice as many double-stand nuclear DNA breaks in the cerebral cortex of adult (180 days) rats as in young rats (4 days) – and old (over 780 days) rats have more than twice the double-strand breaks as adult rats. [note 3] Adult rats show a 28% decrease in NHEJ activity in the cerebral cortex neurons compared to neonatal rats – and old rats show a 40% decrease. [note 4] Declining NHEJ activity with age is at least partially due to ATP decline and cellular damage that SENS is intended to fix. But even if NHEJ activity did not decline with age, nuclear DNA damage in neurons will increase at least in part because NHEJ is so error-prone.

Nuclear DNA damage typically leads to mutation or DNA repair – or apoptosis or cellular senescence when DNA repair fails (a mechanism that is believed to have evolved for protection against cancer). But not all DNA damage is repaired, and NHEJ repair is often defective. Accumulating DNA damage and mutation can lead to increasingly dysfunctional cells.

Cancer is due to nuclear DNA damage, mutations, and epimutations. Dr. de Grey has written that “only cancer matters” for mutation and epimutation to nuclear DNA. His mutation terminology does not even acknowledge DNA damage. He has assumed that damaged DNA either is or becomes a mutation. He has assumed that DNA damage that does not become a mutation is either repaired – or leads to apoptosis or cellular senescence.

Dr. de Grey has made the claim that evolution has required such strong defenses against cancer that residual mutation (and, implicitly, DNA damage) is negligible. But cancer incidence increases exponentially with age up to age 80, so it is likely that the residual increases exponentially at the same time.

As recently as the 1980s it was widely believed that normal aging is associated with extensive neuron loss. Now it is established that functional decline in the aging brain is associated with increased neural dysfunction rather than neurodegeneration. [note 5] This neural dysfunction may or may not be mostly due to cellular damage that SENS is intended to fix – including causes of declining NHEJ activity. How much neuron dysfunction associated with aging is due to accumulating mutations or unrepairable nuclear DNA damage is unknown. SENS assumes without proof that nuclear DNA damage and mutation is negligible as a cause of aging (apart from cancer, apoptosis, and cellular senescence). This may be right or it may be wrong. I believe that without definitive proof, nothing should be assumed, and active investigation to determine the facts should not be neglected.

I believe the situation is not hopeless if nuclear DNA damage proves to be a significant cause of brain aging. Future molecular technologies for detection and repair of nuclear DNA damage could be significantly better than natural DNA repair enzymes. And, to simplify the required effort, the DNA repair technologies could be restricted to genes that are actively transcribed in neurons, rather than needing to repair the whole genome.

Notes

1: Best BP. Nuclear DNA damage as a direct cause of aging. Rejuvenation Res. 2009 Jun;12(3):199-208.

2: Mao Z, Bozzella M, Seluanov A, Gorbunova V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst). 2008 Oct 1;7(10):1765-71.

3: Mandaville BS, Rao KS. Neurons in the cerebral cortex are most susceptible to DNA-damage in aging rat brain. Biochem Mol Biol Int 1996 Oct; 40(3):507-14.

4: Vyjayanti VN, Rao KS. DNA double strand break repair in brain: reduced NHEJ activity in aging rat neurons. Neurosci Lett. 2006 Jan 23;393(1):18-22.

5: Morrison JH, Hof PR. Life and death of neurons in the aging brain. Science. 1997 Oct 17;278(5337):412-9.

10. November 2014 · Comments Off on Ancient Brains · Categories: Cryonics, Neuroscience, Science

Cryonics seeks to preserve terminally ill humans in anticipation of future medical advances that may restore these patients to youthful vigor, cure their devastating diseases, and resuscitate them from cryopreservation itself. At the core of this mission lies the goal of preserving that which we know to be most important to continuity of the person him/herself: the brain.

Absent reversible cryopreservation of the brain (i.e., maintenance of viability), a cryonicist’s best hope for eventual resuscitation lies in preserving brain ultrastructure with as much fidelity as possible. Improvements in cryopreservation solutions, methodologies, and protocols from the field to the operating room have greatly enhanced our ability to meet this objective, as evidenced by microscopic evaluations of tissues vitrified in the lab. More recently, CT scans of patients after neuropreservation have provided valuable feedback as to the efficacy of cryoprotective perfusion in actual Alcor cases. Such progress bodes well for good patient outcomes.

But even our greatest attempts at optimal preservation are thwarted by issues such as long ischemic periods resulting in significant perfusion impairment or even the inability to perfuse at all. So how do we evaluate these patients in light of our objective?

