Sepsis ist eine dysregulierte Reaktion des Organismus auf eine Infektion. Bei Sepsis werden oft Blutungs- und Thromboseereignisse beobachtet, welche in einer Disseminierten Intravasalen Gerinnung (DIG) gipfeln können. Thrombozyten sind die Schlüsselzellen von Thrombose und Hämostase. Bei Sepsis und DIG kommt es häufig zu einem Abfall der Thrombozytenzahl, doch Blutungs- und Thromboseereignisse können unabhängig von der Thrombozytenzahl auftreten, was zusätzlich eine Veränderung der Thrombozytenfunktion nahelegt. In dieser Arbeit wurde deshalb die Thrombozytenfunktion bei 15 Patienten mit Sepsis zu drei Zeitpunkten im Krankheitsverlauf untersucht. Es konnte bei unauffälliger Rezeptorexpression keine Voraktivierung der Thrombozyten mittels Durchflusszytometrie festgestellt werden. Jedoch war die Aktivierung nach Stimulation mit multiplen Agonisten signifikant reduziert. Besonders ausgeprägt war die Hyporeaktivität bei Stimulation des Kollagen-Rezeptors GPVI mit dem Agonisten CRP-XL. Es wurde gezeigt, dass nach GPVI-Stimulation eine reduzierte Phosphorylierung der nachgeschalteten Proteine Syk und LAT im Vergleich zum Gesundspender induziert wird. In Kreuzinkubationsexperimenten hatte die (Co )Inkubation von Thrombozyten in Plasma von Sepsispatienten oder mit Bakterienisolaten aus Sepsis-Blutkulturen keinen Effekt auf die Thrombozytenreaktivität. Allerdings konnte durch Sepsis-Vollblut eine signifikante GPVI-Hyporeaktivität in Thrombozyten von gesunden Probanden induziert werden, was einen zellulären Mediator als Ursache des Defekts nahelegt. In dieser Arbeit wurde gezeigt, dass insbesondere die GPVI-Signalkaskade bei Sepsis massiv beeinträchtigt ist. Der Immunorezeptor GPVI ist ein vielversprechendes Zielmolekül, um die Pathogenese der Sepsis, des Capillary Leak und die immunregulatorische Rolle von Thrombozyten besser zu verstehen. Die GPVI-Hyporeaktivität könnte als zukünftiger Biomarker für die Sepsis-Frühdiagnose genutzt werden. / Sepsis is the dysregulated immune response of a host to infection and the leading cause for intensive care unit (ICU) treatment worldwide. Patients often suffer from bleeding and thrombotic events, which can escalate to a disseminated intravasal coagulation (DIC). Platelets are important regulators of hemostasis and thrombocytopenia is a hallmark of sepsis and DIC. However, bleeding and thrombosis are observed independently from thrombocytopenia suggesting that altered platelet function might contribute. While platelet number has been investigated in multiple studies and is an integral part of the diagnostic SOFA-score, platelet function during sepsis remains ill-defined. We assessed platelet function in 15 patients with sepsis in a single center study at three times during disease: I intensive care unit (ICU) admission day; II day 5-7 at ICU; III day of ICU discharge. Platelets of all patients at time point I and II had an overall unaltered receptor expression shown by flow cytometry, were not preactivated, but showed a markedly impaired response upon stimulation with multiple agonists. The defect was most prominent upon stimulation of the collagen receptor GPVI with the selective agonist CRP-XL. Sepsis platelets failed to induce phosphorylation of downstream effectors Syk and LAT, as shown by immunoblotting. Next, we asked which factor(s) in patients can induce GPVI hyporeactivity. Incubation of platelets from healthy individuals in plasma of sepsis patients did not cause pre-activation or altered platelet responsiveness. However, platelet incubation in sepsis whole blood diminished CRP-XL reactivity, suggesting the contribution of a cellular component. Co incubation of healthy platelets with bloodborne heat-inactivated bacteria or antibiotics did neither lead to platelet preactivation nor impaired platelet reactivity upon GPVI stimulation. Taken together, our results imply that GPVI function is highly deficient in sepsis patients. GPVI is a promising target, which can pave the way for a better understanding of platelet function in innate immunity and the regulation of vascular integrity. GPVI hyporeactivity might serve as a robust biomarker for the early identification of sepsis patients in the future.

