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Evaluating the Implications of COVID-19 on Patients with Sickle Cell Disease

by Harveen Kaur

Abstract

The presence and rapid spread of the novel infectious disease COVID-19, caused by the respiratory pathogen/coronavirus SARS-CoV-2, has encompassed the world as a global pandemic. COVID-19 spreads contagiously through air and droplets of bodily fluids, such as saliva, and has an array of symptoms associated with its contraction and spread, leading to approximately 364 cumulative coronavirus-related hospitalizations per 100,000 individuals only in the United States during early 2021. Additionally, COVID-19 cases are often more severe in individuals that have pre-existing medical conditions, with sickle cell disease being at the top of the list. Sickle cell disease, also referred to as sickle cell anemia, is an inherited blood disorder that abnormally distorts the structure and function of hemoglobin in the body leading to oxygen deficiency. This medical condition has been associated with increasing the risk of developing a severe case of COVID-19, but a definite connection has not yet been confirmed that categorizes the risk factor for various age groups. Studies published in September 2020 report that an international registry, called SECURE-SCD, has been created to track patients suffering from both conditions and their respective health outcomes. By using the data from various documented studies, this article hypothesizes that there is an important connection between sickle cell anemia and COVID-19 that leads to enhanced symptoms and overall higher fatality in patients suffering from the two. Hence, the primary purpose is to dissect and evaluate the specific linkage between the SARS-CoV-2 coronavirus and sickle cell anemia including the risk factors for both medical conditions experienced simultaneously and based on key demographic categories. Establishing the connection between sickle cell disease and COVID-19 is significant to better understand the implications of the virus for those with pre-existing conditions and potentially adjust treatment protocols that are currently being discovered.

Introduction

Infectious diseases have proven to be the ‘silent killer’ of the world throughout history. Ranging from the Black Death bubonic plague, the Spanish flu, and the swine-origin influenza A (H1N1) virus, the slow but steady upward  trend of infectious disease occurrence  enforces the fact that a human-dominated world can never thrive with imbalances in nature [1]. Many of these infectious diseases are zoonoses, diseases in animals that are transmitted to human beings, while others are classified as viruses transmitted by human beings to other human beings [2]. Virulent microorganisms have been present  among humankind for thousands of years, but it is only within the past couple of centuries that they have caused a huge leap in mortality. The most recent example of a globally destructive pathogen is the SARS-CoV-2 coronavirus, which causes the respiratory syndrome [2]. 

COVID-19, a respiratory illness that the world has been facing as a pandemic for over one year, is a contagious infectious disease stemming from the SARS-CoV-2 respiratory pathogen with the most common symptoms as fatigue, cough, and fever [3]. According to the CDC, the virus primarily spreads through respiratory droplets that arise through coughing and/or sneezing [3]. However, recent studies and speculation have stated that transmission may be airborne due to the presence of SARS-CoV-2 viral RNA in air samples within droplet nuclei that stay infectious if left in the air over long distances and large amounts of time [4]. Currently, there is no definitive cure for COVID-19, but many countries including the United States have implemented methodologies to track and contain the virus, especially through vaccine development. Specifically, roughly 78 vaccines for COVID-19 are undergoing clinical trials, with seven vaccines (such as those developed by Pfizer-BioNTech and Moderna) already distributed for full use [5]. These highly infected countries have also encouraged individuals to socially distance themselves from others by at least six feet and wear masks, both of which help control daily cases and fatalities from COVID-19 [6]. Despite these preventative measures, the implications of COVID-19 have affected millions of individuals in the United States and on a global scale. Vulnerable populations that already suffer from pre-existing medical conditions are at high risk to develop and contract a severe case of COVID-19. One such diagnosis that has caused an uproar in the United States is sickle cell disease.

Sickle cell disease (SCD), also called sickle cell anemia, consists of a group of inherited disorders that changes the shape of red blood cells from disc-shaped to crescent-shaped when atypical hemoglobin is present [7]. Through this change, red blood cells are more prone to blocking blood flow and do not move easily in the body, which can lead to an onset of chronic conditions such as strokes and other pain crises [7]. With the spread of COVID-19, however, those suffering from SCD have been studied to have a higher rate of contraction, as well as more severe symptoms and outcomes when coupled with the virus [8]. For example, SCD patients with COVID-19 have a higher overall case fatality (10.9% fatality rate) than non-SCD individuals contracting the virus (3.3% fatality rate) [9]. Patients suffering from SCD have cardiopulmonary comorbidities that COVID-19 heightens, but the specific linkage between the two has yet to be closely compared. However, certain unmistakable trends connecting patient’s age, race and severity of SCD symptoms to their COVID-19 prognosis have been observed [8]. This article focuses primarily on the results from various national and international registries that collect information about COVID-19 and SCD by comparing the effects of suffering from both conditions together, based on age groups and other patient demographics. Establishing and characterizing the connection between the two conditions can create a more accurate solution on how to protect oneself from COVID-19 when suffering from SCD.  

Materials/Methods

The connection between SCD and COVID-19 transmission and symptoms, when specifically focusing on age and race, can be outlined through the data found in three studies. 

The first study, published on the Centers for Disease Control and Prevention, was conducted from March to May 2020 in the United States and was recorded in the Medical College of Wisconsin SECURE-SCD Registry [10]. This particular registry examines COVID-19 cases among those living with SCD in the United States, and the results of the study were limited to a two-month time period in which such patients were analyzed and documented. The study primarily focused on the average age of these patients, the number of SCD-related complications these patients have had in the past, and the resulting intensity of their current COVID-19 symptoms [10].

Another study, published in the journal Haematologica, referenced 10 patients in the UK who had contracted SARS-CoV-2, and all 10 patients had hemoglobin SS disease, which is the most common and most severe type of SCD [11]. The severity of this particular kind of SCD also means that these patients experience the worst symptoms at the highest rates for the condition [12]. With the most recent documentation being published in November 2020, the recovery rates of these patients were analyzed. The figure below from the same study highlighted the steps of evaluation conducted for the patients with SCD who had symptoms that aligned with potentially contracting COVID-19 as well as further standards of treatment and evaluation based on individual patient status [11]. The diagram shows the process of triage for patients with SCD who also contracted COVID-19 based on symptoms and patient accessibility [11].


Figure 1 Determining the appropriate medical response for SCD patients with COVID-19, ranging from patient instructions, types of medical evaluation, and potential therapies [11].

A third study, presented at the American Society of Hematology, was led by Dr. Lana Mucalo from the Medical College of Wisconsin and required the examination of 370 COVID-19 cases in patients already suffering from SCD from an international sickle cell registry [13]. This particular study compared the data found from the patients listed in the international registry to patients suffering from both conditions in the general Black population, keeping race as the measured demographic independent variable [13]. 

Results

The results of the first study, documented by the Medical College of Wisconsin SECURE-SCD Registry in between March 20, 2020 and May 21, 2020, showed that roughly 178 individuals who had SCD — and who were not simply carriers of the sickle cell trait — also contracted COVID-19 during this time. [10] Out of these 178 individuals, 7% (13 individuals) died upon contracting COVID-19 and being hospitalized, and 11% (19 individuals) were admitted to the intensive care unit while suffering from both conditions simultaneously [10]. More than half of the total individuals had suffered an SCD-related complication in the past that led to prior hospitalizations, and the average age of the 178 individuals at the time was roughly 28.6 years [10]. On the contrary, individuals with COVID-19 that don’t suffer from SCD have a death rate of less than 1% for those 20—54 years of age in early 2020, which is dramatically less than rates analyzed for patients with both SCD and COVID-19 [14].  

From the 10 patients in the UK documented in Haematologica, a 54-year-old patient died who suffered from delayed hemolytic transfusion reactions and also had high levels of CRP (C-reactive protein), a marker of negative prognostics for patients suffering from COVID-19 [11]. From all 10 patients, two underwent hydroxyurea therapy, seven had regular blood transfusions, and a total of five patients were managed remotely through telephone contact for their treatment [11]. Medical professionals and experts believed that suffering from the SARS-CoV-2 infection along with SCD would lead to acute chest syndrome (ACS). However, in the 10 patients, only one of them suffered from related respiratory complications; this was the same patient who passed away during clinical monitoring [11]. 

In the final study, the Black population was examined since, especially in the United States, individuals with African-American descent are statistically more likely to have both the sickle cell trait as well as the disease [13]. Additionally, completely separate from SCD, Black Americans are nearly four times more likely to be hospitalized due to COVID-19 as the virus has progressed globally [13]. Using race as the measured variable, this study concluded that those with SCD are roughly 6.2 times more likely to suffer a fatal complication from COVID-19 when compared to the general African-American population in the United States [13]. While the fatality rate based on ages 18-34 and 35-50 for the general Black population is less than 1% for all Black people in both age groups, those with SCD had death rates of 2.6% and approximately 12%, respectively, for the same age groups [13]. Additionally, a significantly larger amount of COVID-19 hospitalizations for Black individuals with SCD occurred among the younger age group, which correlates with larger death rates as age decreases [15].

Discussion

With the knowledge of all three pieces of data, these results illustrate that there is a correlation regarding age and race when examining the compounding effects of SCD and COVID-19. When examining the whole percentage of individuals who were hospitalized and severely affected by suffering from both conditions, the data from the SECURE-SED Registry suggests that such a connection exists. Additionally, data from the registry includes reports of 7% fatalities and 11% admittance to the ICU over 60 days for individuals with both conditions, which is heavily alarming. Even in the UK, the data suggests that there is an evident conjunction between the two conditions, despite the study being conducted with a small sample size. If 1 out of 10 individuals died, a 10% mortality rate from the combined detrimental effects of both conditions is just as concerning, if not more, than the first study. Lastly, the final study examined the effects of racial background on contracting COVID-19 while having SCD, and a significant relationship between the two is evident. In fact, having SCD, particularly for Black Americans, dramatically increases the fatality rate of these individuals from COVID-19 (by up to 11%). Based on these aforementioned studies, strong evidence exists to suggest that SCD and COVID-19 do have detrimental implications for individuals on a global level.

