Natural Killer cells (NK) are critical components of the innate immune system. Often referred to by their morphology, these large granular lymphocytes (LGLs) are bone marrow-derived lymphocytes and can be found throughout the body. NK cells reside in the liver, lymph nodes, spleen, thymus, and mucosal-associated lymphoid tissues (MALT). Importantly, NK cells also circulate throughout the blood where they function as surveyors of the body and are armed to eliminate malignant, infected, damaged, or foreign cells. NK cells function by a dual receptor system. That is, NK receptors are broadly categorized as inhibitory or activating. It is a fine balance, or lack thereof, that dictates the function of an NK cell. Unlike their T and B cell adaptive counterparts, NK cell receptors (NKR) are germline encoded and do not undergo gene rearrangement. NKRs are expressed in a variegated but overlapping fashion such that different cell subsets in the NK compartment elaborate different combinations of activating and inhibitory NKR. Varying the array of NKRs used by each subset increases the potential specificities of the NK compartment, while retaining tolerance to self. Thus, a diverse and balanced NK cell receptor repertoire (NKRR) is extremely important in order for this lineage to respond to various immunologic challenges and to do so in a normal, effective manner. As we have previously shown, aberrations in the expression of NKRs or downstream signaling can lead to severe immune deficiency, as observed in SHIP-deficient mice. We also showed that in the absence of SHIP-1, 2B4 becomes highly upregulated, functioning as a dominant inhibitory receptor and rendering the SHIP-1-deficient NK cell unresponsive to complex tumor targets. Traditionally MHC-I inhibitory ligands are largely responsible for the regulation of NK function. However, we show here that 2B4, which mediates MHC-I-independent inhibition, is required for formation of a normal NKRR, NK homeostasis, and effector functions. Moreover, in the absence of 2B4 and SHIP-1, NK cells have improper licensing, or education. In addition to SHIP-1 and 2B4 we show that the nature of the MHC-I ligands also play a significant role in repertoire formation, NK effector functions, and NK cell education. As described above, NK cells are critical components of the immune system. Understanding how NK cell biology and function are regulated, or affected in the context of pathology is of high significance. NK function is often severely impaired in a diseased state, and more importantly, NK cells are frequently adversely affected by the treatments themselves. Here we sought out to determine the effects of an immunomodulating drug, lenalidomide, on the biology and function of healthy NK cells. Lenalidomide is a unique drug that displays immune enhancing functions yet can be cytotoxic to tumor cells. However, lenalidomide treatment can result in immune suppression and severe cytopenia, and has the ability to impair NK viability. We show here that if used in combination with cytokine treatment (e.g. IL-2 or IL-15), many of these negative affects can be overcome. Furthermore, we show that lenalidomide treatment results in what appears to be an NK activating phenotype with a down-modulation of inhibitory KIRs and upregulation of CD16. Lenalidomide also leads to a sustained and robust activation of STAT5 and consequential increase in perforin and granzyme B. Finally, we find that treatment with lenalidomide in combination with IL-2 or IL-15 enhances the expression of IL-Rβ and IL-2Rγ chains, a presumed mechanism of action, which may provide a positive feedback loop. These findings have important clinical application. We propose that using lenalidomide in combination with IL-15 can augment its immune activating effects, while minimizing unwanted cytopenias.

