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Oral Microbiome Imbalances Could Provide Early Warning of Disease

June 18, 2021

Image caption: Fragments of amyloid precursor protein aggregate in β-amyloid plaques, seen here in dark brown. These plaques have been found in the brains of patients with Alzheimer’s disease. Credit: Wikimedia Commons.

 

By Daniel Erenstein, Neurobiology, Physiology & Behavior ‘21

Author’s Note: I first learned about research on the oral microbiome while covering this year’s annual meeting of the American Association for the Advancement of Science in February. Under the theme of “Understanding Dynamic Ecosystems,” the conference, which was held virtually, welcomed scientists, journalists, students, and science enthusiasts for four days of sessions, workshops, and other talks. The human microbiome, home to trillions of bacteria and other microbes, is as dynamic an ecosystem as they come. This article focuses on the bacteria that live in our mouths and their fascinating role in diseases such as diabetes and chronic kidney disease. With this article, I hope that readers consider further reading on the diverse, lively ecosystems within our bodies. For more stories on the microbiome, The Aggie Transcript is a great place to start.

 

There is more to the oral microbiome than meets the mouth. Established within a few minutes of birth, this diverse community of bacteria, fungi, and other microbes lives on every surface of our mouths throughout our lives [1]. For decades, scientists have researched these microorganisms and their role in dental diseases.

But far less is known about the interactions of these bacteria and their products with other parts of the body, and these interactions could hold a particularly important role in human health.

“We’ve always thought of the mouth as somehow in isolation, that oral health does not somehow impact the rest of the body,” said Purnima Kumar, DDS, MS, PhD, professor of periodontology at Ohio State University, during a session at the American Association for the Advancement of Science annual meeting that took place on February 8 [2].

Scientists, though, are increasingly looking to the oral microbiome for answers to questions about health and disease [3].

“The time is absolutely right for us to start putting the mouth back into the body,” Kumar said during the session “Killer Smile: The Link Between the Oral Microbiome and Systemic Disease.”

Panelists highlighted three systemic diseases — diabetes mellitus, rheumatoid arthritis, and Alzheimer’s disease — and their unexpected connection to disturbances in the oral microbiome [4-6]. A common thread running through all three diseases is an association with periodontitis, a gum disease triggered by the accumulation of bacteria, viruses, and even fungi in dental biofilm, or plaque, on the surface of teeth [7].

In health, there is peace between oral microbes and our immune system. Healthy and frequent communication — molecular diplomacy between bacteria and immune surveillance — maintains stable relations. But microbial imbalances due to bacterial buildup in plaque can cause inflammatory immune reactions, resulting in the gradual breakdown of the barrier between biofilm and gum tissue.

“When you have gum disease, that crosstalk, that communication, that harmony is broken down,” Kumar said.

When bacteria subsequently invade our gum tissues, there are consequences for human disease [8].

Mark Ryder, DMD, professor of periodontology at University of California, San Francisco, studies the role of one such bacterium, Porphyromonas gingivalis, in disease [9]. This bacterium secretes enzymes called gingipains, which are essential for its survival.

In the case of Alzheimer’s disease, these gingipains can travel through the bloodstream, cross the blood-brain barrier, and accumulate in brain regions like the hippocampus, which is involved in memory. There, they help break down an embedded membrane protein called amyloid precursor protein into fragments, which group together in deposits found in people with Alzheimer’s disease.

Further study of gingipains and other microbial products could provide insight into a “critical early event in the initiation and progression of Alzheimer’s disease,” Ryder said.

Similarly, rheumatoid arthritis can be triggered by immune responses to other by-products of P. gingivalis, including protein antibodies that cause joint inflammation, according to Iain Chapple, BDS, PhD, professor of periodontology at the University of Birmingham [5].

Past research on links between the oral microbiome and systemic disease has even shown that these effects can travel a two-way street.

This is apparent in the research of Dana Graves, DDS, DMSc, professor of periodontics at University of Pennsylvania, whose work has examined the effects of diabetes on our microbiome, and vice versa [4].

“Diabetes impacts the mouth in a very profound way,” said Graves, adding that the inflammatory responses to bacteria caused by diabetes lead to disruption of the microbiome.

“This bidirectionality is something we saw first with diabetes, we’ve seen it now with rheumatoid arthritis, and it appears now that we’re starting to see it with chronic kidney disease,” Chapple said. “We need to start really digging down into [biological mechanisms] to understand more about that relationship.”

However, the verdict is still out on how much bacterial products such as gingipains contribute to disease. For Ryder and others, the existing data is insufficient — fully answering that question depends on carefully constructed clinical trials.

“When we’re trying to establish a link between something like the microbiome and the mouth and Alzheimer’s, association studies are important, the actual underlying biological mechanisms are important, but finally what sort of seals the deal, of course, is the actual effects of intervention,” Ryder said.

An ongoing clinical trial with more than 600 patients is evaluating the success of gingipain inhibitors in preventing symptoms of Alzheimer’s disease [10]. The results, expected by the end of this year, could carry implications not just for the treatment of Alzheimer’s disease but any disease with underlying roots in the oral microbiome.

Regardless of the results, it’s clear that breaking out the toothbrush and floss every day is crucial to our overall well-being.

 

References:

  1. Ursell LK, Metcalf JL, Parfrey LW, Knight R. 2012. Defining the human microbiome. Nutr Rev. 70 Suppl 1: S38-S44. https://doi.org/10.1111/j.1753-4887.2012.00493.x.
  2. Kumar P, D’Souza R, Shaddox L, Burne RA, Ebersole J, Graves D, Ryder MI, Chapple I. 2021. Killer Smile: The Link Between the Oral Microbiome and Systemic Disease [Conference presentation]. AAAS Annual Meeting [held virtually]. https://aaas.confex.com/aaas/2021/meetingapp.cgi/Session/27521.
  3. Deo PN, Deshmukh R. 2019. Oral microbiome: Unveiling the fundamentals. J Oral Maxillofac Pathol. 23(1): 122-128. doi:10.4103/jomfp.JOMFP_304_18.
  4. Graves DT, Ding Z, Yang Y. 2020. The impact of diabetes on periodontal diseases. Periodontol 2000. 82(1): 214-224. https://doi.org/10.1111/prd.12318.
  5. Lopez-Oliva I, Paropkari AD, Saraswat S, Serban S, Yonel Z, Sharma P, de Pablo P, Raza K, Filer A, Chapple I, et al. 2018. Dysbiotic subgingival microbial communities in periodontally healthy patients with rheumatoid arthritis. Arthritis Rheumatol. 70(7): 1008-1013. https://doi.org/10.1002/art.40485.
  6. Dioguardi M, Crincoli V, Laino L, Alovisi M, Sovereto D, Mastrangelo F, Lo Russo L, Lo Muzio L. 2020. The Role of Periodontitis and Periodontal Bacteria in the Onset and Progression of Alzheimer’s Disease: A Systematic Review. J Clin Med. 9(2): 495. https://doi.org/10.3390/jcm9020495.
  7. Arigbede AO, Babatope BO, Bamidele MK. 2012. Periodontitis and systemic diseases: A literature review. J Indian Soc Periodontol. 16(4): 487-491. https://doi.org/10.4103/0972-124X.106878.
  8. Curtis MA, Diaz PI, Van Dyke TE. 2020. The role of the microbiota in periodontal disease. Periodontol 2000. 83(1): 14-25. https://doi.org/10.1111/prd.12296.
  9. Ryder MI. 2020. Porphyromonas gingivalis and Alzheimer disease: Recent findings and potential therapies. J Periodontol. 91 Suppl 1: S45-S49. https://doi.org/10.1002/JPER.20-0104.
  10. Cortexyme Inc. 2021. GAIN Trial: Phase 2/3 Study of COR388 in Subjects With Alzheimer’s Disease. ClinicalTrials.Gov. Identifier NCT03823404. https://clinicaltrials.gov/ct2/show/NCT03823404.

The Human-Animal Interface: Exploring the Origin, Present, and Future of COVID-19

June 16, 2021

By Tammie Tam, Microbiology ‘22

Author’s Note: Since taking the class One Health Fundamentals (PMI 129Y), I have been acutely aware of this One Health idea that the health of humankind is deeply intertwined with the health of animals and our planet. This COVID-19 pandemic has been a perfect model as a One Health issue. Through this article, I hope to introduce readers to a fuller perspective of COVID-19 as a zoonotic disease. 

 

The COVID-19 pandemic has escalated into a human tragedy, measured daily by an increasing number of infection cases and a piling death toll. Yet, to understand the current and future risks of the SARS-CoV-2 virus, one must account for the virus’s relationship with animals in the context of its zoonotic nature, as the transmission between animals and humans is often overlooked. Uncovering the range of intermediary hosts of the virus may provide clues to the virus’s origin, point to potential reservoirs for a mutating virus, and help inform future public health policies. As a result, a small but growing body of researchers is working to predict and confirm potential human-animal transmission models.

The origin of the SARS-CoV-2

Currently, the World Health Organization (WHO) and other disease detectives are still working to unravel the complete origin of the virus. Scientists have narrowed down the primary animal reservoir for the virus through viral genomic analysis, between strains of human and animal coronaviruses [1]. They suspect bats to be the most likely primary source of the virus because the SARS-CoV-2 strain is a 96.2 percent match for a bat coronavirus, bat-nCoV RaTG13 [1]. Despite the close match, the differences in key surface proteins between the two viruses are distinct enough to suggest that the bat coronavirus had to have undergone mutations through one or more intermediary hosts in order to infect humans [2]. 

To identify potential intermediate hosts, scientists are examining coronaviruses specific to different animal species [1]. If SARS-CoV-2 is genetically similar to another animal-specific coronavirus, SARS-CoV-2 may also possess similar viral proteins to the animal-specific coronaviruses. With similar proteins, similar host-virus interactions can theoretically take place, allowing for SARS-CoV-2 to infect the animal in question. For example, besides bats, a pangolin coronavirus, pangolin-nCoV, has the second highest genetic similarity to SARS-CoV-2, which positions the pangolin as a possible intermediate host [3]. Because of the similarity, viral proteins of the pangolin coronavirus can interact with shared key host proteins in humans just as strongly as in pangolin [4]. However, more epidemiological research is needed to determine whether a pangolin had contracted coronavirus from a human or a human had contracted coronavirus from a pangolin. Alternatively, the intermediate host could have been another animal, but there are still no clear leads [1]. 

What it takes to be a host for SARS-CoV-2

In any viable host, the SARS-CoV-2 virus operates by sneaking past immune defenses, forcing its way into cells, and co-opting the cell’s machinery for viral replication [5]. Along the way, the virus may acquire mutations—some deadly and some harmless. Eventually, the virus has propagated in a high enough quantity to jump from its current host to the next [5].

Most importantly for the virus to infect a host properly, the virus must recognize the entranceway into cells quickly enough before the host immune system catches on to the intruder and mounts an attack [5]. SARS-CoV-2’s key into the cell is through its spike glycoproteins found on the outer envelope of the virus. Once the spike glycoproteins interact with an appropriate angiotensin-converting enzyme 2 (ACE2) receptor found on the host cell surfaces, the virus blocks the regular functions of the ACE2 receptor, such as regulating blood pressure and local inflammation [6,7]. At the same time, the interaction triggers the cell to take in the virus [5]. 

Since the gene encoding for the ACE2 receptor is relatively similar among humans, the virus can travel and infect the human population easily. Likewise, most animals closely related to humans like great apes possess a similar ACE2 receptor in terms of structure and function, which allows SARS-CoV-2 a path to hijack the cells of certain non-human animals [8]. Despite the overall similar structure and function, the ACE2 receptor varies between animal species at key interaction sites with the spike glycoproteins due to natural mutations that are kept to make the ACE2 receptor the most efficient in the respective animal. Thus, while there are other proteins involved in viral entry into the host cells, the ACE2 receptor is the one that varies between animals and most likely modulates susceptibility to COVID-19 [9]. 

As a result, scientists are particularly interested in the binding of the ACE2 receptor with the viral spike glycoprotein because of its implications for an organism’s susceptibility to COVID-19. Dr. Xuesen Zhao and their team from Capital Medical University examined the sequence identities and interaction patterns of the binding site between ACE2 receptors of different animals and the spike glycoproteins of the SARS-CoV-2 [10]. They reasoned that the more similar the ACE2 receptor of an animal is to humans, the more likely the virus could infect the animal. For example, they found ACE2 receptors of rhesus monkeys, a closely related primate, had similar interaction patterns as humans [10]. Meanwhile, they found rats and mice to have dissimilar ACE2 receptors and poor viral entry [10].

While entrance into the cell is a major part of infection, there are other factors to also consider, such as the ability for viral replication to subsequently take place [11]. With so many different organisms on the planet, the models simply provide a direction for where to look next. SARS-CoV-2 is unable to replicate efficiently in certain animals despite having the entrance key to get in. For example, the virus is able to replicate well in ferrets and cats, making them susceptible to the virus [12]. In dogs, the virus can only weakly replicate. Meanwhile in pigs, chickens, and ducks, the virus is unable to replicate [12]. Outside of the laboratory, confirmed cases in animals include farm animals such as minks; zoo animals such as gorillas, tigers, lions, and snow leopards; and domestic animals such as cats and dogs [13].

The future for SARS-CoV-2

Due to the multitude of intermediary hosts, COVID-19 is unlikely to disappear for good even if every person is vaccinated [14]. Viral spillover from human to animal can spill back to humans. Often, as the virus travels through a new animal population, the virus population will be subjected to slightly different pressures and selected for mutations that will confer a favorable advantage for virus survival and transmission within the current host population [15]. Sometimes, this could make the virus weaker in humans. However, there are times when the virus becomes more virulent and dangerous to humans if it spills back over from the animal reservoir [15]. Consequently, it is important to understand the full range of hosts in order to put in place preventative measures against viral spillover. 

As of now, most of the known susceptible animals usually do not get severely sick with some known exceptions like minks [1]. Nevertheless, people must take precautions when interacting with animals, since research into this area is still developing and there are many unknown factors involved. This is especially important for endangered species to not become sick, because they already face other threats that make them vulnerable to extinction [8]. As a result, some researchers are taking it into their own hands to keep certain animals safe. For example, after the San Diego Zoo’s resident gorillas contracted COVID-19 in January, the zoo proactively began using the experimental Zoetis vaccine to vaccinate their orangutans and bonobos, which are great apes that are considered closely related to humans and susceptible to COVID-19 [16]. Due to an assumed COVID-19 immunity in the gorillas and a limited supply of the Zoetis vaccines, they decided to not vaccinate the gorillas [16]. Now, scientists are trying to modify the Zoetis vaccine for minks, because minks are very susceptible to severe symptoms from COVID-19 and have shown to be able to transmit the virus back to humans [17]. 