Conclusion

After understanding the symptoms of patients already suffering from SCD, adding the effects of COVID-19 can exponentially deteriorate a patient’s health. Through the three different pieces of data, there is a noticeable connection between incidence of SCD and severity of symptoms of COVID-19, the most recent and prevalent globally-spread virus. These connections can be analyzed on the basis of age as well as race, thereby indicating that the hypothesis for a linkage between the two conditions is well-supported. Since COVID-19 treatments and vaccinations are still being actively manufactured, knowing its added implications for those with SCD can be significant when determining a proper healing regimen for the virus. Additionally, understanding the damage done by both ailments combined can help reduce their high mortality rate, thereby creating more successful outcomes for patients for both diseases individually and together. 

Works Cited

[1] Bean, Mackenzie. Becker’s Hospital Review. 2020. 

[2] Tabish, Syed Amin. International Journal of Health Sciences. 2009, 3(2) V-VIII.

[3] Coronavirus Disease 2019 [Online]. 2021. https://www.cdc.gov/dotw/covid-19/index.html (accessed Mar. 17, 2021).

[4] Liu, Jiaye. Emerg Infectious Dis. 2020, 26(6), 1320-1323.

[5] Zimmer, Carl. The New York Times. 2021.

[6] Thu, Tran P. B. Elsevier Pub Health Emerg Cond. 2020, 742.

[7] National Heart, Lung and Blood Institute. US Dept. of Health & Human Services. 

[8] Shet, Arun. American Society of Hematology. 2021.

[9] Minniti, Caterina P. Blood Advances. 2021, 5(1), 207-215.

[10] Panepinto, Julie A. Centers for Disease Control and Prevention. 2020.

[11] Menapace, Laurel A. Haematologica. 2020, 105 (11), 2501-2504.

[12] Rogers, Graham. Healthline. 2019. 

[13] Thompson, Dennis. Medical Xpress. 2020

[14] Razzaghi, Hilda. Centers for Disease Control and Prevention. 2020, 69(12), 343-346.

[15] Mucalo, Lana. American Society of Hematology. 2020.

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Evaluating the effectiveness of CQ and/or HCQ against SARS-CoV-2

by Puneetha Goli

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease (COVID-19). Patients exhibit a spectrum of severity in their symptoms ranging from asymptomatic to breathing difficulties[1]. The purpose of this article is to evaluate the effectiveness of chloroquine (CQ) and its less toxic metabolite hydroxychloroquine (HCQ) as a viable treatment for SARS -CoV-2 through analyzing data from available research studies. In one of the early breakthrough studies published in February 2020, Gao et al. found that a low-micromolar concentration of CQ blocked COVID-19 infection in early in vitro studies and improved lung imaging, indicating a potential correlation between CQ administration and a decrease in the spread of COVID-19[2]. However, more recent studies, including Skipper et al., since then have indicated that there are no significant differences with the administration of the agents, and some even caution against the use of them because of potential long-term side effects [3,4]. The general consensus among the scientific community, however, remains that the use of agents must be rigorously considered on a case-by-case basis and with caution in research or clinical trial settings[4]. Determining the effectiveness of CQ/HCQ will aid in preventing the spread of misinformation. 

 

Introduction 

Responsible for taking the lives of 2,244,713 people worldwide (as of February 3, 2021), the World Health Organization (WHO) classified COVID-19 as a pandemic in March 2020 (5,6). SARS-CoV-2, the virus responsible for COVID-19, is only one of seven identified human coronaviruses and is primarily transmitted from person-to-person through direct contact, saliva, and airborne droplets. It is considered “novel” because it is newly identified and is different from the other coronaviruses that have been linked with causing mild illnesses, such as the common cold [7]. Because of its novel nature, there is much that is unknown about the virus and the disease, and unfortunately misinformation regarding treatments has been spread, specifically the effectiveness of chloroquine (CQ)/ hydroxychloroquine (HCQ) as a treatment [7]. 

CQ and HCQ are antimalarial treatments, and interest in these drugs as possible treatments for COVID-19 was spurred in part from the drugs’ history of in vitro activity against viruses such as influenza. HCQ’s role in modulating the immune system through treating other autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis, could have also played a role [4,7].  

In April 2020, nearly 55 countries, including the US, Mauritius, and Seychelles, were in the process of receiving HCQ from India [8]. Country leaders, including Brazil’s President Jair Bolsonaro and United States’s President Donald Trump, showed support for the drug, some even claiming to have taken it [9,10]. 

In the months since, however, scientists and national agencies have discouraged the use of CQ and HCQ against treatment for COVID-19, claiming that its effectiveness hasn’t been proven yet. Evaluating the research surrounding CQ and HCQ’s effectiveness is critical in order to address misconceptions and misinformation that is being spread as well as to evaluate its role as a potential treatment.

This paper will be reviewing two studies presenting contradictory evidence regarding the effectiveness of CQ/HCQ. In the first study published on March 16, 2020, Gao et al. examines the effectiveness of CQ in the early stages of the COVID-19 pandemic. Later, a study published on October 20, 20202 by Skipper et al., provides a more comprehensive comparison documenting HCQ’s utility.

Materials and Methods 

Gao et al., one of the early breakthrough studies which found favorable results in treating pneumonia associated with COVID-19 with CQ/HCQ, first conducted in vitro studies in which CQ was tested against the COVID-19 infection [2]. After a determination of a half-maximal effective concentration (EC50) and a half-cytotoxic concentration (CC50), multiple clinical trials were then conducted in China.100 patients from more than 10 hospitals in Wuhan, Jingzhou, Guangzhou, Beijing, Shanghai, Chongqing, and Ningbo were treated with chloroquine phosphate and results, including lung imaging findings, were gathered. 

The following section examines the method used in Skipper et al, which found that HCQ did not significantly decrease the severity of symptoms among patients experiencing early, mild COVID-19[3]. The study is the first randomized clinical trial studying COVID-19 treatments among outpatients with early, mild COVID-19 through a randomized, placebo-controlled procedure.

Participants

Participants included non-hospitalized, symptomatic adults that either were lab-tested for COVID-19 or those who demonstrated COVID-19 compatible symptoms and had an epidemiologic link to a lab-confirmed COVID-19 contact. Due to the scarcity of COVID-19 tests during the time the study was conducted, not all of the participants were tested for COVID-19, and the second criterion had to be included to recruit subjects. Further, adults were only enrolled if they had experienced COVID-19 symptoms for 4 or fewer days.

A total of 491 subjects were enrolled in the study (244 assigned to HCQ and 247 assigned to placebo), however only 423 contributed toward data collection (231 assigned to HCQ and 234 assigned to placebo) because they had completed at least 1-follow up survey with symptom data. 

 14-Day Trial

Participants were given either 200-mg tablets of HCQ sulfate or masked placebos of either 400mcg folic acid or lactose by a research pharmacist. Both the HCQ and placebo shared similar physical characteristics and opaque dispensary bottles so that the two tablets were hard to distinguish between. 

On the first day, adults in the experimental group first took 800mg of oral HCQ (4 tablets) and an additional 600mg (3 tablets) after 6 to 8 hours. For 4 more days after, participants continued to take 600 mg daily (for a total of 5 days). Participants in the placebo group were prescribed to take the placebo tablets in a similar regiment. The dose was based on previously published pharmacokinetic measures that serve to maintain the HCQ concentration above the EC50 for SARS-Co-V-2. 


Outcomes and Data Collection

Researchers collected data regarding symptoms and severity through participant-reported surveys on day 1 (baseline), 3, 5 (end of medication), 10, and 14. A 10-point visual analogue scale was used to collect data regarding symptom severity. The study’s primary endpoint was a measure of overall change in symptoms throughout the study’s 14-day period.

Results 

Although both Gao et al. and Skipper et al. examine the effectiveness of CQ and/or HCQ against SARS -CoV-2, the studies report different experimental approaches and results

While Gao et al. didn’t publish the exact data and figures for their study, they concluded that the patients treated with CQ showed better results in inhibiting the exacerbation of pneumonia, facilitating an environment for virus-negative conversion, improving lung imaging, and reducing the disease course [2]. Researchers also noted that no severe adverse reactions as a result of the CQ were found among the 100 patients.

In the Skipper et al. study however, differences in data collected between the placebo and HCQ were not significant [3]. As depicted in Figure 1, data on day 5 of the study showed that 54% of participants in the HCQ group reported symptoms versus 56% in the placebo group. Further, at day 14, 24% in the HCQ group reported symptoms compared to 30% in the placebo group. The study concluded that the proportion of participants that reported symptoms was not significantly different between the HCQ and placebo group.

Figure 1. Percentage of participants reporting COVID-19 symptoms throughout the Skipper et al. study’s 14-day time period [3]. On day 5, the percentage of HCQ participants with symptoms was 2 points less than that of the placebo participants, and by day 14, the difference had increased to 6 points, but still not enough for statistical significance.

