THE FLORIDA AGRICULTURAL AND MECHANICAL UNIVERSITY
COLLEGE OF SCIENCES AND TECHNOLOGY
DESIGN OF AN EXTRACORPOREAL HEMOFILTRATION DEVICE FOR THE TREATMENT OF GRAM NEGATIVE SEPSIS
A Proposal submitted to the
Department of Biological Sciences
in partial fulfillment of the
requirements for the degree of
Master of Science in Biology
The members of the Committee approve the Proposal, entitled, Design of an Extracorporeal Hemofiltration Device for the Treatment of Gram Negative Sepsis, by Aisha Jimenez presented on March 23rd, 2017.
Karam Soliman, Ph.D., Pharmacy
Professor Directing Thesis/Outside Committee Member
Lekan Latinwo, Ph.D., Biology
Adrian McCollum, Ph.D., Biology
Lekan Latinwo, Ph.D., Chair, Department of Biological Sciences
Maurice Edington, Ph.D., Dean, College of Science and Technology
Jackson, David , Ph.D., Dean, School of Graduate Studies and Research
Sepsis is becoming a global concern, due to overuse of anti-biotics and lack of medical protocols to counteract late stage septic shock, where timing is critical. Proliferation of gram negative pathogens in the blood stream can be counteracted by anti-biotics but the endotoxin remains in the blood where it causes massive inflammation, traditionally counteracted with hydrocortisone – often too late ending in multi-organ failure (MOF) and death. New technologies using hemoperfusion cartridges such as torymyxin are being explored to filter the blood of the endotoxins. Here we explore the capacity of POLYMYXIN B (the main LPS neutralization cartridge component) to prevent the inflammatory process from time zero (T0) -48 hours (T48) with parallel studies conducted using a known anti-inflammatory (cardamonin). In this study, we first establish the proinflammatory effects of LPS±IFN in BV-2 cells, where the data show no production of superoxide (extracellular or intracellular), or hydrogen peroxide, but large amounts of nitric oxide tantamount to elevated iNOS protein induction. LPS alone was found effective to elicit these effects also causing IL-6, GM CSF, MCP1.etc Next the effects of Polymyxin B on neutralization capacity of LPS was assessed. The data show approximately a 1:1 (1ug/ml LPS- binds 1ug/ml PMB) binding capacity but the time course data reveals that only the immediate pre-neutralization of LPS with PMB (but not post) can prevent inflammation. In contrast, pre to 6 hours post LPS evoked inflammation was attenuated with cardamonin (a known and potent anti-inflammatory), as expected. The aforementioned data, in particular for PMB, suggest that PMB is either permanently bound to the cell receptor docking domain of LPS (ie Tol like 4) or that LPS binding to receptors is irreversible effective upon impact LPS receptor binding dynamics was determined using FITC-LPS, where the data show no permanent docking of LPS to either the perimeter or the interior of the cells, suggesting LPS acts in a dynamic transient fashion.
In ancient Greece and Rome, when medical hygiene was an equivocal concept, wound infections were regular and greatly feared surgical complications (Geroulanos & Douka, 2006). Early physicians blamed wound infections on putrefaction or sepsis. Despite the term sepsis being closely connected to modern intensive care, its concept is somewhat older. The earlier use of ‘sepsis’ occurred over two and half centuries ago in the poems of Homer, and it was derived from the word “sepo” which translates to “I rot.” According to Thurston (2000), Hippocrates (460-370 B.C.) first introduced the word ‘sepsis, ’ and it was derived from the Greek word ‘sipsi’ (making to rot).
Ibn Sina also observed a correlation between fever and blood putrefaction around 987 BC (Majno, 1991). All these concepts of sepsis that were introduced during classical antiquity were used over the centuries into the early 19th century. Up until then, only a few examples of the disease pathogenesis were well documented. Early efforts by Boerhave (1667-1737), a medical doctor in Leyden, argued that toxic substances in the atmosphere were the primary causes of sepsis. In the early 19th century, Justus von Liebig, a chemist, further expanded this theory by arguing that the contact between oxygen and wounds triggered the progression of sepsis (Francoeur, 2000).
The modern concepts of sepsis, however, were developed by Ignaz Semmelweis (1818-1865). Being a researcher and an obstetrician at the Vienna General Hospital, Semmelweis learned that the death of women delivering children from puerperal fever was a regular complication (Jones, 1933). At Vienna hospital, the obstetric department experienced up to 18% cases of mortality. In the process, Semmelweis observed that since medical students regularly examined pregnant women after pathology lessons, hygiene might be the cause of high cases of Sepsis (Majno, 1991). Since hygienic processes like using surgical gloves and handwashing were uncommon, Semmelweis deduced that sepsis was as a result of decomposed animal matter penetrating the blood circulation system. His intervention helped reduce the mortality levels to approximately 2.5% after he introduced handwashing using chlorinated lime solution prior to obstetric examinations.
Later, Louis Pasteur, a French chemist, discovered that putrefaction was caused by single cell organisms. He called these organisms as microbes or bacteria and correctly elucidated that these microbes were the primary causes of putrefaction. In addition, Pasteur recommended that bacteria in fluids could be killed by using heat or sterilized. Findings by Pasteur and Semmelweis played a key role improving the lessons drawn by Joseph Lister (1827-1912) on the death of amputees at the Glasgow Royal Infirmary. By first experimenting with animals and later humans, Lister examined the impact of skin and disinfecting instruments using carbolic acid and was able to reduce post-amputation mortality significantly. In contrast to Semmelweis, Lister was able to persuade his colleagues on the importance of an antiseptic approach to reducing cases of sepsis. Towards the end of the 1880s, Robert Koch refined Lister’s techniques by introducing steam sterilization.
