Neisseria gonorrhoeae, the causative agent of the STI gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. One important strategy for gonococcal survival in the host is iron acquisition in the face of nutritional immunity. To overcome iron limitation, the gonococcus expresses TonB dependent transporters (TdTs), outer membrane proteins that facilitate nutrient acquisition. Of the TdTs, the transferrin (Tf), lactoferrin (Lf), and hemoglobin (Hb) receptors hijack iron directly from host proteins, and studies have already shown that the Tf receptor is essential for the initiation of human infection. Given that the TdTs are virulence factors, they are widely conserved across strains, and are not subject to antigenic variation, they are ideal targets for novel therapeutics and vaccine development. As such, studies exploring these proteins and their potential as vaccine candidates and antimicrobial targets are needed. In this study we report that loops of the Tf receptor protein TbpA are not strongly immunogenic, and the antibodies raised against them are incapable of inhibiting TbpA-Tf interactions on the gonococcal cell surface. We also report that the loop 3 helix motif of TbpA is a critical functional domain for Tf-binding and iron uptake; however, no single residue was identified that was essential for these functions. In addition, we report the development of a platform for the structure-function analysis of HpuA, a member of the poorly studied Hb receptor. We also present evidence that novel small molecules may be able to inhibit TbpA-Tf interaction, presenting the Tf receptor as a novel, species-specific antimicrobial target. Finally, we demonstrated that a novel drug, OSU-03012, has antimicrobial activity against the gonococcus through down-regulation of DnaK, a protein chaperone. These findings suggest that DnaK, a widely conserved protein, may be a universal target for antimicrobial development. These studies provide insight into the structure function relationship of TbpA, the drug potential of DnaK, and lay the framework for future investigations of the TdTs for use in a multi-antigen vaccine.

Sepsis is the body’s response to an overwhelming infection and is a serious consequence of critical illness. It can cause tissue damage, organ failure, and death. Sepsis continues to have an unacceptably high mortality rate, due to the lack of effective treatments. Specific therapeutic targets for sepsis remain elusive since the complex functional changes that result in a septic state remain poorly understood. Macrophage Scavenger Receptor A (SRA, CD204) is a surface receptor that binds negatively charged, endogenous and exogenous ligands. We have discovered that SRA plays a significant role in the pathophysiology of sepsis. We have shown that mice with SRA have increased inflammation, decreased survival, and increased bacterial burden compared to SRA deficient mice. We have also found an increase in the expression of SRA on monocytes and macrophages in septic wild type mice. To determine the mechanism responsible for increased SRA expression in sepsis we treated a mouse macrophage cell line, (J774a.1), with mediators that stimulate toll like receptors (TLRs), innate immune receptors which are activated in sepsis. The cells were cultured with ultra pure LPS (a TLR 4 ligand), PAM3CSK4 (a TLR 2 ligand), glucan (a Dectin-1 ligand), ultra pure LPS and PAM3CSK4, or ultra pure LPS and glucan for 24 hours. The cells were stained with an SRA antibody, and flow cytometry was used to measure the SRA expression for each treatment group. LPS treatment alone resulted in a significant increase in SRA expression when compared to control cells. Specifically, LPS increased SRA expression by 53.4% compared to media alone (p

The Association of Cancer Development in Patients with Systemic Lupus Erythematosus by Rose Michelle Coley MPH, Walden University, 2011 BS, University of Mount Olive, 2008 Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Public Health Walden University March 2016 Both cancer and autoimmune diseases have been associated with numerous factors that may independently lead to the development of either disease. When these factors overlap the difficulty in assessing association is compounded. The numerous factors that are thought to cause systemic lupus erythematosus (SLE), which leads to the development of cancer, makes the study of an association between the 2 diseases challenging. The purpose of this study was to examine whether the risk of cancer development increased in SLE patients compared to the risk in non-SLE patients. Researchers have not shown consistent relationships of cancer development in patients with SLE; however, consideration of the various factors that contribute to the diseases is necessary to measure an association between the 2 diseases. This study used the Clinical Practice Research database (CPRD), a large, population-based database to test the relationship between SLE and cancer. A matched retrospective cohort study among SLE (n=3025) and non-SLE (n=180555) patients was conducted using the propensity score methodology to help balance the differences between the comparison groups. The propensity score methodology created a similar distribution of observed baseline covariates between the 2 groups. With adjustment for age, the predictor variable of SLE indicates that a patient with SLE is still 2.7 times more likely to develop cancer than is a non-SLE patient. The study outcomes could promote positive social change by reinforcing current recommendations for cancer screenings in persons with SLE, which could enhance the ability to detect cancer early enough to decrease mortality because of cancer in persons with SLE.