Severe sepsis kills an estimated 1,400 people worldwide every day. This often fatal infectious process accounts for an estimated 215,000 deaths in the United States (US) annually. The main goal of this project was to evaluate the impact of the Emergency Department Severe Sepsis Alert and Practice Protocol (EDSSAPP) post implementation, on time to first antibiotic administration, length of stay, and mortality in patients admitted via the ORMC ED with severe sepsis. This study evaluated the time to first antibiotic administration, total ED and hospital length of stay (LOS) and mortality of severe sepsis patients either with a severe sepsis alert (SSA) activated or no alert activated that were admitted to the hospital through the ED. A retrospective review of the electronic medical record (EMR) was conducted to gather the required data across three time cohorts: base line/time zero (T0), six months prior to the implementation of EDSSAPP; Time one (T1) the first six months following initial EDSSAPP implementation; and Time two (T2), six months following reinstatement of the corporate sepsis committee. The most significant finding of this study was the increased number of Severe Sepsis Alerts activated in time cohort T2 (n=113) compared to T1 (n=19). Another important finding was the decreased mortality in T2 (16.4%) compared to T0 (22.7%) and T1 (33%). Overall, the number of ED patients with severe sepsis who received antibiotics within the EDSSAPP required 60 minutes did not consistently improve across the three time cohorts, T0 (81.8%), T1 (71.7%) and T2 (80.6%). The hospital LOS of stay was increased by almost 1.5 days between those patients with a severe sepsis alert activated in T1 (9.00 days) compared to time T2 (10.48 days). There was no significant decrease in the ED LOS across time cohorts and between groups of patients who had a SSA activated versus no alert activated. However, there was a 1 hour and 28 minute decrease in ED LOS in patients who had a severe sepsis alert activated in T1 compared to T0. In addition, there was a 1 hour and 52 minutes decrease in ED LOS between patients who had a SSA activated compared to those who had no alert activated in T2. While EDSSAPP data does not demonstrate the statistically significant results that was expected, the challenges related to adherence by providers to EDSSAPP is as it is seen in the literature. Increased awareness via consistent communication of on-going audit results to ED personnel will heighten their awareness for severe sepsis and EDSSAPP. Improved collaborative efforts with the interdisciplinary team are needed to refocus everyone's efforts to increase early recognition that is followed by appropriate treatment interventions and documentation is essential. Lastly, the development of a formal process to follow up with individual providers as close to real time as possible following a SSA that includes accountability for care provided and related documentation would also contribute to both awareness and adherence.

This thesis consists of two related studies presented as three separate manuscripts (all three have been published in peer-reviewed journals) and a study protocol that has been submitted for peer-reviewed funding. The over-arching theme of this thesis was to better characterize the efficacy of different intravenous fluids used for the resuscitation of intensive care unit (ICU) patients with severe sepsis or septic shock. We performed an extensive search including multiple databases which found 20 randomized controlled trials (RCTs) that examined the effects of different intravenous fluids used in septic patients and met our a priori inclusion and exclusion criteria. In the first manuscript, we described in detail the composition of the 19 unique fluid products that were used in the various studies. This description included the fluid type, trade name, osmolality, tonicity, electrolyte content, molecular composition, pH, and manufacturer. We reviewed manufacturer’s websites, product monographs, and emailed industry representatives or study authors for more information regarding the fluids as required. The results of this study and systematic review led us to the second and third manuscripts which reported on a Bayesian network meta-analysis (NMA) of all fluid type comparisons. Despite multiple well-done RCTs, comparative data regarding the clinical effect of different resuscitative fluids when used for sepsis was incomplete. Most RCTs used 0.9% saline (normal saline) as control fluid and very few studies compared colloids directly. The advantage of using an NMA model in this setting was the ability to include indirect data into the overall point estimates. Data was abstracted from the 14 studies which focused on adult ICU patients and analyzed examining the outcomes of mortality (manuscript #2) and the use of renal replacement therapy (RRT) (manuscript #3). Certainty of evidence was evaluated for both outcomes using the GRADE approach. Results of the analysis clearly document the harm of starch-based fluids when used in septic patients. Albumin containing fluids and crystalloids (such as normal saline and Ringer’s Lactate) are better options. Lower chloride solutions, such as Ringer’s Lactate, showed a signal towards decreased mortality and a decreased use of renal replacement therapy when compared to higher chloride fluids, such as normal saline, however this was based on indirect data, not statistically significant, and warrants direct comparison trials. The final component of this thesis is a pilot study protocol for a study assessing the feasibility of a larger RCT examining the effect of low chloride versus high chloride fluids for resuscitation in patients with sepsis and septic shock. This protocol has been submitted as part of a peer-reviewed grant with the hopes of addressing this clinically important and timely question. / Thesis / Master of Science (MSc) / This thesis examines the ideal intravenous fluid to be given to patients with severe infection causing low blood pressure. A review of the current literature is presented with a protocol for future work.