 

In addition to the proportion of COVID-19 symptoms observed in both groups, Skipper et al. recorded the severity of the symptoms based on a 10-point visual scale. As shown in Figure 2, the participants in the HCQ group reported an average reduction of symptom severity of 2.60 throughout the 14 day period, while placebo participants reported a 2.33-point reduction. Although the HCQ group saw an overall 12% more reduction in symptom severity compared to the placebo group, the difference was, once again, not statistically significant.


Figure 2. Overall scores of symptom severity throughout the Skipper et al. study’s 14-day time period [3]. Although the data showed a 12% relative reduced severity difference for the HCQ in comparison to the placebo, it was not statistically significant.

 

Discussion

Gao et al. is one of the earliest studies that showed favorable results for CQ. The researchers have hypothesized that evidence for the CQ’s anti-viral properties lies in previous studies which have found the drug to increase the endosomal pH needed for the fusion of the virus. In addition the drug is suspected to cause interference with the glycosylation of the SARS-CoV cellular receptors [11,12]. Based on these findings along with the timing of the study (before the WHO classified COVID-19 as a pandemic), the researchers recommended CQ be used to treat patients that experience pneumonia associated with COVID-19 in China.

Therefore, although Skipper et al. points out differences among the HCQ and placebo groups, they weren’t statistically significant, contradictory to the findings of Gao et al.

Conclusion

Differences in results across studies, as sampled through Gao et al. and Skipper et al., have led scientists to continue to question and rethink the effectiveness of HCQ/CQ against COVID-19. While a concrete conclusion regarding the drugs’ fates is yet to be determined, scientists continue to caution the use of HCQ/CQ for public use and rather recommend the drug to preferably only be administered under carefully designed clinical trials, as well as examined on a case-by-case basis [4]. 

With vaccinations becoming more widely available, discussion and debate centered around the administration of HCQ/CQ has largely reduced, and rather, the focus has shifted towards the effectiveness of the vaccines. Although a 2021 study has found the Moderna vaccine to be antibody persistent through 6 months, there isn’t exact data regarding the maximum efficiency of this particular vaccine as well as that for the Pfizer-BioNTech and Johnson & Johnson vaccines, the two others approved by the Food and Drug Administration [13]. Future directions of study can involve a close examination of these vaccines. Results and information collected from the close examination of these vaccines would be critical in planning the continued management of the COVID-19 pandemic in the United States. 

References 

[1]Transmission of SARS-CoV-2: implications for infection prevention precautions. World Health Organization [Online], July 9, 2020, https://www.who.int/news-room/commentaries/detail/transmission-of-sars-cov-2-implications-for-infection-prevention-precautions (Feb. 3, 2021)

[2] Gao, J. et al. BioSci. Trends 2020, 14, 72-73

[3] Skipper, C. P. et al. Ann. Intern. Med 2020, 173, 623-631

[4] Meyerowitz, E. A. et al. FASEB 2020, 34, 6027-6037

[5]World Health Organization. https://www.who.int/ (accessed Feb. 3, 2021)

[6]Archived: WHO Timeline - COVID 19. World Health Organization [Online], Apr. 27, 2020, https://www.who.int/news/item/27-04-2020-who-timeline---covid-19 (accessed Feb. 3, 2021)

[7] Centers for Disease Control and Prevention. https://www.cdc.gov/ (accessed Feb. 3, 2021)

[8] India sending hydroxychloroquine to 55 coronavirus-hit countries. The Economic Times, April 16, 2020. https://economictimes.indiatimes.com/news/politics-and-nation/india-sending-hydroxychloroquine-to-55-coronavirus-hit-countries/articleshow/75186938.cms?utm_source=contentofinterest&utm_medium=text&utm_campaign=cppst (accessed Feb. 3, 2021)

[9]Stargardter, G.; Paraguassu, L. Special Report: Bolsonaro bets 'miraculous cure' for COVID-19 can save Brazil - and his life. Reuters, July 8, 2020. https://www.reuters.com/article/us-health-coronavirus-brazil-hydroxychlo/special-report-bolsonaro-bets-miraculous-cure-for-covid-19-can-save-brazil-and-his-life-idUSKBN249396 (accessed Feb. 3, 2021)

[10] Bruggeman, L. Hydroxychloroquine returns as wedge between President Trump, health advisers. ABC News, July 28, 2020. https://abcnews.go.com/Politics/hydroxychloroquine-returns-wedge-president-trump-health-advisers/story?id=72036996 (accessed Feb. 3, 2021)

[11] Savarino, A. et al. Lancet Infect Dis. 2003, 3, 722-727

[12] Yan, Y. et al. Cell Res. 2013, 23, 300-302

[13] Doria-Rose, N. et al. NEJM. 2021

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Determining the Efficacy of Sourcing IgY Antibodies from Chicken Eggs Using the PierceTM Chicken IgY Purification Kit

by Sachi Kishinchandani

ABSTRACT

IgY is known to be an immunoglobulin that is formed by the maternal immune system and passed down to offspring, in reaction to certain foreign substances. It is hypothesized that IgY can be used to boost human immune systems. Thus, to ask how we can easily gather these proteins for increased human immunity, we analyzed the purification of IgY via the Thermo ScientificTM PierceTM Chicken IgY Purification Kit. Through a Bradford assay, SDS PAGE, and a Densitometry, we determined that it is possible to purify around 40-80 mg of IgY per egg using similar methods. Since each hen produces hundreds of eggs annually, and each egg contains such a large portion of IgY, it is reasonable to conclude that mass purification of IgY is feasible.

INTRODUCTION/BACKGROUND

Our immune system plays a vital role in protecting our body from foreign diseases, viruses, and harmful bacteria. Therefore,the study of its mechanisms and functions is extremely important. One important feature of the immune system across a variety of species is how antibodies are passed from mothers to their newborn offspring [1]. Over time, natural selection has led to adaptation of antibodies to be passed down to offspring for survival, whether it is in the form of the IgG antibodies passing through the placenta in mammals, or birds passing down IgY antibodies in their egg yolk [1].

By studying the IgY antibodies found in chicken eggs, we can study and potentially introduce useful IgY antibodies into humans (for example, via the consuption of IgY Antibodies), in order to bolster our immune systems. IgY antibodies can be used to create immunization in a form that does not specifically require a vaccine [2]. People can be passively immunized against respiratory illness with the help of IgYs to help neutralize respiratory pathogens and disease spread. IgY is an ideal candidate for this antibody insertion due to its structural similarity to the IgG antibodies common in humans, as well as its ability to target specific antigens [3]. IgY has been found to bind specifically to influenza virus Nucleoprotein (NP) by detecting multiple virus subtypes in swine [4]. These traits make IgY a worthwhile subject of study, especially one whose potential outcomes are so beneficial. 

In this series of experiments, we focused on investigating IgY protein concentrations, abundance, and purity in potential sources for IgY protein purification, as the extent of these properties are not widely available in current literature. We used the Thermo ScientificTM PierceTM Chicken IgY Purification Kit to extract the IgY protein from the egg yolk [5]. The extraction of the IgY protein allowed us to conduct a Bradford assay, SDS PAGE, and a Densitometry in order to determine the concentration, abundance and purity of the protein. With the results of this experiment, we determined that we can effectively purify IgY from store-bought chicken eggs.

MATERIALS AND METHODS

PHYSICAL PURIFICATION OF IgY PROTEIN

We purified IgY from store-purchased Eggland Best eggs using the Pierce™ Chicken IgY Purification Kit from Thermo Fisher to physically separate the antibody rich yolk from the white using a series of suspensions and centrifugations with reagents. We conducted this by first separating the egg yolk from the egg whites. We then rolled the egg yolk on a paper towel to remove excess egg-white protein, pricked the yolk sac and then drained an approximate 8 mL of the yolk into a beaker. This step is essential to remove any contaminant protein from the egg white. We then added five times the egg yolk volume of cold Delipidation Reagent (40 mL) from the Thermo ScientificTM PierceTM Chicken IgY Purification Kit to the 8 mL egg yolk. The mixture was stirred until well incorporated and then stored at 4 ºC for a week. 

The mixture was then centrifuged for 15 min at 6,000 × g in a refrigerated centrifuge. The colorless and translucent supernatant was decanted and placed in a secondary conical tube, to which the precipitation reagent solutions from the Thermo ScientificTM PierceTM Chicken IgY Purification Kit were added. The mixture was placed in a 4 ºC incubator overnight, and then centrifuged for 15 min at 6000 rpm in a 4 ºC refrigerated centrifuge. We then isolated the pellet from the conical tube.

Our final step in the purification of the protein was to resuspend the pellet in Phosphate buffered saline (PBS) (100 mM Na3PO4, 150 mM NaCl, pH 7.2). We added the PBS to the pellet and stirred the mixture until it was well incorporated. 


BRADFORD ASSAY

Our next step was determining the concentration of the IgY protein using a Bradford assay. The assay uses acidic Coomassie dye to bind to proteins and result in a visible color change from brown to blue, the intensity depending on the amount of protein present. We measured out 5 mL of Bradford reagent into 8 glass test tubes. We pipetted Bovine Serum Albumin (BSA) protein into the first six test tubes, and IgY into the remaining two test tubes. With the BSA protein test tubes, we measured the absorbance at 595 nm for each solution with a spectrophotometer, and created a standard curve of the absorbance in relation to BSA protein amounts. We then measured the absorbance of our protein to be 0.383. 