The discovery that cationic lipids can independently stimulate the immune system has generated interest in their potential as vaccine adjuvants. Here, we show that the cationic lipid R-DOTAP can independently stimulate type 1 interferon production in dendritic cells in both primary culture and immortalized cell culture. Levels of type 1 interferon production are cell line-dependent and limited in vitro by lipid-induced cell death. We show that cationic lipids can independently activate TLR-7 and TLR-9, suggesting a mechanism for type 1 interferon induction. This TLR-stimulatory activity is not restricted to R-DOTAP and can be extended to other similar cationic lipids in a lipid-specific and TLR-specific manner.

Clinicians lack appropriate non-invasive methods to be able to predict, diagnose, and reduce the risk of rejection in the years following kidney transplantation. Protocol biopsies and monitoring of serum creatinine levels are the most common methods of monitoring graft function after transplant; however, they have several negative aspects. Use of traditional factors regarding donors and recipients such as Human Leukocyte Antigen (HLA) DNA typing, pre-transplant anti-HLA antibody levels, and basic demographics (age, ethnicity/race, gender), has proved inadequate for post-transplant graft monitoring past the first few years. We propose that by utilizing immunologic factors available to clinicians across the United States, development of a non-invasive model for predicting renal graft outcome will provide a useful tool for post-transplant patient monitoring. We advocate an expanded model which incorporates both the traditional factors, as well as new factors, which have shown promise in predicting kidney outcome and are widely available for testing using commercial kits. These additional factors include major histocompatibility complex class I chain-related gene A (MICA) typing of donor and recipient, degree of matching for killer cell immunoglobulin-like receptors (KIRs) between donor and recipient, detection of MICA antibodies, and soluble CD30 level (sCD30). This proposed graft-function prediction model is the first to include all of these factors. Using multi-center data from adult recipients of standard-criteria deceased-donor (SCD) kidneys, we were able to construct models, containing the traditional factors only, for prediction of outcome at 1 year and 3 years post-transplant. Using single-center data from adult recipients of standard-criteria deceased-donor kidneys, we developed comparison models containing traditional factors only, as well as, expanded models containing the new suggested variables for prediction of outcome post-transplant. These additional variables, when incorporated into the expanded models provided greater positive predictive values, greater negative predictive values, and lower false negative rates for graft outcome at 1 year and at 3 years post-transplant than the models utilizing traditional factors only. Our results indicate that evaluation of sCD30, MICA and KIR as part of routine protocol testing, is helpful to clinicians for predicting risk of kidney graft rejection.

