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Focus of Project

 

The United States’ economic debt is increasing at an unprecedented rate, which experts say will soon lead to an abrupt economic collapse. This exponential rise in the debt is not projected to stop any time soon: While in the mid-1900s, debt averaged around 35% of the Gross Domestic Product (GDP), today it is around 78% of GDP (Swagel), and projections indicate that the national debt will continue to double every administration (Chappell). According to experts, left unchecked, this exponential rise in the national debt will soon spell the collapse of the United States’ economy, as foreign investors begin to pull out of American investments and the value of the dollar plummets (Gilchrist).  This debt is constituted almost entirely by one thing alone: Medicare/Medicaid (Mulvaney). These United States’ health care entitlement programs, as the expert consensus indicates, is due to collapse in roughly 8 years due to its own financial insolvency (Pear). Experts agree that this economic disaster cannot possibly be prevented by any political solutions, as any effort to reform the Medicare system has time and again been met with government inaction and often complete shutdown (Krawzak). Historical analysis of similar situations in other countries demonstrates similar outcomes of political inaction; political action simply cannot solve this problem (Pierson). Moreover, according to economic experts (Feldstein), this problem cannot be solved by increasing taxes or shifting the allocation of government revenue either, as the financial unsustainability of Medicare is increasing at a rate faster than the government could possibly increase its sources of revenue: at the inception of Medicare, one elderly individual was supported by almost five taxpayers, while today, that ratio has dropped to one elderly individual supported by fewer than three taxpayers, and is projected to drop to one elderly individual supported by only two taxpayers by 2030 (Verma). The only solution to the rapidly approaching economic collapse of Medicare, therefore, is a scientific one. The cost of elderly healthcare expenses -- indeed, the cost of lifetime expenses -- is constituted almost entirely by care in the last few years of life (approximately 80% of lifetime medical expenses). Ironically, these expenses predominantly consist not of the procedures and operations performed to recover elderly patients from infection and injury -- all of which have been all but perfected by modern western medicine -- rather, they consist of healthcare measures aimed at keeping elderly patients stabilized during postoperative care. Despite the near-perfection of surgical and medical practice in the United States and in the West as a whole, there is yet no clinical avenue by which to ameliorate post-operative autoimmune responses, which is the primary (almost the only) cause of mortality post-surgery. In other words, the advancements in western medicine have been rendered almost completely moot, especially among elderly patients, by the current complete lack of methods to deal with harmful autoimmune response, a process known as sepsis (Gyawali). Sepsis represents the single most humiliating medical failure of western medicine: various studies indicate that 30-50 percent of patients with severe sepsis die despite expensive treatment, and this number skyrockets the older the patient becomes (Paoli). In other words, when a patient (especially an elderly patient) develops sepsis, even after successful treatment of the initial infection or injury, survival is up to the flip of a coin. Moreover, sepsis accounts for the single most costly elderly healthcare expense and, if means of mediation are not developed, will single-handedly result in the collapse of the funding structure of the American Medicare system (Torio). Sepsis is, without a doubt, the most devastating medical and economic problem faced by the United States today, and this is what my project has addressed. Given the severity and prevalence of this issue, completely solving sepsis in one project alone would be infeasible, but I have developed a novel framework and methodology which future scientists can proceed to apply clinically in order to eliminate the danger of sepsis once and for all.

 

 

Central Achievement

 

I have discovered a biochemical method of inhibiting and reversing the process of sepsis. Firstly, I have recognized the single largest biochemical contributor to the process of sepsis, which is the binding of a ligand called eCIRP to an extremely proinflammatory receptor called TREM-1. Secondly, I targeted this binding with the novel small peptides called M3 and LP17. By using small peptides M3 and LP17 to inhibit eCIRP binding to TREM-1, the process of sepsis is downregulated to an extent that has never before been achieved. By developing peptides M3 and LP17 into a pharmaceutical treatment, this discovery opens up clinical avenues for the unprecedented inhibition of sepsis.

 

Methods of ameliorating sepsis remains the single weakest link in western medicine, with only rudimentary and largely ineffectual treatments currently in practice. Medical procedures and surgeries that in and of themselves have been all but perfected by the western medical world, are rendered useless by the fatal impacts of the West’s near-complete lack of strategies to deal with the sepsis that results from the implementation of such practices. Despite the overwhelming consensus among physicians with respect to the severity of this problem and the desperate need to discover a means of prevention, previous efforts before my research were unsuccessful. (Rello). Previous methods of sepsis prevention consisted solely of the ineffectual use of antibiotics to combat the initial opportunistic infection, which do little to nothing to combat the deleterious effects of sepsis itself (Rello). As stated, my research has successfully discovered a biochemical means of downregulating the process of sepsis itself and preventing associated mortality.

