Doctor of Philosophy, The Ohio State University, 2019, Microbiology

The cell envelope of bacteria mediates their interaction with the outside world and determines what can enter the cell. Gram-negative bacteria have a cell envelope defined by two membranes: an inner membrane, which surrounds the cytoplasm, and an outer membrane, which together with the inner membrane delimits an additional cellular compartment termed the periplasm. The inner membrane of Gram-negative bacteria is primarily composed of a phospholipid bilayer. The outer membrane, in contrast, contains phospholipids in its inner leaflet, and the essential glycolipid lipopolysaccharide (LPS) in the outer leaflet. The presence of LPS in the outer leaflet renders the outer membrane relatively impermeable, and therefore grants the cell resistance to noxious compounds in the environment, such as antibiotics. LPS is synthesized in the cytoplasmic face of the inner membrane, though it can also undergo non-stoichiometric modifications in the periplasmic face, and must thereafter be transported across the rest of the cell envelope. Transversal of the inner membrane by LPS is mediated by the ATP-binding cassette transporter MsbA. LPS extraction from the inner membrane, and subsequent transport across the rest of the cell envelope, is mediated by the LPS transport, or Lpt, complex. The Lpt complex is composed of eight proteins: a dimer of LptB in the cytoplasm that binds and hydrolyzes ATP to drive LPS transport; two transmembrane domains, LptF and LptG, which form a cavity in the inner membrane that accepts LPS and extracts it; LptC, LptA, and LptD, which form a bridge across the periplasm to allow the hydrophobic portion of LPS to traverse the aqueous periplasm; and LptE, which in conjunction with LptD facilitates the transport of LPS across the outer membrane. Here, we describe work in which we dissect the molecular mechanisms by which the Lpt system's inner membrane complex, LptB2FGC, interacts with LPS. In chapter two, we describe the identification of a residue within LptG, (open full item for complete abstract)

Committee: Natividad Ruiz (Advisor); Irina Artsimovitch (Committee Member); Ross Dalbey (Committee Member); Patrice Hamel (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

The innate immune response to infection involves a complex series of signaling events, threat recognition molecules, and effector proteins that act together to eliminate pathogens. As part of the patrolling immune cells, monocytes are often the first responders to infection and are specifically equipped to recognize a diverse range of pathogens and quickly respond to threats. One of these mechanisms involves a series of proteins known as inflammasomes that are critical for producing an adequate immune response to fend off pathogen infection while simultaneously being regulated to prevent hyper inflammation and inflammatory diseases such as sepsis. Inflammasomes are multimeric complexes that become activated upon pathogen detection by an upstream sensor molecule that can be in response to both bacterial and viral infection. Once activated, a highly regulated signaling cascade occurs that results in the eventual enzymatic activation of the pore forming protein Gasdermin D (GSDMD). Once activated, GSDMD traffics to cellular membranes to form pores often leading to an inflammatory form of cell death known as pyroptosis. While cell death is necessary to maintain homeostasis and cell turnover, dysregulated cell death in the case of GSDMD induced pyroptosis can lead to severe inflammatory diseases such as sepsis and acute respiratory distress syndrome (ARDS). Despite significant advances in unraveling the mechanism of GSDMD activation and designing targeted therapeutics against inflammation, gaps in knowledge still persist as evident by the high mortality rate of sepsis and inflammatory disorders. As such, it is essential to improve our understanding about the signaling events leading to GSDMD activation, the impact of active GSDMD on inflammation and cell death, and the regulators involved with GSDMD activation. Our lab has recently discovered a novel avenue of GSDMD mediated cell injury through the release of monocyte derived exosomes (EVs) containing active GSDMD that a (open full item for complete abstract)

Committee: Anasuya Sarkar (Advisor); Yutong Zhao (Committee Member); Susheela Tridandapani (Committee Member); Jacob Yount (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2021, College of Sciences and Health Professions