Background: Sepsis is the most common cause of death in critically ill patients in settings other than cardiovascular intensive care units (ICUs). Research shows that early detection is the best way to prevent sepsis progression and improve patient outcomes. Nurses can play a critical role in the treatment of sepsis using their knowledge and resources to detect the presence of sepsis at the earliest possible point in the progression of the syndrome. Baccalaureate nursing students were surveyed to assess students' beliefs and knowledge of sepsis and to examine the gaps in students' abilities to identify sepsis. Methodology: An instrument, consisting of 46 items, was developed and administered as a survey. The survey contained demographic questions, belief statements, knowledge questions on sepsis, and an unfolding case study designed to gauge students' understanding and recognition of sepsis. Data were analyzed for descriptive statistics. Participants were undergraduate nursing students recruited from baccalaureate programs across three campuses at the University of Central Florida. Results: The sample consisted of 40 participants. Over 75% (n=31) of participants were females, 42.5% (n=17) were over 27 years old, and 45% (n=18) had five to six years of previous college experience. Only 22% (n=11) of participants selected the three best measures to screen for sepsis at the bedside and 60% (n=24) identified the correct definition of sepsis. In the knowledge application section, 40% (n=16) of participants identified the correct patient in the beginning of the case study (i.e., most likely for developing sepsis or showing signs and symptoms of sepsis). Discussion: Most students reported that they were relatively comfortable with their abilities to identify sepsis in the clinical setting. However, there were some clear gaps in students' understanding of sepsis, particularly related to general knowledge about sepsis and recommended bedside screening measures. Education on sepsis is key to provide timely care to septic patients and to provide them with the best care possible. Conclusion: This study identified gaps in baccalaureate nursing students' understanding of sepsis. Addressing these knowledge deficits could provide students with the ability to identify sepsis earlier and improve patient outcomes in their future practice.

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yes / Identifying and explaining the pathophysiology of sepsis, as well as the importance of monitoring for indicators of patient deterioration in sepsis.

According to the third international consensus definition (sepsis-3), sepsis is characterized as life-threatening organ dysfunction resulting from an uncontrolled host response to infection. Sepsis stands as a prominent contributor to worldwide mortality. A study revealed approximately 50 million reported cases of sepsis and 11 million associated deaths worldwide, constituting nearly 20% of all global fatalities. Various biomarkers have been investigated for sepsis prognosis including Procalcitonin (PCT), C-reactive protein (CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and protein C. In addition to proteomic markers genomic biomarkers have also been investigated for sepsis. For instance, research indicates a substantial rise in plasma cell-free DNA (cfDNA) and total circulating histones levels during sepsis, correlating with its severity and mortality. The complexity arises in creating a measurement tool for sepsis, given the diverse nature of these biomarkers, each requiring distinct detection methods. The objective of this doctoral thesis is to develop a low-cost fully integrated microfluidic device for detecting a genomic biomarker (cfDNA) and a proteomic biomarker (total circulating histones) using a new method for integration of hydrogels inside microfluidic devices during the fabrication process. This method involves using porous and fibrous membranes as scaffolds to support gels. The scaffold facilitates the drying and reconstitution of these gels without any loss of shape or leakage, making it advantageous in various applications, especially in point-of-care (POC) devices where long-term storage of gels inside the device is required. This hydrogel integration method was applied to demonstrate gel electrophoretic concentration and isoelectric trapping of cfDNA and histones respectively in rehydrated agarose gates with proper pH embedded in a porous membrane in a microfluidic device. Then, these two detections were performed in a single fully integrated microfluidic device. Additionally, nonspecific fluorescent dyes were incorporated within the device, eliminating the necessity for off-chip sample preparation. This enables direct testing of plasma samples without the need to label DNA and histones with fluorescent dyes beforehand. In all the fabrication steps of the microfluidic device, xurography, a cost-effective and rapid fabrication method, was utilized. This device demonstrated the effective separation of cfDNA and histones in the agarose gates in a total time of 20 minutes, employing 10 and 30 Volts for cfDNA and histone accumulation, respectively. This device could be further developed to create a POC device for the quantification of cfDNA and histones simultaneously in severe sepsis patients plasma sample. / Thesis / Doctor of Philosophy (PhD)