The change in sepsis interpretation was further redefined by Lennhartz, a Germany physician at Eppendorf Hospital, by changing the traditional views into bacterial pathogenesis. However, it was Lennhartz’s student, Hugo Schottmuller that paved way to the modern definition of sepsis as a bacterial infection in the human bloodstream in a manner that it led to objective and subjective symptoms. As such, for the first time, the source of sepsis infection came into focus. According to Schottmuller, sepsis therapy was not directed against the bacteria, but against the bacterial toxins released into the bloodstream (Carswell et al., 1975). Despite the antiseptic procedures being making great breakthroughs, the number of persons developing sepsis increases, often resulting in septic shock. However, the introduction of antibiotics and intensive care units during World War II contributed to the reduction of sepsis-related deaths.
Over the decades, several definitions of sepsis have been postulated in attempts to understand the condition from a clinical perspective better. One of the earliest definitions presented sepsis as a systematic host response to an invading pathogen (Riedemann, Guo, & Ward, 2003). Precisely, the classical approach to sepsis was presented by William Osler (1849–1919), an American physician, in his seminal work when he noted that a patient appeared to die from the body’s response to the infection than from the pathogen itself. In the mid-20th century, this concept was reinforced in a medical review by Germs (1972) when he observed that sepsis was as a result of the host response to disease. More recently, the general sepsis concept has been considered to be a form of blood poisoning, where there is the presence of toxins in tissues of blood.
Given the inconsistencies of these earlier medical definitions of sepsis (Abraham et al., 2000) and other countless attempts to develop diagnostic assays and tools to identify sepsis (Vincent, 2000), there was a need for a consensus conference focused on improving the clinical definition of sepsis. In the early 1990s, the Society of Critical Care Medicine (SCCM) and the American College of Chest Physicians (ACCP) jointly worked towards a consensus definition of sepsis (ACCP, 1992). Table 1 shows the frequently agreed definitions of sepsis in critical care across the world by critical care providers and other intensivists.
|Sepsis||More than two SIRS criteria caused by suspected or known infection|
|Severe Sepsis||Sepsis with acute organ dysfunction (hypotension and hypoperfusion) caused by sepsis|
|Septic shock||Sepsis with refractory or persistent hypotension or tissue hypoperfusion in spite of adequate fluid resuscitation|
|MODS||Evidence of organ dysfunction in an acutely ill patient in a way that homeostasis cannot be preserved without intervention.|
However, these early 1990 definitions had clinical limitations, and they were later revisited in 2001 (Levy et al., 2001). Irrespective of the several limitations that were identified concerning the present definitions of sepsis, the 2001 conference failed to generate superior alternative sepsis concepts. One of the approaches entailed expanding the initial definition to include systemic inflammatory response syndrome (Bone et al., 1992). Nonetheless, this approach was perceived to broaden the diagnostic criteria and make sepsis less specific than it was a decade ago (Riedemann et al., 2003). Moreover, some proposed criterion overlapped with definitions that had earlier been identified with organ dysfunction, a critical component essential in distinguishing between septic shock and severe sepsis (Francoeur, 2000).
Conceivably, an essential outcome of the 2001 Consensus Conference was the postulation for a sepsis staging system called “Predisposition, Infection, Response and Organ dysfunction” (PIRO) (Levy et al., 2011). Primarily, this PIRO concept was similar to cancer staging, and it appeared that the criteria made it possible to differentiate different groups of patients with sepsis (Howell et al., 2011). Concerned with the limitations of the present definitions of sepsis (Vincent et al., 2013), the Society of Critical Care Medicine (SCCM) and the European Society of Intensive Care Medicine (ESICM) convened a 19-member task force 2014 to develop better definitions. Multiple large electronic health records were used in assessing ecologic validity (Seymour et al., 2016) in addition to using Delphi consensus methods and literature reviews (Shankar-Hari et al., 2016), on the clinical criteria describing and defining sepsis.
After compiling their research, the task force formulated major findings and recommendations. The key findings from the evidence analysis identified limitations of previous sepsis definition, including an excessive focus on inflammation, and inadequate sensitivity and specificity about systemic inflammatory response syndrome (SIRS) criteria. Also, the task force argued against the misleading model that sepsis is limited to a continuum of severe sepsis to septic shock (Shankar-Hari et al., 2016). At the moment, the task force noted that multiple terminologies and definitions for sepsis, organ dysfunction and septic shock existed to only add to the confusion and discrepancies about the observed mortality and reported incidence. In conclusion, the 19-member task force noted that the concept of severe sepsis was redundant (Seymour et al., 2016).
In their recommendations, the task force pointed out that sepsis should be defined as a life-threatening organ dysfunction resulting from dysregulated host response to infection. In efforts to achieve clinical operationalization, organ dysfunction is represented as an increase in sequential organ failure assessment (SOFA) score of 2 points or more. On its part, septic shock can be defined as a subset of sepsis where specifically metabolic, cellular, and circulatory abnormalities are related to high risks of mortality than an individual case of sepsis. In the clinic, septic shock patients can be identified by the vasopressor need to regulate a mean arterial pressure of 65 mm Hg or more, and serum lactate levels of more than 2 mmol/L, in the absence of hypovolemia (Shankar-Hari et al., 2016). In conclusion, the both the ESICM and the SCCM reached a consensus that the clinical criteria and updated definitions should replace previous sepsis concepts, in efforts to ensure timely management of patients with sepsis or those at risk of developing sepsis.
The updated clinical consensus of sepsis has made it possible for various epidemiological studies to be undertaken. Generally, sepsis occurs nearly in 2% of all hospitals across the developed countries. In the intensive unit, the risk of sepsis occurs between 6% and 30% of the hospitalized patients, although with substantial variations as a result of heterogeneity among various ICUs (Vincent et al., 2006). For example, sepsis can occur at high proportions of trauma or medical ICU patients in urban hospital settings but comprise a relatively small proportion of ICU patients in a surgical ICU or community cardiac (Gaieski et al., 2013). Generally, nearly 50% of severe sepsis patients need intensive care services (Iwashyna et al., 2012).