Perhaps the best place to start is the extreme. Let us consider, for example, a prehistoric human brain discovered in 2008 at a construction site in York, UK. A paper published in 2011 in the Journal of Archaeological Science (“Exceptional preservation of a prehistoric human brain from Heslington, Yorkshire, UK”) provides gross and histological observations as well as preliminary results of chemical assays in order to determine the extent and cause of preservation of the brain. Low-powered reflected light microscopy and electron microscopy were performed to explore the surviving morphology and histology of the brain, while highly sensitive neuroimmunological techniques and proteomic analyses were employed to explore brain chemistry.

Examination of the skull indicated death by an abrupt trauma to the neck followed by deliberate dismemberment of the head between veretebrae C2 and C3. Significantly, the authors report “no trace of microbial activity, bacterial or fungal, with none of the porosity or ‘tunneling’ that is characteristic of putrefactive microorganisms.” Examination of the brain masses revealed recognizable sulci and gyri, but neither macroscopic nor CT evaluation could differentiate between grey and white matter.

Histological examination of the brain masses showed “a homogenous, amorphous substance that had not retained any cellular or matrix structure.” Transmission electronic microscopy (TEM) also did not detect any surviving cellular structure, although it did reveal what appeared to be “numerous morphologically degraded structures characteristic of the myelin sheath of nerve fibres.”

Preliminary biomolecular analysis found only 5% of the brain was detectable as hydrolysable amino acids, in contrast to fresh brain tissue of which proteins represent more than 1/3 of dry weight. When compared with a fresh brain, the Heslington brain was also depleted in polar amino acids and enriched in hydrophobic amino acids. Very little undegraded solventsoluble brain lipid was preserved (0.8%- 1.1% wet weight compared with 17.1% for rat brain). In addition, there was an almost complete absence of phospholipids and only a trace of cholesterol, while degradation products of a wide range of lipids were found in abundance.

Ultimately, the authors determined that the preservation of this brain was due to decapitation (thus eliminating the movement of putrefying bacteria from the gut to the brain) followed by inhibition of postmortem putrefaction achieved through rapid burial into fine-grained wet sediment. They go on to argue that this type of preservation is not as unusual as one might think, citing several similar examples of preserved prehistoric human brains, almost always found in wet burial environments.

While interesting in its own right, few would argue that the Heslington brain represents a state of preservation amenable to resuscitation. The ability to infer anything beyond gross macro structure has been obliterated and the normal chemical constituents of the brain have dissolved almost completely into the surrounding environment. Clearly, much of the look of a brain can be retained while none of the person’s identity remains (or is recoverable).

Let us then look at a situation that hits a little closer to home. Published in Forensic Science International in 2007, an article entitled “Autopsy at 2 months after death: Brain is satisfactorily preserved for neuropathology” provides us with considerable food for thought. In this example, a 77-year-old woman’s whole body was stored postmortem in a 3°C cooling chamber for 2 months prior to chemical fixation of her brain at autopsy.

The authors describe moderate autolysis of internal organs of the body, indicating the start of decomposition and putrefaction, as well as reduced tissue consistency and superficial areas of disintegration of the brain. Overall gross morphology was sufficiently preserved to allow macroscopic examination and application of neuropathological methods for diagnosis of neurological disorders. Importantly, they also report that “histologically, normal brain structures including all major parenchymal cell types (neurons, astrocytes, oligodendrocytes, microglia), neuropil, axons, and myelin sheaths were preserved.”

In this case, the use of cold temperatures (3°C) drastically slowed, but did not stop, deterioration of the brain. However, enough of the brain’s chemical constituents and physical structure remained to provide the basis for possible future resuscitation. And while this woman’s brain was preserved by chemical diffusion over the course of 9 weeks (allowing for continued degradation of subcortical tissues during the course of fixation), the use of cryogenic temperatures to quickly preserve her brain would also have been possible, as has been the situation for many “straight frozen” Alcor patients who were received in similar condition.

Exactly where the line between recoverability and non-recoverability — resulting in information-theoretic death — exists is yet to be determined. And while we push, rightfully, for ever greater preservation methods, we do well to remember that those preserved under lessthan- optimal conditions are by no means lost causes. Preserved information, even in fractured and distorted form, may well be adequate to infer the original state.