Gangliosides are involved in various biological functions, like modulation of LPS/TLR4 signaling pathway, but specific mechanism is still unknown. Gangliosides are desialylated from various sialidases. However, sialidase specificity toward individual ganglioside is unknown. In this study, sialidase substrate specificity was investigated and endogenous ganglioside carbohydrate profile change upon LPS stimulation in THP1 macrophages was explored. First, membrane-mimic system with liposome ganglioside conjugates containing different lipids with was developed to evaluate substrate specificity of sialidase and the lipid effect on the enzyme activity. Their desialylation profiles with Arthrobacter ureafaciens and H1N1 sialidase were determined using HPLC. Diversity of substrate preference was found for sialidase was found. In addition, the apparent Km and Vmax of different sialidases were found to be dependent on the type of lipid. Secondly, major gangliosides (GM3, GM2, GM1, GD3) in THP1 macrophages were quantified, where gangliosides were extracted from total cell, cell membrane, and lipid rafts. Ceramide chain was digested with ceramide glycanase, and carbohydrate unit of gangliosides were labeled with 2-anthranilic acid (2-AA). They were quantified using HPLC, in comparison to the 2-AA labeled glucose polymer ladder. A comparison in gangliosides was made in amount between the total cell membrane and lipid rafts. Next, the expression change of ganglioside was determined upon LPS stimulation of THP1 macrophages. With LPS stimulation, all gangliosides showed a decline with the most significant decrease of GM3, and least reduction of GD3 in cell lysate, lipid raft, and GM2 on the cell membrane. A total sialidase was found to be increased upon LPS stimulation. Moreover, endogenous ganglioside change was observed with Neu3 sialidase, which further investigated with sialidase inhibitor (DANA) on cell surface and in total. Finally, the sialidase specificity of end (open full item for complete abstract)

Committee: Xue-Long Sun Ph.D (Advisor); David Anderson Ph.D (Committee Member); Bin Su Ph.D (Committee Member); Chandra Kothapalli Ph.D (Committee Member); Aimin Zhou Ph.D (Committee Member) Subjects: Biochemistry; Molecular Biology

MS, University of Cincinnati, 2021, Medicine: Immunology

NETosis is a regulated form of cell death that neutrophils initiate in order to trap and kill bacteria. During this process neutrophil extracellular traps (NETs) are released. The traps consist of extracellularized DNA coated with antimicrobial proteins. Additionally, the release and rupture of granulocytes helps kill foreign bodies. Little is known about NET formation in the context of fetal infection and inflammation. During in utero infection, fetal lungs and other mucosal tissues come in contact with bacteria and bacterial products present in the amniotic fluid. To study the functionality of fetal neutrophils, two Rhesus macaque models of chorioamnionitis were used. Pregnant animals were injected intra-amniotically with lipopolysaccharide (LPS) or Escherichia coli (E. coli), and fetal lung tissues were harvested after 16 or 48 hours respectively. NETs were identified by immunohistochemistry detection of extracellular citrullinated histone in neutrophils, identified as CD68+HLA-DRneg cells. Compared to controls, the IA LPS fetal lungs did have significantly greater mean NET area. To compare the functionality of fetal neutrophils to adult neutrophils, fetal lung samples were matched with their corresponding chorioamnion. In LPS animals, there was a trend towards higher NET formation in the chorioamnion, suggesting that fetal neutrophils may be capable of NET formation but not as much as adult neutrophils. We also compared the LPS treatment to the E. coli treatment since NETs are classically thought to trap and kill live bacteria. We found no difference between the two treatments demonstrating that fetal neutrophils respond to bacterial products.

Committee: Claire Chougnet Ph.D. (Committee Chair); Ian Lewkowich (Committee Member); Jonathan Katz Ph.D. (Committee Member) Subjects: Immunology

MS, University of Cincinnati, 2020, Medicine: Immunology

Chorioamnionitis (chorio) occurs in 10% of all preterm births (<37weeks) and around 40% of very preterm births (<32 weeks). There is a massive increase in the concentration of inflammatory cytokine in the amniotic fluid during chorio. Although the fetal lung and all mucosal tissues are directly exposed to this inflammatory environment, the innate response induced in the fetus is not well understood. We use an experimental model of chorio with lipopolysaccharide (LPS) injection intra-amniotic (IA) given to pregnant rhesus macaque to stimulate an inflammatory response similar to human chorio. The amniotic fluid cytokines (IL-1, IL-6, TNF-a) were increased by more than one log. Previous data suggest that one of the immune e populations affected in the fetal lung was the mucosal-associated invariant T cell (MAIT). Using immunofluorescence we established a massive increase in MAIT cells in lung, expressing TCR Va7.2 and co-expressing CD161, IL-12R and IL-18R. MAIT cells increased 10x after 16hr and 48hr IA exposure compared to controls, and began to decrease 120hr post LPS. The absence of Ki67 but high expression of CCR5 suggested they migrated to the lung. Corresponding analysis of fetal peripheral blood polynuclear cells (PBMC) showed the frequency of blood CCR5+ MAIT cells decreased significantly following LPS exposure. MAIT cells are known for being activated through their TCR or through cytokine alone so we next looked at expression of these cytokines in the lung of IA LPS animals. We found significant increases in both IL-12p40 and IL-18 mRNA in lung MAIT cells. In accordance, IA LPS animals appeared activated because of their high rate of expression of their functional markers granzyme B and PD-1. We next looked at the role of IL-1 and TNFa signaling in the accrual of MAIT cells. IL-1ß injection caused increased amount of MAIT cells into fetal lung similar to LPS IA. Lastly, we tried to block the effects of LPS with treatments of subcutaneous and IA IL-1RA and ant (open full item for complete abstract)