Population burden studies undertaken by Iwashyna et al. (2012) documented that sepsis accounts for 5.2% of U.S. hospitals costing up to $20 billion in 2011. The reported incidence was also supported by Gaieski et al. (2013) when they noted that the incidence of sepsis was a key indicator of aging populations with greater recognition, comorbidities (Dellinger et al., 2013), and in some western nations reimbursing suitable coding (Rhee et al., 2014). Even is the realistic incidence non-established, close estimates are in consensus that sepsis is a leading cause of critical illness and mortality across the world (Vincent et al., 2014; Fleischmann et al., 2015). Iwashyna et al. (2010) also noted that patients that survive sepsis often experience long-term cognitive, psychological, and physical disabilities with significant social and health care implications.
Initial studies have placed more emphasis on the incidence of sepsis compared to the occurrence of septic shock and severe sepsis. According to Bone et al. (1992), this focus is perhaps suitable considering that sepsis may be present in nearly all patients that require hospitalization, while severe sepsis is present in about 50% and 75% of critically ill patients. In addition, it is patients with high severity and organ dysfunction of acute disease that consume most of the resources, besides being at high risk of complications and even death. In western countries, the incidence of severe sepsis lies between 50 and 100 cases per 100,000 people (Levy et al., 2003).
Vincent et al. (2013) reported the incidence of sepsis to be 3 to 4 times more, indicative the relative percentage of persons who develop organ dysfunction and develop severe cases of septic shock or severe sepsis. In the USA, there has been a significant longitudinal change in the incidence of sepsis in the past decades. In the past decade, a study on US hospitalizations by Vincent et al. (2013) noted an increase in cases of sepsis among hospitalized patient. The incidence rate was noted to increase by 8.7% per year and that the moment there over a million cases of sepsis among inpatients on an annual basis in the USA. Several studies have reported that the incidence of sepsis and severe sepsis is increasing in excess of the growth population (Vincent et al., 2013; Seymour et al., 2013;). Similar findings have been reported to exist in Croatia, Austria, and the UK (Angus et al., 2013; Wiersinga et al., 2014).
In the developing world, the incidence of sepsis, septic shock, and severe sepsis are less well documented (Deutschman et al., 2014). Instead, there is more data available on infectious diseases that remain a constant battle for which there several high incidence cases. Since infectious diseases are largely the cause of sepsis, the condition is presumably of greater or similar importance in these areas of the world than in developed countries. In the developing nations, the cases of sepsis are more frequent in younger generations were responsible organisms are Gram-negative enteric pathogens and atypical malaria pathogens like Plasmodium falciparum and P. vivax. As earlier discussed, in sepsis cases, patients die from host immune responses in attempting to fight an infection, rather than from the infection itself.
In the last decade, therapeutic interventions have been focused on selective removal of endotoxin from blood to reduce the burden of sepsis (Davies & Cohen, 2011; Ronco & Klein, 2014). Specifically, polymyxin B is an antibiotic with a capacity to bind and inactivate endotoxins. However, the systematic use of the drug has its limitations in terms of associated nephrotoxicity and neurotoxicity. At the start of the early 1990s, a new affinity column that was characterized by polymyxin B and covalently immobilized to a polystyrene-derived fiber was developed in Japan (Shoji et al., 1998). Once blood passes through the column through an extracorporeal circuit, the endotoxin was efficiently removed with no detectable polymyxin B in the elute (Hanasawa, 1988).
A pilot trial undertaken by Vincent et al. (2005) documented that in septic patients, hemoperfusion via the column that contained polymyxin B immobilized fibers (PMX-HP) contributed to significant improvements in hemodynamic parameters. In another trial by Cruz et al. (2009), 64 sepsis patients were treated in an EUPHAS trial and later underwent abdominal surgery, in a two 2 hour session of PMX-HP. The trial took place for 28 days, and the absolute risk was significantly (p=0.03) reduced from 53% to 32%. In a recently completed clinical trial (Payen et al., 2015), sepsis patients that underwent surgical operations for abdominal infections were randomized to PMX-HP or standard treatment. The study indicated that PMX-HP was not linked to any survival benefits, and neither did it reduce the risk of developing organ failure. Essentially, both studies showed that only patients that had abdominal sepsis and adequately underwent surgical control to prevent primary infection were enrolled. As such, PMX-HP was mainly used to clear residual circulating endotoxins.
Until recent years, PMX-HP has been largely used among surgical patients with intra-abdominal sepsis (Abe et al., 2012). Additional trials have shown some survival benefits among patients with abdominal sepsis after undergoing PMX-HP and surgical treatments (Kush et al., 2005). In the study, scholars also examined if using PMX-HP can reduce vasopressor requirement and improve hemodynamic stability (Tsushima et al., 2002). The first interim analysis indicated potential survival benefits and the study was terminated earlier. Despite the observations, Yatera et al. (2011) noted that initial trials have often been underpowered to show reduced mortality levels. Besides, other scholars express concern that high mortality rates among control groups limit any possibility to translate the findings into other intensive care units (Fujitami et al., 2011; Mitaka et al., 2009).
A later analysis by Puyen et al. (2015) among in patients with peritonitis as a result of biliary and gut perforations, was designed to detect variations in a 28-day mortality study. The study found mortality rate of 27.8% and 19.3% in the treatment and control groups, respectively. The difference was not statistically significant (p=0.14). At the moment, an ongoing American trial aimed at examining the use of Polymyxin B Hemoperfusion in a Randomized Controlled Trial among patients with Septic shock and Endotoxemia, all patients have been assigned to standard treatment or PMX-HP (Klein et al., 2016). The study continues to show few cases of postoperative benefits with polymyxin B, similar to findings from a recent Japanese retrospective propensity analysis (Ishizuka et al., 2016).