Doctor of Philosophy / Department of Industrial & Manufacturing Systems Engineering / David H. Ben-Arieh / Chih-Hang Wu / Patients who suffer from sepsis or septic shock are of great concern in the healthcare system. Recent data indicate that more than 900,000 severe sepsis or septic shock cases developed in the United States with mortality rates between 20% and 80%. In the United States alone, almost $17 billion is spent each year for the treatment of patients with sepsis. Clinical trials of treatments for sepsis have been extensively studied in the last 30 years, but there is no general agreement of the effectiveness of the proposed treatments for sepsis. Therefore, it is necessary to find accurate and effective tools that can help physicians predict the progression of disease in a patient-specific way, and then provide physicians recommendation on the treatment of sepsis to lower risk for patients dying from sepsis. The goal of this research is to develop a risk assessment tool and a risk management tool for sepsis. In order to achieve this goal, two system dynamic mathematical models (SDMMs) are initially developed to predict dynamic patterns of sepsis progression in innate immunity and adaptive immunity. The two SDMMs are able to identify key indicators and key processes of inflammatory responses to an infection, and a sepsis progression. Second, an integrated-mathematical-multi-agent-based model (IMMABM) is developed to capture the stochastic nature embedded in the development of inflammatory responses to a sepsis. Unlike existing agent-based models, this agent-based model is enhanced by incorporating developed SDMMs and extensive experimental data. With the risk assessment tools, a Markov decision process (MDP) is proposed, as a risk management tool, to apply to clinical decision-makings on sepsis. With extensive computational studies, the major contributions of this research are to firstly develop risk assessment tools to identify the risk of sepsis development during the immune system responding to an infection, and secondly propose a decision-making framework to manage the risk of infected individuals dying from sepsis. The methodology and modeling framework used in this dissertation can be expanded to other disease situations and treatment applications, and have a broad impact to the research area related to computational modeling, biology, medical decision-making, and industrial engineering.

Sepsis claims over 215,000 lives in the US annually. Inducible glucocorticoids (iGC) is produced during sepsis. However, the precise effects of iGC in sepsis remain unclear due to a lack of appropriate animal models. Glucocorticoid (GC) insufficiency is associated with a marked increase in mortality and occurs in 60% of severe septic patients. Yet the conclusion of GC therapy on septic patients is still controversial. Scavenger receptor class B type I (SR-BI) in the adrenal mediates the selective uptake of cholesteryl ester from lipoproteins for GC synthesis. SR-BI-/- mice completely lack iGC during sepsis and are highly susceptible to septic death, which presents SR-BI-/- mice as a GC insufficient model. However, SR-BI-/- mice display multiple defects contributing to septic death, making it difficult to study iGC by using these mice. Therefore, we utilized adrenal-specific SR-BI-/- mice (ADR-T SR-BI-/-) generated by adrenal transplantation. As expected, the ADR-T SR-BI-/- mice failed to generate iGC under cecal ligation and puncture (CLP)-induced sepsis and showed a significantly higher mortality than the control mice, demonstrating that iGC is essential for preventing septic death. High blood urea nitrogen (BUN) was observed in the ADR-T SR-BI-/- mice but not in the control mice in CLP, indicating that iGC protects kidney injury in sepsis. Plasma IL-6 was remarkably higher in the ADR-T SR-BI-/- mice than the control mice, demonstrating an anti-inflammatory effect of iGC in sepsis. The ADR-T SR-BI-/- mice also displayed significantly lower phagocytic activity of monocytes and neutrophils in the blood and lower activation of T cells in the spleen compared to the control mice in CLP, suggesting that iGC is immunomodulatory in sepsis. Low-dose GC supplementation significantly improved the survival of SR-BI-/- mice in CLP, but did not increase the survival rate of SR-BI+/+ mice in CLP, indicating that GC supplementation improves the survival specifically in mice with adrenal insufficiency. Overall, we revealed that iGC is essential for sepsis survival. iGC prevents kidney damage, modulates inflammatory responses and exerts immunomodulatory functions in sepsis. GC supplementation specifically improves survival of individuals with adrenal insufficiency in sepsis.