Committee: Claire Chougnet Ph.D. (Committee Chair); Jonathan Katz Ph.D. (Committee Member); Ian Lewkowich Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy, Case Western Reserve University, 2020, Pathology

Neuronal mitochondrial dysfunction and neuroinflammation are two prominent and potentially etiologic features in neurodegenerative diseases. Despite this, the interplay between both aspects is unclear. Mitofusin 2 (Mfn2) is a GTPase on the outer mitochondrial membrane that promotes mitochondrial fusion, a process essential to mitochondrial health and function. In the present study, we overexpressed Mfn2 exclusively in neurons of mice to augment neuronal mitochondrial fusion in a widely used model of peripheral lipopolysaccharide (LPS)-generated neurodegeneration-associated neuroinflammation. Astonishingly, neuronal Mfn2 overexpression almost completely abolished LPS-induced lethality. These mice further showed alleviation of sickness behavior, bodyweight loss, and ischemic heart pathologies compared to non-transgenic littermates. Moreover, peripheral LPS triggered mitochondrial fragmentation in brain neurons of non-transgenic mice that was completely abrogated by neuronal Mfn2 overexpression. Immunological assays revealed that LPS-induced proinflammatory interleukin (IL)-1β and microglia activation were explicitly reduced in brains and spinal cords of mice with neuronal Mfn2 overexpression. Genome-wide expression analyses identified highly increased expression of microglia inhibitory chemokine CX3CL1 in brains of mice with neuronal Mfn2 overexpression, likely explaining protection from microglia activation and IL-1β-mediated neuroinflammation. To examine the impact of neuronal mitochondrial fusion in a more disease-relevant system, we crossed neuronal Mfn2 overexpression mice with the PS19 tau transgenic mouse model for tauopathy. Excitingly, neuronal Mfn2 overexpression alleviated muscular and cognitive deficits in the model. PS19 mice with neuronal Mfn2 overexpression also displayed a reduction in hippocampal tau pathology and microglia activation compared to tau transgenic mice. In addition, neuronal Mfn2 overexpression reduced abnormal mitochondrial morphologies (open full item for complete abstract)

Committee: Xinglong Wang (Advisor); Brian Cobb (Committee Chair); Shu Chen (Committee Member); Jason Mears (Committee Member); Clive Hamlin (Committee Member) Subjects: Biology; Immunology; Neurosciences; Pathology

PHD, Kent State University, 2019, College of Arts and Sciences / School of Biomedical Sciences

Neuroinflammation, a hallmark of many neurodegenerative diseases is mediated by microglia, the primary immune cells of the central nervous system (CNS). Activated microglial cells respond to neuronal injury and remove cellular debris, infectious agents via phagocytosis conferring neuroprotection. However, the chronic activation of these cells impairs neuronal function through the excessive release of NO and proinflammatory cytokines TNF-α and interleukins (IL-6, IL-1β and IL-12) which contributes to neuroinflammation and subsequent neurodegeneration in the brain. Thus, suppressing microglial activation is an effective therapeutic strategy to combat neuroinflammation associated with degenerative brain diseases. While anti-inflammatory agents are required to treat neurodegeneration, they may not be sufficient on their own as the disorder is multifaceted and complex but may be effective as part of a combination therapy. Therefore, improved treatment options focused on combinatory neuroprotective effects of simvastatin (Sim) and brain derived neurotrophic factor (BDNF), seem most beneficial in restoring CNS damage, as Sim is known to inhibit inflammation, promote cell survival and BDNF is a predominant neurotrophic factor that mediates survival and growth of a variety of neurons in the CNS. However, the delivery of combination therapeutics that hold promise for the treatment of neurological disorders lack clinical efficacy due to their inability in reaching high enough concentrations in the brain primarily due to the blood brain barrier (BBB), blood-cerebrospinal fluid (CSF) barrier, presence of efflux systems, enzymatic degradation, and several other factors such as rapid clearance from circulation and off-target effects. This calls for the need to develop an efficient drug delivery system (DDS) to overcome obstacles that impede CNS drug delivery and alternate approaches to bypass the BBB. Since microglia function as macrophages, the DDS must be also be effectively rem (open full item for complete abstract)