Neutrophils are the innate immune system's first line of defense in response to an infection. During an infection in the tissue, chemical cues called chemoattractants are released, which signal neutrophils to exit circulation and enter the tissue. Once in the tissue, neutrophils directionally migrate in response to the chemoattractant and toward the site of infection in a process called chemotaxis. At the site of infection, they initiate antimicrobial responses to clear the infection and resolve inflammation, restoring homeostasis. However, neutrophils are exposed to multiple chemoattractants and must prioritize these signals in order to correctly migrate to the appropriate site. The ability of neutrophils to properly undergo chemotaxis in the presence of infection and inflammation is crucial for resolution of inflammation and pathogen clearance. It has been recently shown that when pre-conditioned with bacterial endotoxin (LPS), innate immune function can become dysregulated. Neutrophils start to display altered antimicrobial response as well as dysfunctional migration patterns. This behavior has been seen in patients with sepsis, where a person's immune system overreacts to an infection, leading to systemic inflammation throughout the body, causing tissue damage, multiple organ failure, and in many cases, death. We explore the effects of inflammation on neutrophil migratory patterns and decision-making within chemotaxis. Additionally, to understand how inflammation within disease impacts chemotaxis, we measure the difference between neutrophils from healthy individuals and those from septic patients. We approached this using a combination of experimental and computational techniques. We developed a microfluidic assay to measure neutrophil decision-making in a competitive chemoattractant environment between an end-target (fMLP) and intermediary (LTB4) chemoattractant. Additionally, we probed for the expression level of molecules related to neutrophil chemotaxis. We also built a system of ordinary differential equations to model the dynamics of the molecular interactions underlying neutrophil chemotaxis. Our results showed that when neutrophils were induced into a highly inflammatory state, they prioritized pro-inflammatory signals over pro-resolution signals and displayed dysfunctional migration patterns. Similarly, neutrophils from patients with sepsis also displayed dysregulated migration patterns. This aberrant neutrophil chemotaxis may be implicated in the pathogenesis of sepsis, where accumulation of neutrophils in off-target organs is often seen. These results shed light onto the directional migratory decision-making of neutrophils exposed to inflammatory signals. Understanding these mechanisms may lead to the development of pro-resolution therapies that correct the neutrophil compass and reduce off-target organ damage. / Doctor of Philosophy / Neutrophils are innate immune cells that act as the first line of defense toward an infection. During an infection, chemical signals are released, stimulating neutrophils to migrate toward that specific site of infection. Once the cells are in the tissue, they can clear the pathogen and resolve inflammation. However, when neutrophils are migrating in the tissue, they are overwhelmed with multiple signals, directing them toward different sites. These signals must be prioritized by the cell so they can properly migrate toward the correct location. It has been recently shown that neutrophils that have been preconditioned into inflammatory states will display dysfunctional migration patterns. They are unable to migrate to the site of infection and instead migrate to healthy tissue, where they can cause damage. This has been shown in patients with sepsis, which is a condition where a person's immune system overreacts to an infection, causing inflammation throughout the body, leading to tissue damage and multiple organ failure. Our work explores the impact of inflammation on neutrophil migration patterns and the ability of the cell to properly prioritize when stimulated by multiple chemical signals. Additionally, we look at how neutrophils from healthy individuals differ from neutrophils from patients with sepsis, to understand how inflammation within disease impacts cellular migration. We approach this both experimentally and computationally. We designed a microfluidic assay to measure neutrophil migration in the presence of two competing chemical signals. We also measured the expression levels of molecules relevant to cell migration. We also built a mathematical model to investigate the molecular interactions underlying these processes. These results shed light on how inflammation impacts neutrophil migration and its role in inflammatory diseases.