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My research, firstly, has identified this receptor’s primary ligand as Cold Inducible RNA-binding Protein (eCIRP), a piece of knowledge crucial to the successful attenuation of the harmful pathway. Secondly, it has revealed two small peptides capable of downregulating eCIRP-mediated sepsis, known as M3 and LP17, by inhibiting eCIRP binding to TREM-1 on two fronts. This discovery creates countless novel avenues for both the prevention and retroactive alleviation of sepsis: with the crucial discovery of the anti-inflammatory effects of M3 and LP17, future researchers can now spend resources on the development of clinical and pharmaceutical applications of these peptides.

 

Societal Issue

 

As mortality statistics indicate, manifestations of systemic inflammation are presently one of the most prevalent and unpreventable causes of death in the United States. In 1995, complications due to sepsis resulted in more than 215,000 deaths, equating to roughly 10% of all deaths in the United States that year, and these mortalities have only been growing since (Angus). The continued inability of the industrialized world to adequately treat sepsis compromises all otherwise-perfected areas of medicine, rendering pointless all western medical and surgical advances, and will lead to the collapse of the American Medicare system within the next decade if not dealt with.  Moreover, due to the current lack of means of either prevention or successful retroactive treatment, the mortality rate for sepsis is exceedingly high at an average of roughly 30%, reaching as high as roughly 50% among elderly patients (patients age 85 or above). Similarly, the vast majority of sepsis cases occur in elderly patients, with an incidence rate of almost 27 per 1000 elderly individuals (Angus), a rate that is more than 20 times higher than the rate of murder in the most violent city in the United States (Schiller). In other words, once patients become afflicted with severe sepsis, their survival is almost entirely up to chance, and the older one gets the worse one’s likelihood of survival becomes. The elderly, therefore, are being continually killed by what should be routine and harmless procedures. When they are not killed by sepsis resulting from these routine procedures, moreover, they are forced to incur the tremendous personal costs of intensive care treatment: A Premier analysis indicated that costs for elderly patients can reach as high at $70,000 for hospital-associated sepsis per patient, a cost that the vast majority of elderly patients, most of whom have little more in assets than their home and social security payouts (Grad), cannot possibly afford. The cost of this sepsis treatment, therefore, is then cast upon the Medicare (DeVore). Experts, even the optimistic, agree that Medicare is in rapid decline due to financial insolvency, and will collapse in as soon as seven years. Almost all of Medicare expenditure is not constituted by medical or surgical treatment (these make up a negligible amount of elderly healthcare costs) rather by ineffectual and drawn-out treatment of sepsis which occurs after these procedures. In other words, especially for the elderly, sepsis is a death sentence, both for the elderly patients whose survival is left entirely up to chance and for the American Medicare system, which is predicted by experts to suffer a collapse due to sepsis alone. Physicians still did not yet have the means of preventing or even substantially downregulating the base process of sepsis. The medical community desperately requires a method of controlling the biological mechanisms of sepsis (Rello), which my research provides.

 

Literature Review

 

My research has built upon prior basic science research in the field of sepsis in order to develop a biochemical methodology to solve the problems that had been identified by previous scientists. Although previous scientists have furthered basic understanding of the process of sepsis, none have succeeded in developing a methodology to solve the problem. 

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Sepsis is the single most medically and economically devastating problem facing the medical world. A review article (Gyawali) published in March of 2019 underscores the most pertinent facts about the biological process, “sepsis remains one of the major causes of morbidity and mortality in critically ill patients. The annual incidence of severe sepsis and septic shock in the United States is up to 300 cases per 100,000 people. Sepsis is also the most expensive healthcare problem in the United States...more than 30 million people are affected by sepsis every year worldwide, resulting in potentially 6 million deaths annually. Mortality rates from sepsis, as per the data from the Surviving Sepsis Campaign 2012, were approximately 41% in Europe versus approximately 28.3% in the United States” (Gyawali). Sepsis is simultaneously one of the most abundant and the deadliest medical condition, resulting in both an extremely high rate of incidence (30 million people per year) and rate of mortality (ranging from 28.3% to 41%). Sepsis single-handedly accounts for over 10% of total deaths every year (6 million sepsis deaths out of roughly 57 million total deaths annually) (Ritchie). In fact, sepsis kills about 10 times as many people per year than every form of cancer combined. 