SDS PAGE

Because current literature information on IgY proteins states that two chains of IgY should occur around 70 kDa and 30 kDa [6], we decided to make our gel a 12% acrylamide. With our SDS PAGE, we first created a standard mixture of 1 L Running buffer, 10 mL of 1x Separating buffer and 10 mL 1x Stacking buffer. We also created a 1 mL of 10% APS solution for activating the polymerization of the gel by dilution of stock APS available in the lab. We then added 100 uL of APS and 10 uL of TEMED into both the Stacking and Separating buffers. We then began to pipette the Separating buffer solution in between the glass plates until a marked line, allowing the Separating buffer to set with isopropyl alcohol before adding the Stacking buffer on top. While the Stacking buffer was still liquid, we inserted the comb to create the wells in the gel. 

We stored the gel for four days in a moist paper towel, then conducted SDS PAGE with 5 uL of our protein and 5 uL of sample buffer in each of our wells, with the Broad Range 5 Microliters NEB in one of the wells for reference. We let the SDS PAGE run for 90 min --the first 10 min at 120 V and the rest of the time at 180 V--until the bands reached the bottom of the gel, and then stained the gel with Coomassie blue solution. We then destained the gel using the premade solutions Destain #1 (50% methanol, 10% acetic acid) and Destain #2 (7% methanol, 10% acetic acid). 

DENSITOMETRY

This part of the experiment was to determine the concentration of the IgY bands. The densitometry was conducted in a similar fashion to the SDS PAGE. In order to address the possible presence of a dimer in our previous SDS-PAGE gel, we increased the concentration of BME in two of the wells with our IgY protein in the hopes of removing any remaining disulfide bonds. Instead of creating a gel for this experiment, we used a BIO-TEC premade gel. We created 5 solutions of different concentrations of BSA: 0.2 ug, 0.5 ug, 1 ug, 1.5 ug, and 2 ug. We then added 5 uL of these solutions, mixed with 5 uL 2x Laemmli buffer into separate wells. We also added our purified IgY protein into the gel, with two wells made with 5 uL of protein and 5 uL 2x Laemmli buffer each, and two wells with 5 uL protein and 5 uL 10% BME Laemmli buffer each. We ran the gel at 300 V for 15 min, until the dye front hit the bottom of the gel. We stained and destained the gel exactly as we did for the SDS PAGE. We then used a machine to analyze the concentrations and Molecular Weight (MW) of the bands of IgY in comparison to BSA.

RESULTS & DISCUSSION

The overall purpose of the experiments was to determine the practicality of extracting large quantities of protein from store-bought chicken eggs. From the experiments, we were able to determine the following:

PHYSICAL IgY PURIFICATION

The weight of our purified protein match the expected result of using the kit. We were able to purify 70.2756 mg of IgY solution from a single store-bought egg (Table 1).

Table 1. The total mass of IgY protein solution as a result of the initial purification of IgY protein with Thermo ScientificTM PierceTM Chicken IgY Purification Kit. 

BRADFORD ASSAY

Once our protein was purified, we were able to determine the concentration of the protein from a Bradford assay, comparing the absorbance of our protein to the absorbance of BSA. 

Figure 1. The absorbance of BSA over the concentration of BSA measured via a spectrophotometer, used as a standard curve to determine that the concentration of our IgY protein is 2.43 ug/uL.

Table 2. Calculated concentrations of our IgY solution as determined by a Bradford Assay. Conducted by measuring the absorbance of different volumes of the IgY solution in a spectrophotometer. These data were used to find the average concentration of the IgY protein to be 2.43 ug/uL. 

With these calculated concentrations (Table 2), we found that the average concentration of our IgY sample is 2.43 ug/uL. This matched expected results as previous studies have estimated that the concentration of IgY purified from egg yolk should be 2-4 mg/mL [7]. 

SDS-PAGE

Knowing the concentration of the protein helped us conduct SDS-PAGE, which would help us find out the size and purity of our purified IgY protein. 

Figure 2. SDS-PAGE of our IgY protein with clear bands around 27 kDa and 130 kDa. Done in a 12% acrylamide gel that was stained with Coomassie blue solution. The left-most well on the right end of the figure indicates standard protein molecular weights from the NEB Protein standard solution. The remaining wells have 5uL of 2.34 ug/uL of IgY and 5uL of 2X Laemmli buffer, as indicated at the bottom of each column in the figure.  

Figure 3. Graph of the log (molecular weight) of NEB Protein Standard as a function of relative distance. The curve between the distances 2.5 and 4.4 cm was used to find the MW of the heavy band to be 117.7 kDa. The curve between the distances 14.7 and 18.6 cm were used to find the MW of the second band to be 27.28 kDa.

Once we conducted our SDS-PAGE and stained/destained it, we found that the gel showed significant IgY purity because the most significant bands were at around 27 kDa and 130 kDa. We also found a light band around 43 kDa (Figure 2), which was confirmed to be the contaminant ovalbumin by our instructors (see acknowledgments). 

We calculated the MW of the bands to be 27.28 kDa and 117.7 kDa. In order to do so, we calculated the equation of the line between the points 2.5 and 4.4 cm to be logMW = -0.7169 x + 2.2932. When we plug 3.1 in for x, the MW ends up being 117.7 kDa. We also calculated the equation of the line between points 14.7 and 18.6 cm to be logMW = -0.02987x + 1.971. Plugging in 17.9 for x, we get MW = 27.28 kDa (Figure 3). 

We expected to have the band at 27.28 kDa for the IgY light chain, but our other expected value for the IgY proteins was around 70 kDa for the heavy chain, not 117.7 kDa, based on current literature on IgY. Because this 117.7 kDa band was almost exactly twice the weight of our expected heavy chain weight, we hypothesized that our protein’s disulfide bonds had not broken down completely. In order to understand whether this high molecular weight was a result of the small amount of BME in the 2x Laemmli solution, or was simply a contaminant, we conducted a densitometry with two of the wells containing our protein along with a higher concentration of BME. 

DENSITOMETRY

Figure 4. The results of our Coomassie stained and de-stained IgY and BSA Densitometry [left-most well: NEB marker. Well 2-3: 10% BME + IgY. Well 4-5: IgY + normal Coomassie. Wells 6-10: decreasing concentrations of BSA] The red arrow points to the heavy IgY chain, which is a band we did not observe in the previous (SDS PAGE) experiment. The blue arrow points to the light IgY chain. The yellow arrow points to the BSA chain. 

SDS-PAGE was run, this time with more BME and varied BSA amounts to both analyze the light/heavy chains and to possibly get a reading of the bands that would help us determine the concentration of protein in each band. Figure 4 shows there is clearly a large amount of BSA in wells 6-10, which would have been difficult to analyze our IgY density with, so we were unable to carry out analysis of the IgY. 

We found that the gel with the higher percentage of BME had a more prominent band around 72 kDa, along with bands around 27 kDa and 130 kDa. The finding of a band around 72 kDa and 27 kDa was consistent with current literature on IgY protein chains. The bands around 27 kDa and 130 kDa were consistent with our previous SDS PAGE (Figure 4). We still assume that the band around 130 kDa could be the IgY protein’s heavy chain that remains a dimer, however, there is a possibility that this could be a contaminant.  

This experiment does not tell us the identity of each of the protein bands found in the densitometry. If we had more time, we would have run the experiment again, using lower BSA concentrations and using the BSA band densities to make a standard curve in order to determine the total integrated density of the protein. From there, we would have calculated the density and concentration of the IgY, which would have told us the purity of the protein in relation to BSA. However, since we were unable to conduct this experiment, we suggest that further experiments should be performed to determine whether the IgY was effectively purified. 

CONCLUSION

Based on preliminary results, we can reasonably conclude that our IgY purification was successful and we were able to extract around 24.3 mg of pure IgY protein from the 8 mL of store-bought chicken eggs. Current literature indicates that the expected concentration of protein from each egg yolk is 2-4 mg/mL, which was what we observed in our results. Other studies using similar methods to our experiment have also found that it is possible to purify around 40-80 mg of IgY per egg using similar methods [7]. The reason we predict 40-80 mg per egg yolk is because we only extracted 8 mL of yolk, whereas the average egg yolk is about 15 mL, [8] so the total protein amount would be around 40 mg for 15 mL of yolk. The 24.3 mg of IgY extracted from one egg shows that eggs are a reliable source of IgY protein. Since each hen produces hundreds of eggs annually, and each egg contains such a large portion of IgY, it is reasonable to conclude that mass purification of IgY is feasible. 

The extraction of IgY has important implications for further application. In the future, we hope to see multiple pathways for immunization built from IgY. One is the immunization of humans through digestion of IgY antibodies [9]. Another is epitope mapping, [10] identification and characterization of antibody binding sites on cells, which could help us understand the structures of antigen binding sites to combat diseases. The results of our purification of IgY seem promising for the future of IgY extraction and execution of further research. 

ACKNOWLEDGEMENTS 

We thank Dr. Thomas and Dr. Catanese for all their support and mentorship throughout the course. OURI, CUR, and the Biosciences Department for funding BIOS 211.  Anika Sonig and Aaron Lin for their partnership through this project and aid with the creation of the figures. BIOS 211 TAs for their guidance. 

Works Cited

(1) Pereira, E P V et al. International immunopharmacology 2019 vol. 73, 293-303. doi:10.1016/j.intimp.2019.05.015  

(2) Aymn Talat Abbas, Sherif Aly El-Kafrawy, Sayed Sartaj Sohrab & Esam Ibraheem Ahmed Azhar, Human Vaccines & Immunotherapeutics. 2019, 15:1, 264-275, doi: 10.1080/21645515.2018.1514224  

(3) Nagaraj et al. International Journal of Food Microbiology. 2016, 237, 136-141.

(4) da Silva M.C, Schaefer R, Gava D, Journal of Immunological Methods. 2018, 461, 100-105. https://doi.org/10.1016/j.jim.2018.06.023. 