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a disease of equids. Following EAV infection, up to 70% of stallions may become carriers and continuously shed the virus in their semen for varying time periods. The long-term carrier stallion has an important role in the transmission and maintenance of EAV in horse populations. Recently, it has been demonstrated a correlation between in vitro susceptibility of CD3+ T lymphocytes to EAV infection and establishment of long-term persistent infection among stallions following natural infections. In this study, we investigated whether stallions with in vitro EAV susceptible CD3+ T lymphocytes are at higher risk of becoming long-term carriers compared to those with the resistant phenotype following experimental infection with the KY84 strain of EAV. Furthermore, we investigated whether there is a significant effect of EAV infection on semen quality during acute phase of the infection. The data suggested that the establishment of the long-term carrier state seems to be associated with the in vitro CD3+ T lymphocyte susceptible phenotypes and that reduced semen quality resulted from the combined effect of fever and scrotal edema observed following EAV infection rather than the direct effect of the virus.

This thesis represents the first anthropological perspective to be offered on the nature of the Klondike Gold Rush population. In order to better understand the experience of the average gold rusher, morbidity and mortality patterns are examined for the residents of the Yukon Territory following the discovery of gold in the region (1898-1904). Infectious diseases such as measles, pneumonia, smallpox and typhoid fever are the primary focus of this study, however local factors such as the severe climate and the seclusion of the gold fields from the outside world also offers an interesting opportunity to examine the consequences of leading a particularly harsh and physically demanding lifestyle in an inhospitable environment.

This thesis represents the first anthropological perspective to be offered on the nature of the Klondike Gold Rush population. In order to better understand the experience of the average gold rusher, morbidity and mortality patterns are examined for the residents of the Yukon Territory following the discovery of gold in the region (1898-1904). Infectious diseases such as measles, pneumonia, smallpox and typhoid fever are the primary focus of this study, however local factors such as the severe climate and the seclusion of the gold fields from the outside world also offers an interesting opportunity to examine the consequences of leading a particularly harsh and physically demanding lifestyle in an inhospitable environment.

Liquid chromatography tandem mass spectrometry has become a powerful tool for investigating biological systems. Herein we describe the development of both isotope dilution mass spectrometry methods and targeted metabolomics methods for the study of metabolic and cell-cell signaling applications. A putative yeast enzyme was characterized by discovery metabolite profiling, kinetic flux profiling, transcriptomics and structural biology. These experiments demonstrated that the enzyme shb17 was a sedoheptulose bisphosphatase that provides a thermodynamically dedicated step towards riboneogenesis, leading to the redefinition of the canonical pentose phosphate pathway. An extension of metabolic profiling and kinetic flux profiling methods was developed for a set of symbiotic marine microorganisms. Carbon flux from the most abundant photosynthetic organism, Prochlorococcus, to a symbiotic Alteromonas was observed in liquid coculture. These methods enable a more biologically relevant assay for marine species and will lead to a better understanding of carbon flux in the oceans. Energy taxis refers to the active migration of bacteria in response to electron transport system related signals. The second messenger cyclic-di-GMP provides a link between the metabolic signals and motility. Quantitation of c-di-GMP helped characterize the nature of this regulation. Autoinducer-2 is a small sugar produced by a large variety of bacteria that is proposed to be a universal quorum sensing signal. The quorum sensing function of autoinducer-2 is disputed because it is produced by an enzyme of the activated methyl cycle, leading to an alternate hypothesis that it is simply a metabolic byproduct. Herein a method for the detection of autoinducer-2 is developed to enable studies of its signaling role and biosynthetic regulation. These studies demonstrated that autoinducer-2 does not function as a signal in all species. Further, metabolic experiments indicated that the metabolic impact of LuxS dysfunction was small and could be mitigated by recycling oxidized glutathione. Together, these data indicate that neither hypothesis is adequate. Evidence is provided that autoinducer-2 suppresses the immune system, by the interruption of cytokine signaling, implying that autoinducer play a protective role during host colonization.