Toxoplasmosis is an important cause of congenital disease, and it is one of the most common opportunistic infections in patients with acquired immunodeficiency syndrome. The need for a reliable experimental model of this infection is crucial not only for achieving a better understanding of the patho-physiology of the disease, but also for developing better methods for evaluating new therapeutic regimens. The purpose of the present study was to investigate the role of CD4+CD25+ T regulatory lymphocytes in mice infected with Toxoplasma gondii. T regulatory (Treg) cells have been shown to play an important role in our immune system in controlling the activity of other T lymphocytes. These cells are differentiated from other T lymphocyte populations based on the co-expression of CD4 and CD25 and expression of the Foxp3 gene. The results of several recent studies have suggested that certain pathogens may be able to increase their survival in the host by exploiting T reg cell activity. T regulatory cells have been shown to control the persistence of the protozoan parasite, Leishmania major, in mice; however, this population of cells plays only a limited role during murine infection with Trypanosoma cruzi. In the present study we have investigated the role of Treg cells during murine infection with the ME49 strain of T. gondii. In vivo depletion of Treg cells was accomplished by injecting mice with a monoclonal antibody (Mab) isolated from the 7D4 rat hybridoma cell line. This Mab is specific for the interleukin-2 receptor chain (also known as CD25). Female Swiss Webster mice of approximately 6-7 weeks of age were depleted of Treg cells by intraperitoneal injection of 400µg of Mab, mice were injected once 7days prior to infection, and a second time 1 day prior to infection, with 20 tissue cysts of T. gondii. Mouse weight and tissue cyst numbers were monitored to evaluate the impact of Treg depletion on the outcome of infection. Our results suggest that depletion of Treg cells has little measurable impact during the acute stage of infection with the ME49 strain of T. gondii. Further studies will be required to determine what role, if any, these cells play in the chronic stage of murine toxoplasmosis.

Immunosenescence results in reduced immune response to infections with Streptococcus pneumoniae as well as to pneumococcal polysaccharide vaccines. The antibody response to the capsular polysaccharide (CPS) provides protection against S. pneumoniae infection. CPS immunoresponse is T cell independent and needs the macrophage-derived cytokines such as IL-12, IL-6 and IL-1β to elicit an antibody response. We showed a cytokine dysregulation, i.e. a decrease in IL-12, IL-6 and TNF-α but an increase in IL-10, in the aged (18-24 months old comparable to >65 years in human) compared to young adult mouse (8-12 weeks less than 65 years old) splenic macrophages (SM) or bone marrow derived macrophages (BMDM) activated via TLR4, TLR2 or TLR9 as well as heat killed Streptococcus pneumoniae (HKSP). There is also an age-associated defect in splenic B cells in the production of IgG3 upon stimulation with these ligands. A microarray analysis in SM followed by validation by both qt-RTPCR and western blots indicated that this age-associated defect in aged SM, BMDM and B cells was due to a heightened activity of the PI3K-Akt signaling pathway. We hypothesized that the senescence of immune responses in macrophages and B cells is due to an increase in activity of PI3K/Akt and decrease in the activity of GSK-3, the downstream kinase. Inhibition of the PI3-kinase with either LY294002 or Wortmannin restored the TLR2, 4, 9 and HKSP induced cytokine phenotype of the aged to that of the young adult in both the SM and BMDM and an enhanced IgG3 production in aged mice. We also showed that inhibition of glycogen synthase kinase-3 (GSK-3) the downstream target of the PI3K-Akt signaling pathway with SB216763 in SM, BMDM and B cells resulted in an enhancement in production of IL-10, IL-6 and IL-1β by macrophages and in B cell activation. Treatment of B cells with SB216763 in the presence of ligands for TLR-1/2, 4 or 9 as well as HKSP under in vitro conditions led to enhanced production of IgG3 and IgA, plasma cell formation and a slight increase in the proliferation of the B-cells with no adverse effects on the viability of the cells. Therefore, targeting the PI3K-AKT-GKS-3 signaling pathway could rescue the intrinsic signaling defect in the aged macrophages, increase IL-12 and IL-6, and enhance anti-CPS antibody responses.

Inflammatory Bowel Diseases, Crohn's Disease and Ulcerative colitis, are idiopathic chronic conditions with multifactorial determinants. In general, terms, intestinal inflammation results from abnormal host-microbe interactions. Alterations in homeostasis involve host genetic factors, environmental cues and unique luminal microbial niches. We have examined the coordinated expressions of several molecular targets relevant to the mucosal immune system and identified signature biomarkers of IBD. Qualitative and quantitative changes in the composition of microbiota can be related to unique immuno-phenotypes. This in turn can have more systemic effects that involve energy metabolism. Adiponectin, an adipose tissue derived adipokine, can restore cellular ATP levels and fulfills innate immune functions. We have concluded that IBD might represent a state of adiponectin resistance relating to chronic inflammation and obesity status. Lastly we hypothesized that activation of xenobiotic pathway (AHR-aryl hydrocarbon receptor) can further modulate host immune and metabolic responses, and thus contribute to IBD phenotypes. We found that IBD is associated with robust mucosal, aryl hydrocarbon receptor pathway and related to proinflammatory cytokine secretion. We conclude that IBD heterogeneity is reflected through distinct immunophenotypes. Furthermore, environmental cues that involve the AhR receptor and adipose tissue derived adiponectin are important regulators of the inflammatory process in IBD.