Besides the virus mutating into different variants through basic genetic mutations, people must be cautious of potential new coronaviruses which can infect humans [18]. The human population has encountered other novel coronaviruses over the past several years, so it is not out of the question. In animals, if two coronaviruses of a human and an animal infect the same animal host, it could cause a recombination event and create a new hybrid coronavirus [19]. 

For the SARS-CoV-2 virus, Dr. Maya Wardeh and their team at the University of Liverpool found over 100 possible host species where recombination events could take place [18]. These hosts are animals who can contract two or more coronaviruses with one of them being the SARS-CoV-2 virus. For instance, the lesser Asiatic yellow bat, a well-known host of several coronaviruses, is predicted to be one of these recombination hosts [18]. Also, species closer to home such as the domestic cat is another possible recombination host [18]. While it will take many different rare events, from co-infection to human interaction with the particular animal for recombination to be possible, scientists are on the lookout.

Even without a full picture, the Center for Disease Control (CDC) understands the potential risks of animal reservoirs and advises COVID-19-infected patients to stay away from animals—wildlife or domestic—to prevent spillover [20]. COVID-19 has also brought to light zoonotic disease risks from illegal animal trades and wet markets. Once research into the human-animal transmission model becomes more well-developed, public health officials will have a clearer picture as to how the pandemic spiraled to its current state and help develop policies to prevent it from happening again. 

 

References: 

  1. Zhao J, Cui W, Tian BP. 2020. The Potential Intermediate Hosts for SARS-CoV-2. Frontiers in Microbiology 11 (September): 580137. https://doi.org/10.3389/fmicb.2020.580137. 
  2. Friend T, Stebbing J. 2021. What Is the Intermediate Host Species of SARS-CoV-2? Future Virology 16 (3): 153–56. https://doi.org/10.2217/fvl-2020-0390.
  3. Lam TT, Jia N,  Zhang YW, Shum MH, Jiang JF,  Zhu HC,  Tong YG, et al. 2020. Identifying SARS-CoV-2-Related Coronaviruses in Malayan Pangolins. Nature 583 (7815): 282–85. https://doi.org/10.1038/s41586-020-2169-0. 
  4. Wrobel AG, Benton DJ, Xu P, Calder LJ, Borg A, Roustan C, Martin SR, Rosenthal PB, Skehel JJ, Gamblin SJ. 2021. Structure and Binding Properties of Pangolin-CoV Spike Glycoprotein Inform the Evolution of SARS-CoV-2. Nature Communications 12 (1): 837. https://doi.org/10.1038/s41467-021-21006-9. 
  5. Harrison AG, Lin T, Wang P. 2020. Mechanisms of SARS-CoV-2 Transmission and Pathogenesis. Trends in Immunology 41 (12): 1100–1115. https://doi.org/10.1016/j.it.2020.10.004. 
  6. Hamming I, Cooper ME, Haagmans BL, Hooper NM,Korstanje R, Osterhaus  ADME, Timens  W, Turner  AJ, Navis G, van Goor H. 2007. The Emerging Role of ACE2 in Physiology and Disease. The Journal of Pathology 212 (1): 1–11. https://doi.org/10.1002/path.2162.
  7. Sriram K, Insel PA. 2020. A Hypothesis for Pathobiology and Treatment of COVID‐19 : The Centrality of ACE1 / ACE2 Imbalance. British Journal of Pharmacology 177 (21): 4825–44. https://doi.org/10.1111/bph.15082. 
  8. Melin AD, Janiak MC, Marrone F, Arora PS, Higham JP. 2020. Comparative ACE2 Variation and Primate COVID-19 Risk. Communications Biology 3 (1): 641. https://doi.org/10.1038/s42003-020-01370-w. 
  9. Brooke GN, Prischi F. 2020. Structural and Functional Modelling of SARS-CoV-2 Entry in Animal Models. Scientific Reports 10 (1): 15917. https://doi.org/10.1038/s41598-020-72528-z.
  10. Zhao X, Chen D, Szabla R, Zheng M, Li G, Du P, Zheng S, et al. 2020. Broad and Differential Animal Angiotensin-Converting Enzyme 2 Receptor Usage by SARS-CoV-2. Journal of Virology 94 (18). https://doi.org/10.1128/JVI.00940-20.
  11. Manjarrez-Zavala MA, Rosete-Olvera DP, Gutiérrez-González LH, Ocadiz-Delgado R, Cabello-Gutiérrez C. 2013. Pathogenesis of Viral Respiratory Infection. IntechOpen. https://doi.org/10.5772/54287. 
  12. Shi J, Wen Z, Zhong G, Yang H, Wang C, Huang B, Liu R, et al. 2020. Susceptibility of Ferrets, Cats, Dogs, and Other Domesticated Animals to SARS–Coronavirus 2. Science 368 (6494): 1016–20. https://doi.org/10.1126/science.abb7015.
  13. Quammen D. And Then the Gorillas Started Coughing. The New York Times. Accessed February 19, 2021. Available from: https://www.nytimes.com/2021/02/19/opinion/covid-symptoms-gorillas.html. 
  14. Phillips N. 2021. The Coronavirus Is Here to Stay — Here’s What That Means. Nature 590 (7846): 382–84. https://doi.org/10.1038/d41586-021-00396-2. 
  15. Geoghegan JL, Holmes EC. 2018. The Phylogenomics of Evolving Virus Virulence. Nature Reviews Genetics 19 (12): 756–69. https://doi.org/10.1038/s41576-018-0055-5. 
  16. Chan S, Andrew S. 2021. Great Apes at the San Diego Zoo Receive a Covid-19 Vaccine for Animals. CNN. Accessed March 5, 2021. Available from: https://www.cnn.com/2021/03/05/us/great-apes-coronavirus-vaccine-san-diego-zoo-trnd/index.html. 
  17. Greenfield P. 2021. Covid Vaccine Used on Apes at San Diego Zoo Trialled on Mink. The Guardian.Accessed March 23, 2021. Available from: http://www.theguardian.com/environment/2021/mar/23/covid-vaccine-used-great-apes-san-diego-zoo-trialled-mink. 
  18. Wardeh M, Baylis M, Blagrove MSC. 2021. Predicting Mammalian Hosts in Which Novel Coronaviruses Can Be Generated. Nature Communications 12 (1): 780. https://doi.org/10.1038/s41467-021-21034-5.
  19. Pérez-Losada M, Arenas M, Galán JC, Palero F, González-Candelas F. 2015. Recombination in Viruses: Mechanisms, Methods of Study, and Evolutionary Consequences. Infection, Genetics and Evolution 30 (March): 296–307. https://doi.org/10.1016/j.meegid.2014.12.022.
  20. Centers for Disease Control and Prevention. 2020. COVID-19 and Your Health. Accessed February 11, 2020. Available from: https://www.cdc.gov/coronavirus/2019-ncov/daily-life-coping/animals.html.

Human Cryopreservation: An Opportunity for Rejuvenation

June 15, 2021

By Barry Nguyen, Biochemistry & Molecular Biology ‘23

Author’s Note: I became interested in ways to bypass built-in lifespans after taking HDE 117, a longevity class with Dr. James Carey. During the course of the class, I was exposed to many different ways to extend the human lifespan. However, I was most interested in cryogenics and its prospects of human rejuvenation, prompting me to explore the possibilities of human cryopreservation. 

 

Summary

This paper is focused on exploring the prospects of human cryopreservation. The first section discusses cryogenics and its relevance in the discussion of human cryopreservation. The following section utilizes empirical modeling to support the relationship between temperature and reaction rates. Next, the paper discusses the cryopreservation procedure itself and explores how the definition of death can be reimagined. We then transition to discussing cryopreservation’s possibility for rejuvenation. Specifically, we redefine the definition of aging itself and discuss aging phenomena on the molecular scale and use both of these as a basis for the discussion of immortality. The succeeding section is concerned with the limitations of human cryopreservation. Finally, the paper concludes with a brief discussion of the possible future of cryogenic technology. 

Cryogenics

Cryogenics is a field of study focused on material behaviors at very low temperatures, ranging from -150°C to -273°C. At these extremely low temperatures, chemical properties are altered, and molecular interactions are halted [1]. By halted, it is not correct to say that all molecular interactions have been stopped. Rather, the molecular interactions have come as close to theoretically possible to ceasing and are at the lowest possible energy state. At these temperatures, chemical properties are also altered and unique phenomena emerge, allowing for extensive applications, most notably human cryopreservation. Because heat is related to the motion of particles, at these temperatures the biochemical activities within living systems are effectively reduced [9]. The prospects for preserving an individual at extremely cold temperatures have been increasing throughout the years as research within the field continues to develop. As of now, human cryopreservation seems more of a speculation than reality. Freezing an individual is one thing, but there is no guarantee that the individual will wake up from such an extensive period of suspension. Although extremely low temperatures serve as an appropriate basis for human cryopreservation, many more factors must be considered to avoid consequences that may occur during the procedure and after revival.

Empirical Modeling

The rates of biochemical processes at extremely low temperatures can be modeled mathematically [4]. The Arrhenius equation, proposed by Arrhenius in 1889, establishes a relationship between temperature and reaction rates. In figure 1, K is the reaction rate, Ea is the activation energy, A is the frequency factor (related to the orientations of molecules necessary to produce a favorable reaction), R is the universal gas constant, and T is the temperature. Manipulating the equation, we produce a form that directly shows the relationship between the reaction rate and temperature, as depicted in Figure 2. We will use the enzyme lactate dehydrogenase to illustrate the relationship between K and T [4]. With its activation energy defined as 54, 810 J/mol, we can explore the enzyme’s reaction rate at a 10°C difference. With T1 and T2 at 40°C and 30°C respectively, we get a reaction rate ratio of 2.004. This tells us that a 10°C difference is enough to cut the reaction rate of the enzyme exactly in half.  

The relationship between reaction rates and temperature, as expressed by the Arrhenius equation, lends weight to the viability of cryopreservation. If a 10°C difference is enough to cut a reaction rate in half, imagine how much the reaction rate would be reduced within cryonically preserved individuals at extremely low temperatures. Furthermore, the biochemical processes that are occurring in the body at these levels are paused—not in the sense of being physically stopped, but rather the time needed for the processes to go to completion is relatively infinite. 

Figure 1. The Arrhenius equation Figure 2. Manipulation of the Arrhenius equation to compare reaction rates at two different temperatures

 

Human Cryopreservation

By understanding that at these extremely low temperatures, biochemical reaction rates are suppressed, the practice of cryogenically preserving a whole individual became a reality [14]. For this process to begin, the individual must be induced in the death state. Once an individual enters the initial stages of death, the human body initiates its decomposition phase. The body’s cell walls begin to break down and in turn, release digestive enzymes that process the tissues in the body [11]. Because the body begins to break down at such a rapid pace, it is imperative that the patient, once induced in the death stage, be worked on immediately.

 The process of chilling the human body to extremely low temperatures is a delicate and slow process and is very important in the initial steps of the cryopreservation procedure. Once the patient arrives in the death state, the circulation and respiration of the cryonic subject is restored and they are ready to be cooled [4]. First, the subject’s blood is replaced with 10% cryoprotectants to prevent ice formation. A small percentage of cryoprotectants are added initially to avoid an elevated osmotic shrinking response. Once the intracellular and extracellular cryoprotectant volume reaches equilibrium, the cells are ready for cooling which is done at a very slow pace (1°C/min) [5].

The cryoprotectant used typically consists of nutritional salts, buffers, osmogens, and apoptosis inhibitors, ingredients necessary in the maintenance of isotonic concentrations of the cell [5]. In doing so, cells within the human body can avoid swelling and shrinking. Additionally, another key formulation of cryoprotectant mixture is non-penetrating cryoprotectants which are typically large molecular polymers. These play a large part in the inhibition of ice growth and prevention of injury due to being subjected to the extreme cold [5]. 

To understand the prospects of human cryopreservation, it is helpful to redirect ourselves back to the definition of death. In 1988, the scientific community reviewed and redefined the definition of death from being in cardio-respiratory arrest to brain death [8]. In cryonically preserved patients, the extremely cold temperatures are thought to preserve the neural structures, which store long-term memory and the identity of the person. In this way, utilizing extremely low temperatures to preserve neural structures and prevent them from being compromised is a prospect worth noting. Individuals who are cryonically preserved should not be viewed as being dead or alive, but rather be viewed as being temporarily suspended in time [8]. The normal cycles of biological processes such as growth and decay are paused, providing an opportunity for resuscitation and reanimation in the future [10]. To give a new perspective, cryopreservation can be viewed similarly to frozen embryos: just as embryos preserved in extremely cold temperatures gain life once implanted in a uterus, the cryopreserved patient may reenter the living state through the process of human reanimation. 

Prospects for Immortality

The process of human cryopreservation aims to allow individuals to escape imminent death by first being induced into a transient death state [8]. Essentially, individuals are given the opportunity to bypass human mortality. Dr. James Hiram Bedford, a former psychology professor at UC Berkeley had his life threatened by renal cancer. He decided to undergo the cryopreservation process and became the first human to be cryonically preserved in 1967 [13]. By agreeing to enter this process, he hoped that, in the future, technology would be advanced enough to revive him and cure his illness. Ever since interests in cryopreservation have increased substantially, and as of 2014, about 250 corpses have been cryogenically preserved in the US [13].

Shifting Views on Aging

Aging is a degradative process that entails a whole array of pathologies. If we were to view aging as a disease itself that can be treated, cryopreservation opens a wide range of possibilities. Specifically, the process of cryopreservation allows an individual to avoid the effects of aging pathologies by having the opportunity to be treated once technology has advanced enough. This provides hope to bypass the mechanically built-in lifespans of humans, and essentially, provides prospects for immortality.