(5) ThermoScientific PierceChicken IgY Purification Kit manual https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011404_Pierce_Chicken_IgY_Purifi_UG.pdf

(6) Sudjarwo, S. A., Eraiko, K., Sudjarwo, G. W., & Koerniasari, Journal of advanced pharmaceutical technology & research. 2017, 8(3), 91-96. https://doi.org/10.4103/japtr.JAPTR_167_16 

(7) Pauly, Diana, et al. Journal of Visualized Experiments. 2011, www.ncbi.nlm.nih.gov/pmc/articles/PMC3197133/.    

(8) Egg Cooking Tips: Cooking With Eggs: Egg Farmers Of Alberta. eggs.ab.ca/eggs/egg-cooking-tips/. (Accessed November 24, 2021)

(9) Constantin, C.; Neagu, M.; Diana Supeanu, T.; Chiurciu, V.; A. Spandidos, D, Exp Ther Med, (2020), 20, 151–158. https://doi.org/10.3892/etm.2020.8704. 
(10) Lu, Y.; Wang, Y.; Zhang, Z.; Huang, J.; Yao, M.; Huang, G.; Ge, Y.; Zhang, P.; Huang, H.; Wang, Y.; Li, H.; Wang, W, Journal of Immunology Research (2020). https://doi.org/10.1155/2020/9465398.

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Combatting E. Coli through Secretion Inhibitors

by Akshay Sethi

Introduction:

Escherichia coli (E. coli) is a rod-like anaerobic bacterium that infects the gastrointestinal tract of humans (Fig. 1).

Fig 1: (E. coli bacterial cells)

Most E. coli are harmless, but there are pathogenic strains such as Shiga toxin-producing E. Coli (STEC) that can cause diarrhea, abdominal cramping, and/or acute renal failure. [1] E. coli is found to colonize the human body hours after childbirth and does not pose harm unless the gastrointestinal barrier is broken or the host is immunocompromised. The microorganism is a threat to humans. Gram-negative bacteria possess cell envelopes and an outer membrane that can protect the inner membrane and its organelles from antimicrobial enzymes and antibiotics (Fig. 2). [2]

Fig 2: (Gram-negative bacteria have more components, including an outer membrane, to protect it)

The combination of a low concentration of E. coli needed to infect the host and its resistance to antibiotics makes the microorganism dangerous. [3]. E. coli has a core genome, or series of DNA strands that are common in all strains, but the bacteria also has a non-shared gene pool that differentiates each strain from anotherThere are at least six types of deadly pathotypes of E. coli., all of which cause enteric disease, or diseases relating to the gastrointestinal tract, and potentially lead to long-term injury. 

E. coli is not highly transmissible, as the risks come from contaminated food such as undercooked meat, unpasteurized milk, dirty water, or contaminated stool. [4] Therefore, the best prevention for the risk of infection is cleanliness such as washing one’s hands, as an individual must ingest the pathogen to contract it. [4] 

Novel therapries and treatments for E. coli have shown limited success. [8] Much of the successful experimentation has come from the process of injecting fluid with secretion inhibitors to stop production of key components of the bacterium. The main system for infecting host cells, T3SS, is the main target of secretion inhibitors and has been the focus of modern research on E. coli. [10]

Discussion:

Harms in E. coli:

 E. coli varies in its threat to humans based on the amount of Shiga toxins it produces. [3] Shiga toxins act as ribotoxins that inhibit protein synthesis, leading to altered gene expression or even apoptosis. This would lead to bloody diarrhea, or worse, the Hemolytic Uremic Syndrome (HUS). [5] HUS is one of the more dangerous outcomes of contracting harmful E. coli, where infected people suffer red blood infection and potential total kidney failure. [5] Global estimates project about 10% of STEC infections lead to HUS. HUS is the most common cause of acute renal failure in children, one of the two high-risk groups to contracting an E. coli infection. [5] The syndrome can also cause neurological failures in 25% of cases, resulting in symptoms such as seizure, coma, or stroke. [5]  

Shiga toxins are found in over 200 strains of E. coli. [7] Shiga toxins found in E. coli are found in the pathogenic serotypes of E. coli as Stx1. [4] A serotype is a specific group of bacteria sharing a common antigen, which is an immunogenic particle that incites an antibody response. Another serotype of E. coli contains  Stx2, which is a more potent Shiga toxin and a part of a different antigen serogroup. A serogroup consists of microorganisms differing in their composition of antigens. [4]  The Stx toxin is comprised of two key subunits—the A subunit and B subunit. The A subunit’s function is to inhibit protein synthesis by damaging the ribosome in an infected cell. [4] The B subunit binds to the globotriaosylceramide (Gb3), which is found in endothelial cells lining cardiac muscles and blood vessels. [4] Among biological substances, Shiga toxins are amongst the most poisonous and are lethal to various animals in small doses. E. coli carrying the Shiga toxin serotypes have been shown to lead to the worst outcomes of contracting the pathogen. Across different species, the Shiga toxins have been found to cause acute renal damage and in some cases, total renal failure. [7] Within population-based observations, along with experimentation on mice and baboons, Shiga toxins have been linked to HUS. [4] 

The pathway for Stx serotypes of E. coli to lead to HUS begins with the consumption of contaminated substances. E. coli rapidly replicates within the host and the Shiga toxins latch onto Gb3 before being enveloped and packaged into a microvesicle. This vesicle is then absorbed into the kidney endothelial cells, where the toxins cause necrosis, eventually leading to acute renal failure (Fig. 3). [6] 

Fig 3: (Stx binding to the GB3 on the cell membrane to take over the ribsomal components of the cell)

Besides the kidney, the brain is the second most affected organ in severe E. coli infection. The Shiga toxins attack neurons within the brain and can cause damage or partial failure to the central nervous system. Central nervous system damage can lead to seizure, shock, or paralysis in some cases. [7] 

Classifications of Shiga toxins go more in-depth than Stx1/Stx2 to distinguish between the potency of the toxin and the binding receptor. [7] Some types of Stx can cause severe loss of body mass, along with renal failure. [7] One common symptom of E. coli infection shared between most classifications is colon damage. Shiga toxins inhibit the function of the large intestine, which in turn causes severe symptoms such as bloody diarrhea. [7]

In the past couple of decades, Shiga toxin-carrying E. coli has been found in animal-to-animal transmission. [7] In an isolated study, STEC infection was found in zoonotic transmission, and a common source was domestic animals, such as house cats and dogs. Other sources included wild animals, but at similar rates. Most commonly found were feral hogs and hyenas. Scientists have looked to treat E. coli because of the long-lasting impact of the infection on the human body, including high blood pressure, heart disease, or kidney problems. [1]


Secretion Inhibitors:

One of the newly discovered methods in treating E. coli is anti-virulence therapy. This therapy takes advantage of the type III secretion system (T3SS) in E. coli that primarily exists to grow and multiply the bacteria within hosts. [8] The T3SS acts as a needle that will “inject” effector proteins into the epithelial cells of the gastrointestinal tract. Effector proteins are biomolecules that can attach to proteins and affect the production and type of product that the protein will create. These proteins function as ligands as well as secretory proteins, attaching mainly to host cells to infect them. The ligands will lower the activation energy to conduct the process of making bacterial cells, and therefore increase the rate of production. [8] The impetus for targeting T3SS by scientific researchers is that it is the main mechanism that both Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) use to infect and populate the host effectively. EHEC is the strain of E. coli that can cause HUS, while EPEC is less severe in bodily harm but is the leading strain for diarrheal deaths. [8] 

The method in which the treatment is proposed to work is unique. Instead of regulating or limiting the growth of new cells, the anti-virulent treatment targets and inhibits a virulence factor. [8] Virulence factors are what cause bacteria to cause disease in eukaryotic, multicellular organisms. [8] These factors will include molecules that will aid bacteria in infecting the host organism’s cells. [12] The factors are categorized as either secretory, membrane associated, or systolic. Systolic factos will lead to the bacteria changing its physiological or physical structure. [12] Secretory virulence factors assist the bacterium in counteracting the host’s immune response by releasing chemicals. [12] 

One inhibitor of the T3SS is salicylidene acyl hydrazide, which has been shown to be effective against EPEC, EHEC, and Salmonella. [9] Although effective, the salicylidene acyl hydrazide was discovered to indiscriminately bind to human proteins, which may negatively affect patient metabolism. [10] It is important to note that the effect of salicylidene acyl hydrazide on T3SS is broadly accurate in preventing the secretion system from functioning. [10] 

One important study explores the ways a secretion system inhibitor, Aurodox, can affect the T3SS function in different bacteria. [8] Aurodox is the novel system that incorporates salicylidene. [8] Although it does not affect the growth of bacteria, Aurodox has been shown to inhibit the secretion of T3SS. The effectiveness of Aurodox on inhibiting the T3SS system depends on the concentration of the Aurodox injected into the in vivo sample. [8] Researchers have found that in key strains of E. coli, Aurodox did not inhibit growth of the bacterium while inhibiting the mechanism of T3S production (Fig. 4). [8 ]

Fig 4: (Aurodox will inhibit T3S secretion, but not the growth of different bacterial strains)

In the study, researchers found that Aurodox has the function of inhibiting EHEC to infect epithelial cells in infected mice. Although results were inconclusive at the cellular level, Aurodox was shown to reduce colon damage in the mice. [8] The expression of T3SS in various proteins involved in DNA-binding and altering were also shown to have been inhibited by Aurodox. 