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However, despite the indubitable severity of the problem of sepsis, there is exceedingly little the western medical world can do to reduce its rates of morbidity and mortality. A review article (Rello) published in 2017 summarizes the consensus that is already ubiquitous in the medical community (Rello): As Dr. Rello states, “at present there is no specific treatment.” The article goes on to discuss that the only current management methods for sepsis “focus on containing the infection through source control and antibiotics plus organ function support,” with no treatment of the process of sepsis itself. In other words, physicians attempt to treat the infection that causes sepsis, but do not focus on the biological process of sepsis itself. However, as this review article describes, “the response to the infection is inadequate and may lead to organ dysfunction.” Therefore, once sepsis begins, using a source control method of treatment, survival of the patient is up to chance. I was able to identify from the most recent literature receptors and ligands that may be responsible for upregulating systemic inflammation, from which I could develop a biochemical means of preventing sepsis through completely novel inhibitory peptides that I synthesized, using not only biological assays but using advanced computational modelling as well. No other peptides to inhibit the eCIRP/TREM-1 binding had ever previously been identified.

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Although the peptides on which my research is centered -- receptor TREM-1 and its ligand eCIRP -- had previously been identified, my research has, one, uncovered that eCIRP is a ligand of TREM-1, and two, developed a methodology to overcome an inability to inhibit eCIRP/TREM-1 binding. In prior research, the TREM-1 receptor had been linked to inflammation attenuation. As a paper from the American Institute for Clinical Research stated, “TREM-1 is constitutively expressed on most monocytes/macrophages and neutrophils and is upregulated by various stimuli such as the TLR ligands LPS and lipoteichoic acid (LTA) and the proinflammatory cytokine TNF. Hence, it has been suggested that TREM-1 is mainly involved in acute inflammatory reactions but not in chronic inflammatory disorders. The natural ligand of TREM-1 is still unknown” (Schenk). In other words, TREM-1, as an independent actor in the process of acute systemic response to stimuli has been known for a considerable period of time. However, the primary natural ligand involved in the stimulation of its proinflammatory pathways has remained an enigma, without which any hope of its control or inhibition is futile. My research builds on this previously-established knowledge by introducing both the natural ligand of TREM-1 and its potential inhibitors, offering not only greater comprehension of a biological pathway so crucial in the process of sepsis, but also clinical avenues for control thereover. 

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Similarly, the independent attenuation of inflammation caused by the eCIRP ligand has previously been identified without any relationship to the TREM-1 receptor and without any means of inhibiting its binding. Prior research from the Feinstein Institute for Medical Research observed, “...the detection of cold-inducible RNA-binding protein (eCIRP) in the blood of surgical ICU individuals. In animal models of hemorrhage and sepsis, eCIRP is up-regulated in several organs and released into the circulation. Under hypoxic stresses, eCIRP in macrophages is translocated from the nucleus to the cytosol and actively released” (Liao). While this research was crucial in establishing a better knowledge of the effects of eCIRP, it required further study on two fronts: firstly, it did not address closely enough the effects of eCIRP in human models, leaving questions about the similarity between animal models and humans generally unanswered. Secondly, it did not identify the primary receptor involved in eCIRP-induced pathways of upregulation. Thirdly, it did not suggest any means of inhibiting eCIRP binding. By addressing all of these issues, my research finally provides a clinical application for eCIRP in inhibiting the biochemical processes of sepsis.

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While first steps had clearly been taken in the understanding of eCIRP and TREM-1 pathways before my research, no work into the possibility of their inhibition had been undertaken. In other words, my research does not simply expand general biological knowledge about the two molecules, but offers solutions to their control, any proposal for which has not yet been suggested, yet which is fundamental to the attenuation of sepsis.

 

Significance

 

The revolutionary ability to use small inhibitory peptides M3 and LP17 to downregulate inflammation will finally provide the medical world with a biochemical avenue by which to control sepsis. Sepsis is the single most pervasive challenge facing healthcare providers all over the world. However, previous efforts in source control treatments have yielded little to no actual results in reducing the severity of this problem. The only way to solve the grave medical challenge that is sepsis is to prevent it before it begins, and my research has proven able to do exactly that. The cutting edge ability to control and utilize small peptides M3 and LP17 in a clinical environment will lead future scientists to finally eradicate sepsis.

 

Research Hypothesis

 

Given the structural similarities between the peptides M3 and eCIRP and LP17 and TREM-1, I hypothesized that they M3 and LP17 could be used as inhibitors of eCIRP/TREM-1 binding, thereby preventing the stimulation of important proinflammatory pathways involved in systemic inflammation and sepsis.