With the identification of different pathogenic aspects of sepsis, there are varied therapeutic interventions that have been developed in attempts to improve patient survival. Past attempts were meant to neutralize mediators of the overexuberant inflammatory response using monoclonal antibodies against IL-1 receptors (Abraham, 1995), TNF (Fisher et al., 1994;1996), and or antibodies to endotoxin (Ziegler et al., 1991). However, these initial interventions failed to demonstrate significant improvement in patient survival but instead emphasized extreme redundant and complex nature of the innate cytokine responses during sepsis. In addition, broad-spectrum interventions to suppress inflammatory responses through the use of high dose steroids also failed to result in any significant improvement in patient survival (Bone et al., 2014).
Annane et al. (2009) found that a low dose of steroid, such as hydrocortisone 200 mg per day, even if it failed to lead to an actual reduction in mortality, it did reduce time to reversal shock and was subsequently used as an adjunctive therapy in sepsis. Additional therapies like toll-like receptor 4 antagonists have been recently introduced and used to target upstream of the inflammatory response cascade. Nonetheless, in recent reviews, Tidswell et al. (2010) reported in Phase II clinical trial about the lack of reduced mortality, even if cases of low mortality were observed in higher-dose groups. The only FDA therapy that is approved for sepsis— activated protein C has been used to target the micro-thrombosis that occur during sepsis, and it has been reported to anti-inflammatory effects (Bernard et al., 2001). To this point, however, there is no conclusive proof to support this claim.
The early goal-directed therapy was not limited to infectious and inflammatory responses to the pathophysiology of sepsis. Instead, the overall goal of the guidelines is to maintain substantial control of infection, organ perfusion, control hyperglycemia, and limit barotrauma as a result of mechanical ventilation. Even so, early goal-directed therapy (EGDT) has worked to reduce mortality in clinical cases with septic shock and severe sepsis (Rivers et al., 2001), and also evolved to inform sepsis survival guidelines (Dellinger et al., 2001). This intervention has worked to address the need for active and timely supportive care for sepsis patients. If indicated, other precise interventions like using activated protein C or low-dose steroids are recommended. A recent review by Levy et al. (2010) documented a decrease in sepsis mortality with increased compliance with therapy guidelines.
2.1 Sequence of Events in Sepsis
A series of immunologic response triggers sepsis condition first resulting into Systemic inflammatory response syndrome (SIRS). Immunologic responses are triggered by various microbial elements including Gram-negative bacteria’s proteases, exotoxins, formyl peptides, and endotoxin (Lindenauer et al., 2013). Also, Gram-positive components can trigger the body’s immunologic reactions, including lipoteichoic acid, peptidoglycans, hemolysins, enterotoxins, streptococcal pyrogenic exotoxin A, superantigens, and exotoxins (Lindenauer et al., 2013). In addition, inflammatory responses can also be triggered by fungal cell wall materials. Following infection, a sequence of events follow, and these sequences are largely clinical conditions that result from the host’s immune responses to infection, characterized by coagulation and inflammation. The responses include a full range of immunological triggers from SIRS to organ dysfunction to multiple organ failures and eventual death (Rivers et al., 2001).
The authors confirm that the sequence of events during sepsis is a complex biological phenomenon that requires more research to understand how patients go from SIRS to septic shock. In the case of septic shock, patients display a biphasic immune response (Lindenauer et al., 2013). At the initial stages, a patient manifests overwhelming inflammatory responses to infections. The cause of this inflammation has been linked to pro-inflammatory cytokines like IL-6, Interferon gamma (IFN-γ), Tumor Necrosis Factor, IL-1, and IL-12. In the process, the body starts to regulate these responses by producing anti-inflammatory cytokines (IL-10), IL-1 RA, IL-1 receptor type II, and soluble inhibitors which are manifested during the immunodepression period (Lindenauer et al., 2013). In the event of continued hypo-responsiveness, there is increased the risk of nosocomial infections and even death. Figure 1 shows sequences of sepsis progression from infection with lysed bacterial cells to multiple organ failures.
Figure 1: Sequence of events in Sepsis pathogenesis
The produced pro-inflammatory cytokines include TNF, IL- 1, 6, and 12, and IFN-γ. Once generated, these cytokines work either directly to affect organ functions, or they can act indirectly through secondary mediators. Some of the common secondary mediators acted upon include complement system, prostaglandins, platelet activating factor, leukotrienes, thromboxanes, and nitric oxide (Payen et al., 2015). IL-1 and TNF in addition to endotoxins, work to trigger the release of tissue factors via endothelial cells that lead to disseminated intravascular coagulation and fibrin deposition. The release of these primary and secondary mediators trigger the activation of the coagulase cascade system, produces leukotrienes and prostaglandins, and the complement cascade (Lindenauer et al., 2013).
In the process, clots lodge inside blood vessels, and this can lower profusion of organs and can result in multiple organ failures. With time, the activation of the coagulation cascade system depletes the host’s capacity to make clot leading to disseminated intravascular coagulation and adult respiratory distress syndrome (Kumar et al., 2011). The overall impact of this cascade system is an unbalanced state where inflammation is dominant over anti-inflammation, while the coagulation system becomes dominant over fibrinolysis. Subsequently, hypoperfusion, tissue injury, ischemia, and microvascular thrombosis result. As a result, severe sepsis, shock, and multiple organ failures might result, leading to death (García et al., 2009).
2.2 Pathogenesis of Sepsis
Independent of any pathogenic infection, the systematic inflammatory response syndrome (SIRS), displays similar pathophysiologic properties with minor variations in inciting cascades. Scholars consider the SIRS syndrome a self-defence mechanism (Levy et al., 2013). That is, inflammation during the onset of sepsis is the body’s response to nonspecific pathogen insults that emerge as a result of infectious stimuli, traumatic injury, or chemical processes (Ollendorf et al., 2012). Often, the inflammatory cascade is a complex procedure that entails both cellular and humoral responses, cytokine cascades, and complement system (Angus et al., 2016). The pathogenesis process can be presented as a three-stage process as outlined below.