Source: Dissertation Abstracts International, Volume: 70-07, Section: B, page: 4122. Adviser: John Michael Swint. Includes bibliographical references

Population´s Awareness in Case of Epidemic Infection Herd Occurrence This diploma work deals with the problem of biological weapons. It consists of a theoretical and a practical part. The theoretical part describes biological weapons in general, the options of their use and the principles of population´s protection. Further, I focussed on selected diseases which are considered to present biological weapons. The information I acquired is based on technical literature, the internet and the current legislation. The practical part of my diploma work deals with the opinions and attitudes of the Czech population concerning biological weapons and the potential risks of being exposed to biological weapons. Further I tried to find out whether the Czech population is aware of the basic issues of the problem in question. At the same time I focussed on comparing the state of awareness in the age groups of the economically active population on the one side and senior citizens on the other side. The research results have shown that the issue of biological weapons tends not to be appreciated adequately and that the opinions found in both age groups do not show significant differences. Further, an analysis of the acquired data has shown that the rate of awareness of the basic issues of the problem is low and that the population lacks sufficient information how to respond to a biological weapon attack in case of suspected assault. Differences in the level of awareness in the studied age groups have not been confirmed. The research has also shown that the respondents feel they do not have access to sources of sufficient and adequate information on biological weapons. Based on the acquired facts I have written a brochure providing information on biological weapons as well as on the recommended measures in case of suspected exposure. I have also elaborated a proposal concerning the implementation of the project and the distribution of the brochure.