Adult immunizations effectively reduce morbidity, mortality, and transmission rates of multiple diseases; however, outpatient providers often a struggle to convince patients to accept vaccinations. This project’s aim is to address vaccination rates in our adult population, focusing first on the influenza vaccine in year one (2016), and then on pneumococcal vaccine in year two (2017), by 1) using a strong quality improvement strategy (known as a Champion Team) and 2) implementing a clinic program consisting of provider training, improved documentation, and informative posters targeted at patients. A quality improvement strategy known as a “Champion Team” provided a strong mechanism through which we developed and implemented the interventions across both years. Specifically, the Champion Team consisted of key stakeholders (nurses, residents, physician faculty, and informatics expert) who identified, developed, and evaluated the program. Programming included an annual health care professional training session for each vaccine (early fall of 2016 and 2017 for flu, spring 2017 for pneumococcal), improved documentation strategies and nursing uptake, and informative posters in the clinic. We assayed data from our patient electronic health record to evaluate: the percentage of our patient population for whom an immunization was documented relative to the number of unique patients seen in our clinic during that time frame. This approach in year one showed a marked increase in influenza vaccination rates in our clinic. During the 2014/2015 and 2015/2016 flu seasons our clinic vaccination rates were 39.98% and 42.05% respectively. After implementation of our champion team and clinic wide program to increase rates in 2016 our vaccination rates for the 2016/2017 flu seasons was 50.88%. Pneumonia data for a full year are under analyses and will be included in this presentation. We anticipate a similar increase in rates for our pneumococcal vaccinations. Our Champion Team and clinic wide program were perceived as relatively low-effort interventions yet appeared to increase vaccinations over the course of this study. The replication of these findings across pneumonia data (pending) and, in future work, with the herpes zoster vaccine (planned for Year 3), will increase our confidence that increases in rates were attributable to these very accessible interventions.

<p>Among food allergies, peanut has attracted the most research attention because the allergy is typically lifelong, often severe and potentially fatal. Furthermore, other than epinephrine, there are no treatments available to date. A decade of research has provided a great deal of insight into the factors that promote and regulate the <em>development </em>of allergic responses. However, less in known about the factors involved in the <em>elicitation</em> of the most common and severe manifestation of peanut allergy, namely anaphylaxis. The research in this thesis centers on the investigation of cellular and molecular pathways leading to peanut-induced anaphylaxis (PIA) as well as potential therapeutic targets. Specifically presented are: i) the development and characterization of a mouse model of PIA (Chapter 2), ii) the role of molecules including histamine, leukotrienes (LT) and platelet-activating factor (PAF) (Chapter 3) and, iii) the relative contribution of mast cells, basophils and macrophages as well as IgE and IgG₁(Chapter 4). Our data show that oral sensitization to peanut in C57BL/6 mice generated local and systemic markers of type-2 immunity that was associated with robust and consistent clinical anaphylaxis following antigen challenge. In this context, concurrent blockade of PAF and histamine receptors markedly decreases the severity of these reactions. Moreover, they demonstrate that distinctive immune effector pathways involving activation of mast cells (via IgE and IgG₁) and macrophages (via IgG₁) cooperate to elicit a broad range of systemic reactions to peanut. These findings highlight that concomitant and progressive recruitment of immune-effector pathways leads to a full range of anaphylactic reactions and therefore, therapeutic strategies for PIA may need to target several pathways or, alternatively shared components within these pathways. Combination therapy blocking both PAF and histamine may represent such as a therapeutic approach.</p> / Doctor of Philosophy (Medical Science)