On a larger scale, as we age, the probability of dying increases significantly [7]. To put it simply, as we age, there are more health factors in place to compete for our lives and the chance of survival through older ages decreases. In such cases, aging can be correlated with functional decline. Similarly, on the molecular scale, aging can be seen as a direct consequence of telomere shortening [6]. Telomeres are nucleoprotein structures that exist at the ends of chromosomes and are essential to the integrity of our DNA. During the process of DNA replication, telomeres protect the ends of chromosomes and prevent loss of genetic information [16]. However, as we age, and as our body continues to undergo DNA replication, the telomeres shorten leading to the joining of ends of various chromosomes, pathological cell division, genomic instability and apoptosis. 

In short, the health consequences that come with aging are inevitable but human cryopreservation can be seen to offset these inevitable aging phenomena. The process allows an individual who is suffering from a presently incurable disease to be temporarily frozen in time. In this way, they may be revived when society is advanced enough to deal with the disease successfully. In essence, the human cryopreservation process can be seen to bypass inevitable health consequences, providing rejuvenating possibilities for any individual.

Technological Limitations 

Although successfully preserving an individual through extreme temperatures is certainly an exciting prospect, little evidence exists to indicate that successful preservation and remanimation is possible [15]. At present, there are many challenges that need to be overcome to even support the viability of such an extensive process. According to Professor Armitage, the director of tissue banking at the University of Bristol, preserving the whole human body is an entirely new challenge [15]. Society is not even at the stage of cryopreserving organs. Organs, alone, are very complex, containing different types of cells and blood vessels that all need to be preserved. Similarly, Barry Fuller, another professor at the University of College London, has stated that before exploring the prospects of human cryopreservation, society must be able to demonstrate that human organs can be cryopreserved for transplantation [15]. Hence, as of current, there is close to zero evidence that a whole human body can survive cryopreservation. 

In the previous section, we discussed the arrhenius equation which derived the relationship between temperature and metabolic rates. However, the equation itself does not explore the consequences of raising the temperature of the human body during reanimation. While thawing, the frozen tissues and cells can experience physical disruptions which can damage them [3]. To a greater extent, an individual’s epigenetic markers can even be affected, causing epigenetic reprogramming, which can change the expression of certain genes. However, the biggest hurdle is the successful preservation of the brain. The human brain is arguably one of the most important organs in the body, and cryopreservation must be successful in preserving the integrity of the neural structures. Prospects of successfully cryopreserving whole human brains are slim due to minimal research. Moreover, experiments with frozen whole animals’ brains have not been reported since the 1970s [3]. Obviously, research on this matter is severely limited.

Discussion 

Despite the overwhelming uncertainties surrounding human cryopreservation and society’s current limits, the prospects of being able to defy death or possibly avoiding it in the future are becoming a topic of increasing interest. When an individual is brought to the brink of death, the uncertainties around the cryopreservation procedure, specifically its unproven track record of success, seem inconsequential in the long run. If society were to overlook the field of preservation based purely on unsubstantiated results and the unlikelihood of success, advancements would never occur. All in all, the increase in technological advancements and research within cryogenics is making the prospects of reviving a frozen individual in the future ever so likely. 

 

References:

  1. Britannica, T. Editors of Encyclopaedia. “Cryogenics.” Encyclopedia Britannica, May 26, 2017. https://www.britannica.com/science/cryogenics.
  2. “What Is Cryogenics? “Gaslab.com. Accessed May 2, 2021. https://gaslab.com/blogs/articles/what-is-cryogenics. 
  3. Stolzing, Alexandra . “Will We Ever Be Able to Bring Cryogenically Frozen Corpses Back to Life? A Cryobiologist Explains.” The Conversation, March 26, 2019. https://theconversation.com/will-we-ever-be-able-to-bring-cryogenically-frozen-corpses-back-to-life-a-cryobiologist-explains-69500.
  4. Best, Benjamin P. “Scientific Justification of Cryonics Practice.” Rejuvenation Research 11, no. 2 (2008): 493–503. https://doi.org/10.1089/rej.2008.0661. 
  5. Bhattacharya, Sankha. “Cryoprotectants and Their Usage in Cryopreservation Process.” Cryopreservation Biotechnology in Biomedical and Biological Sciences, 2018. https://doi.org/10.5772/intechopen.80477.
  6. Blasco, M. A. “Telomere length, Stem Cells and Aging.” Nature Chemical Biology, 3, no.10 (September 2007): 640–649. doi:10.1038/nchembio.2007.38
  7. Carey, J.R. 2020, June 13. Limits of morbidity compression. Longevity (HDE/ENT 117) lecture notes, UC Davis.
  8. Cohen, C. “Bioethicists Must Rethink the Concept of Death: the Idea of Brain Death Is Not Appropriate for Cryopreservation.” Clinics 67, no. 2 (2012): 93–94. https://doi.org/10.6061/clinics/2012(02)01. 
  9. Jang, Tae Hoon, Sung Choel Park, Ji Hyun Yang, Jung Yoon Kim, Jae Hong Seok, Ui Seo Park, Chang Won Choi, Sung Ryul Lee, and Jin Han. “Cryopreservation and Its Clinical Applications.” Integrative Medicine Research 6, no. 1 (2017): 12–18. https://doi.org/10.1016/j.imr.2016.12.001. 
  10. Lemke, Thomas.“Beyond Life and Death. Investigating Cryopreservation Practices in Contemporary Societies,”  Soziologie, 48. No. 4 (April 2019):450-466.
  11. Lorraine. “The Stages of Human Decomposition.” Georgia Clean Services.” Georgia Clean, April 6, 2020. https://www.georgiaclean.com/the-stages-of-human-decomposition/.
  12. Luke Davis. “The Difference between Cryonics and Cryogenics,” August 10, 2020. https://logicface.co.uk/difference-between-cryonics-and-cryogenics/.
  13. Moen, Ole Martin. “The Case for Cryonics.” Journal of Medical Ethics 41, no. 8 (2015): 677–81. https://doi.org/10.1136/medethics-2015-102715. 
  14. Purtill, Corinne. “Fifty Years Frozen: The World’s First Cryonically Preserved Human’s Disturbing Journey to Immortality.” Quartz. Quartz. Accessed May 2, 2021. https://qz.com/883524/fifty-years-frozen-the-worlds-first-cryonically-preserved-humans-disturbing-journey-to-immortality/.
  15. Roxby, Philippa. “What Does Cryopreservation Do to Human Bodies?” BBC News. BBC, November 18, 2016. https://www.bbc.com/news/health-38019392.
  16. Trybek, Tomasz, Artur Kowalik, Stanisław Góźdź, and Aldona Kowalska. “Telomeres and Telomerase in Oncogenesis (Review).” Oncology Letters 20, no. 2 (2020): 1015–27. https://doi.org/10.3892/ol.2020.11659.

Potential Therapeutic Effects of sEH Inhibition in Neurological Disorders

May 29, 2021

By Nathifa Nasim, Neurobiology, Physiology & Behavior ‘22 

Author’s note: I was recently introduced to this topic and the potential for sEH inhibition in the context of Alzheimer’s while at Dr. Lee-Way Jin’s lab in the MIND Institute. Further research into the topic outside the lab led to the realization of the broader implications of sEH inhibition across numerous neurological disorders through its role in inflammatory pathways. The paper aims to illustrate the therapeutic potential sEH inhibition in neurological disorders through inflammation mediation.

 

Introduction

Neuroinflammation is a symptom of nearly all neurological disorders, and occurs when the immune system of the brain is activated. Microglia, an immune cell in the brain, release inflammatory mediators typically associated with neuroprotection and neurogenesis. However, incessant inflammation can be associated with harmful effects and neurological diseases. Therefore, treatments that target inflammatory processes are of interest to researchers as they have the potential to alleviate numerous disorders. The soluble epoxide hydrolase enzyme (sEH), an enzyme that modulates various physiological processes through the regulation of inflammatory pathways, is one such possible therapeutic agent. Research on the effect of sEH inhibition provides an avenue for treating different neurological disorders such as brain injury, depression, and Alzheimer’s. 

sEH and the Role of EETs

The sEH enzyme is important primarily through its ability to metabolize fatty acids in inflammatory pathways. It is found across mammalian tissues, such as in the brain or the liver, where it is highly expressed. As a member of the epoxide hydrolase family of enzymes, sEH’s C-terminus is responsible for the enzyme’s characteristic epoxide hydrolase activity, converting epoxides by adding a water molecule. The N-terminus, on the other hand, has phosphatase activity instead [2]. Due to the different activities of the two sides of the enzyme, the N-terminal phosphatase domain has been linked to cholesterol metabolism, while the C-terminal domain has been associated with inflammatory and cardiovascular effects.  

The epoxide hydrolase activity of sEH is performed on epoxyeicosatrienoic acids (EETs), epoxy fatty acids that act as lipid messengers in the body. Due to their nature as lipid messengers, EETs have numerous roles, such as being anti-inflammatory messengers and increasing vasodilation. An increase in EET levels provides anti-inflammatory, antihypertensive, neuroprotective, and cardioprotective effects [2]. These protective effects of EET may be especially acute in the brain. For example, the inhibition of the cytochrome p450 enzyme results in an absence of EET, which reduces cerebral blood flow by 30 percent, indicating a critical need for EET in brain circulation [4].

Inhibition of sEH, a negative regulator of EET, increases these protective effects of EETs. The enzyme lowers EET activity via conversion to dihydroxyeicosatrienoic acids (DHETs), which have less anti-inflammatory effects, and can be pro-inflammatory instead. Numerous studies have knocked out or selectively inhibited sEH which stabilizes or increases EET activity because it is no longer being converted into DHET. It is through sEH’s metabolism of EETs that it is linked to numerous diseases and various physiological effects, as EETs are critical in controlling inflammation. Research on the possible therapeutic effects of sEH is therefore primarily focused on increasing fatty acid/EET levels through inhibition of sEH. 

Inflammation in Brain Injury

Neuroinflammation is a critical aspect of traumatic brain injury (TBI), or brain damage via physical injury. TBI results in cerebral inflammation, which activates microglia to produce post-traumatic inflammatory mediators and reactive oxygen species (ROS). While inflammatory mediators function as an immune response after injury, their overproduction after TBI can lead to neural damage, apoptosis (cell death) and/or brain edema (swelling due to the accumulation of fluids). This additional damage occurring in the brain stimulates an increased microglia response, resulting in a positive feedback mechanism [5]. 

Inhibition of this post-traumatic stress-induced inflammation is a common mechanism in treating TBI. Given sEH’s role in inflammation, deletion of sEH via genetic knockouts has been shown to improve these effects. Deletion of sEH decreases the number of activated microglia post-TBI, and as a result, reduces the release of inflammatory mediators, thereby reducing edema, apoptosis, and inflammation [5]. The rise in EET levels associated with sEH inhibition also results in anti-inflammatory effects, and possibly an increase in neurotrophic factors, or growth factors that have a neuroprotective effect [5]. 

sEH and Alzheimer’s

Neuroinflammation is also linked to both the onset and progression of Alzheimer’s. Prolonged activation of glial cells— astrocytes and microglia—increase proinflammatory molecules which lead to a “neurotoxic” environment that aggravates the disease [6]. Alzheimer’s patients have nearly twofold higher sEH levels in the astrocytes, leading to lower EET levels and thus lower anti-inflammatory effects [7]. Given the importance of inflammation in Alzheimer’s, inhibition of sEH should increase EET levels, promoting anti-inflammatory effects crucial to treating the disease. 

One experiment treated 5xFAD mice, transgenic mice that express Alzheimer’s phenotypes, with an sEH inhibitor TPPU. The results indicated that not only did the mice have increased EET levels, the inhibitor had also affected gene expression in the hippocampus by downregulating proinflammatory genes. This effectively “calmed” the overactive immune response correlated with Alzheimer’s. Further testing uncovered that these treated mice had fewer, smaller amyloid plaques, or proteins that are considered to play a critical role in Alzheimer’s and are often regarded as a biomarker for the disease, and fewer microglia surrounding these plaques [6]. As prolonged activation of microglia leads to proinflammatory effects and reduced phagocytosis of amyloid plaques, lowering the immune response of these glial cells could theoretically reverse these inflammatory effects. The reduction effects of EETs on ROS may also be involved, as ROS may contribute to neurotoxicity [7]. These results were achieved with TPPU and verified with another sEH inhibitor; it also aligned with results seen in genetic knockouts of sEH [6]. 

sEH in Peripheral Organs

In addition to the brain, peripheral organs may also play an important role in sEH’s effects on neurological disorders. In addition to astrocytes and other glial cells, sEH is most expressed in the liver. With the liver being the largest gland and responsible for a variety of metabolic activities, hepatic sEH (sEH of the liver) has an influence throughout the body. In a study on major depressive disorder (MDD) by Qin et al., researchers used mice in a chronic mild stress model (CMS), where they were exposed to varying stressors to mimic the effects of depression in an animal model. They found that chronic stress increased sEH levels in mice liver, suggesting hepatic sEH levels are linked to stress and depression [8]. Increased sEH levels in these mice via targeted gene therapy not only led to depressive-like behaviors, but there was also a decrease in proteins that modulate synaptic plasticity, suggesting that sEH parallels the effects of stress at the molecular level. On the other hand, deleting the gene that codes for sEH in the liver induced an antidepressant effect in the CMS mice [8]. In other words, sEH induced depressive-like effects, and inhibiting sEH activity led to antidepressant-like effects, even in stressed model mice. These findings were paralleled in MDD human patients who had lower EET levels compared to the control group, suggesting higher sEH activity in patients with depression, again similar to the Alzheimer’s patients [8]. 

Conclusion

Given the anti-inflammatory abilities of EETs, further development of sEH inhibitors has the capacity to affect the treatment of multiple neurodegenerative diseases which are associated with inflammation. In addition to those mentioned, other neurological conditions and disorders have also exhibited elevated sEH levels, such as Parkinson’s, schizophrenia, and seizures [6]. As these diseases have different pathologies, the cause for elevated sEH levels may vary and is still under research. Nevertheless, the implication of sEH in a variety of diseases expands the therapeutic range of sEH inhibitors.

As seen in the aforementioned MDD study by Qin et al., peripheral organs’ usage of sEH may also be involved with neurological disorders, such as in the case of hepatic sEH. This not only points to a possible liver-brain axis or connection between the two organs, it also opens another avenue of research into the effects of sEH across the body. Diseases such as depression and heart disease have been implicated with sEH in previous studies, for example, and at present, sEH inhibition has been successful in decreasing blood pressure levels [9]. Development and further research into sEH inhibition and effects therefore have the potential to touch numerous conditions and parts of the human body. 