Originally published as an article (in the Cooler Minds Prevail series) in Cryonics magazine, March, 2013

20. October 2014 · Comments Off on Consciousness, Natural Selection, and Knowledge · Categories: Neuroscience, Science

Cryonics Magazine, February 2013

This is the first entry in a new series of short articles about neuroscience and its implications for the field of human cryopreservation and life extension. In this article I discuss the relationship of the brain to consciousness and knowledge acquisition before venturing into more specific and practical topics

What is consciousness? Most of us understand the word in context, but when asked to define it we are suddenly at a loss for words or at best we offer a description that seems wholly inadequate. Scientists, philosophers, and religious scholars have debated the source, meaning, and nature of consciousness for all of recorded history. But with the rise of neuroscience over the past few decades, it now seems as though explaining the nature and mechanisms of conscious experience in neurobiological terms may be an attainable goal.

The recent work on consciousness by neuroscientists has left certain philosophers more frustrated than ever before, including the likes of Thomas Nagel and David Chalmers. They suspect that consciousness may be quite different and separate from the brain circuitry proposed to underlie it.

Consciousness has appeared to be a strange and undefinable phenomenon for a very long time. Daniel Dennett captured the feeling very nicely in the 1970s:

“Consciousness appears to be the last bastion of occult properties, epiphenomena, immeasurable subjective states — in short, the one area of mind best left to the philosophers. Let them make fools of themselves trying to corral the quicksilver of “phenomenology” into a respectable theory.”(1)

Consciousness no longer appears this strange to many researchers, but the philosophers just mentioned continue to hold that it may not be reduced to brain processes active in cognition. A common philosophical complaint is that any neurobiological theory of consciousness will always leave something out. What it will always leave out is the feeling itself — the feeling of what it is like to be aware, to see green, to smell flowers, and so on (Nagel 1974; Chalmers, 1996). These are so-called qualia — the experiences themselves — and these are what are important about consciousness. The philosopher making this argument may go on to conclude that no science can ever really explain qualia because it cannot demonstrate what it is like to see green if you have never seen green. Ultimately, they argue, consciousness is beyond the reach of scientific understanding.

By contrast, neuroscientists take for granted that consciousness will be domesticated along with the rest of cognition. Indeed, this work tends to assume that neuroscience will not only identify correlates of consciousness, but will eventually tell us what consciousness is. By and large, these neuroscientific efforts have been directed toward cortical regions of the brain, cortical pathways, and cortical activity. This is due, in part, to the prevalence of clinical studies of human patients with region-specific cortical lesions that are correlated with deficits in specific kinds of experiences. This tendency to focus on the cortex may also reflect the common knowledge that humans possess the highest level of consciousness of all animals and have proportionally more cortex than our closest relatives (and — so the supposition goes — therein lies the difference in levels of consciousness).

Another theory of consciousness, offered by Dr. Gerald M. Edelman, aims to resolve this “divorce” between science and the humanities over theories of consciousness. The premise of Edelman’s theory is that the field of neuroscience has already provided enough information about how the brain works to support a scientifically plausible understanding of consciousness. His theory attempts to reconcile the two positions described earlier by examining how consciousness arose in the course of evolution.

In his book on the topic, Second Nature: Brain Science and Human Knowledge, Edelman says:

“An examination of the biological bases of consciousness reveals it to be based in a selectional system. This provides the grounds for understanding the complexity, the irreversibility, and the historical contingency of our phenomenal experience. These properties, which affect how we know, rule out an all-inclusive reduction to scientific description of certain products of our mental life such as art and ethics. But this does not mean that we have to invoke strange physical states, dualism, or panpsychism to explain the origin of conscious qualia. All of our mental life, reducible and irreducible, is based on the structure and dynamics of our brain.

In essence, Edelman has attempted to construct a comprehensive theory of consciousness that is consistent with the latest available neuroanatomical, neurophysiological, and behavioral data. Calling his idea Neural Darwinism, Edelman explains that the brain is a selection system that operates within an individual’s lifetime. Neural Darwinism proposes that, during neurogenesis, an enormous “primary repertoire” of physically connected populations of neurons arises. Subsequently, a “secondary repertoire” of functionally defined neuronal groups emerges as the animal experiences the world. A neural “value system,” developed over the course of evolution and believed to be made up of small populations of neurons within deep subcortical structures, is proposed to assign salience to particular stimuli encountered by the animal in order to select patterns of activity.

For example, when the response to a given stimulus leads to a positive outcome the value system will reinforce the synaptic connections between neurons that happened to be firing at that particular moment. When a stimulus is noxious, the value system will similarly strengthen the connections between neurons that happened to be firing at the time the stimulus was encountered, thus increasing the salience of that stimulus. When a stimulus has no salience, synaptic connections between neurons that fired upon first exposure to that stimulus will become weaker with successive exposures.