Conclusion:

These results are promising for the inhibition of T3SS expression in anti-virulent treatment. As noted previously, past antibacterial treatments had induced the SOS response in the EHEC strain of E. coli, which was not the case for the anti-virulent Aurodox. [8] This is significant because the SOS response as a function leads to the release of Shiga toxins. Without the release of the deadly toxins, EHEC infection can be inhibited.

Aurodox as a treatment poses an interesting future for combatting E. coli. While Aurodox can effectively inhibit the secretory function of E. coli through the T3SS system, it does not halt production of the bacterium. This poses a question of whether to accept the cohabitation of EHEC and EPEC strains, among others that are considered harmful in the present. [8] If Aurodox can effectively neutralize the harmful effects of the strains, then there is little reason to eradicate the bacterium from the host. 

Drawbacks:

One drawback to current antibiotic treatments which anti-virulent therapy attempts to solve is the SOS response by E. coli. [8] The anti-virulent therapy can cause minor DNA damage, signaling an SOS response. The SOS response will occur after the use of antibiotics and eventually lead to the overproduction of Stx in the intestines. This uptick in Stx production will lead to a higher chance of severe symptoms of the anti-virulence treatment. 

Antibiotic drawbacks are more feared by the scientific community because E. coli is the most common pathogen in humans. In a study analyzing 150 different food samples to test the antibiotic sensitivity pattern of the bacteria, the highest percentage of drug-resistant E. coli was found in the most common foods with E. coli present, which included raw meat, eggs, and salad. [11] To combat this, scientists propose that the general population practice good hygiene, and farmers should only use reserve antimicrobial drugs to reduce the possibility for antimicrobial resistance. [11] Anti-virulence treatment does not deal with antimicrobial drugs, and is more likely to reduce unwanted side effects of general treatment of E. coli as compared to other proposed solutions. 

[1] E. coli. (2018). Retrieved 20 December 2021, from https://www.who.int/news-room/fact-sheets/detail/e-coli

[2] Kaper, J., Nataro, J., & Mobley, H. (2004). Pathogenic Escherichia coli. Nature Reviews Microbiology, 2(2), 123-140. doi: 10.1038/nrmicro818

[3] Fact Sheet: Escherichia coli - Microbial Identification - MALDI ToF. (2021). Retrieved 20 December 2021, from https://wickhamlabs.co.uk/technical-resource-centre/fact-sheet-escherichia-coli/

[4] Melton-Celsa, A. (2014). Shiga Toxin (Stx) Classification, Structure, and Function. Microbiology Spectrum, 2(4). doi: 10.1128/microbiolspec.ehec-0024-2013

[5] Gram-negative Bacteria Infections in Healthcare Settings | HAI | CDC. (2021). Retrieved 20 December 2021, from https://www.cdc.gov/hai/organisms/gram-negative-bacteria.html

[6] E. coli: What is It, How Does it Cause Infection, Symptoms & Causes. (2021). Retrieved 20 December 2021, from https://my.clevelandclinic.org/health/diseases/16638-e-coli-infection

[7] Kim, J., Lee, M., & Kim, J. (2020). Recent Updates on Outbreaks of Shiga Toxin-Producing Escherichia coli and Its Potential Reservoirs. Frontiers In Cellular And Infection Microbiology, 10. doi: 10.3389/fcimb.2020.00273

[8] https://doi.org/10.1128/IAI.00595-18

[9] Zambelloni R, Marquez R, Roe AJ. 2015. Development of antivirulence compounds: a biochemical review. Chem Biol Drug Des 85:43–55.

[10] Dai Wang, Caroline E. Zetterström, Mads Gabrielsen, Katherine S.H. Beckham, Jai J. Tree, Sarah E. Macdonald, Olwyn Byron, Tim J. Mitchell, David L. Gally, Pawel Herzyk, Arvind Mahajan, Hanna Uvell, Richard Burchmore, Brian O. Smith, Mikael Elofsson, Andrew J. Roe, Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds*, Journal of Biological Chemistry, Volume 286, Issue 34, 2011, Pages 29922-29931, ISSN 0021-9258, https://doi.org/10.1074/jbc.M111.233858. (https://www.sciencedirect.com/science/article/pii/S0021925819760700)

[11] Rasheed MU, Thajuddin N, Ahamed P, Teklemariam Z, Jamil K. Antimicrobial drug resistance in strains of Escherichia coli isolated from food sources. Rev Inst Med Trop Sao Paulo. 2014;56(4):341-346. doi:10.1590/s0036-46652014000400012

[12] Sharma AK, Dhasmana N, Dubey N, et al. Bacterial Virulence Factors: Secreted for Survival. Indian J Microbiol. 2017;57(1):1-10. doi:10.1007/s12088-016-0625-1

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The Cardiovascular Complications of COVID-19

by William Zhang

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic known as the coronavirus disease 2019 (COVID-19). While initially thought to be a pulmonary virus, scholars have recently argued that the virus may primarily affect the cardiovascular system instead. [1, 2] As such, SARS-CoV-2 remains a pathological mystery for the most part. This paper aims to review the interactions between SARS-CoV-2 and the cardiovascular system as well as how potential SARS-CoV-2 treatments can affect pre-existing cardiovascular complications by looking at the results of published studies. One specific study, published by Shi et al. in the March 2020 issue of JAMA Cardiology, retrospectively assessed the cardiovascular characteristics of 416 COVID-19 patients at the Renmin Hospital of Wuhan University. It was found that approximately one-fifth of the study cohort suffered from COVID-related cardiac injuries. Those who suffered cardiac injuries were of older age, and were associated with increased mortality, necessity for mechanical ventilation, and more severe complications such as acute respiratory distress syndrome (ARDS). [3] On a cellular level, COVID-19 causes cardiac damage through its affinity to the ACE2 protein that is highly expressed in cardiomyocytes. [3,4] Ultimately, a stronger understanding of COVID-19’s cardiovascular pathology is essential for the development of more effective countermeasures and treatments as well as a more specific identification of COVID-19 risk groups. 


Introduction

Since the reporting of its first case in Wuhan, China in December 2019, the novel coronavirus COVID-19 caused over a million confirmed deaths globally. The coronavirus outbreak was declared a pandemic by the World Health Organization on March 11, 2020 and continues to spread rapidly throughout the world with over 122 million confirmed cases as of March 19, 2021. [5] On a macroscopic level, the COVID-19 pandemic has caused global economic downturns due to businesses temporarily shutting down to comply with quarantine. For the average person, the COVID-19 pandemic not only poses physiological risks due to its high mortality rate, but also psychological dangers that can arise through prolonged quarantine and concern for loved ones that may be part of the COVID risk group. As such, an increased pathological understanding of COVID-19 is essential for the development of stronger preventative as well as curative approaches in order for our daily lives to return to normalcy.

COVID-19 is a member of a broader category of viruses known as coronaviruses, which are RNA viruses coated in layers of proteins that typically infect avians and mammals. According to the World Health Organization, COVID-19 is primarily spread through the respiratory tract. When an infected person expels viral droplets through coughing or sneezing, the virus remains in the air. When the airborne virus comes into contact with open orifices such as the eyes, mouth, or nose, it can enter an uninfected person and initiate a new viral infection. [6] While self-diagnosis is possible through detection of critical symptoms such as bluish lips and an inability to stay awake, confirmative diagnosis is performed through real-time reverse transcriptase polymerase chain reaction (RT-PCR) tests on nasopharyngeal swab specimens to detect viral RNA fragments. [7]

Due to COVID-19’s extensive interactions with the human respiratory system, the American Lung Association has classified it as a primarily pulmonary disease. [8] However, the pathology of COVID-19 remains mostly unknown and needs further research. In a recently published study by Reynolds et al., it was found that some COVID-19 patients exhibit hypoxemia due to abnormal vasodilation leading to ventilation-perfusion mismatches, which differs from acute respiratory distress syndrome-induced hypoxemia, where patients receive less oxygenated blood due to alveolar collapse. [9] Researchers began to suspect cardiovascular involvement because the primary receptor for COVID-19 viral entry, angiotensin-converting enzyme 2 (ACE2), is heavily expressed in cardiomyocytes, which renders them susceptible to viral infection and cellular damage. [4] Existing clinical studies also support this approach. One study, conducted by Klok et al., shows that 31% of the 184 Dutch ICU patients enrolled in the study suffered from thromboembolic complications such as ischemic stroke and deep-vein thrombosis. [10] This suggests that COVID-19 has significant cardiovascular effects, and a deeper understanding of its cardiovascular pathology can be groundbreaking for advancing COVID-19 countermeasures.

This paper focuses on the study published by Shi et al. in JAMA Cardiology in order to demonstrate how COVID-19 damages the cardiovascular system as well as how it exacerbates the patients’ pre-existing comorbidities. In this study, approximately 82 of the 416 enrolled patients suffered from cardiovascular complications. Patients with cardiac injury had higher mortality rates (51.2% vs. 4.2%), more need for mechanical ventilation (noninvasive 46.3% vs. 3.9%, invasive 22.0% vs. 4.2%), but also higher median age (74 vs. 60). [3] From these data, it can be seen that the cardiovascular factors that affect a COVID-19 patient’s health are complex, which warrants further investigation. With a stronger understanding of COVID-19’s cardiovascular complications, healthcare professionals can treat risk groups more effectively and therefore lessen the severity of COVID-19 infections in demographics such as elders and patients with pre-existing medical conditions. In addition, we can also better grasp the long-term effects of COVID-19 infections and more readily prepare against them.