The research regarding Alzheimer’s and TBI both implemented pharmacological sEH inhibitors, in which only the C-terminal hydrolase domain of the enzyme was affected while the N-terminal phosphatase activity was left intact [5,6]. As the N-terminal has been associated with cholesterol metabolism, its role in neurological disorders provides another possible area of study of sEH in treating these disorders. Genetic deletion of the gene that codes for sEH, as seen in the Qin et al. study, deletes both the hydrolase and phosphatase activity of the enzyme. Therefore, studies implementing sEH knockouts may have benefitted from the loss of the phosphatase activity as well as the hydrolase [9]. As a result, further research into different aspects of the enzyme broadens the possibility of its usage and potential. 

 

References:

  1. Wee Yong V. 2010. Inflammation in neurological disorders: a help or a hindrance? Neuroscientist. 16(4):408-20. doi: 10.1177/1073858410371379
  2. Harris TR, Hammock BD. 2013. Soluble epoxide hydrolase: gene structure, expression and deletion. Gene. 526(2):61-74. doi: 10.1016/j.gene.2013.05.008. 
  3. Flores DM, Natalia CS, Regina PA, Regina CC. 2020. Soluble Epoxide Hydrolase and Brain Cholesterol Metabolism. Frontiers in Molecular Neuroscience. 12: 325. doi: 10.3389/fnmol.2019.00325    
  4. Sura P, Sura R, Enayetallah AE, Grant DF. 2008. Distribution and expression of soluble epoxide hydrolase in human brain. J Histochem Cytochem. 56(6):551-9. doi: 10.1369/jhc.2008.950659. 
  5. Hung TH, Shyue SK, Wu CH, Chen CC, Lin CC, Chang CF, Chen SF. 2017. Deletion or inhibition of soluble epoxide hydrolase protects against brain damage and reduces microglia-mediated neuroinflammation in traumatic brain injury. Oncotarget. 8(61):103236-103260. doi: 10.18632/oncotarget.21139.
  6. Ghosh A, Comerota MM, Wan D, Chen F, Propson NE, Hwang SH, Hammock BD, Zheng H. 2020. An epoxide hydrolase inhibitor reduces neuroinflammation in a mouse model of Alzheimer’s disease. Sci Transl Med. 12(573):eabb1206. doi: 10.1126/scitranslmed.abb1206.
  7. Griñán-Ferré C, Codony S, Pujol E, Companys-Alemany J, Corpas R, Sanfeliu C, Pérez B, Loza MI, Brea J, Morisseau C, Hammock BD, Vázquez S, Pallàs M, Galdeano C. 2020. Pharmacological Inhibition of Soluble Epoxide Hydrolase as a New Therapy for Alzheimer’s Disease. Neurotherapeutics 17:1825–1835. doi: 10.1007/s13311-020-00854-1
  8. Qin X, Wu Z, Dong JH, Zeng YN, Xiong WC, Liu C, Wang MY, Zhu MZ, Chen WJ, Zhang Y, Huang QY, Zhu XY. 2019. Liver Soluble Epoxide Hydrolase Regulates Behavioral and Cellular Effects of Chronic Stress. Cell Reports. 29(10): 3223-3234. doi:10.1016/j.celrep.2019.11.006.
  9. Spector AA, Fang X, Snyder GD, Weintraub NL. 2004. Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Progress in Lipid Research. 43(1): 55-90. Doi:10.1016/S0163-7827(03)00049-3.

Modified Mu Opioid Receptors Lead to Analgesia Without Physical Dependence

May 14, 2021

By Neha Madugala, Neurobiology, Physiology, and Behavior ‘21

Author’s Note: I wrote this literature review for my UWP104F class to assess new opioid-based medications for pain-relief. While opioids are the best known pain relievers we currently have, they have the severe risks of addiction and overdose. This paper analyzes literature that attempts to amplify the analgesic (pain-relief) properties of opioids, while minimizing their addictive potential. 

 

Introduction

As the opioid epidemic grows, opioids are becoming increasingly synonymous with addiction and overdose. While opioids have immense pain-relief properties, their use has been limited due to their major risks. The potential for addiction lies in the interaction between opioids and the mu-opioid receptor. Scientists are working to create a modified receptor that can have these analgesic effects but have limited risk. These findings could aid in the development of safer but effective pain-relief medications. 

Background

Drug addiction is a chronic relapsing disorder that occurs through repeated exposure to the drug, leading to physiological changes in brain chemistry [6]. While initial use is associated with improved well-being and feelings of euphoria, this repeated use in addition to environmental factors and genetics can lead to modifications in the endogenous opioid system, or the body’s natural pain killers, and alterations of stress physiology through hormonal imbalance [6, 7]. These homeostatic changes deregulate brain reward pathways, resulting in tolerance and dependence [7]. 

Opioids have a significantly higher rate of relapse compared to other addictive drugs since they induce strong physical dependence [7] and craving [7]. As a result, scientists have extensively studied the mu-opioid receptor (MOPr), which directly binds to opioids and indirectly binds to other addictive drugs, such as alcohol, cannabinoids, and nicotine [6]. Current research is focused on understanding the mechanism of the MOPr. While scientists agree that this mechanism plays a role in physical dependence, it is unclear what part of this pathway is responsible for these observed effects. 

Opioids act as agonists at the MOPr. Agonists modify a receptor through intrinsic efficacy and affinity. Affinity determines how well a ligand is able to bind to the active site of a receptor and intrinsic efficacy is a measure of how well a ligand is able to stabilize the receptor in its active conformation [1]. Intrinsic efficacy is particularly important for the MOPr because the MOPr is strongly agonist-dependent [6]. For instance, morphine acts as a full agonist at the MOPr, inducing the maximal effect. As a result, morphine is commonly used to study the function of this receptor and its role in addiction. 

At the MOPr, opioids bind to the active site inducing a conformational change and activating the receptor. The MOPr is a G protein-coupled receptor (GPCR), which is a type of receptor where activation leads to secondary pathway signaling. As a result, binding of a ligand to the MOPr causes secondary pathways to be activated, resulting in downstream signaling effects. Specifically, the MOPr is a Gi-coupled GPCR, so it inhibits the enzyme adenylyl cyclase, which converts ATP to cAMP. As a result, opioid binding inhibits the production of cAMP. 

This effect is brief. MOPr activation promotes translocation of β-arrestin from the cytosol to the plasma membrane. MOPr is rapidly phosphorylated by GPCR kinases (GRKs). This phosphorylation increases the affinity of β-arrestin to the MOPr [6]. β-arrestin binds to the MOPr. This binding uncouples the MOPr from the Gi-coupled GPCR, which halts the inhibition of cAMP production. This uncoupling further halts the signaling pathways and results in desensitization of the MOPr, since the opioid can no longer induce downstream effects.

β-arrestin also recruits components of the endocytic machinery [2] to engulf the MOPr into the cell. The desensitized receptor is internalized within the cell via endocytosis [2]. Endocytosis plays an important role in engulfing the desensitized receptor, which is no longer functional, and placing it back into the plasma membrane resensitized, or functional. This rapid resensitization process is important for having a consistent supply of available receptors for ligand binding. 

Naloxone acts to block agonists of the MOPr and is used as a treatment for drug overdose, and to induce withdrawal in experimental models. As an antagonist, naloxone has zero intrinsic efficacy, so it does not modify the constitutive activity of the receptor.  Essentially antagonists do not activate any additional pathways but have an affinity for the active site of the respective receptor [1]. As a result, naloxone competes with agonists for the binding site and prevents agonists from inducing an effect on the MOPr. Their ability to compete is dependent on their level of affinity. Opioid antagonists are used in drug experiments to quickly stop drug administration in the brain to assess signs of physical dependence. This method is efficient because it can prevent agonists from working, even when the agonist is present in the bloodstream. For this reason, antagonists are also administered following overdose to reverse opioid-induced respiratory depression, where the brain stops sending signals to the body to breathe [4]. 

β-arrestin and Physical Dependence

Enkephalins, an endogenous opioid, at the MOPr activate the Gi-coupled GPCR and β-arrestin in equal amounts. As a result, enkephalins are “unbiased.” However, many addictive opioids, such as morphine, act as biased agonists signaling the Gi-coupled GPCR and β-arrestin asymmetrically [1]. At the MOPr, scientists hypothesize that “G-protein signaling [is] responsible for opioid-induced analgesia, while [β-arrestin is] responsible for the adverse effects of mu-receptor activation” [9]. 

In a study in the Nature Journal of β-arrestin-2 knockout mice by Bohn et al., they studied the development of antinociception tolerance following daily administration of a moderate dose of morphine (10 mg/kg) for nine days [3]. They conducted this experiment to determine whether chronic morphine use can diminish antinociception over time, a sign of addiction. They used wild-type mice as controls. To control for genetic variation, they crossed over eight generations of mice that were heterozygous for β-arrestin-2. This allowed them to develop wild-type and knockout mice (through the homozygous progeny) that were “age-matched, 3—5-month-old male siblings weighing between 20 and 30 g [3]. The wild-type mice had a significantly diminished response to morphine administration by day five. In contrast, the knockout mice had comparable antinociception throughout the entire experimental period [3]. These findings suggest that β-arrestin presence has a significant role in the development of antinociceptive tolerance. 

Moreover, β-arrestin plays multiple roles in the MOPr trafficking process. To further assess these results, researchers studied one aspect of β-arrestin, promoting endocytosis. Kim et al. hypothesized that over time morphine binding to the MOPr led to diminished antinociception, tolerance, and dependence due to morphine’s inability “to promote substantial receptor endocytosis” [10]. They generated knockout mice by genetically modifying the MOPr at exon 3 to create an experimental receptor (rMOP-R) that would be more effective at resensitizing the receptor through endocytosis [10]. They used wild-type mice as a control. 

The mice with the rMOP-R had enhanced and prolonged analgesic effects compared to the wild-type mice with the MOP-R. They suggested that these results were due to the rMOP-R being active for longer due to quicker resensitization [10]. Furthermore, they assessed tolerance and dependence over both a short-term experiment (one day) and a long-term experiment (five days). The wild-type mice displayed both acute and chronic antinociceptive tolerance to morphine, as well as withdrawal responses to any administration of naloxone following morphine administration. The knockout mice did not develop acute or chronic antinociceptive tolerance to morphine and showed much fewer withdrawal responses to administration of naloxone following morphine administration [10]. 

Kim et al. did these experiments with endogenous opioids (DAMGO), morphine, and methadone with acts similarly to endogenous opioids. They found a significant difference between the wild-type and knockout mice for morphine administration only [10]. This supports their hypothesis since DAMGO and methadone already have enhanced endocytosis. 

These results suggest that enhanced endocytosis of the MOPr can help alleviate tolerance and signs of physical dependence while maintaining the antinociceptive effects. While β-arrestin functions to recruit the endocytic machinery, it also turns off the MOPr by desensitizing the receptor. Since the MOPr is strongly agonist-dependent, morphine acting at this receptor results in an increased period of desensitization. By diminishing this off period through enhanced endocytosis, the rMOP-R receptor leads to quicker resensitization, alleviating signs of physical dependence [10]. 

To further explore this hypothesis, Berger and Whistler conducted a similar study to Kim et al. by comparing the knockout mice with rMOP-R to wild-type mice with the MOPr in a conditioned place preference (CPP) paradigm and self-administration study. A CPP paradigm is when the mice are placed in a room with two sides that are decorated in distinct manners. On one side they are administered morphine, while they are not on the other side. Preference for morphine is determined by which room they spend more time in. For the CPP paradigm, the rMOP-R mice displayed greater CPP at lower doses and the wild-type mice displayed greater CPP at higher doses [2]. This indicates that the knockout mice had a large rewarding effect at low doses, suggesting that smaller doses of morphine elicited a larger effect in the knockout mice compared to the wild-type mice. 

Furthermore, they assessed how these mice lines differed for additional determinants of addiction in a self-administration study. For each operant session, the mice were allowed to administer morphine on the first day, morphine and water for the next four days, and were given only water on the last two days of the experiment [2]. They assessed four factors: “high motivation to obtain drug,” “futile drug-seeking,” “persistent drug-seeking in the face of adverse consequences,” and “reduced preference for alternative rewards” [2]. 

Berger and Whistler found that when morphine was administered into the lever at regular intervals, the knockout mice learned these intervals while the wild-type mice attempted to administer the drug more often by pressing the lever, even though it was in between the intervals [2]. They further assessed lever presses when accompanied by an electric shock or in the presence of an alternate reward, saccharin. The wild-type mice administered the drug more often and showed a greater preference for morphine respectively for these experiments compared to the knockout mice [2]. These results indicate that the enhanced endocytosis and quicker resensitization of the MOPr helps alleviate the adverse effects of addiction beyond physical dependence. 

G-Protein Biased Agonists 

Based on these findings, researchers hypothesize that creating drugs that are G-protein biased agonists could help improve the antinociceptive effects of opioids while diminishing the adverse effects of delayed desensitization due to β-arrestin [9]. There is ongoing research to develop G-protein-biased agonists of the MOPr. For instance, oliceridine, a G-protein-biased agonist, is currently in Phase 3 of clinical trials [8]. However, current research is bringing into question whether only β-arrestin is responsible for the adverse effects of addiction [8]. 

There is now evidence suggesting that “MOPr activation in [the preBotzinger (preBotC) and Kolliker-Fuse (KF) neurons] … inhibits neuronal activity via G protein signaling” [8]. The preBotC and KF neurons are located in regions of the brain associated with respiratory control [8]. They also found that the activation of GRKs, which promotes arrestin binding to Gi-coupled GPCRs, is mediated by G protein signaling [8].

In a study in the British Journal of Pharmacology of morphine-induced respiratory depression independent of β-arrestin-2 by Kliewer et al., they used β-arrestin knockout mice to assess respiratory depression when administered an opioid. This experiment was conducted in three laboratories, located in Jena, Germany; Sydney, Australia; and Bristol, United Kingdom [11]. Each laboratory used a similar experimental set-up with slight variations. 