During the first stage, an insult occurs from infective microbes like bacteria toxins or injury. In the process, local cytokines are produced at the site of injury (Lindenauer et al., 2013). The produced cytokines trigger inflammatory responses, in efforts to promote the process of wound repair and recruitment of reticular endothelial systems. Initially, this process is fundamental in promoting normal host defense mechanisms and create homeostasis, and if absent the process is not compatible with life (Levy et al., 2013). Local inflammation on subcutaneous and skin tissues displays cardinal signs of rubor, tumor, dolor, calor, and functio laesa. During rubor, the skin surface appears red as a result of vasodilation caused by vasodilating substances like prostacyclin and nitric oxide (Kumar et al., 2011).
Moreover, tumor or swelling results from the disruptions of vascular endothelial tight junctions and extravasation of protein-rich fluid into the interstitium, triggering the activation of white blood cells passing the vascular space into the tissues to help clear the infected site and promote tissue repair. On its part, dolor is a systematic pain that represents the impact that inflammatory mediators have on the local somatic sensory nerves (Lindenauer et al., 2013). Often, the pain generated stops the host from utilizing this part of the body as the immune response tries to repair the injury (Ollendorf et al., 2012). Calor refers to increased heat as a result of elevated blood flow and increased local metabolism as the white cells became localized and activated at the injured tissue site (Levy et al., 2013). Lastly, functio laesa refers to the loss of function which is common clinical findings of organ dysfunction and a hallmark of inflammation with the infection specific to a precise organ like acute kidney failure or acute respiratory failure. Importantly, at the local level, the chemokine and cytokine release attracts activated leukocytes to the region which can cause local tissue abscess or destruction, or cellular injury leading to pus production (Kumar et al., 2011), all which are by-products of effective inflammatory responses.
In the second stage of the immunologic reactions, there is the production of small quantities of cytokines into the circulation system, leading to improved local responses. The increased circulation promotes growth factor stimulation and the recruitment of platelets and macrophages (Riedemann et al., 2003). At this phase, the host experiences an acute phase that is often well controlled and regulated by a reduction of proinflammatory mediators and the release of endogenous antagonists—the primary objective of homeostasis (Kumar et al., 2011). Besides, the stage is often characterized by low-grade fever and minimal malaise. Failure to reach homeostasis triggers the third stage of immunologic reactions (Ollendorf et al., 2012).
In the third phase, if the injured site fails to attain homeostasis, and if the inflammatory stimuli progress to send mediators into the circulation, there is a significant systematic reaction. The release of additional cytokines leads to tissue destruction rather than protection (Lindenauer et al., 2013). One of the impacts of this activation is the several humoral cascades and the activation of the reticular endothelial system, followed by subsequent loss of circulatory integrity and end-organ dysfunction (Angus et al., 2016). The progression of SIRS to organ dysfunction and potentially multiple organ dysfunction syndromes (MODS) has been discussed by Bone et al. (2014) as a multi-hit theory. The theory argues that the events that initiate and trigger the SIRS cascade work to prime the pump. With added events, the exaggerated or altered responses takes place resulting in the progression of illness (Ollendorf et al., 2012). The approach to preventing multiple hits is sufficient identification of the cause of SIRS to suppress the inflammatory cascade elaborated below.
2.2.1 Inflammatory Cascade
Infection, inflammation, or trauma leads to activation of the inflammatory cascade as initially discussed. In the process, proinflammatory responses take place but are almost immediately suppressed through the production of anti-inflammatory responses (Levy et al., 2013). In addition, this SIRS often manifests as an increased systemic responses to both proinflammation and anti-inflammation processes. When SIRS is triggered by infective stimuli, the inflammatory cascade is largely initiated by either exotoxin or an endotoxin (Kumar et al., 2011). In addition, endothelial cells, platelets, mast cells, monocytes, and tissue macrophages are able to generate multitudes of cytokines. For example, the cytokines like tissue necrosis factor alphas, and IL-1 are released first, and in the process, they initiate several cascade systems (Riedemann et al., 2003).
The presence of exotoxin or endotoxin, and the release of TNF- α and IL-1 results in the cleavage of the nuclear factor-kB (NF-kB) inhibitors. Upon the removal of the inhibitors, NF-kB is capable of initiating the production of messenger ribonucleic acid (mRNA) that induces the production of additional proinflammatory cytokines (García et al., 2009). The primary proinflammatory mediators during sepsis include IFN-γ, IL-6, and IL-8, and they are induced by NF-kB (Ollendorf et al., 2012). In vitro research indicates that glucocorticoids might function as inhibitors on NF-kB. Research has also documented that IL-1 and TNF-α can be released in large quantities within an hour of an infection, and have both local effects and systemic effects (da Silva et al., 2015).
In vitro research has also reported that these two cytokines— IL-1 and TNF-α— at individual levels can trigger the production of significant hemodynamic responses, although they also cause severe hypotension and lung injury when produced together (Ollendorf et al., 2012). Often, IL-1 and TNF-α are responsible for the release of stress hormones like vasopressin, norepinephrine, and activation of the renin-angiotensin aldosterone system, and fever episodes (da Silva et al., 2015). Additional cytokines, such as IL-6, stimulates the release of acute-phase reactants like procalcitonin and C-reactive protein. Importantly, infections have been noted to induce the release of TNF-α and subsequent inducement and release of IL-6 and IL-8 than trauma does. The authors attribute these mechanisms to high fever linked to infections rather than injury or trauma (Kumar et al., 2011). The proinflammatory interleukins either function directly on tissue or work through the secondary mediators to activate the coagulation cascade, the also the complement cascade and result in the release of leukotrienes, prostaglandins, platelet activating factor, and nitric oxide (Ollendorf et al., 2012).