Committee: Moses Oyewumi Ph.D. (Committee Chair); Werner Geldenhuys Ph.D. (Committee Member); Fayez Safadi Ph.D. (Committee Member); Denise Inman Ph.D. (Committee Member); Douglas Delahanty Ph.D. (Committee Member) Subjects: Biomedical Research; Nanoscience; Nanotechnology; Pharmacology

PhD, University of Cincinnati, 2017, Medicine: Immunology

The protective benefits of commensal bacteria in positively calibrating the responsiveness of systemic immune cells to bolster antiviral immunity is now increasingly recognized. However, key gaps in knowledge remain regarding the broader applicability of this immune modulation to other pathogen types, and whether non-bacterial commensal microbial species share similar beneficial roles. Here we show that in contrast to dampened antiviral immunity, commensal bacteria eradication bolsters protection against disseminated Candida albicans fungal infection by unleashing neutrophil mobilization and accumulation. This discordance in how commensal bacteria control antifungal compared with antiviral immunity highlights intrinsic differences in responsiveness for distinct immune cell subsets. For example, we show pathogen-specific CD8 T cells that protect against viruses are suppressed after either fungal C. albicans or viral influenza A infection in the absence of intestinal bacteria. Furthermore, the protective benefits of enteric bacteria in averting mortality induced by viral infection or local tissue injury are each recapitulated by diverse strains of commensal fungi that commonly colonize the mammalian intestinal tract. Using antigen-specific tools to characterize how endogenous T cells respond to commensal fungi revealed the systemic accumulation of a new lineage of fungal-specific KLF2+CD44+ CD4 T cells that have potent suppressive properties in vitro. This lineage of suppressive CD4 T cells may play fundamental roles in restraining proliferation of effector T cells with commensal specificity to avert immunopathology such as inflammatory bowel disease. Interestingly, tolerance to commensal fungi may also require cell intrinsic regulation as commensal-specific CD4 T cells were also enriched for expression of the inhibitory molecule programmed death-1 (PD-1). Given the necessity we show for PD-1 in silencing the activation of high-affinity self-reactive CD4 T cells to a (open full item for complete abstract)

Committee: Sing Sing Way Ph.D. (Committee Chair); Theresa Alenghat Ph.D. (Committee Member); George Deepe M.D. (Committee Member); Gurjit Khurana Hershey M.D. (Committee Member); Marc Rothenberg M.D. Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2017, Biological Sciences

Polychlorinated biphenyls (PCBs) are environmental pollutants and endocrine disruptors, harmfully affecting reproductive, endocrine, neurological and immunological systems. This has implications for processes such as wound healing, which is modulated by the immunological response of the body. Conversely, while PCBs can be linked to diminished wound healing, outside of PCB pollution systems, exercise has been shown to accelerate wound healing. However, the potential for moderate intensity exercise to modulate or offset the harmful effects of a toxin like PCB are yet unknown. Exploration of this possible moderation on local immune response was achieved by measuring wound size and analyzing the concentrations of proinflammatory cytokines, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-a (TNF-a) in wounds (inflicted by punch biopsy) in mice that were not exercised as compared with those previously exercised at moderate intensity by running on a treadmill for 30min/day and then injected intraperitoneally with Aroclor 1254 (industrial mixture of PCB congeners) in doses of either 0, 100, 500 and 1000 ppm (wt/wt of mice). Mice were euthanized at Day 3 or Day 5 (n = 3-6) and skin excised from the wound area was homogenized and analyzed for cytokine content. Systemic effects of exercise on immune function in PCB exposed animals were examined by lipopolysaccharide (LPS) challenge (intraperitoneal injections) and analyzed by measuring the average body temperatures using a thermal imaging camera. Wound healing data revealed that in animals not exercised only the greatest dose of PCB (1000 µg/g) showed a pattern for faster wound healing. Exercise produced a pattern of more rapid wound healing rates compared to the animals administerd similar doses, except for animals administered 100 µg/g PCB. Concentrations of pro-inflammatory cytokines revealed patterns t (open full item for complete abstract)