They all found a “dose-dependent depression of respiratory rate by morphine” [11]. The laboratory in Jena used a nose-out plethysmography system; the laboratories in Sydney and Bristol used whole-body plethysmography [11]. (Plethysmography methods assess the difference in the volume of air present in the lungs prior to and post-exhaling.) 

While biased agonism has been suggested as a possible mechanism to isolate the analgesic properties of opioids from the adverse effects, these studies point out weaknesses in this model. First, the initial study assessing β-arrestin-2 knockout mice has not been extensively repeated to verify accuracy and the pathway for respiratory depression cannot be isolated to only β-arrestin [11]. This new evidence indicates that both G-protein signaling and β-arrestin play a role in the physical dependence on opioids. However, more research is needed to determine the extent of how much each protein influences these physiological symptoms. 

Discussion

While the original findings indicated that only β-arrestin is responsible for the adverse effects of opioid use, new research suggests that these effects cannot be easily isolated to just β-arrestin. The results indicate that β-arrestin plays a role in tolerance and dependence, while G-protein plays a role in respiratory depression during an overdose. More research on G-protein signaling and its connection to the dopamine reward pathway is necessary to understand the extent of its involvement in the MOPr trafficking system. Furthermore, more research is needed to replicate the studies done on β-arrestin-2 knockout mice because these studies have not been extensively replicated, bringing into question the accuracy of these past findings. Also, our understanding of the role of β-arrestin-2 in the desensitization of the MOPr is based on research done in HEK 293 cells, found in the human embryonic kidney; these results have not been replicated in neurons [5], yet the MOPr trafficking system occurs within neurons. Overall, further research is needed to establish the reliability of past findings on β-arrestin and to understand the adverse effects of G-protein signaling. The latter is especially important because current research is focused on developing G-protein-biased agonists to act as analgesics; however, these new analgesics may still pose the risk of respiratory depression.  

Conclusion

Current research of the mu-opioid receptor trafficking system indicates that both β-arrestin and Gi-coupled GPCR are responsible for the adverse effects of opioids, including tolerance, dependence, and respiratory depression. More extensive research is required to determine the exact roles of the β-arrestin and Gi-coupled GPCR, while also verifying the results of past studies. These findings can help determine the risk of respiratory depression in the development of new analgesics and extend our understanding of the development of tolerance and withdrawal in addiction. Moreover, these results can help diminish the risk of more potent opioids, while acting as effective pain medications. 

 

References

  1. Berg, Kelly A, and William P. Clarke. “Making Sense of Pharmacology: Inverse Agonism and Functional Selectivity.” International Journal of Neuropsychopharmacology, vol. 21, no. 10, 2018, pp. 962-977. NIH, https://pubmed.ncbi.nlm.nih.gov/30085126/. 
  2. Berger, Amy C, and Jennifer L. Whistler. “Morphine-Induced Mu Opioid Receptor Trafficking Enhances Reward yet Prevents Compulsive Drug Use.” EMBO Molecular Medicine, vol. 3, no. 7, 2011, pp. 385-97. NCBI, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394511/.
  3. Bohn, Laura M., et al. “Mu-Opioid Receptor Desensitization by β-arrestin-2 Determines Morphine Tolerance but not Dependence.” Nature, vol. 408, 2000, pp. 720-23. NIH, https://pubmed.ncbi.nlm.nih.gov/11130073/. 
  4. Bubier, Jason A., et al. “Genetic Variation Regulates Opioid-Induced Respiratory Depression in Mice.” Nature Research, vol. 10, no. 14, 2020, pp. 1-15. Nature, https://www.nature.com/articles/s41598-020-71804-2.
  5. Connor, Mark, et al. “Mu-Opioid Receptor Desensitization: Is Morphine Different?” British Journal of Pharmacology, vol. 143, 2004, pp. 685-696. Nature Publishing Group, doi:10.1038/sj.bjp.0705938.
  6. Contet, Candice, et al. “Mu Opioid Receptor: A Gateway to Drug Addiction.” Current Opinion in Neurobiology, vol. 14, 2004, pp. 370-78. Science Direct, https://www.sciencedirect.com/science/article/pii/S0959438804000728. 
  7. Gerrits, Mirjam, et al. “Drug Dependence and the Endogenous Opioid System.” European NeuroPsychoPharmacology, vol. 13, 2003, pp. 424-434. Elsevier Ltd., doi:10.1016/j.euroneuro.2003.08.003.
  8. Gillis, Alexander, et al. “Critical Assessment of G Protein-Biased Agonism at the Mu-Opioid Receptor.” Cell Press Reviews, vol. 41, no. 12, 2020, pp. 947-59. Elsevier Ltd., https://doi.org/10.1016/j.tips.2020.09.009.
  9. Groom, Sam, et al. “A Novel G Protein-Biased Agonist at the Mu Opioid Receptor Induces Substantial Receptor Desensitisation Through G Protein-Coupled Receptor Kinase.” British Pharmacological Society, 2020, pp. 1-15. Wiley Online Library, doi:10.1111/bph.15334. 
  10. Kim, Joseph A., et al. “Morphine-Induced Receptor Endocytosis in a Novel Knockin Mouse Reduces Tolerance and Dependence.” Cell Press Journal, vol. 18, no. 2, 2008, pp. 129-35. NIH, https://pubmed.ncbi.nlm.nih.gov/18207746/. Kliewer, Andrea, et al. “Morphine-Induced Respiratory Depression is Independent of β-arrestin-2 Signalling.” British Journal of Pharmacology, vol. 177, 2020, pp. 2923-2931. Wiley Online Library, https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15004.

It’s Not You, It’s Your Microbes: The Association Between Microbiota and Depressive Behavior in Mice

May 7, 2021

By Reshma Kolala, Medical & Molecular Microbiology ‘22

Author’s Note: A recent switch into the Microbiology major prompted me to explore recent developments in the field. I came across this study that examined the role of gut microbiota in brain function and mood regulation. With the globally rising prevalence of depression, this study provides some potential insight into the development of the disorder on a physiological level and provides a novel approach to anti-depression therapeutics. 

 

Afflicting nearly 350 million individuals annually, depression is a leading cause of disability worldwide. Despite the widespread effort to uncover the environmental and genetic basis of the disorder, the pathophysiology of depression remains elusive. This is attributed to the fact that, similar to other mental disorders, depression is the result of a complex interplay between several biological and societal factors [1]. Several studies have found that the pathology of depression is influenced by dysfunction in neuromodulatory systems, such as the endocannabinoid system (ECS). The ECS is composed of endocannabinoids (eCB), lipid-based neurotransmitters that regulate mood, emotions, and stress responses [2,3]. Another physiological factor that contributes to depression is the impairment of the hippocampal region of the brain, specifically hippocampal impaired neurogenesis, which contributes to depressive-like behaviors in rodents [4]. This is due to the fact that adult hippocampal neurogenesis has been shown to help mediate stress responses and depressive behavior. The dysfunction of these critical processes has recently been investigated in relation to symbiotic microbiota. 

It has been well established that the diversity of intestinal microbiota contributes to enhanced host function (particularly in immunity, metabolism, and the central nervous system) allowing an individual to better combat disease and regulate metabolic function [5,6,7]. Previous studies have demonstrated that dysbiosis, or altered intestinal microbial composition, has been found in depressed patients when compared to healthy controls [8].  It has also been observed that microbiota modulate anxiety symptoms in mice via the release of bacterial metabolites that may affect critical pathways in the brain [9]. Finally, colitis, a digestive disease characterized by inflammation in the colon, is influenced by gut microbiota and is commonly observed in depression patients [10]. Overall, these studies imply a potential association between intestinal microbial composition and depressive-like behaviors. The following study aims to examine the direct effect of gut microbiota on depressive behaviors in mice, allowing for a broader understanding of the physiological basis of depression and provide new avenues for therapeutics and potential treatment [11].

Researchers used unpredictable chronic mild stress (UCMS), a mouse model of stress-induced depression. To simulate stress, mice in the UCMS group were exposed to various stressors including cage tilting, altered cage bedding, foreign odor, and altered light/dark cycle. The mice in the UCMS group were exposed to two stressors a day for eight weeks. As expected, UCMS mice exhibited depressive-like behaviors such as decreased feeding and self-grooming behavior, consistent with apathetic behavior in those diagnosed with depression. UCMS mice also exhibited reduced hippocampal neurogenesis, confirming a previous study by Snyder et al. that noted this observation in rodents with depression [3].

Once depressive-like behaviors were established in UCMS mice, researchers conducted a fecal microbiota transplant (FMT) from mice exposed to stressors to mice that have not been exposed to any stressors. FMT’s are an innovative form of treatment in which a stool sample is collected from one individual and transplanted in the colon of another individual. This can be administered in various ways, such as a colonoscopy, oral capsules, or via a tube that stretches from the nose into the stomach or bowel [12]. In this study, mice received transplants via an oral gavage which involves the passage of a feeding needle down the esophagus. The purpose of an FMT is to populate the recipient intestine with diverse microorganisms that preferentially provide some benefit to the host. When the microbiota from the UCMS mice was transplanted into the healthy mice, the healthy mice exhibited decreased hippocampal neurogenesis and mimicked the depressive-like behaviors exhibited in the UCMS mice, although the healthy mice had not been exposed to any stressors. 

The effect of the FMT on recipient mice illustrated the influence of intestinal microbial composition on the host. Researchers in this study hypothesized that this was due to alterations in the host’s metabolism. To investigate this further, the concentration of multiple small molecule metabolites in bodily fluids was measured. This revealed a significant decrease in levels of several short-chain fatty acids which may have resulted from dysbiosis-induced changes. As fat is primarily broken down in the small intestine via chemical and mechanical processes, an altered microbial composition in the intestinal tract would unsurprisingly influence fat breakdown. More specifically, there was a decrease in the concentration of an eCB precursor, fatty acids containing arachidonic acid (AA), in recipient mice. As dysregulation of the ECS has been studied in association with depression, this finding in recipient mice aligns with the typical model of depression. To further understand the role of impaired eCB signaling in the recipient mice, researchers observed whether enhancing eCB signaling via dietary supplementation could alleviate the depressive-like behaviors observed in the recipient mice. It was found that recipient mice that were orally administered AA had reversed the depressive-like behaviors indeed by UCMS microbiota. Additionally, AA supplementation aided hippocampal neurogenesis.

To determine how UCMS microbiota affected the microbial composition of recipient mice, fecal microbiota from UCMS mice was sequenced using 16s rRNA. As 16s rRNA is present in all bacteria, the 16s rRNA gene is highly conserved and therefore, a useful tool to identify microbes within complex biological mixtures. The analysis revealed increased levels of Ruminoccacaae and Porphyromonodaceae and a decrease in Lactobacillacae. This finding supports previous studies that report an association between decreased Lactobacillacae and stress in mice. The differences in the microbial composition of recipient mice and donor UCMS mice were maintained eight weeks after transplantation. To test the influence of decreased Lactobacillacae in recipient mice, Lactobacillacae was orally administered similarly to AA supplantation. Dietary complementation of Lactobacillacae had a similar effect as AA supplantation, where depressive-like behaviors and impaired hippocampal neurogenesis were reversed.

Using mice, researchers discovered that the onset of depressive-like behaviors is triggered by a reduction in lipid metabolites. These lipid metabolites, more specifically endocannabinoids, bind to receptors in regions of the brain that control emotion and memory. Surprisingly, the concentrations of endocannabinoids are biochemically influenced by the gut microbiota. Although the mechanism by which this occurs has yet to be understood, these studies have elucidated the impact of gut microbiota beyond digestive function, revealing the extensive scope of microbial composition on healthy host function. This study specifically illustrates the importance of balanced gut microbiota for healthy neural and metabolic function and supports the potential use of dietary or probiotic supplementation as a treatment option for those diagnosed with depression. However, it is important to note that this area of research is relatively new and further studies are required to determine the translational capacity of studies related to the gut-brain axis from mice to humans. With consideration of the limitations of this study, this finding does still provide an intriguing avenue of treatment for mood disorders by introducing a novel physiological approach to mediate depressive-like symptoms. 

 

References

  1. Limbana, T., Khan, F., & Eskander, N. (2020). Gut Microbiome and Depression: How Microbes Affect the Way We Think. Cureus, 12(8). https://doi.org/10.7759/cureus.9966
  2. Hill, M. N., Hillard, C. J., Bambico, F. R., Patel, S., Gorzalka, B. B., & Gobbi, G. (2009). The therapeutic potential of the endocannabinoid system for the development of a novel class of antidepressants. Trends in pharmacological sciences, 30(9): 484–493. https://doi.org/10.1016/j.tips.2009.06.006
  3. Freitas, H. R., Ferreira, G., Trevenzoli, I. H., Oliveira, K. J., & de Melo Reis, R. A. (2017). Fatty Acids, Antioxidants and Physical Activity in Brain Aging. Nutrients, 9(11): 1263. https://doi.org/10.3390/nu9111263
  4. Snyder, J., Soumier, A., Brewer, M. et al. (2011) Adult hippocampal neurogenesis buffers stress responses and depressive behavior. Nature 476: 458–461. https://doi.org/10.1038/nature10287
  5. Belkaid, Y., & Hand, T. W. (2014). Role of the microbiota in immunity and inflammation. Cell, 157(1), 121–141. https://doi.org/10.1016/j.cell.2014.03.011
  6. Cani P. D. (2014). Metabolism in 2013: The gut microbiota manages host metabolism. Nature reviews. Endocrinology, 10(2): 74–76. https://doi.org/10.1038/nrendo.2013.240
  7. Sharon, G., Sampson, T. R., Geschwind, D. H., & Mazmanian, S. K. (2016). The Central Nervous System and the Gut Microbiome. Cell, 167(4): 915–932. https://doi.org/10.1016/j.cell.2016.10.027
  8. Jiang, H., Ling, Z., Zhang, Y., Mao, H., Ma, Z., Yin, Y., Wang, W., Tang, W., Tan, Z., Shi, J., Li, L., & Ruan, B. (2015). Altered fecal microbiota composition in patients with major depressive disorder. Brain, behavior, and immunity, 48: 186–194. https://doi.org/10.1016/j.bbi.2015.03.016
  9. Bercik, P., Denou, E., Collins, J., Jackson, W., Lu, J., Jury, J., Deng, Y., Blennerhassett, P., Macri, J., McCoy, K. D., Verdu, E. F., & Collins, S. M. (2011). The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice. Gastroenterology, 141(2): 599–609. https://doi.org/10.1053/j.gastro.2011.04.052
  10. Kennedy, P. J., Clarke, G., Quigley, E. M., Groeger, J. A., Dinan, T. G., & Cryan, J. F. (2012). Gut memories: towards a cognitive neurobiology of irritable bowel syndrome. Neuroscience and biobehavioral reviews, 36(1): 310–340. https://doi.org/10.1016/j.neubiorev.2011.07.001
  11. Chevalier, G., Siopi, E., Guenin-Macé, L. et al. (2020). Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system. Nat Commun 11: 6363. https://doi.org/10.1038/s41467-020-19931-2
  12. Gupta, S., Allen-Vercoe, E., & Petrof, E. O. (2016). Fecal microbiota transplantation: in perspective. Therapeutic advances in gastroenterology, 9(2): 229–239. https://doi.org/10.1177/1756283X15607414

After Eureka Comes Death

April 30, 2021

As insulin prices skyrocket, diabetics turn to increasingly dangerous solutions to manage their illnesses

By Jesse Kireyev, History ‘21

Author’s Note: There’s an indescribable type of heartbreak that comes from hearing a close diabetic family member or friend tell you they cannot afford their next dose and won’t be able to for weeks. A day or two of missed insulin shots could easily end in death as it did for at least one individual mentioned in this article. It’s an especially American experience to be gripped in fear for your loved one’s life because the barebones that they need to survive is out of reach, and despite the relatively high prevalence of diabetes in the population, it’s an issue that’s starkly ignored. Mothers, fathers, siblings, and children wilting away in hospital rooms don’t grab headlines as easily as the latest political trauma, and so too often they get entirely ignored. While I chose to conclude the article on a hopeful note, it’s vital to emphasize that while we wait far too patiently for that hope to materialize, more diabetics die. Many of those who outwaited their hope will never have it again. Americans can’t wait much longer.