Sepsis is a systemic inflammatory response to infection, which may ultimately lead to multi-organ failure. Sepsis causes millions of deaths each year and creates tremendous financial burdens on the health care system, yet there is no effective cure for sepsis. Even years after the onset of sepsis, patients who have clinically recovered still die from sepsis-related complications due to chronic immune dysfunction. Neutrophils, the most dominant leukocytes in human circulatory systems, play a critical role in not only promoting inflammation to fight against microbe invasion but also facilitating inflammation resolution to restore immune homeostasis. While dysfunctional/exhausted neutrophils have been implicated in the long-term morbidity and mortality of sepsis, the cause of neutrophil exhaustion and the system to rejuvenate the dysregulated immunity are understudied. To fill in the missing piece here, we conducted our trilogy-like projects. First, we established an in vitro culture system to mimic sepsis-like conditions: murine neutrophils prolonged-stimulated with LPS exhibit exhaustion-related phenotype with the elevated expression of both proinflammatory and immunosuppressive makers on the cell surface as well as dysregulated swarming patterns. We found that by knocking out TRAM (TICAM2), an adaptive molecule regulating TLR4 downstream MyD88-independent signaling pathway, neutrophils exhibit attenuated exhaustion on both phenotypic and functional levels. Of note, TRAM contributes to the development of exhausted neutrophils through activating Src family kinases (SFKs)-STAT1 cascade, and deficiency in TRAM provides protective effects on systemic inflammation, reduces tissue injury, and improves survival in a murine colitis-induced sepsis model. Next, in my second project, we reported that neutrophils can be clustered into three subpopulations even at their naïve state based on the single-cell RNA sequencing (scRNAseq) analyses. Of note, neutrophils in one of the clusters are more mature but less apoptotic with the elevated expression of resolving-associated markers Cd86 and Cd200r, hence we termed these neutrophils as 'resolving neutrophils'. We found that the resolving neutrophil population can be expanded via pharmacologically reprogramming with sodium 4-phenylbutyrate (4-PBA) or genetic deletion of TRAM. Resolving neutrophils not only secrete more pro-resolving mediators, such as ResolvinD1 and SerpinB1, but also exert enhanced phagocytic and bactericidal capacities. Mechanistically, we discovered that the development of resolving phenotype in neutrophils is mediated by the PPARγ/LMO4/STAT3 signaling circuitry, which is constitutively suppressed by TRAM. To explore the translational applications of resolving neutrophils, in my third and final project, we conducted adoptive transfer experiments to examine the effects of TRAM-deficient resolving neutrophils in cecal slurry (CS)-induced septic mice. We found that TRAM-deficient mice are more resilient to severe sepsis with reduced tissue injury and less compromised lung integrity as compared to wild-type (WT) mice, and splenic neutrophils from TRAM deficient septic mice better preserve resolving-related features. Moreover, transfusing TRAM deficient neutrophils in WT septic mice renders therapeutic effects with alleviated lung and kidney damage. We also observed TRAM-deficient neutrophil-mediated resolving memory propagation in vitro to promote resolving features of neutrophils, monocytes, and T cells, as well as to strengthen endothelial cell barrier function. In terms of the mechanism, we reported that TRAM is critical for the secretion of neutrophil elastase, a potent protein to compromise endothelium; hence, endothelial cells cocultured with TRAM deficient neutrophils maintain higher levels of adhesion/tight junction markers than cocultured with WT neutrophils. Taken together, our trilogy projects better define exhausted and resolving neutrophils. And most importantly, our works demonstrate that TRAM, an underappreciated molecule, is responsible for inducing neutrophil exhaustion and suppressing resolving neutrophil generation. / Doctor of Philosophy / A 'good' inflammation upon the infection should include two steps: the initial proinflammatory response to combat invading pathogens followed by the later resolution process to repair damage and restore the balance of the host's immune system. The harmony of these reactions is essential to maintain immune homeostasis, and the disruption of immune homeostasis may lead to different pathogenic conditions, including sepsis. Neutrophils are the most dominant white blood cells in human circulation, and they play a critical role in both promoting proinflammatory response and facilitating inflammation resolution. While the dysfunction of neutrophils is associated with the pathogenesis of sepsis and implicated in long-term sepsis-related death, approaches to rejuvenate dysregulated/exhausted neutrophils to restore immune homeostasis in septic patients are still lacking. In our projects, we better defined the characteristics of exhausted neutrophils in a sepsis-mimicking condition and unveiled the underlying mechanisms of neutrophil exhaustion. In addition, we demonstrated that neutrophils with pro-resolving features can be expanded concurrently with the decrease of exhausted neutrophils by a genetic modification approach. Finally, we showed that neutrophils with pro-resolving features can offer therapeutic effects in sepsis mice to alleviate tissue injury and organ dysfunction.