 

Methodology

 

BIOVIA Discovery Studio 3D Rendering and Structural Homology Comparison:

Through the Discovery Studio modelling software, the 3D physical structures of M3 and eCIRP were compared and their similarity in homology was quantified as well as qualitatively oriented and visualized in order to structurally illustrate the potentiality of M3 as an inhibitor of eCIRP binding.

 

Enzyme-Linked Immunosorbent Assay: Human monocyte HL-60 cells were stimulated with rmeCIRP (μg/mL) with and without TREM-1 inhibitors LP17 and M3 at various doses. IL-1β and IL-6 concentrations present in the culture supernatants were subsequently measured with ELISA. Note: the HL-60 cells used in this assay were from a commercially purchased cell line from the American Type Culture Collection (ATCC), catalog number CCL-240.

 

Western Blot Assay: RAW 264.7 cells were stimulated with or without TREM-1 inhibitor M3. Gel electrophoresis was run on protein samples derived from cells and concentrations of various downstream signals of the TREM-1 pathway were measured.

 

Biacore (SPR) Assay: Binding affinity of eCIRP and TREM-1 as well as M3 and TREM-1 were determined through surface plasmon resonance. In each analysis, one ligand, a fixed, immobilized substance, and one analyte, a substance allowed to flow over the ligand, were allowed to bind and dissociate. During the test, the Biacore Machine calculates a Kinetic Dissociation (KD) constant that illustrates the binding affinity between the two molecules. The KD values calculated for the eCIRP/TREM-1 relationship (natural ligand/receptor) and for the M3/TREM-1 relationship (potential inhibitor/receptor) were then compared to quantitatively evaluate the efficacy of M3 as an inhibitor of the eCIRP/TREM-1 complex.

 

 

Results

 

BIOVIA Results: As a preliminary analysis of the potential for M3 inhibition of eCIRP/TREM-1 binding, BIOVIA Discovery Studio software was used to render small peptide M3 and eCIRP visually and compare their structural homology. Qualitative analysis indicated M3 to be sufficiently homologous to serve as a potential competitive inhibitor. This initial analysis was substantiated quantitatively by biochemical BioCore assay.  

 

Figure 1: Computationally rendered structures: M3 and eCIRP (superimposed)  

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BiaCore Results: As a secondary analysis of the potential for M3 inhibition of eCIRP/TREM-1 binding, a surface plasmon resonance assay utilizing the Biacore hardware was performed. This assay both visualized and quantified the binding affinity between both M3 (the inhibitory peptide) and TREM-1 and eCIRP (the natural ligand) and TREM-1. Through comparison of the calculated dissociation coefficients (KD) for both complexes, a primary hypothesis about the inhibitory potential of M3 was determined. From the determined coefficients, it was found that the binding affinity between M3 and TREM-1 was remarkably close to that between eCIRP and TREM-1, suggesting that such a competitive relationship in TREM-1 receptor binding would most likely exist between M3 and eCIRP, a hypothesis later substantiated by an ELISA analysis.

 

Figure 2: Binding affinities between M3 and TREM-1 vs eCIRP and TREM-1 

 

 

  

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ELISA Results: Through an enzyme-linked immunosorbent assay (ELISA), the inhibitory impacts of the peptides M3 and LP17 were tested: five groups in total were made per assay, each containing eight wells of a particular solution. Two were control groups, one positive and one negative, followed by three experimental solutions of eCIRP/M3 or eCIRP/LP17 in a dose dependent manner. Each group contained HL-60 (human monocyte) cells, all of which expressed TREM-1. The positive control contained a solution of solely eCIRP, while the negative control contained a PBS buffer without any eCIRP. By using the secretion of certain proinflammatory cytokines, specifically IL-1β and IL-6, as a metric for the upregulation of proinflammatory pathways, both peptides M3 and LP17 individually were observed to yield significantly decreased concentrations of both aforementioned cytokines when introduced to the assay in increasing concentrations. Once the concentration of LP17 and M3 reached 139 μg/mL and 100 μg/mL respectively, level of both IL-1β and IL-6 differed statistically insignificantly from the negative control, indicating a near-complete inhibition of proinflammatory pathways triggered by eCIRP/TREM-1 binding.

 

Figure 3: ELISA results

 

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Western Blot Results: Through western blot analysis, concentrations of downstream signals of TREM-1 in RAW 264.7 cells stimulated with a sham solution, eCIRP, or eCIRP and M3. Computational pixel analysis of results indicate that there is a significant inhibitory trend between small peptide M3 and eCIRP/TREM-1 binding, even further suggesting its potential as a clinical mediator of sepsis. 