 

Oakland, California, has always been a hub of counterculture. Set in the heart of the Bay Area, Oakland has hosted dozens of America’s most famous hip hop musicians, visual artists, social justice advocates, and tech pioneers. This cultural backdrop has facilitated the creation of the Open Insulin Project: a communal space where diabetics and biohackers meet twice a week to try to create an open source guide that would let type 1 diabetics produce insulin at home. 

Diagnosis of this condition was once a death sentence. Prior to the discovery of artificially produced insulin, a child diagnosed with type 1 diabetes had only about a year or two to live after diagnosis [1]. Type 1 diabetes is an autoimmune disease that develops when the immune system starts treating beta cells in the pancreas as a threat and attacks them. The beta cells are responsible for insulin production, so as they are attacked, the pancreas becomes unable to produce insulin. The reasons for why this happens are unknown — current theories suggest environmental and hereditary factors may play a big role, but lifestyle factors do not seem to affect it significantly. This differs from type 2 diabetes, which causes your body to resist the effects of insulin rather than making it unable to produce it [2]. Insulin is a hormone that your body naturally produces to regulate the amount of glucose, or blood sugar, in your system. As type 1 diabetes develops and insulin production stops altogether, the body loses its ability to regulate blood sugar unless insulin is supplemented through injection. 

The grim sites of hospital wards full of dying children pushed three scientists at the University of Toronto—Sir Frederick Banting, Charles Best, and JJR Macleod—to discover insulin in 1921. The theory of injecting insulin to regulate blood sugar wasn’t developed until a year later by James Collip, but the first patients treated with insulin injections suffered from severe allergic reactions due to the insulin’s impurity. Collip discovered a way to purify it, putting insulin to use to save children dying from diabetic ketoacidosis—a dangerous diabetic complication that can often end in a coma or death that develops when the body, lacking glucose, begins to produce ketones that acidify the blood. Shortly after discovering and producing insulin, the researchers refused any compensation for their discovery and gave exclusive production rights to a chemical manufacturing firm in Indianapolis, which was to sell insulin at three cents per  unit. As a New York Herald article from 1923 put it, Sir Frederick Banting set out to make insulin “available for even the poorest sufferers from diabetes” [3]. Despite Banting’s intentions, the price of insulin has since soared.

The current cost to produce a single vial of human insulin is somewhere in the range of two-and-a-half to three-and-a-half dollars, and a year’s supply of the medicine could be sold to type 1 diabetes patients for as little as seventy-two dollars, a cost that is not only affordable to consumers but still produces a large profit margin for the pharmaceutical companies creating it [4]. And yet insulin prices remain far higher than they theoretically should be. A survey of medical prices conducted by the Health Care Cost Institute showed that in 2016 type 1 diabetics paid an average of $5,705 for insulin, over seventy-nine times the cost of what medical researchers believe they should be paying [5]. American diabetes patients paid an average of $300 for a vial of Humalog, a specific type of insulin, in 2019. That same vial costs just thirty-two dollars  in Canada [6]. As a result, more people are trying to fight back against these skyrocketing costs. 

Drug development can drain millions of dollars from a pharmaceutical company’s budget, meaning companies have to sell their drugs at high prices to recoup the costs of development. New developments to certain medications can temporarily raise their prices, which is a reason often used by pharmaceutical companies to defend their price increases, but that’s not the case with insulin. Dr. Nicholas Argento said in an interview with Business Insider, “The products that are out are not really new. They may have tweaked the manufacturing process and[…]they have better delivery pens and the like, but the increase in price has been astronomical” [7].

While the base drug itself hasn’t seen significant development in decades, the patent on it still remains, and keeps getting extended. “Drugs are kept on patent by making somewhat fairly small fluctuations or modifications to the particular thing, like insulin,” said Dr. Huising. This effectively prevents the production of a cheaper, generic version of the drug, leaving diabetics to rely on insulin produced by only three suppliers in the United States. Canadian patients are able to buy insulin at lower costs due to increased market price regulation, as well as the fact that Canada allows generic insulin into the market, side-stepping the patent issues that generic insulin would face in the United States. And the patents still have a while to go before expiring: just last year, French pharmaceutical company Sanofi, one of the three insulin suppliers in the United States, got its patent extended to 2031. The medical advocacy non-profit, Initiative for Medicine, Access, and Knowledge, points to this as one of the main culprits behind the price increases, stating in its 2018 report, “The U.S. cannot fix the drug pricing crisis until it solves the drug patent problem” [8]. 

When comparing the price of insulin produced by each of these three companies, it is clear that each company sells its product at almost the exact same price, and price increases between the three companies have remained almost identical over the past few decades. While the companies deny any collusion or price fixing, by law, pharmaceutical corporations are not required to provide the reasons behind price increases, and they can raise them without limit [9]. The issue has become so dire that in 2018, the Congressional Diabetes Caucus released a report stating that the current system is “unfairly putting insulin out of reach, placing millions of lives at risk” [10].

Placing millions of lives at risk is not an over exaggeration: without enough insulin, blood sugar rapidly increases. A diabetic with high blood sugar runs the immediate risk of developing diabetic ketoacidosis, alongside long-term cardiovascular and nervous system problems, which can significantly shorten lifespans. According to Dr. Kasia Lipska, an assistant professor at the Yale School of Medicine, “About 1 in 5 people with type 2 need insulin to prevent short-term and long-term complications like blindness, kidney failure, and dialysis and heart disease” [11]. For other types of non-gestational diabetes, the risk is more immediate: “Insulin is a life-saving drug, people need it,” says Dr. Huising. “If you have type 1 diabetes and no insulin, you die.”

These problems can emerge from missing just a few doses of insulin, something that many diabetics increasingly have to resort to due to its cost. Lipska et al. published a study in the journal JAMA Internal Medicine that found that over a fourth of diabetes patients have had to cut back on insulin dosage due to the high price of the medicine. The human costs of insulin prices are very real. In a CBS interview, the mother of a man who died because he couldn’t afford his medicine spoke out against the costs. Alec, a young man with diabetes, began to ration his insulin when faced with a $1,300 price tag. Unfortunately, after struggling under a diabetic coma, the lack of insulin cost him his life. “I wanted to be there with him, to hold his hand, or to call for help. And then I think about if he had never moved out, if he had lived at home, somebody would have seen the signs,” she said. “I’ll probably feel guilty every day for the rest of my life” [12].

It is within this context that a number of rogue diabetics in Oakland have begun to try to synthesize their own insulin supply. The Open Insulin Project was started in 2015 by Anthony Di Franco, a type 1 diabetic who struggled for years with being able to buy his insulin. The project utilizes biohacking—a movement that applies do-it-yourself, rule-averse hacker practices to the exploitation of genetic material—to create a homebrew form of insulin for type 1 diabetics. The project’s motives are exceptionally ambitious, given that almost none of the people involved are trained biochemists. Di Franco and other project leaders such as David Anderson lack experience in biological or chemical sciences, and neither work in relevant fields—Di Franco is a computer scientist, and David Anderson is pursuing a degree in business economics [13]. The Project aims to one day create a fully safe and functional form of insulin, and recent developments in the chemical process have shown some promise. Currently, the Project is hoping to convert proinsulin—insulins’ chemical precursor—to full insulin, after which they will attempt to scale their process up [14]. But some have their doubts. Hank Greely, a professor at the Center for Law and Biosciences at Stanford University, warns that “manufacturing pharmaceuticals is difficult, painstaking, and dangerous. If you get the dosing or the strength on the insulin wrong, it’s death. If you let contaminants into the insulin, it’s possible death. If your insulin breaks down too quickly in storage, it’s death” [13].

Dosing is a difficult challenge in a homebrew environment, where biohackers might not be able to access the proper equipment to create safe and stable insulin with consistent doses and without contaminants. Immunogenic reactions—wherein a foreign molecule entering the body provokes an immune system response—predominates the list of concerns over impurities. “You’re injecting something. If there is an impurity there that is a foreign molecule, then your immune system might start to respond,” Dr. Huising said. “Doing it at scale with a quality that is consistent is extremely challenging to do. It’s not that hard to make it if you’re a trained biochemist, but making it with a quality and consistency that is compatible with injecting it as a drug over multiple batches is hard to achieve.”  Nor does the Project have access to most of the biochemistry equipment Dr. Huising insists is necessary to create insulin safe for injection. “The motivation behind wanting to make insulin is clear,” he said. “But doing it homebrew style is just dangerous and irresponsible.” 

Due to this, the Open Insulin Project may face legal challenges from the FDA and other regulatory agencies, challenges that the Project may not have the money or resources to address.  This is not a problem that has escaped the minds of those running the Project, as they currently are trying to figure out the legal issues surrounding human testing and safety of human consumption. That is why the project has been focusing on creating a do-it-yourself guide to synthesizing insulin at home—while the FDA can regulate distribution of medicine; the first amendment stops them from regulating the distribution of a guide on how to make the medicine. 

But the situation may soon change enough that the need for the Open Insulin Project will fade away entirely. Over the past few years, the FDA has been pursuing paths to change insulin regulatory procedures, introduce generic insulin to the market, and lower the costs of the drug—policies that the last three presidential administrations have publicly advocated for. Dr. Huising has hope that this public pressure might help insulin prices fall within the next few years—“Even in the past couple of years, there has been talk in Washington about how big pharma does overcharge. I don’t think that’s necessarily a left or a right talking point,” he said. “There is a recipe there for improvement, where we demand that insulin is made available at prices that don’t force people to self censor or self limit how much insulin they dose themselves with.” 

 

References

  1. Editor. Diabetes history. Diabetes.co.uk. 2019 Jan 15. https://www.diabetes.co.uk/diabetes-history.html
  2. Causes of type 1 diabetes – JDRF. Jdrf.org. 2017 Oct 17. https://www.jdrf.org/t1d-resources/about/causes/
  3. Moulton Weekly Tribune. Newspaperarchive.com. https://moultonpl.newspaperarchive.com/moulton-weekly-tribune/1923-12-07/page-8/
  4. Gotham D, Barber MJ, Hill A. Production costs and potential prices for biosimilars of human insulin and insulin analogues. BMJ global health. 2018;3(5):e000850.
  5. U.S. insulin costs per patient nearly doubled from 2012 to 2016: study. Reuters. 2019 Jan 22. https://www.reuters.com/article/us-usa-healthcare-diabetes-cost-idUSKCN1PG136
  6. Goldman B. The soaring cost of insulin. CBC News. 2019 Jan 28. https://www.cbc.ca/radio/whitecoat/blog/the-soaring-cost-of-insulin-1.4995290
  7. Business Insider. Why insulin is so expensive. 2019 Feb 12. https://www.youtube.com/watch?v=7Ycd8zEdoVk
  8. I-mak.org. 2018. https://www.i-mak.org/wp-content/uploads/2018/08/I-MAK-Overpatented-Overpriced-Report.pdf
  9. Thomas K. Drug makers accused of fixing prices on insulin. The New York times. 2017 Jan 30. https://www.nytimes.com/2017/01/30/health/drugmakers-lawsuit-insulin-drugs.html
  10. Skyrocketing insulin cost: Congressional Diabetes Caucus highlights need and ways to bring prices down. House.gov. 2018 Nov 1. https://diabetescaucus-degette.house.gov/media-center/press-releases/skyrocketing-insulin-cost-congressional-diabetes-caucus-highlights-need
  11. Adam.com. http://pennstatehershey.adam.com/content.aspx?productId=35&gid=4470
  12. CBS This Morning. Mother says son died “because he could not afford his insulin.” 2019 Jan 4. https://www.youtube.com/watch?v=Zp_1ohad0Tg
  13. Osterath B. Deutsche Welle (www. dw.com). 2019. Do-it-yourself insulin: Biohackers aim to counteract skyrocketing prices. https://www.dw.com/en/do-it-yourself-insulin-biohackers-aim-to-counteract-skyrocketing-prices/a-48861257
  14. Di Franco A. New frontiers for the New Year. Openinsulin.org. 2018 Dec 31. https://openinsulin.org/our-blog/new/

COVID-19 Testing: Three Tools for Public Health

April 23, 2021

By Jessica Lee, Biochemistry & Molecular Biology ‘21

Author’s Note: Inspired by the success of the asymptomatic testing at UC Davis, I wrote this article exploring the different types of diagnostic and antibody tests for SARS-CoV-2, focusing on mechanisms and relative sensitivities and specificities. 