In the process, high mobility group box 1 (HMGB1), a protein present in the nuclei and cytoplasm in most cells, is generated by innate immune cells or passively released by damaged cells in response to injury or infection, as the case with SIRS (Lindenauer et al., 2013). Hence, the elevated levels of tissue or serum HMGB1 largely result from many triggers of SIRS. HMGB1 primarily acts as a potent proinflammatory cytokine and is involved in delayed cases of sepsis and endotoxin lethality (Dellinger et al., 2013). In research on patients with traumatic brain injury, scholars performed a multivariate analysis of selected plasma HMGB1 levels as independent factors for 1-year mortality. However, therapeutic interventions are still in progress to examine approaches that can be used to block HMGB1, with a focus based on improving the outcome of SIRS and sepsis syndrome (Davies et al., 2011).
Several pro-inflammatory polypeptides are also released within the complement cascade system. For instance, protein complement C3a and C5a are the most studied and are believed to contribute directly to the release of additional cytokines, in addition to causing vascular permeability and vasodilatation (Lindenauer et al., 2013). Leukotrienes and prostaglandins provoke endothelial damage, and this process triggers the sequence of events related to multiorgan failure (da Silva et al., 2015). In cases of critically ill patients with SIRS, polymorphonuclear cells (PMNs) have been reported shown to be highly resistant to activation compared to PMNs from healthy hosts. However, when stimulated, these events demonstrate exaggerated processes of microbicidal responses (Kumar et al., 2011). In the process, this can represent autoprotective mechanisms where PMNs in an already injured host might avoid excessive side effects of inflammation, thereby reducing the risk of further host injury or death (Dellinger et al., 2013).
Understanding the correlation between coagulation and inflammation is fundamental to understanding the progression of SIRS and sepsis pathogenesis. TNF-α and IL-1 largely affect endothelial surfaces, and this leads to the expression of tissue factors (García et al., 2009). In the process, tissue factors initiate the production of thrombin, and facilitates coagulation in the process, in addition to promotion of the proinflammatory mediators (Finfer et al., 2012). The process of fibrinolysis is impaired by TNF-α and IL-1 through the production of plasminogen anti-inflammatory mediators, namely activated protein- C and antithrombin. In the event that this coagulation system is not checked, the cascade leads to complications of microvascular thrombosis and eventual organ dysfunction (Reacher et al., 2008). As such, the complement system plays a vital role in coagulation cascade as infection-linked pro-coagulant activities are generally more severe compared to those that are generated by trauma (Gaieski et al., 2013).
The cumulative impact of inflammatory cascade is an unbalanced state with coagulation and inflammation dominating. In efforts to counteract the acute inflammatory responses generated during SIRS, the body initiates the reverse process through counter-inflammatory response syndrome (CARS) (García et al., 2009). In this process, IL-4 and IL-10 are the major cytokines that work to ensure a decrease in the production of TNF-α, IL-1, IL-6, and IL-8 (Payen et al., 2015). In essence, these anti-inflammatory and proinflammatory activations mirror other homeostatic processes, such as complement suppression, complement activation, anticoagulation, and coagulation (García et al., 2009).
Clearly, the normal homeostatic processes attempt to keep these very toxic inflammatory processes in check. Inflammation is an essential component of host defense and serves a very strongly positive survival function in suppressing and then eliminating local infection and tissue injury (Payen et al., 2015). It is only when this localized aggressive injury process gains access to the whole body through the blood stream and lymphatics that a SIRS develops (García et al., 2009). Also, the acute phase response generates antagonists against TNF-α and IL-1 receptors. The generated antagonists either work by binding the cytokines, and thereby activating them, or blocking the receptors (Payen et al., 2015). Other factors, such as comorbidities can also influence the patient’s capability to respond suitably. Creating a homeostatic balance between CARS and SIRS works to improve patient outcome and tissue insult by pathogens. Scholars like Gleason et al. (2009) report that as a result of CARS, the sepsis medications meant to inhibit proinflammatory mediators can result in deleterious immunosuppression.
2.2.3 Microbial Components
Following the microbial intoxication or microbial infection, the immune response triggers a complex series of events that can overwhelm the inflammatory response (Payen et al., 2015). Moss et al. (2013) noted that dilatation of peripheral vasculature takes place and the process becomes leaky leading to peripheral pooling of organs and leads to hypoperfusion, hypotension, and blood. The production of large amounts of proinflammatory cytokines can be triggered by several microbial components, including lipopolysaccharide (LPS), binding protein, and CD-14. For example, Gram-negative bacteria generate endotoxin that is also called LPS (Aikawa, 1996).
In most infections, LPS is the most common Gram-negative bacterial trigger of cytokine release that microbial trigger bind to cell receptors on the host macrophage and activates regulatory proteins, including nuclear factor kappa B (NFκB). The regulatory proteins are activated by LPS by interacting with several receptors. In the process, the CD-14 receptors pool the LPS-LPS binding protein complexes on the surface of the cell, and the production of toll-like receptors (TLR) work to translate the signals into the cells (Payen et al., 2015).
In Gram-positive bacteria, microbial components include super-antigens, cytolysins or hemolysis, peptidoglycans, and lipoteichoic acid. The superantigen includes staphylococcal enterotoxin and TSS toxin produced by S. aureus, and the streptococcal pyrogenic exotoxin A (SpeA) produced by Streptococcus pyrogens. Rather than binding in the groove of the major histocompatibility complex (MHC), the produced super-antigens binds on the outer part of the antigen-presenting cells of the MHC class II molecules, as well as on the outer surfaces of the T-cell receptors, available on the T-cells (Payen et al., 2015). The binding triggered by superantigen causes massive proinflammatory cytokine release and production and T-cell activation, all which lead to peripheral pooling of blood in interstitial space, dilatation of the peripheral vasculature, endothelial cell damage, fever, organ hypoperfusion, organ dysfunction, shock, and death (Aikawa, 1996). Different from most antigens that only activates few T-cells, or 1 in 10,000 T cells during the immune response, the superantigens activates several T cells (1 in 5 T cells), and this causes vigorous and at times life-threatening immune responses evident in patients with sepsis.