Committee: Lee Meserve Dr. (Advisor); Howard Cromwell Dr. (Other); Todd Keylock Dr. (Committee Member); Scott Rogers Dr. (Committee Member); Vipaporn Phuntumart Dr. (Committee Member) Subjects: Immunology; Kinesiology; Molecular Biology; Physiology; Toxicology

Master of Science, The Ohio State University, 2017, Animal Sciences

ABSTRACT It is well understood that above optimum temperatures during incubation can lead to increased moisture loss and dehydrated chicks. For the experiments reported herein, moisture loss was measured without the confounding effects of temperature. Moisture loss was induced in chick embryos from days 11 through hatch by drilling 2 small holes in the air sac of eggs. On the day of hatch, chicks were further divided into three treatment groups, two controls and one treatment consisting of an injection with Salmonella enteritidis lipopolysaccharide (LPS). The Salmonella LPS is derived from the outer membrane of this gram-negative bacteria which is commonly found throughout poultry houses (Wang et al., 2003). The LPS injection was used to mimic a potential bacterial challenge to trigger a response from the chick's immune system. The two control treatments consisted of one group receiving a PBS injection alone (no LPS) and a non-injected control group of eggs. At 24 and 48 hours post injection, tissue samples were collected for RT-PCR analysis of the interleukin 1 (IL1) and interleukin 10 (IL10) response in chicks with normal and increased moisture loss during incubation. The objectives of this study were to 1.) Measure moisture loss differences in egg and chick weights between the treatment groups and 2.) Study effects of induced moisture loss on the immune response when a potential pathogen is introduced.

Committee: Mike Lilburn (Advisor); Ramesh Selvaraj (Committee Member); Francis Fluharty (Committee Member) Subjects: Animal Sciences

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2014, College of Sciences and Health Professions

Inflammation is an obligatory attempt of the host immune system to protect the body against infection. However, unregulated synthesis of pro-inflammatory products can have detrimental effects. Though mechanisms which contribute to inflammation are well appreciated, those that resolve inflammation are poorly understood. Therefore, understanding the molecular basis of such pathways will provide an entirely novel approach to treat and prevent inflammatory diseases. Transcript-selective translational control can regulate the expression of a set of inflammatory genes. We have identified one such mechanism in a novel animal model which relies on the abrogation of ribosomal protein L13a-dependent translational silencing by creating macrophage-specific L13a-knockout mice where resolution of inflammation is severely compromised. We have used these knockout mice to study two different kinds of inflammation: LPS induced systemic inflammation and dextran sodium sulphate (DSS) induced experimental colitis. Upon LPS induced endotoxemia, these mice displayed high mortality rates and severe symptoms of inflammation such as infiltration of immune cells in the peritoneum and major organs leading to tissue destruction. These animals also exhibited high serum levels of TNF-a, blood urea nitrogen (BUN), aspartate aminotransferase (AST) and several other markers of inflammation. Macrophages from these knockouts showed unregulated synthesis of several chemokines (e.g., CXCL13, CCL22, CCL8 and CCR3) and increased polysomal abundance of these mRNAs due to the abrogation of their translational silencing. Upon DSS induced colitis, these knockout mice demonstrated higher susceptibility to colitis displaying reduced survival, significant weight loss, enhanced rectal bleeding and diarrhea. Histopathology analysis of tissue sections from the knockouts showed disruption of epithelial crypts in the colon with infiltration of macrophages in colon and spleen sections. Additionally, elevated levels of (open full item for complete abstract)

Committee: Barsanjit Mazumder Ph.D. (Advisor); Crystal Weyman Ph.D. (Committee Member); Anton Komar Ph.D. (Committee Member); William Baldwin M.D., Ph.D. (Committee Member); Roman Kondratov Ph.D. (Committee Member); Aimin Zhou Ph.D. (Committee Member) Subjects: Animals; Biology; Immunology; Molecular Biology