 

The COVID-19 pandemic has demonstrated the importance of widespread and accurate diagnostic testing in controlling community spread. Together with mask mandates, social distancing, and quarantining, COVID-19 testing can slow the spread of a disease that has killed over 2.5 million people [1]. Approximately 80 million COVID-19 tests have been reported at this time, and yet, many people are confused as to how COVID-19 tests work and how each type of test differs in mechanism, sensitivity, and specificity [2]. 

Depending on the type of COVID-19 diagnostic test, they will either detect SARS-CoV-2 nucleic acid, protein, or antibodies generated as a consequence of infection [3]. Samples can be collected from patients via nasal swab, saliva collection, or blood collection [3]. This article will review current COVID-19 tests as well as address potential confusion arising from false negative and false positive results. 

 

Diagnostic Tests

Diagnostic tests are administered to patients to determine if they are infected with SARS-CoV-2 at time of sampling. Such tests may be administered to either symptomatic or asymptomatic patients as a diagnostic tool or preventative public health measure. Various types of diagnostic tests have been developed since the emergence of SARS-CoV-2 as an infectious agent; however, all tests rely on one of two underlying technologies [4]. Molecular tests detect segments of the viral genome while antigen tests detect the presence of viral proteins [4]. However, both molecular tests and antigen tests differ from antibody tests which detect previous SARS-CoV-2 infections. 

 

Molecular Tests

Molecular tests primarily rely on polymerase chain reaction (PCR) technology to detect relatively low quantities of SARS-CoV-2 genome [4]. A PCR has four primary components: DNA template, DNA-dependent DNA polymerase, primers, and nucleotides [5]. SARS-CoV-2 has a positive-sense single stranded RNA genome meaning the genome can directly be translated into protein by host translation machinery [6]. However, since PCRs require a DNA template, the genome must be converted from RNA to DNA via the enzyme reverse transcriptase [5]. Due to this reverse transcription step, this specific type PCR is called reverse transcription, or RT-qPCR. The resulting DNA product then serves as the template for PCR during which a DNA-dependent DNA polymerase recognizes the template and synthesizes the complementary strand via incorporation of nucleotides. The primers, short DNA fragments about 20 nucleotides in length, are the component of PCR that confers specificity to the assay through their complementarity to a specific region in the DNA template [5]. When all reaction components are combined and cycled through specific temperatures, the result is the exponential increase in the number of copies of the target DNA [5]. Thus, the assay can produce a positive result with very small quantities of original template. 

The U.S. Centers for Disease Control (CDC) has established two sets of oligonucleotide primers for the detection of the nucleocapsid (N) gene of SARS-CoV-2 [7]. Thus, if SARS-CoV-2 genome is present in a sample, the primers will hybridize to the N gene, resulting in the amplification of the region and yielding a positive result. Other primers for the detection of the RNA-dependent RNA polymerase and envelope genes have also been developed by the World Health Organization (WHO) [7]. Since the primers are essential to establishing high specificity, it is important that the primers only bind to the intended target. If the primer is too promiscuous, the assay might produce a false positive. Alternatively, if the primer is too stringent, the assay might produce a false negative. There is also concern that if the region in which the primers bind mutates, the assay may no longer consistently detect such mutants [7].

Samples for molecular tests may be collected via nasal swab or saliva and may be pooled for more efficient testing [8]. If a sample pool is positive for SARS-CoV-2, the individual samples are then tested to determine which individuals are positive. Care must be taken, however, with pooled sampling because samples with low viral loads may not be detected due to decreased sensitivity [8]. 

Molecular tests are generally characterized by high sensitivity, high specificity, and moderate price (~$100/test), earning their status as the “gold standard” of COVID-19 testing [9]. However, depending on the laboratory, results may take up to a week to be returned to the patient [3].

 

Antigen Tests

Antigen tests are immunoassays that detect viral proteins by binding viral protein to SARS-CoV-2 antibodies [10]. Generally, antibodies specific to the N protein are produced and purified for use in antigen tests [10]. There is more variation in how antigen tests work as compared to molecular tests; however, generally, the antibodies are conjugated to a tag which can either be read by a machine or is visible to the naked eye. The Abbott BinaxNow COVID-19 Ag Card is even similar to an over-the-counter pregnancy test–a positive result is indicated by a line in a test window [10]. Samples are collected via nasal or nasopharyngeal swab and are flowed over test strips loaded with conjugated antibodies [3,9]. 

Antigen tests often produce results within 15-30 minutes–much faster than many of the molecular tests [3]. Thus, many health care providers use antigen tests as point-of-care “rapid” tests [9]. Although antigen tests are generally cheaper than molecular tests at about $5-50 per test and are highly specific, they are moderately less sensitive than molecular tests [9]. If the antigen level of a specimen is low due to collection before symptom onset or in late infection, a false negative may be more likely than if a molecular test was administered [9]. However, for both molecular and antigen tests, the probability of a false positive is low due to their high specificity [9]. 

 

Antibody Tests

Instead of diagnosing active SARS-CoV-2 infections, antibody tests indicate if the patient has an adaptive immune response to a SARS-CoV-2 infection [3]. Samples are collected via fingerstick–a device which pricks the finger to produce a few drops of blood–or blood draw and detect the presence of antibodies generated by SARS-CoV-2 infection [3]. There are many different types of antibody tests, but all can be sorted into two categories: binding antibody detection or neutralizing antibody detection tests [11]. Assays that detect antibodies through binding of an antigen use purified spike or nucleocapsid protein from SARS-CoV-2 to determine if the patient has previously been infected [11]. The mechanism of these tests is very similar to antigen tests which also depend on the selective binding of antibodies to antigens. Neutralizing antibody detection tests determine the ability of antibodies in a sample to prevent infection in cell culture [11]. This category of tests give a more accurate assessment of a patient to resist reinfection.    

 The CDC’s binding antibody test uses purified spike protein for the detection of IgG and IgM antibodies [12, 13]. While both IgG and IgM antibodies are usually generated 1-3 weeks after exposure to an antigen, IgG antibodies persist longer–often months post infection [11]. Due to the lag between peak viral production and peak antibody production, antibody tests cannot be used as diagnostic tools; however, they are useful for identifying recovered individuals for surveillance purposes[13]. Furthermore, widespread serology surveillance allows public health officials to monitor where COVID-19 cases are concentrated and inform public health policy [14].  

Some antibody tests have high specificity and sensitivity; although, this is highly dependent on the type of test and period of time post infection. Tests may result in a false negative if there has not been sufficient antibody production or if antibody levels have decreased below the limit of detection [13,11]. For example, if one compares a test that only detects IgM antibodies to a test that only detects IgG antibodies, one might see differential results if administered months after infection. Some antibody tests have a slight cross-reactivity with SARS1 and MERS-CoV sera which could result in false positives; however, there is minimal cross-reactivity with commonly circulating coronaviruses [15]. While nucleic acid detection tests remain the gold standard for diagnosis of acute SARS-CoV-2 infection, antibody tests can be valuable tools for clinical and surveillance efforts [12]. 

 

Asymptomatic Testing 

Many U.S. colleges have implemented regular, asymptomatic testing of their students and employee populations [16]. Regular testing can allow early cases of COVID-19 to be identified and allow for efficient contact tracing, limiting the spread of COVID-19. At UC Davis, students and employees who go on campus are required to get tested at least every week at the free asymptomatic testing clinic [17]. With the ability to screen thousands of samples each day, UC Davis has expanded its saliva testing program to include the greater Davis community, catching at least 850 asymptomatic cases that otherwise might have spread to other individuals [17]. 

Clearly, the importance of widespread testing of both symptomatic and asymptomatic individuals cannot be understated. Testing is one of the most important tools public health officials have to monitor and control the ongoing pandemic.

 

References

  1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Accessed April 13, 2021. Available from: https://covid19.who.int/. 
  2. Centers for Disease Control and Prevention. COVID Data Tracker: United States Laboratory Testing. Accessed April 13, 2021. Available from: https://covid.cdc.gov/covid-data-tracker/#testing_totalpositivity.
  3. U.S. Food and Drug Administration. Coronavirus Disease 2019 Testing Basics. Accessed April 13, 2021. Available from: https://www.fda.gov/consumers/consumer-updates/coronavirus-disease-2019-testing-basics#:~:text=There%20are%20two%20different%20types,tests%20and%20antibody%20tests.
  4. U.S. Food and Drug Administration. A Closer Look at COVID-19 Diagnostic Testing. Accessed April 13, 2021. Available from: https://www.fda.gov/health-professionals/closer-look-covid-19-diagnostic-testing.
  5. NCBI. Polymerase Chain Reaction (PCR). Accessed April 13, 2021. Available from: https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/.
  6. Ahlquist P, Noueiry AO, Lee WM, Kushner DB, Dye BT. 2003. Host factors in positive-strand RNA virus genome replication. J Virol [Internet]. 77(15), 8181–8186. https://doi.org/10.1128/jvi.77.15.8181-8186.2003.
  7. Wang R, Hozumi Y, Yin C, Wei GW. 2020. Mutations on COVID-19 diagnostic targets. Genomics [Internet]. 112(6):5204-5213. doi:10.1016/j.ygeno.2020.09.028.
  8. Centers for Disease Control and Prevention. 2020. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Available from: https://www.fda.gov/media/134922/download.
  9. Centers for Disease Control and Prevention. 2020. Interim Guidance for Antigen Testing for SARS-CoV-2. Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-guidelines.html.
  10. Andrea Prinzi, MPH. 2020. How the SARS-CoV-2 EUA Antigen Tests Work. American Society for Microbiology. Available from: https://asm.org/Articles/2020/August/How-the-SARS-CoV-2-EUA-Antigen-Tests-Work.
  11. Centers for Disease Control and Prevention. 2021. Interim Guidelines for COVID-19 Antibody Testing. Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antibody-tests-guidelines.html.
  12. Centers for Disease Control and Prevention. 2020. Serology Testing for COVID-19 at CDC. Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/serology-testing.html. 
  13. U.S. Food and Drug Administration. 2020. Serology/Antibody Tests: FAQs on Testing for SARS-CoV-2. Available from: https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/serologyantibody-tests-faqs-testing-sars-cov-2. 
  14. Centers for Disease Control and Prevention. 2021. COVID-19 Serology Surveillance Strategy. Available from: https://www.cdc.gov/coronavirus/2019-ncov/covid-data/serology-surveillance/index.html.
  15. Freeman B, Lester S, Mills L, et al. 2020. Validation of a SARS-CoV-2 spike protein ELISA for use in contact investigations and serosurveillance. Preprint. bioRxiv. 2020;2020.04.24.057323. doi:10.1101/2020.04.24.057323.
  16. Anderson, N. 2020. Welcome to college. Now get tested for the coronavirus — again and again. The Washington Post. Available from: https://www.washingtonpost.com/local/education/welcome-to-college-now-get-tested-for-the-coronavirus–again-and-again/2020/09/04/2d087722-ed2f-11ea-b4bc-3a2098fc73d4_story.html.
  17. Hubler, S. 2021. A California University Tries to Shield an Entire City From Coronavirus. The New York Times. Available from:  https://www.nytimes.com/2021/01/30/us/college-coronavirus-california.html.

 

Online References

  1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Accessed 2021. 
  2. Centers for Disease Control and Prevention. COVID Data Tracker: United States Laboratory Testing. Accessed 2021. 
  3. U.S. Food and Drug Administration. Coronavirus Disease 2019 Testing Basics. Accessed 2021.
  4. U.S. Food and Drug Administration. A Closer Look at COVID-19 Diagnostic Testing. Accessed 2021. 
  5. NCBI. Polymerase Chain Reaction (PCR). Accessed 2021. 
  6. Ahlquist P, et al. 2003. J Virol [Internet]. 77(15), 8181–8186. 
  7. Wang R, et al. 2020. Genomics [Internet]. 112(6):5204-5213. 
  8. Centers for Disease Control and Prevention. 2020. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. 
  9. Centers for Disease Control and Prevention. 2020. Interim Guidance for Antigen Testing for SARS-CoV-2. 
  10. Andrea Prinzi, MPH. 2020. American Society for Microbiology. 
  11. Centers for Disease Control and Prevention. 2021. Interim Guidelines for COVID-19 Antibody Testing. 
  12. Centers for Disease Control and Prevention. 2020. Serology Testing for COVID-19 at CDC. 
  13. U.S. Food and Drug Administration. 2020. Serology/Antibody Tests: FAQs on Testing for SARS-CoV-2. 
  14. Centers for Disease Control and Prevention. 2021. COVID-19 Serology Surveillance Strategy. 
  15. Freeman B, et al. 2020. Preprint. bioRxiv. 2020;2020.04.24.057323. 
  16. Anderson, N. 2020. The Washington Post.
  17. Hubler, S. 2021. The New York Times.

Talking with a pediatric oncology nurse about COVID-19 and how it has forever changed the culture of oncology healthcare

April 16, 2021

By Grace Wensley, Biological Sciences ‘21

Author’s Note: As I saw how the COVID-19 pandemic has so greatly affected the elderly population and immunocompromised adults, I wondered why there wasn’t as much as a conversation about immunocompromised children. I interviewed a pediatric oncology nurse working at Children’s Hospital in Oakland, California, and discovered how difficult the pandemic has been on pediatric oncology patients and the healthcare culture shift that has emerged from it. 

This pandemic has taken quite a toll on the healthcare system, and this interview displayed the hardships faced among the pediatric oncology sector. Nurse Kelli Hemmingsen-Smith  discusses how various COVID-related protocols can disrupt pediatric oncology patients’ treatments. Additionally, she describes the emotional impact of the pandemic from limited visitors allowed, a scary environment due to all doctors and nurses wearing masks, and extended inpatient stays for some patients.

 

This interview has been lightly edited for clarity and brevity.

 

Health Care Practices Pre-COVID-19

Grace Wensley: When would your oncology patients wear masks prior to COVID?