With either type of bacteria—Gram-negative or positive— the immune response starts with an overwhelming inflammatory response as a result of increased production of IL-1 and TNF-alpha. IL-1 and TNF-alpha or microbial triggers cause the cleavage of NF-kB inhibitors. In addition, mRNA can also be induced by NF-kB to trigger the production of proinflammatory cytokines, including IFN-γ, IL-6, and IL-12. On its part, IL-6 stimulates the release of acute-phase reactants—procalcitonin and C-reactive protein. The proinflammatory cytokines act directly or indirectly using secondary mediators like nitric oxide, prostaglandins, the complement cascade, and leukotrienes. As such, microbial components work to trigger the release of TNF-alpha and IL-1, which in turn affects the endothelial cells that affect tissue expression. Tissue factors initiate the release of thrombin and promotion of coagulation (Payen et al., 2015). In the event that IL-1 and TNF-alpha impair the process of fibrinolysis via the production of plasminogen activator inhibitor-1, microvascular thrombi can result, leading to organ dysfunction and failure, or even death.
2.2.4 Toll-Like Receptors (TLR-4, NF-kB signaling pathway)
The expression of TLRs occur in various cell types but in two general cellular locations namely the intracellular compartments (endosomes and endoplasmic reticulum) and in the plasma membrane. Toll-like receptors detect and respond to a diverse number of molecules including nucleic acids (TLR3), proteins (TLR5), and lipids (TLR1, 2,4,6) (Payen et al., 2015). Increasing scientific findings continue to argue that TLRs are capable of recognizing both exogenous ligands (like those produced by microbes) or endogenous ligands such as the extracellular matrix molecules (fibronectin, heat-shock proteins, and hyaluronan) and damage-associated molecular patterns (Akira et al., 2006).
Generally, the structure of TLR is that they are transmembrane proteins that have a series of leucine-rich repeats, in the N-terminal extracellular domain and a cytoplasm portion similar to the IL-1 receptor. As a result, they are referred to as Toll-IL-1 receptor homology domains (Hack et al., 1997). The IL-1R and TLRs work by activating both MAP kinase and NF-kappa B pathways through myeloid differential factor 88, a key adaptor molecule recruited in a Toll-IL-1 receptor (TIR) domains of TLRs. As a result, the simulation of TLR by cognate ligands and proinflammatory response genes including cytokines like IL-6, IL-12, and TNFα, are induced through interferon regulatory factors IRF 3 and/or IRF7 (Aikawa, 1996). Hence, stimulation of TLRs contributes to a robust but precise activation of the innate immune response.
The generation of chemokines and inflammatory cytokines is a direct result of the activation of TLR signaling. Once simulated via microbial pathogens, TLRs can induce the generation of inflammatory cytokines and interferons. The cytokines can activate surrounding cells leading to a production of adhesion molecules or chemokines, thereby triggering the recruitment of various inflammatory cells into the infected site to clear invading pathogens (Payen et al., 2015). The production of these cytokines, including IL-6 and TNFα, is essential for adequate functioning of both the adaptive and innate immune responses. Among other functions, they work to control the duration, amplitude, and direction of immune responses and the modeling or remodeling of tissues, including constitutive, unscheduled, and programmed cells. In most cases, unscheduled remodeling triggers infection, repair, wound healing, and inflammation (Hack et al., 1997). Nonetheless, dysregulation or overactivation of TLRs signaling also triggers severe disease like autoimmune diseases, atherosclerosis, and sepsis.
2.2.5 Cytokines/Signalling Amplification (IL-1, IL-6, TNF-alpha)
The key role of cytokines during sepsis is to regulate the different inflammatory responses, and this includes the migration of immune cells to the site of infection. The migration is essential in containing localized infection and also preventing it from becoming systematic disease (Hack et al., 1997). Nonetheless, the dysregulation of cytokine generation and release can cause endothelial dysfunction characterized by capillary permeability and increased vasodilation. The outcome is signal amplification and leakage syndrome that is clinically linked to edema, macromolecular extravasation, hemoconcentration, and hypotension which are key findings in septic patients (Hack et al., 1997).
Often, the dysfunctional epithelial barriers making it possible for pathogens and their products to invade the host system further, resulting in the disturbance of regulatory mechanisms, and finally causes remote organ dysfunctions equilibrium (Junger, 1996). Furthermore, increasing evidence shows that inflammatory and immune responses are tightly interwoven with various physiologic processes within the human host, like neuroendocrine activation, metabolism, and coagulation (Hack et al., 1997). For instance, an inflammation-induced dysregulation of the coagulation system by IL-1 and IL-6 can significantly result in signal amplification and aggravate the deleterious effects of sepsis, and also contribute to lethal disseminated intravascular coagulation.
Once released, IL-1, IL-6, and TNF-alpha act as proinflammatory cytokines that contribute to ensuring activation of the innate or adaptive immune responses. The signal amplification is characterized by the additional production of effector cytokines and immunoregulatory cytokines (Cavaillon et al., 1992). This sequential release of specific cytokines if called cytokine cascade (Blackwell and Christman, 1996). In the early 1990s, sepsis was thought to be linked to exacerbated release of proinflammatory cytokines, like macrophage migration inhibitory factor, interferon (IFN)-γ, interleukin (IL)-1, IL-6, IL-12, and tumor necrosis factor (TNF)-α. As a result, the term cytokine storm was formulated (Aikawa, 1996).
Nevertheless, recent studies on sepsis pathophysiology mechanisms show that the profound proinflammatory responses are counteracted by specific anti-inflammatory cytokines, which include IL-4, IL-10, and , transforming growth factor (TGF)-β that attempt to restore immunologic equilibrium (Junger, 1996; van der Poll & Opal, 2008). Recently, increasing studies have been a focus to identify unifying mechanisms that employ genome-wide expressions data in both early and late sepsis stages. For example, Tang et al. (2010) documented that sepsis contributes to immediate upregulation of Pattern recognition receptors (PRRs) and also the activation of signal transduction cascades. Even so, an essential inflammatory markers like TNF-α, IL-1, and IL-10, failed to show any consistent pattern of their gene expression and that they remain highly variable in persons (Hack et al., 1997).