Methods

The following methods are paraphrased and taken from the study published by Shi et al. in JAMA Cardiology. [3]

Participants

The study was conducted with COVID-19 patients admitted to the Renmin Hospital in Wuhan, China between January 20, 2020 and February 10, 2020. These patients were diagnosed with COVID-19 with the guidance of the World Health Organization. For the purposes of this study, the enrolled patients who did not present cardiac damage biomarkers such as high-sensitivity troponin I (hs-TNI) and creatine kinase (CK-MB), which are substances that are released into the bloodstream when the heart is under stress, were excluded from the study.

Data Collection

Metrics relevant to the study were collected by researchers from electronic medical records. The data includes: demographics, clinical history, lab test results, and results from cardiac investigations (biomarkers and EKG). Cardiac damage biomarkers were measured upon patient admission, while radiologic data were collected through chest radiography and CT scans. Patients were divided into two groups, one with cardiac injury and another without cardiac injury, where cardiac injury is defined as the expression of hs-TNI above the 99th-percentile upper reference limit. The clinical outcomes of the patients were monitored until February 15, 2020, which was the final date of the follow-up.

COVID-19 diagnoses were confirmed with the Viral Nucleic Acid Kit (Health), which extracted viral nucleic acids. The nucleic acids were then subjected to the 2019-nCoV kit (Bioperfectus), which tests for the N gene and the ORF1ab gene via RT-PCR. Positivity in both tests determine a successful diagnosis of COVID-19.

Statistical Analysis

Categorical variables involved in the study were compared to each other using the Fisher exact test or the χ2 test, while continuous variables were compared to each other using the t-test or the Mann-Whitney U-test. Continuous data were expressed as means (with standard deviation) or medians (with interquartile range), and categorical data were presented as proportions. Survival data were presented as Kaplan-Meier curves, and the survival of patients with cardiac injury versus patients without cardiac injury were analyzed through the log-rank test. Multivariate Cox regression models were used to determine the independent risk factors for death during hospitalization. For all the statistical analyses, P < .05 was considered significant.


Results

The data, figures, and results as presented in this section are all paraphrased and taken from the work of Shi et al. as published in JAMA Cardiology. [3]



Table 1. Baseline Characteristics and Laboratory and Radiographic Findings of 416 Patients With COVID-19. [3]

The retrospective chart study conducted by Shi et al. yielded a large set of patient data demonstrating the interactions between COVID-19 and the cardiovascular system (Table 1). Shi et al. separated the patients into two categories: with cardiac damage and without cardiac damage, and the two patient groups are compared against each other. For this study, cardiac injury was defined as the presence of the cardiac biomarker, hs-TNI, above the 99th percentile.

Statistical analyses show a significantly higher median age for the former group (74 vs. 60, p < 0.001), which implies stronger vulnerability for the elderly against the cardiovascular effects of COVID-19. The statistics for signs and symptoms upon admission are similar between the two groups, but it should be noted that patients who present with chest pain, a common sign of cardiovascular distress, are much more likely to experience cardiac damage from COVID-19 (13.4% vs. 0.9%, p < 0.001). 

Patients with COVID-19 comorbidities appear to have a higher risk of suffering cardiac damage. For example, a higher proportion of patients with cardiac damage suffer from hypertension as opposed to those that are infected by COVID-19 but did not experience cardiovascular symptoms (59.8% vs. 23.4%, p < 0.001). This phenomenon seems to remain consistent in this patient population across most other severe comorbidities known to medical professionals with the exception of pregnancy (0% vs. 2.1%).

Figure 1. Kaplan-Meier survival curves for COVID-19 patients. Mortality over time for patients are graphed from A. time of symptom onset and B. time of admission. In B, the maximum number was 16 days for the population with cardiac injury. C. A comparison of outcomes between patients with and without cardiac injury through log-rank test both starting from time of symptom onset and time of admission.

After analyzing the patients’ pre-admission statistics, Shi et al. also studied the progression of COVID-19 in the two patient populations by plotting their mortality against time (Fig. 1). Patients who suffered from cardiovascular damage from COVID-19 were seen as more severe cases, as it had taken them significantly shorter time to go from symptom onset to follow-up (mean, 15.6 [range 1-37] days vs. 16.9 [range 3-37] days, p < 0.001) as well as admission to follow-up (mean, 6.3 [range 1-16] days vs. 7.8 [range 1-23] days, p < 0.001). Mortality rate was also higher among the patients that experienced cardiac injury as opposed to the non-cardiac injury group (51.2% vs. 4.5%, p < 0.001). 


Table 2. Multivariate Cox Regression Analysis on the Risk Factors Associated With Mortality in Patients With COVID-19

In order to further determine the risk behind cardiovascular damage behind COVID-19, Shi et al. performed a Cox regression analysis on various risk factors and their impact on patient mortality (Table 2). The Cox regression analysis is a model that determines the the effect of multiple variables on a given event through the hazard ratio, and as seen from Shi et al.’s data, the leading risk factor of COVID-19 mortality is ARDS with an average hazard ratio of 7.89 (p < 0.001), followed by cardiac injury with an average hazard ratio of 4.26 (p < 0.001). 

Discussion

Shi et al.’s work shows that despite COVID-19’s nature as a primarily pulmonary disease, its cardiovascular complications are severe and cannot be overlooked. Even though ARDS overshadows cardiac injury as the primary risk factor for COVID-19 mortality, Shi et al. reports that patients with cardiac injury are more likely to need advanced intervention such as noninvasive and invasive mechanical ventilation. [3] Other researchers’ works have agreed with these observations, bringing the cardiovascular complications of COVID-19 to the attention of emergency care workers and even discussing the potential of chronic cardiovascular damage. [2, 11] Zheng et al. have also noted the complex interactions between COVID-19 antivirals and the cardiovascular system, citing “cardiac insufficiency, arrhythmia” among other forms of antiviral-induced cardiac toxicity as a cause for concern for patients with pre-existing cardiovascular complications. [11] While much of COVID-19’s pathophysiology remains unexplored, current research is bringing light to the importance of long-term care for COVID-19 patients even after discharge as well as the necessity for more effective treatment plans that address the severity of cardiovascular damages.

The main observation from Shi et al.’s publication is the association between cardiovascular damage from COVID-19 and its risk of mortality; two risks correlated with COVID-related cardiac injuries are comorbidities and old age. Shi et al.’s findings bring a new understanding as to how the elderly are a risk group beyond their possession of a generally weaker immune system that renders them more vulnerable to viral infections. [12] As the human body ages, so does its organs, and a weaker heart is more likely to be exploited by COVID-19 specifically. While the precise interaction between COVID-19 comorbidities and the virus itself remains unclear, Shi et al. has also cemented a strong association between the two through a cardiovascular perspective. Alongside other present research such as Klok et al.’s study on thromboembolic crises in COVID-19 patients, [10] the results highlighted in this paper will encourage further research to be done on the cardiovascular pathophysiology of COVID-19 in order to better serve known risk groups beyond knowing that the elderly and those with underlying illnesses are more susceptible to severe COVID-19 symptoms.

However, it must be noted that Shi et al. acknowledged the limitations of the study. The ongoing nature of the clinical observations may lead to further conclusions being drawn in the future, and larger patient populations must be observed in order to draw more general conclusions. [3] While the work of Shi et al. supports theories that COVID-19 can directly damage the heart due to its affinity for ACE2, [13] there have also been studies that disagree with the potential for COVID-19 to directly damage the heart. For instance, a study by Xu et al. shows that signs of cellular inflammation have been found in COVID-19 patients without significant cardiac injury, and Shi et al. cited Xu et al.’s work as a potential indication of COVID-19’s indirect involvement in cardiac injuries. [3, 14] As such, the cardiac pathophysiology of COVID-19 remains a mystery, but is certainly an aspect of the disease that necessitates further research. 

Conclusion

Shi et al.’s study was conducted in order to gain a better understanding of the pathophysiology of COVID-19 after observing potential cardiovascular correlations in the patient body. Through clinical observations as well as retrospective chart studies, Shi et al. have found that not only does COVID-19 worsen with pre-existing cardiovascular comorbidities, the presentation of new cardiac injury in COVID-19 patients is strongly associated with mortality. Despite the virus’s tendency to primarily attack the pulmonary system, Shi et al. has shown that the cardiovascular system is also a risk factor to consider, as patients with cardiovascular symptoms typically need more intensive care and intervention. These results are in agreement with existing studies showing that COVID-19 cell entry is dependent on the ACE2 protein that is heavily expressed in cardiomyocytes, which implies that cardiomyocytes are at great risk of being a target for COVID-19. Overall, Shi et al.’s findings suggest that the human heart is an important subject of study in COVID-19 pathophysiology due to its association with increased severity of symptoms. As research progresses, cardiovascular breakthroughs can help with the treatment and control of COVID-19 in the long term. 