MS, University of Cincinnati, 2013, Medicine: Immunology

Sepsis is a life-threatening event predominantly caused by gram-negative bacteria. Bacterial infection causes a pronounced macrophages (MΦs) and dendritic cells (DCs) activation which leads to excessive pro-inflammatory cytokine IL-1β, IL-6, and TNFα production (cytokine storm), resulting in endotoxic shock. Previous experimental studies have revealed that inhibition of NFκB signaling ameliorates disease symptoms, however, the contribution of myeloid p65 in endotoxic shock remains elusive. In this study, we demonstrate increased mortality in p65Δmye mice compared to WT mice upon ultra pure LPS (U-LPS) challenge. We show that increased susceptibility to endotoxic shock was associated with elevated serum level of IL-1β and IL-6 and increased recruitment of neutrophils to the peritoneal cavity following U-LPS challenge. Mechanistic analyses revealed that LPS-induced pro-inflammatory cytokine production was ameliorated in p65-deficient non-inflammatory bone marrow-derived macrophages (BMDMs), however, p65-deficient inflammatory peritoneal MPhi;s exhibited elevated IL-1β and IL-6. We show that the elevated IL-1β secretion in part was due to increased caspase-1 expression. Our data demonstrate a differential role of NFκB signaling in non-inflammatory and inflammatory MPhi;s in regulating host response toward LPS challenge and provide an insight to the MΦs/NFκB axis in sepsis.

Committee: Simon Hogan Ph.D. (Committee Chair); Kasper Hoebe Ph.D. (Committee Member); Nives Zimmermann Ph.D. (Committee Member) Subjects: Immunology

Master of Science (MS), Ohio University, 2014, Biomedical Engineering (Engineering and Technology)

The endotoxin lipopolysaccharide (LPS), a major component of Gram negative bacterial outer membrane, is a well-known inducer of inflammatory responses such as the release of cytokines in various types of cells including monocytes and macrophages. LPS can be specifically recognized by Toll-like receptor 4 (TLR4), in association with its accessory protein – myeloid differentiation factor (MD2) [1]. TLR4 in conjunction with other TLRs, plays a critical role in innate immunity, mediates pro-inflammatory cytokine production, and induces adaptive immunity of the host upon bacterial and viral infection. Intensive research aimed at identifying TLR4 inhibitors has been performed to identify possible treatments for inflammatory diseases. In the thesis, we investigated the effects of a series of 1,4- and 1,5-disubstituted-1, 2, 3-triazole derivatives on LPS-induced expression of the pro-inflammatory cytokine Interleukin 6 (IL-6), by phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages. The effect of these compounds on THP-1 derived macrophage viability was also assessed. A subset of 1,2,3-triazole derivatives that are potent inhibitors of LPS-induced IL-6 expression was identified and a preliminary structure-activity relationship analysis (SAR) was performed. These studies represent an initial step toward identifying 1,2,3-triazole derivatives that have the potential to become therapeutics for pathological inflammation.

Committee: Douglas Goetz PhD (Advisor); Stephen Bergmeier PhD (Committee Chair); Fabian Benencia PhD (Committee Member); Monica Burdick PhD (Committee Member) Subjects: Biomedical Engineering

Master of Science (MS), Wright State University, 2010, Microbiology and Immunology

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a disrupter, of B-cell differentiation, induces binding of the aryl hydrocarbon receptor (AhR) nuclear complex to dioxin responsive elements (DRE) within the mouse immunoglobulin heavy chain regulatory region (3'IgHRR), and produces a marked inhibition of 3'IgHRR activation, IgH expression, and antibody secretion in a well-characterized mouse B-cell line (CH12.LX). The mouse 3'IgHRR consists of at least four enhancers (hs3a; hs1,2; hs3b; hs4), and is highly homologous with the three enhancers (hs3; hs1,2; hs4) of the human 3'IgHRR. A polymorphism of the human hs1,2 enhancer (resulting in varying numbers of tandem repeats containing a DRE and κB site) has been correlated with several autoimmune diseases. Although the human and mouse hs1,2 enhancers are share a ~90% identity, luciferase reporter studies in mouse CH12.LX B-cells showed that TCDD inhibited LPS stimulation of the mouse hs1,2 enhancer but co-treatment with TCDD and LPS synergistically activated human hs1,2 enhancer activity. To evaluate transcriptional differences between the human and mouse hs1,2 enhancers, our objectives were to characterize the IM-9 cells as a potential human B-cell model, and to evaluate TCDD-induced transcriptional regulation of the polymorphic human hs1,2 enhancer in a human cell line. We confirmed AhR expression and TCDD-induced CYP1A1 induction in IM-9 cells. Then we transiently transfected IM-9 cells with the human hs1,2 reporters and determined that TCDD activates the human hs1,2 enhancer in IM-9 B-cells, as seen in CH12.LX B-cells. However, the TCDD-induced fold-activation in human IM-9 cells appeared less compared to results in mouse CH12.LX B-cells perhaps due to differences between the mouse and human AhR. Our data suggests that the TCDD-induced inhibition of the mouse hs1,2 enhancer versus the activation of the human hs1,2 enhancer may be related to an inhibitory BSAP site located on the mouse hs1,2 enhancer that is absent from th (open full item for complete abstract)