Kelli Hemmingsen-Smith, RN: I’m trying to remember what life was like before we wore masks. Normally we would ask them to wear a mask any time they were neutropenic—so any time their absolute neutrophil count (ANC), the amount of white blood cells that are actually neutrophils, was under 500. Part of wearing a mask is for [going] outside because of the spores found in the dirt, on top of sick contacts. We try to be a bit more lenient so that kids can have somewhat of a normal life. But, 99 percent of kids have a central line (a catheter placed in a large vein for fast blood draws and drug administering) so what happens at our hospital is if you have a fever and you have a central line, you have to come to the hospital, get a dose of antibiotics that last for 24 hours and then wait. If you get another fever you do the same thing again. If you get to the hospital and you have a fever, and you’re neutropenic, you have to be admitted and stay until your fever resolves and your counts recover which could take weeks. So while we aren’t requiring kids to wear masks in certain situations [prior to COVID] a lot of family members will enforce mask-wearing because if your kid gets a fever, we know it’s probably a virus, but we can’t risk it so this is how we treat it. If it’s not a virus though, kids go septic really fast so they’ve kind of taken it into their own for mask-wearing.

GW: When these patients are neutropenic is that usually due to their chemotherapy? 

KH: Yes, pretty much all the time. Kids get their chemo. About 7-10 days later, they reach the nadir, which is where your white blood cell counts are at the lowest that they should be, and then they slowly start to recover after that. Also, if they’re sick, that can also cause them to be neutropenic because their immune systems are non-functioning, especially in the beginning days of leukemia, a cancer of your immune system. We can have kids who are waiting for their counts to recover before they start certain chemo, and they can be delayed because their counts aren’t recovering. Sometimes, it can just be because they have a cold. 

GW: Do patients’ counts have to recover between each dose of chemo? 

KH: Depends on the chemo. Not always. Depending on where they are in their cycles. 

GW: If their counts aren’t recovering, and they keep getting sick and can’t recover, does their treatment get delayed? 

KH: Yes. So mask-wearing is a big deal. Like I said, although we required [mask wearing] only for certain instances, kids getting sick that are in treatment is a huge deal and parents are very aware of that.

GW: In these situations of patients being required to wear masks pre-COVID, would their doctors and nurses also be wearing masks? 

KH: Pre-COVID, never. I would actually be very surprised if our culture didn’t change in the fact that we always wear a mask to be perfectly honest, because when you really think about it, it does make sense. When they are neutropenic in the hospital, they get a private room. Our unit has an entire HEPA-filtered unit, so instead of them having to stay in their rooms they can come out, and there is a playroom. Prior to COVID, the only thing that changed from a nursing standpoint if the kid was neutropenic was what room they were put in and that was it. 

 

Healthcare Practices During COVID-19

GW: Has there been a big delay in cancer treatment schedules due to how the hospital has had to adjust infrastructure with COVID? 

KH: We never limited anything. But the only way that it has really impacted our kid’s treatment is when they are getting procedures. They have to have been tested for COVID within four days of their procedure, and for a lot of our patients, it is hard to get to us twice—it is hard enough to get to us once. We do have a lot more people missing procedures, or we don’t get the results of the COVID tests back fast enough. Now, it’s a little different, because we have a rapid in-house test that takes 2-3 hours. Now, if you missed [the test] and didn’t come, we can usually make it work, but in the beginning, that wasn’t always the case. 

I think where we see the delay the most is if a kid’s family gets COVID. In that case, we hold [off on] chemo because the patient could then test positive for COVID. We automatically hold chemo, get a COVID test, and while they are doing the quarantine we are still holding chemo because we just don’t know. That is 14 days. Then if they get it, that’s another 14 days that we are on hold. These are kids that are usually at the end of their treatments [in clinic] which is what we call the maintenance phase which is where you take oral chemo every day. So a month of not taking it is a lot. How does that affect survival, relapse? We don’t know.

GW: Have there been instances where you admit a patient from the clinic to inpatient out of fear that by going home, they could potentially get COVID?

KH: Yes. We had a kid whose housing situation doesn’t allow them to fully quarantine so he just stayed inpatient until we could find a safe place for him to go. We had another patient who was going for a bone marrow transplant, and her family got COVID. We had to hold her marrow transplant until we knew that she had been away from them long enough. She stayed inpatient, and no one could come to visit. But what was really interesting, was once they did the transplant, she got COVID. So we don’t know if it was just “chilling” and when we blasted her immune system it came on. So there is a lot that we don’t know. Would it be fine for the kids to take oral chemo the whole time until their ANC dropped? Maybe. But nobody knows.

GW: Have any of your patients had COVID?

KH: Yes, and what is very interesting about the kids who have gotten it is that the younger kids have had pretty much zero symptoms, and the only reason we know that they have it is because they have to be tested prior to anesthesia. They get anesthesia a lot because they get chemo through lumbar punctures [spinal tap] which we administer anesthesia for. We automatically hold chemo when they test positive. When their symptoms resolve we restart chemo. A lot of time during that time, their ANC will drop due to the virus, and then we wait longer if their ANC did drop to restart the chemo until it was at the right numbers. They haven’t really been super symptomatic.  I’ve noticed that a couple of the kids that have been getting it have elevated liver enzymes, but that can be caused by chemo too, so it’s kind of one of those where there isn’t really a correlation. I’ve noticed it with two kids. 

GW: What is the visitor policy currently? 

KH: Inpatient is one person per patient, and that person can alternate and stay overnight with them. In the clinic, we only allow one parent at a time. Before COVID, anyone could come—we couldn’t care less. Now in the clinic, it’s no siblings and only one parent, unless it’s a consent conference or a diagnosis, then we make allowances. 

GW: Do you think the strict visitor policy will remain post-COVID?

KH: I would hope that would change because that’s a big hardship. If you have a kid that’s young, and can’t really be left alone, then the parents don’t ever get an overlap period to communicate things like, “Hey this cup is what he’s been taking his medicine in.” I would hope that would change. They can’t see their siblings and that’s a big deal for kids.

GW: How have wearing masks affected patient and provider care? 

KH: Even now, anytime an employee is in their room, they all have to put masks on. Us being in masks and gaining rapport with children who are really scared of us is really hard. It’s such a simple thing, but we give a lot of toys now. They can’t go in the playroom. There are no volunteers. When kids are awaiting COVID results prior to surgery, it sometimes takes hours to get them back and that whole time they are fasting. Kids get grumpy. While waiting for surgeries, a lot of the time one parent is outside sitting in the car all day on speaker. It’s hard. 

 

Conclusion:

The feeling I observed from the entire interview was that many of these hardships related to COVID-19 will in time go away, but mask-wearing is here to stay. It took a global pandemic to make light of it, but any type of illness can significantly affect the trajectory of a patients’ treatment and masks can help prevent this, so as Hemmingsen-Smith said, “It just makes sense.”

Epigenetics as a Tool for Personalized and Targeted Care

April 9, 2021

By Parmida Pajouhesh, Neurobiology, Physiology & Behavior ‘23

Author’s Note: For as long as I can remember I wanted to attend medical school and become a pediatrician. More recently, I have been exposed to the study of epigenetics, which has unveiled the importance of prioritizing prevention of disease and furthered my interest in the field of medicine. In hopes of practicing internal medicine in the future, I wanted to investigate how providing care and support to the patient on an individualized level is crucial for effective treatment in the long term. 

 

Put simply, epigenetics means “on top of” genetics, derived from the Greek prefix “epi.” Epigenetics describes the relationship between one’s gene activity and their environment, whereby the activity of the genes is altered but the DNA sequence is not directly modified. Genes can be turned “on” or “off” and changes to our epigenome are fortunately reversible. Researchers studying this phenomenon have been trying to answer the following questions: how does our knowledge of epigenetics help to further advance the study of illness and disorders, such as cancer and schizophrenia? How can understanding this phenomenon help scientists, doctors and researchers provide personalized care for patients?

Epigenetic changes, which impact our phenotype without directly affecting our genotype, have been previously tied to aging, our environment and even lifestyle [1]. Prenatal and early postnatal environmental factors have influenced an offspring’s risk and vulnerability to developing a health condition [2]. For instance, in the 1940s, children who were born during a period of increasing famine had exceedingly greater rates of coronary heart disease after maternal exposure to famine, as opposed to those not exposed. This was linked to a decrease in DNA methylation—an epigenetic alteration—of the gene responsible for insulin growth [2]. 

These epigenetic modifications continue to take place throughout an individual’s lifespan. Exposure to pollution can alter methyl tags on DNA and make an individual even more susceptible to neurodegenerative diseases [3]. The foods we consume can have an impact on our epigenome; one study has shown that a high-fat, low-carb diet could open up chromatin and thus improve mental ability [1]. 

 

A closer look into our epigenome

Common and widely studied epigenetic alterations include histone modifications and DNA methylation. These mechanisms regulate the expression of genes as well as “cellular and biological functions related to homeostasis, allostasis and disease” [4]. DNA methylation adds a methyl group (-CH3) to cytosine at a promoter region containing repetitive sequences of CpG (cytosine–phosphate–guanine) dinucleotides. Proteins then bind to the methylated CpG islands, which correlates with transcriptional repression and affects gene expression [4]. This form of methylation has been previously linked to cancer [5]. 

As for the modification of histones, ubiquitin—a molecule which is attached to a protein destined for degradation—has been linked to neurological disorders including Parkinson’s disease and Angelman syndrome [5]. Likewise, histone methylation has been linked to several biological processes: DNA repair, stress responses, development, differentiation and aging [4]. If any one of these processes is altered, whereby histone methylation is either activated or inhibited, this can result in the development and progression of disease. For example, H3K4me2, a post-translational dimethylation at the lysine 4 residue of the histone H3 protein, located at the promoter of active genes, is downregulated in cases of lung, kidney, prostate and pancreatic cancers [6]. Histone modification contributes to the cell cycle, growth, DNA replication and other processes. Therefore, abnormal histone modifications can lead to the development and progression of tumors [6]. 

 

Personalized medicine and targeted care

Inevitably, individuals are prone to changes in their epigenome, which makes providing targeted care an even more challenging task. These changes occur not only between individuals but also within a single individual over time. Therefore, genomic approaches that include identifying specific variations in DNA and RNA sequences can help to bridge the gap between epigenetics and personalized care. Health care professionals have shifted their attention towards diagnostic tests that use genomic data to more accurately assess the extent of a patient’s risk for disease or illness, to determine appropriate dosage amounts and to make conclusions about the benefits of a specific drug or treatment. According to a study by Mahmood Rasool that highlights epigenetics as a contributing factor to personalized and individualized care, “various factors such as nutrition, age, body weight, sex, genetic behavior, infections, co-medications and organ function are important considerations that are unavoidable during the course of treatment for a disease” [4]. 

 

Combatting epigenetic changes. What’s next?

Changes due to our environment are unpredictable; therefore, we must take precautions as early as possible. Being wary of how our lifestyle can impact the activation of our genes is crucial to our health and development. We must take preventative measures early in our life and determine which lifestyle changes will benefit us in the long term. Exposure to hazards in our environment is not fully noticeable until years and sometimes decades later. If we place emphasis on the prevention of disease early on, we are much less likely to encounter abrupt and irreversible effects to our well-being. While epigenetic biomarkers are being evaluated for use in environmental risk assessment, more immediate lifestyle changes include reducing exposure to harmful air pollutants, implementing specific dietary changes and altering medication use that will provide long-term benefits, as opposed to only short-term relief [4]. Our diet can result in profound changes in our epigenome, leading to human disease. For instance, lacking essential amino acids in your diet can result in colon cancer, which “impairs biosynthesis of the active precursor for DNA methylation.” Similarly, exposure to nicotine and other toxins can cause epigenetic changes in smokers, “affecting the genes involved in normal pulmonary function.” Exercise can also have important effects on the skeletal-muscle epigenome [7].

With this being said, we must recognize the importance of integrative medicine in primary care. Physicians, and even specialists, consider the patient as a whole person and are cognizant of their lifestyle, diet, genetic background and even mental health. This holistic approach to medical care provides patients with a greater sense of what they need to accomplish to keep their body and mind healthy. This not only strengthens the connection between practitioner and patient, but it demonstrates the importance of taking preventative measures prior to development of the illness. Understanding epigenetics can increase our awareness of how physical space alters our well-being and reinforce that providing holistic and preventative care reduces the negative impacts of epigenetic changes. 

 

References (online)

  1. Baccarelli, Andrea, and Valentina Bollati. “Epigenetics and environmental chemicals.” Current opinion in pediatrics vol. 21,2 (2009): 243-51. doi:10.1097/mop.0b013e32832925cc
  2. Heerboth, Sarah et al. “Use of epigenetic drugs in disease: an overview.” Genetics & epigenetics vol. 6 9-19. 27 May. 2014, doi:10.4137/GEG.S12270
  3. Moosavi, Azam, and Ali Motevalizadeh Ardekani. “Role of Epigenetics in Biology and Human Diseases.” Iranian biomedical journal vol. 20,5 (2016): 246-58. doi:10.22045/ibj.2016.01
  4. Rasool, Mahmood et al. “The role of epigenetics in personalized medicine: challenges and opportunities.” BMC medical genomics vol. 8 Suppl 1,Suppl 1 (2015): S5. doi:10.1186/1755-8794-8-S1-S5
  5. Tollefsbol, Trygve O. “Dietary epigenetics in cancer and aging.” Cancer treatment and research vol. 159 (2014): 257-67. doi:10.1007/978-3-642-38007-5_15
  6. Li, Simin, et al. “Association between H3K4 Methylation and Cancer Prognosis: A Meta-Analysis.” Thoracic Cancer, vol. 9, no. 7, 2018, pp. 794–99. Crossref, doi:10.1111/1759-7714.12647.
  7. Feinberg, Andrew P. “The Key Role of Epigenetics in Human Disease Prevention and Mitigation.” New England Journal of Medicine, edited by Dan L. Longo, vol. 378, no. 14, 2018, pp. 1323–34. Crossref, doi:10.1056/nejmra1402513.

 

References (print)

  1. Baccarelli and Bollati. Current opinion in pediatrics vol. 21,2 (2009): 243-51. 
  2. Heerboth, et al. Genetics & epigenetics vol. 6 9-19. 27 May. 2014.
  3. Moosavi and Ardekani. Iranian biomedical journal vol. 20,5 (2016): 246-58. 
  4. Rasool, et al. BMC medical genomics vol. 8 Suppl 1,Suppl 1 (2015): S5. 
  5. Tollefsbol. Cancer treatment and research vol. 159 (2014): 257-67. 
  6. Li, et al. Thoracic Cancer, vol. 9, no. 7, 2018, pp. 794–99. 
  7. Feinberg. New England Journal of Medicine, edited by Dan L. Longo, vol. 378, no. 14, 2018, pp. 1323–34.

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