Importantly, the mapping of the world to the neural substrate is degenerate; that is, no two neuronal groups or maps are the same, either structurally or functionally. These maps are dynamic, and their borders shift with experience. And finally, since each individual has a unique history, no two individuals will express the same neural mappings of the world.

This brings us to the three tenets of Edelman’s theory:

1. Development of neural circuits leads to enormous microscopic anatomical variation that is the result of a process of continual selection;

2. An additional and overlapping set of selective events occurs when the repertoire of anatomical circuits that are formed receives signals because of an animal’s behavior or experience;

3. “Reentry” is the continual signaling from one brain region (or map) to another and back again across massively parallel fibers (axons) that are known to be omnipresent in higher brains.

Edelman thus believes that consciousness is entailed by reentrant activity among cortical areas and the thalamus and by the cortex interacting with itself and with subcortical structures. He suggests that primary consciousness appeared at a time when the thalmocortical system was greatly enlarged, accompanied by an increase in the number of specific thalamic nuclei and by enlargement of the cerebral cortex — probably after the transitions from reptiles to birds and separately to mammals about a quarter of a billion years ago. Higherorder consciousness (i.e., consciousness of consciousness), on the other hand, is due to reentrant connections between conceptual maps of the brain and those areas of the brain capable of symbolic or semantic reference — and it only fully flowered with hominids when true language appeared. Regarding language and its relationship to higher-order consciousness, Edelman explains:

“We do not inherit a language of thought. Instead, concepts are developed from the brain’s mapping of its own perceptual maps. Ultimately, therefore, concepts are initially about the world. Thought itself is based on brain events resulting from the activity of motor regions, activity that does not get conveyed to produce action. It is a premise of brain-based epistemology that subcortical structures such as the basal ganglia are critical in assuring the sequence of such brain events, yielding a kind of presyntax. So thought can occur in the absence of language….

The view of brain-based epistemology is that, after the evolution of a bipedal posture, of a supralaryngeal space, of presyntax for movement in the basal ganglia, and of an enlarged cerebral cortex, language arose as an invention. The theory rejects the notion of a brainbased, genetically inherited, language acquisition device. Instead, it contends that language acquisition is epigenetic. Its acquisition and its spread across speech communities would obviously favor its possessors over nonlinguistic hominids even though no direct inheritance of a universal grammar is at issue. Of course, hominids using language could then be further favored by natural selection acting on those systems of learning that favor language skills.”

Such a theory is attractive because it does not simply concentrate on conscious perception, but it also includes the role of behavior. We do well to keep in mind that moving, planning, deciding, executing plans, and more generally, keeping the body alive, is the fundamental business of the brain. Cognition and consciousness are what they are, and have the nature they have, because of their role in servicing behavior.

An important element of Edelman’s theory that consciousness is entailed by brain activity is that consciousness is not a “thing” or causal agent that does anything in the brain. He writes that “inasmuch as consciousness is a process entailed by neural activity in the reentrant dynamic core it cannot be itself causal.” This process causes a number of “useful” illusions such as “free will.”

Edelman’s theory of consciousness has further implications for the development of brain-based devices (BBDs), which Edelman believes will be conscious in the future as well. His central idea is that the overall structure and dynamics of a BBD, whether conscious or not, must resemble those of real brains in order to function. Unlike robots executing a defined program, the brains of such devices are built to have neuroanatomical structures and neuronal dynamics modeled on those known to have arisen during animal evolution and development.

Such devices currently exist — such as the “Darwin” device under development by The Neurosciences Institute. Darwin devices are situated in environments that allow them to make movements to sample various signal sequences and consequently develop perceptual categories and build appropriate memory systems in response to their experiences in the real world.

And though Edelman recognizes that it is currently not possible to reflect the degree of complexity of the thalmocortical system interacting with a basal ganglia system, much less to have it develop a true language with syntax as well as semantics, he nevertheless suggests that someday a conscious device could probably be built.

More ambitiously, Edelman also thinks that contemporary neuroscience can contribute to a naturalized epistemology. The term “naturalized epistemology” goes back to the analytical philosopher Willard Quine and refers to a movement away from the “justification” (or foundations) of knowledge and emphasizes the empirical processes of knowledge acquisition. Edelman is largely sympathetic towards Quine’s project, but provides a broader evolutionary framework to epistemology that also permits internal states of mind (consciousness).

1 Daniel C. Dennett, “Toward a Cognitive Theory of Consciousness,” in Brainstorms: Philosophical Essays on Mind and Psychology (Montgomery, VT: Bradford Books, 1978).