Committee: Courtney E Sulentic PhD (Advisor); Michael Leffak PhD (Committee Member); Nancy Bigley Phd (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Environmental Science; Immunology; Microbiology; Molecular Biology; Pharmacology; Toxicology

Master of Science, The Ohio State University, 2010, Microbiology

Francisella tularensis is a CDC Category A biological agent and a potential bioterrorist threat. There is no licensed vaccine against tularemia in the United States. A long-standing issue with potential Francisella vaccines is strain phase variation to a grey form that lacks protective capability in animal models. Comparisons of the parental strain (LVS) and a grey variant (LVSG) have identified lipopolysaccharide (LPS) alterations as a primary change. The LPS of the F. tularensis variant strain gains reactivity to F. novicida anti-LPS antibodies, suggesting structural alterations to the O-antigen. However, biochemical and structural analysis of the F. tularensis LVSG and LVS LPS demonstrated that LVSG has less O-antigen but no major O-antigen structural alterations. Additionally, LVSG possesses structural differences in both the core and lipid A regions, the latter being decreased galactosamine modification. Recent work has identified two genes important in adding galactosamine (flmF2 and flmK) to the lipid A. Quantitative real-time PCR showed reduced transcripts of both of these genes in the grey variant when compared to LVS. Loss of flmF2 or flmK caused less frequent phase conversion but did not alter intramacrophage survival or colony morphology. The LVSG strain demonstrated an intramacrophage survival defect in human and rat but not mouse macrophages. Consistent with this result, the LVSG variant demonstrated little change in LD50 in the mouse model of infection. Furthermore, the LVSG strain lacks the protective capacity of F. tularensis LVS against virulent Type A challenge. These data suggest that the LPS of the F. tularensis LVSG phase variant is dramatically altered. Understanding the mechanism of blue to grey phase variation may lead to a way to inhibit this variation, thus making future F. tularensis vaccines more stable and efficacious.

Committee: John Gunn PhD (Advisor); Mark Wewers MD (Committee Member); Robert Munson PhD (Committee Member) Subjects: Biomedical Research

Master of Arts, The Ohio State University, 2010, Psychology

Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, that may contribute to loss of function and cell death. Cell loss in the hippocampus and cortex is characteristic of Alzheimer's disease (AD), Parkinson's disease (PD) is marked by the loss of cells in the substantia nigra (SN), and both the locus coeruleus (LC) and the raphe nucleus are known to be affected in these diseases. Caffeine, an adenosine antagonist, is protective against the risk of AD and PD. The experiments described in this thesis were designed to address the following two hypotheses: If neuroinflammation contributes to the onset/progression of behavioral, cellular and molecular characteristics of AD and PD in a time-dependant manner in the temporal lobe and brainstem, then neuroinflammation created in a young rat by i.c.v. infusion of LPS should reproduce a subset of time-dependant characteristics of AD and PD in these brain regions. If the actions of adenosine at its receptors contribute to the behavioral, cellular and molecular characteristics of AD and PD reproduced by LPS-induced neuroinflammation and natural aging in rats, then modulation of the consequences of neuroinflammation by the adenosine receptor antagonist caffeine should attenuate the disease-like characteristics produced by neuroinflammation and aging. In order to test these hypotheses, young rats (3 months) were implanted with a cannula into the IVth ventricle that was connected to an osmotic minipump filled with either lipopolysaccharide (LPS) or the vehicle (aCSF) for 2, 4 or 8 weeks and modulation by caffeine upon experimentally produced neuroinflammation in LPS-infused young rats and naturally occurring inflammation in aged rats was evaluated. We predicted that LPS-infusion would produce microglia activation (defined as presentation of MHC II) in the hippocampus and brainstem, regions that degenerate in AD and PD, that would increase in intensity over (open full item for complete abstract)