Works Cited

[1] Kavanaugh, K. Is COVID-19 Primarily a Heart and Vascular Disease? Infection Control Today, Sep. 8, 2020, https://www.infectioncontroltoday.com/view/is-covid-19-primarily-a-heart-and-vascular-diseases

[2] Long, B et al. Am. J. Emerg. Med. 2020, 38, 1504-1507

[3] Shi, S et al. JAMA Cardiol. 2020, 5, 802-810

[4] Pérez-Bermejo, JA et al. bioRxiv. [Online] 2020. https://www.biorxiv.org/content/10.1101/2020.08.25.265561v1.full. (Accessed October 31st, 2020)

[5] COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University. https://www.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6. (Accessed October 31st, 2020)

[6]: Coronavirus disease (COVID-19): How is it transmitted? https://www.who.int/news-room/q-a-detail/coronavirus-disease-covid-19-how-is-it-transmitted. (Accessed October 31st, 2020)

[7]: Country & Technical Guidance - Coronavirus disease (COVID-19). https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance-publications. (Accessed October 31st, 2020)

[8]: Coronavirus Disease (COVID-19). https://www.lung.org/lung-health-diseases/lung-disease-lookup/covid-19#:~:text=COVID%2D19%20is%20a%20lung,other%20than%20supportive%20care%20available. (Accessed October 31st, 2020)

[9]: Reynolds, AS et al. Am. J. Respir. Crit. Care Med. 2020, 202, 1037-1039

[10]: Klok, FA et al. Thromb. Res. 2020, 191, 145-147

[11]: Zheng, Y et al. Nat. Rev. Cardiol. 2020, 17, 259-260

[12]: Meng, H et al. Psychiatry Res. 2020, 289, 112983

[13]: South, AM. et al. Am. J. Physiol. Heart Circ. Physiol. 2020, 318, H1084-H1090 

[14]: Xu, Z et al.Lancet Respir. Med. 2020, 8, 420-422

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Epigenetic and Metabolic Shifts in Immune Tolerance

by Daanish Sheikh

INTRODUCTION

Although it is well established that the adaptive immune system can mount an improved immune response upon reinfection with most pathogens, newer insight into innate immune system components suggest that macrophages can be “trained” to also elicit a stronger second immune response, known as immune training. However, the opposite effect, a reduced immune response, has also been observed when monocytes are pre-stimulated with bacterial cell wall component lipopolysaccharide (LPS) [1]; this phenomenon has been termed immune tolerance. Much like how exhausted CD8+ T cells following chronic infections are unable to produce cytokines at optimal levels, tolerized myeloid cells seem to produce fewer cytokines and have diminished phagocytic activity. Monocyte derived macrophage impairment characteristic of immune tolerance has also been linked with immune paralysis following sepsis [2] and in patients recovering from tuberculosis (TB) infection [3-4]. Both TB and sepsis survivors have an increased risk of later secondary infection and an increased risk of mortality [5-6]. Long lasting epigenetic changes are presumed to be responsible for the immune suppression that is observed in these patients [7]. These epigenetic changes are induced by metabolic shifts that upregulate glycolysis, the citric acid cycle, and oxidative phosphorylation, which in turn are activated via the cellular metabolism regulating pathways governed by PI3K, Akt, and mTOR. Pathogen-associated molecular patterns (PAMPs) are recognized by macrophage cell surface pattern recognition receptors whose downstream pathways include these metabolism regulating enzymes. The most well characterized PAMP in endotoxin tolerance involves LPS, which activates the macrophage via cell surface Toll-like receptor 4 (TLR4). PAMP-mediated immune activation also induces epigenetic remodeling of various inflammatory response genes and tolerizing genes. This paper will explore the epigenetic and metabolic shifts that are characteristic of immune tolerance or lead to its development, including the metabolic rheostats by which said long-term epigenetic shifts are mediated.

Myeloid Tolerance and TLR4 Pathway Mediation 

We begin with a brief overview of the pathways downstream of TLR4 and then explore how the pathway varies in immune tolerant cells. Resembling conditions present in sepsis, long term exposure to LPS can result in myeloid immune tolerance; in fact, this model remains the most well characterized example. In LPS tolerance models, immune activation is initially induced via TLR4 exposure to LPS and is transiently characterized by increased energy metabolism via the mTOR and NFAT pathways. TLR4 mediated immune activation occurs via two pathways; first, activation initially results in the production of pro-inflammatory cytokines such as TNF, IL-1, IL-12, and CCL3 via a kinase cascade that includes myeloid differentiation factor 88 (MyD88), various interleukin receptor associated kinases (IRAKs 1,2, and 4), TNF receptor associated factor 6 (TRAF6), and transcription factors NF-κB and AP-1. Meanwhile via a second pathway, the initial activation of TLR4 also upregulates the secretion of interferon ß (IFNß) via a pathway including Toll/IL-1 receptor domain-containing adaptor (TRIF), IKKi kinase, TANK-binding kinase (TBK1), and transcription factor IRF3 [8]. IFNß secretion from this second pathway increases expression of interferon-inducible cytokines that are essential in an immune response [9] (Figure 1). Meanwhile, upregulation of the mTOR and NFAT pathways causes shifts in cellular metabolism that emphasize glycolysis, the TCA cycle, and the electron transport chain, allowing greater energy metabolism by the cell to meet the high energy demands of immune activation [10].

Figure 1: Simplified depiction of the TLR4 pathway and two of its downstream transcription factors

Myeloid immune tolerance is characterized by diminished expression of TLR4, decreased interaction of MyD88 and TRIF with TLR4, and diminished NF-κB and AP-1 signaling. As such, proinflammatory cytokine production is significantly reduced [11]. Upregulation of the mTOR energy metabolism pathways during immune activation is catalyzed via association of upstream kinase PI3K with the TLR4; this pathway is fairly well characterized but summarized here for clarity. PI3K, upon activation via association with active TLR4 and MyD88, phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to form PIP3, which in turn associates with phosphate dependent kinase-1 (PDK-1) to activate protein kinase B (PKB or Akt). Phosphorylated Akt activates the mTOR protein resulting in the aforementioned downstream upregulated metabolic effects [12]. However, diminished expression of TLR4 and MyD88 binding in myeloid tolerance terminates this metabolic upregulation, further exacerbating tolerance.

Metabolic and Epigenetic Modification in Myeloid Tolerance

Some of the causes of myeloid immune tolerance can be attributed to pathways that branch off the TLR4 activation pathway. For instance, PI3K, the kinase implicated in upregulating metabolism, is also implicated in pathways that result in production of anti-inflammatory cytokines [13]. p50, a subunit of NF-κB, is essential for the development of immune tolerance by homodimerization rather than the canonical heterodimerization with p65, another subunit [14]. Other transcription factors that are commonly observed in resting macrophages can also be characteristic of tolerized macrophages. Transcriptional repressor Bcl-6 inhibits the production of inflammatory gene products by regulating the TLR4 transcriptome via association with transcription factor Pu.1. Pu.1 and NF-κB association with inflammatory gene promoters upregulate transcription of inflammatory genes by conferring a tri-methylation to the 4th lysine residue of the H3 histone protein in chromatin (H3K4me3) near those genes [25]. Because Bcl-6 recruits histone deacetylases and histone demethylases to the same site, the same inflammatory genes are downregulated in its presence [24]. Other prominent transcription factors responsible for myeloid tolerance include RelB (a subunit of NF-κB) and high mobility group box 1 protein (HMGB1). RelB complexes with H3K9 methyltransferase G9a to form heterochromatin at the IL-1ß promoter, inhibiting the transcription of IL-1ß, while HMGB1 binds to the TNF-α promoter and recruits RelB complex assembly to downregulate TNF-α transcription [26-27]. It seems clear that transcription factor regulators of myeloid cell activity are highly varied and impact individual gene products allowing a significantly greater degree of control over specific effects of myeloid tolerance as opposed to the widely ranging effects of regulating the TLR4 pathway upstream.

However, over a longer period of stimulation, metabolic shifts in NFAT and mTOR also induce the epigenetic changes that eventually lead to immune tolerance [15-16]. There are three primary links between these metabolic and epigenetic aspects of the cell: Sirtuin1 (Sirt1), alpha-ketoglutarate dependent dioxygenases (αKG-DD), and NuRD10. Activation of Sirt1 occurs at increased concentrations of NAD+, and high dosage LPS exposure drives myeloid cells to upregulate a de novo NAD+ synthesis pathway [17]. Sirt1 is part of the NAD-dependent deacetylase sirtuin family and silences several proinflammatory genes including Tnf and Il1b. Sirt1 also deacetylates RELA, the gene that codes for NF-κB subunit p65 while also binding to RelB, another subunit [18-20]. This SIRT1-RelB complex then serves to assemble a repressor complex that via chromatin methylation later results in endotoxin tolerance18. The second rheostat αKG-DD is a group of epigenetic enzymes whose activity is balanced by αKG and succinate levels in the cell; succinate, as well as various other TCA metabolites including malate, itaconate, fumarate, and 2-hydroxyglutarate, inhibit the activity of these enzymes [21-22]. However, further elucidation of the timing, duration, and relevant metabolites of said TCA cycle shifts to induce epigenetic remodeling by these enzymes is needed. Finally, the NuRD complex is responsible for histone deacetylation and DNA hypermethylation that prevent T-cells from targeting native cells or macrophages from excess inflammation [23]. Upon immune activation, large quantities of reactive oxygen species (ROS) are produced via the electron transport chain; mice with cancer induced immune exhaustion have large quantities of ROS and similar epigenetic changes as to what is induced by NuRD. High levels of mitochondrial ROS also seem to be associated with CD8+ T cell immune exhaustion during TB infection which leads to immune activation via the same Toll-like receptors as LPS [20].

Figure 2: Epigenetic rheostats that link metabolic shifts in the Krebs cycle to immune suppression

CONCLUSION

Although immune tolerance acts as a suppressive mechanism to prevent an excessive inflammatory response, immune tolerance increases the likelihood of secondary infections months following the initial immune challenge; as observed by the long-term morbidity and mortality following chronic infections such as TB and sepsis. Further research considering the possible rescue of immune tolerance phenotypes when cells are exposed to drugs modulating the three aforementioned rheostats should be undertaken to explore the clinical possibilities of reversing immune tolerance and thereby restoring optimal immune function after chronic infection. 

 

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28.Images created with Biorender

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