Figure 4: Western blot bands

 

Figure 5: Western blot computationally determined pixel concentrations from bands

 

 

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Discussion

 

From my research, the biochemical and computational data all overwhelmingly point to M3 and LP17 being inhibitors of eCIRP/TREM-1 binding, one of the most substantial contributors to inflammatory upregulation during sepsis. M3 and LP17 both possess remarkably high structural similarity to the binding regions of eCIRP and TREM-1 respectively, and have tested a binding affinity almost as high as their proinflammatory counterparts. Moreover, in vitro assay analysis (ELISA and western blot) on both murine and human monocytes demonstrates the ability of the two peptides to inhibit the secretion of downstream TREM-1 signals IL-1, IL-6, AKT, and MAPK to a point statistically indistinguishable from healthy cells. All of these results open up undeniable clinical avenues for peptides M3 and LP17 in clinical treatment of sepsis.

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Citations:

 

Angus, D. C. (2001). Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Critical Care Medicine, 29(7), 1303–1310. doi: 10.1097/00003246-200107000-00002

Chappell, B. (2019, February 13). U.S. National Debt Hits Record $22 Trillion. Retrieved from https://www.npr.org/2019/02/13/694199256/u-s-national-debt-hits-22-trillion-a-new-record-thats-predicted-to-fall.

DeVore, S. (2019, March 21). Premier Inc. Analysis: Hospital-Associated Sepsis Decreased by 15%... Retrieved from https://www.premierinc.com/newsroom/press-releases/premier-inc-analysis-hospital-associated-sepsis-decreased-by-15-from-2015-2018.

Feldstein, M. (2010, July 20). The 'Tax Expenditure' Solution for Our National Debt. Retrieved from https://www.nber.org/feldstein/wsj07202010.html.

Gilchrist, K. (2017, February 6). We're going to have a dollar collapse like the 1980s, strategist says. Retrieved from https://www.cnbc.com/2017/02/06/were-going-to-have-a-dollar-collapse-like-the-1980s-analyst-says.html.

Grad, S. (2018, February 21). Income and Assets of Social Security Beneficiaries by Type of Benefit. Retrieved from https://www.census.gov/library/working-papers/1988/demo/SEHSD-WP1988-22.html.

Gyawali, B. S. (2019, March 21). Sepsis: The evolution in definition, pathophysiology, and management. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429642/.

Krawzak, P. M. (2019, January 8). White House to put Medicare cuts on hold during shutdown. Retrieved from https://www.rollcall.com/news/whitehouse/white-house-put-medicare-cuts-hold-shutdown.

Liao, Y. (2017, June 26). The role of cold‐inducible RNA binding protein in cell stress response. Retrieved from https://onlinelibrary.wiley.com/doi/full/10.1002/ijc.30833.

Mulvaney, M. (2018). Historical Tables. Retrieved from https://www.whitehouse.gov/omb/historical-tables/.

Paoli, C. J. (2018, December). Epidemiology and Costs of Sepsis in the United States-An Analysis Based on Timing of Diagnosis and Severity Level. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250243/.

Pear, R. (2018, June 5). Medicare's Trust Fund Is Set to Run Out in 8 Years. Social Security, 16. Retrieved from https://www.nytimes.com/2018/06/05/us/politics/medicare-social-security-finances.html.

Pierson, P. (2009). The new politics of the welfare state. Oxford: Oxford Univ. Press.

Rello, J. (2017, November). Sepsis: A Review of Advances in Management. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/29022217.

Ritchie, H. (2019, September 11). How many people die and how many are born each year? Retrieved from https://ourworldindata.org/births-and-deaths.

Schenk, M., Bouchon, A., Seibold, F., & Mueller, C. (2007, October). TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1974863/.

Schiller, A. (2019, May 1). Top 30 Highest Murder Rate Cities in the U.S. in 2019. Retrieved from https://www.neighborhoodscout.com/blog/highest-murder-rate-cities.

Swagel, P. L. (2018, June 26). The 2018 Long-Term Budget Outlook. Retrieved from https://www.cbo.gov/publication/53919.

Torio, C. M. (2016). National Inpatient Hospital Costs: The Most Expensive Conditions by Payer, 2013. Retrieved from https://hcup-us.ahrq.gov/reports/statbriefs/sb204-Most-Expensive-Hospital-Conditions.jsp.

Verma, S. (2019, April 22). 2019 Medicare Trustees Report - cms.gov. Retrieved from https://www.cms.gov/Research-Statistics-Data-and-Systems/Statistics-Trends-and-Reports/ReportsTrustFunds/Downloads/TR2019.pdf.