The findings resound that the host response to sepsis is not a simple model with initial proinflammatory phase, that is followed by anti-inflammatory responses, but rather a highly dynamic and interactive process that can reflect heterogeneous genome-specific pathways (Junger, 1996). A tightly controlled balance in cytokine network during signalling amplification, which includes anti-inflammatory cytokines proinflammatory cytokines, soluble inhibitors of proinflammatory cytokine-like IL-1R2, IL-1, and TNF receptors are crucial in the elimination of invading pathogens and also restricting excessive tissue-damaging inflammatory responses (Hack et al., 1997; Van der Poll & van Deventer, 1997).
2.2.6 Nitric Oxide Production and Regulation
Nitric oxide (NO) refer to a free radical that is generated by a family of enzymes called NO synthases. The process occurs via oxidation of one of the guanidine nitrogen atoms of I-arginine forming NO and citrulline. At least three isoforms of NO synthase exist in mammalian cells. (i) inducible NOS (iNOS or NOS II) that is found in macrophages, smooth muscles, hepatocytes, and different tissues. (ii) neuronal NOS (NOS I or nNOS) found in skeletal muscle and neuronal cells. (iii) endothelial NOS (NOS II or eNOS) that are localized in endothelial cells. In immune reactions, all NOS enzymes contain different cofactors in their active form, namely tetrahydrobiopterin, flavin mononucleotide, flavin adenine dinucleotide, and calmodulin.
During sepsis, nNOS and eNOS are constitutively expressed and are activated when there is an increase in intracellular Ca2+, and they take part in the regulation of neurotransmission and vascular tone, respectively. While iNOS is functionally Ca2+ independent and takes part in the immune defense, it is expressed in many cell types after pro-inflammatory cytokine treatment or endotoxin treatment. To some extent, eNOS and nNOS are thought to generate Ca2+ that is dependent on NO formation, while the expression of iNOS leads in the sustained generation of huge quantities of NO that are considered to mediate the alteration in tissue damage and vascular system that often manifest during septic shock and multiple organ dysfunction syndromes.
Recent research, however, has shown that overproduction of NO during septic shock pathogenesis and MODS works to initiate vasodilatation and tissue damage. During endotoxemia, the levels of nitrate production have been reported to increase and that urinary and plasma nitrite, in addition to NO concentrations, are elevated in patients with septic shock and sepsis. The level of NO also increases with the presence of shock and among patients that have MODS. In mammalian tissues, the only known sources of nitrate and nitrite production are via the conversion of I-arginine to NO using NO synthase, or the breakdown of NO into nitrate and nitrite. Scholars note that there is a qualitative and quantitative clinical relationship between NO production, hemodynamic dysfunction, endotoxemia, and human septic shock.
In addition, iNOS is generated and produced in different tissues in endotoxemia and is proposed to be part of an adaptive response to the host defense mechanisms geared towards limiting tissue infections and injury. After being expressed, the inducible enzyme of activated macrophages is able to produce high concentrations of NO over a long period that has both tumoricidal and bactericidal effects. During the endotoxemia, increases in iNOS mRNA or protein and iNOS expression have been reported in different cell types, including fibroblasts, pancreatic islets, cardiac myocytes, chondrocytes, kidney cells, hepatocytes, Kupffer cells, macrophages, and endothelial cells.
In Gram-positive associated sepsis, LTA alone or together with peptidoglycan can induce iNOS activity and protein in vascular smooth muscles and macrophages. Even if peptidoglycan does not initiate iNOS in cultured macrophages, it combines with LTA to cause iNOS protein expression and-and activity. Precise factors that induce iNOS during septic shock have been noted to vary between tissues and they include proinflammatory cytokines, microbial products, and microbes and a strong synergy exists between these agents. For instance, in macrophages, the LPS has been reported to induce iNOS and create a strong synergy with IFN-γ, while in hepatocyte cells, LPS alone has not been reported to have any effect, other than enhancing the induction by IFN-γ, TNF-α, and IL-1β. On the same note, while IL-1 β works to enhance iNOS expression in islets of Langerhans, hepatocytes, smooth muscle cells, and chondrocytes, it has no impact on macrophages.
Hence, the key important aspect of NO overproduction is to the development of endotoxic shock, in efforts to block the expression of iNOS and also using NO synthase inhibitors. Administration of iNOS during septic shock has been shown to reverse the endotoxin-induced hypotension and hypo-responsiveness, in addition to restoring blood pressure, to vasoconstrictor agents. Besides, NOS inhibitors have been reported to improve hepatic and renal functions after treatment with LTA and peptidoglycan or with endotoxin treatment. Such approaches have been reported to increase survival rates among septic shock patients. Therefore, iNOS works to defend septic patients against tumor cells and infectious agents, although excessive iNOS in vasculature tissues can result in shock and ultimate tissue damage.
The goal of my research is to study the capacity and time limits on Polymyxin B (PMB) lipopolysaccharide (LPS) neutralization, using an in vitro model.
The specific aims of the study are to:
- Define the parameters of the monocyte model in the presence of lps
- Evaluation of free radicals
- Evaluation of mRNA whole transcriptome
- Evaluation of proinflammatory proteins
- Establish Binding Capacity of LPS by Polymyxin B (Toramyxin Component)
- Evaluation of the neutralization of LPS Pre and Post
- Evaluation of LPS-Toll Receptor Binding
Specific Aim 1
- Evaluation of free radicals
LPS treated Microglia Cells
General parameter test 1: Nitric oxide
- Evaluation of mRNA whole transcriptome
- Evaluation of proinflammatory proteins
Specific Aim 2
- Evaluation of the neutralization of LPS Pre and Post
- Evaluation of LPS-Toll Receptor Binding
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