The overall aim of this thesis was to determine how natural killer (NK) cells regulate virus infections in vivo. Anti-viral mechanisms by which NK cells control murine cytomegalovirus (MCMV) infection in the spleens and livers of adult C57BL/6 mice were first studied, revealing different mechanisms of control in different organs. Three days post-infection, MCMV titers in the spleens of perforin-deficient (perforin 0/0) mice were higher than in wild type controls, but no elevation of liver titers was found in perforin 0/0 mice. NK cell depletion in MCMV-infected perforin 0/0 mice resulted only in an increase in liver viral titers but not in spleen titers. Depletion of IFN-γ in adult C57BL/6 mice by injections with mAbs to IFN-γ resulted in an increase in viral titers in the liver but not in the spleen. Analyses using IFN-γ-receptor-deficient (IFN-γR0/0) mice, rendered chimeric with C57BL/6 bone marrow cells, indicated that even though the donor spleen cells could respond to IFN-γ, the depletion of NK cells in a recipient environment where the host cells could not respond to IFN-γ caused an increase in MCMV titers in the spleens but had little effect in the liver. IFN-γ has the ability to induce a variety of cells to produce nitric oxide (NO), and administrating the nitric oxide synthase (NOS) inhibitor Nω-monomethyl-L-arginine (L-NMA) into MCMV-infected adult C57BL/6 mice resulted in MCMV titer increases in the liver but not in the spleen. These data indicate that in adult C57BL/6 mice, there is a dichotomy in the mechanisms utilized by NK cells in the regulation of MCMV in different organs. In the spleen NK cells exert their effects in a perforin-dependent manner, suggesting a cytotoxic mechanism, whereas in the liver the production of IFN-γ by NK cells may be a predominant mechanism in the regulation of MCMV synthesis. These results may explain why the Cmv-1r (Cmv-1-resistant) locus, which maps closely to genes regulating NK cell cytotoxic function, confers an NK cell-dependent resistance to MCMV infection in the spleen but not in the liver. The ability of adoptively transferred cells to protect suckling mice from MCMV was another model used to study the mechanisms utilized by NK cells in the regulation of MCMV. Adoptive transfers of 129, C57BL/6 and perforin 0/0 spleen cells or lymphokine-activated killer (LAK) cells into 4 - 6 day old MCMV-infected C57BL/6 suckling mice significantly lowered the splenic MCMV titers in these mice compared to the infected controls. Adoptive transfers of C57BL/6 spleen cells into MCMV-infected 129 suckling mice also decreased the amount of MCMV in the 129 suckling mice, but C57BL/6 spleen cells could not regulate MCMV synthesis when adoptively transferred into 129/IFN-γR0/0 suckling mice. These results suggest that, in the suckling mouse model, the regulation of MCMV by the adoptively transferred NK cells is via an IFN-γ-dependent, perforin-independent, Cmv-1-independent mechanism. The Cmv-1 gene locus resides within the NK gene complex, in close proximity to the Ly49 NK cell receptor family. Analyses were carried out to determine if any of the 4 known Ly49 NK cell receptors (Ly49A, C, D and G2) played a role in the control of MCMV synthesis by NK cells. Studies comparing the expression of the different Ly49 NK cell subsets in the spleen and the peritoneal cavity revealed that there were differences in the distribution of the Ly49 receptors on NK1.1+ cells. Three days post-MCMV infection, the percentage of NK1.1+- Ly49+ NK cells in the spleen and the peritoneal cavity were different than in naive controls. Within the splenic NK1.1+ population, increases in NK1.1+ -Ly49A+ and NK1.1+-Ly49G2+ cells but decreases in NK1.1+-Ly49C+ and NK1.1+-Ly49D+ cells were observed. These changes in the spleen were accompanied by a concomitant decrease in NK1.1+ - Ly49A+ cells and increases in NK1.1+-Ly49C+, NK1.1+-Ly49D+ and NK1.1+-Ly49G2+ cells within the NK1.1+ population in the peritoneal cavity. These data suggest that 3 days post-MCMV infection, there may be movement of NK cells between the different organs. The role of Ly49 NK cell receptors in the regulation of MCMV was tested using adult C57BL/6 mice depleted of single or multiple Ly49 NK cell subsets. These in vivo depletions did not affect the ability of the residual NK cells to regulate MCMV synthesis. LAK cells sorted into the different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice lowered the splenic MCMV titers in these mice. Together, these results indicate that even though there is a redistribution of the Ly49 NK cell subsets during MCMV infection, the presence or absence of anyone of the 4 tested Ly49 NK cell receptors does not affect the regulation of MCMV by NK cells. However, there remain a possibility that one of the undefined Ly49 receptors or an untested NK cell receptor may be important in the control ofMCMV. Most of the cloned NK cell receptors have been shown to bind to MHC class I molecules, and MHC class I antigens have been implicated as modulators of target cell sensitivity to NK cell-mediated lysis. The regulation of virus infections and the fate of NK cells and their natural targets was examined in β2-microglobulin-deficient mice [β2m (-/-)], which have defective MHC class I expression. Infections with either the NK cell-sensitive MCMV or the NK cell-resistant lymphocytic choriomeningitis virus (LCMV) significantly augmented NK cell activity in either C57BL/6 or β2m (-/-) mice. Depletion of NK cells in vivo with antiserum to asialo GM1 markedly enhanced the synthesis of MCMV but had no effect on the synthesis of LCMV in either strain of mouse. Adoptively transferred β2m (-/-) spleen cells lowered splenic MCMV titers in C57BL/6 suckling mice, not unlike adoptively transferred C57BL/6 spleen cells. Analysis of naturally NK cell-sensitive thymocyte targets from these virus-infected β2m (-/-) mice revealed no cell surface expression of class I MHC detectable by conformation-dependent or -independent antibodies, but the virus infections enhanced class I expression on thymocytes from C57BL/6 mice. The sensitivity of C57BL/6 thymocytes to NK cell-mediated lysis was markedly reduced after in vivo poly inosinic:cytidylic (poly I:C) treatment or viral infection; in contrast, the sensitivity of the β2m (-/-) thymocytes was significantly less affected by poly I:C or viral infection. These data indicate that the normal expression of MHC class I antigens on NK cells or their targets is not required for the anti-viral functions of NK cells against an NK-sensitive virus (MCMV) nor do they protect an NK-resistant virus (LCMV) from the anti-viral activity of NK cells. Together, the data presented in this thesis help to further our understanding of the mechanisms utilized by NK cells in the control ofMCMV in both adult and suckling mice, and also help clarify the roles played by Ly49 NK cell receptors and MHC class I molecules in the regulation of MCMV.