Committee: Gary Wenk PhD (Advisor); Gary Berntson PhD (Committee Member); John Bruno PhD (Committee Member) Subjects: Psychology

Doctor of Philosophy, The Ohio State University, 2007, Animal Science

Failure to terminate the inflammatory response results in chronic inflammation that may lead to disease or cancer, especially in epithelial cells. We explored mammary epithelial cells as a model to identify mechanisms of anti-inflammatory activity in epithelia alone in the absence of immune cells. Bacterial endotoxin (ET) added to SCp2 mammary secretory epithelial cells: (1) induced both interleukin-6 (IL-6) secretion and nitric oxide (NO) production, but with unexpected delay in expression of mRNA for iNOS compared to IL-6; and (2) NFκB activation by 1 h after ET application (post-ET) that was transient for NFκB/p65 but persisted for NFκB/p50. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in ET-induced IL-6 and iNOS regulation via NFκB activation. Serum supplementation but not soluble extracellular matrix (EHS) enhanced ET-induced IL-6 mRNA expression and protein secretion without affecting iNOS mRNA expression or NO production, confirming the different modes of regulation of IL-6 and iNOS expressions. Culturing SCp2 cells on a confluent monolayer of SCg6 mouse mammary myoepithelial cells increased IL-6 secretion dramatically even in the absence of ET, with ET treatment further increasing IL-6 secretion but having little effect on induction of NO production over that for SCp2 cells alone; showing importance of microenvironment and cell-cell interaction in regulation of inflammation and likely its link to cancer in epithelia. ET-induced inflammation in SCp2 cells was used to screen and identify anti-inflammatory fractions of methanol extracts of wild Lebanese Centaurea ainetensis, used in Lebanese folk medicine to treat inflammatory diseases. A partially purified solid phase (SPE columns) methanolic elution fraction of C. ainetensis followed by methanol gradient elution on reverse phase HPLC chromatography (RP-HPLC) strongly inhibited ET-induc (open full item for complete abstract)

Committee: Floyd Schanbacher (Advisor); Charles Brooks (Other); James DeWille (Other); Joy Pate (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2005, Molecular, Cellular, and Developmental Biology

Monocytes and macrophages express innate immune receptors such as FcgammaR and Toll-like receptor 4, which when engaged by their respective ligands; IgG immune complexes (ICs) and bacterial lipopolysaccharide (LPS), become activated to produce inflammatory mediators. While production of inflammatory mediators is critical for elimination of invading pathogens, this process must be tightly regulated to prevent collateral tissue damage. The goal of this thesis project is to analyze molecular mechanisms that regulate macrophage inflammatory responses to ICs and LPS. In the first part of this project we have analyzed the role of the inositol phosphatase SHIP-2 in FcgammaR-mediated macrophage responses and have established for the first time that SHIP-2 regulates these responses. During the course of these studies it was found that peripheral blood monocytes (PBM) express little to no SHIP-2. However, upon LPS treatment SHIP-2 expression is robust in PBM. A similar regulation of SHIP-1 was also found in PBM. These latter findings suggested a role for inositol phosphatases in LPS signaling. Hence, in the second part of this project we have examined the role of the inositol phosphatase SHIP-1 in LPS signaling. Our experiments established that SHIP serves to promote macrophage inflammatory responses. Thus macrophages from SHIP-deficient animals were hyporesponsive to LPS. A comparison of the signaling events in LPS-stimulated SHIP-sufficient and SHIP-deficient cells revealed that the serine/threonine kinase Akt was hyperactivated in SHIP-deficient macrophages. These results suggested a role for Akt in dampening pro-inflammatory responses to LPS. Therefore, in part three of this project we analyzed the role of Akt in LPS-induced cytokine responses. Here, using multiple independent experimental models we have demonstrated that Akt promotes the production of the anti-inflammatory cytokine IL-10 in response to LPS stimulation. Although Akt did not appear to have an effect on the (open full item for complete abstract)

Committee: Susheela Tridandapani (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy, The Ohio State University, 2004, Pharmacy

Atherogenesis shares many features with inflammatory and immunal reactions. Bacterial endotoxin (LPS), together with T-lymphocytes cytokine interferon-gamma (IFNγ), causes inflammation and immune response. Because of its strategic anatomic position, endothelial cells are a primary target for injury and cardiovascular risk factors. A simple method for isolating and culturing mouse aortic endothelial cells (MAEC) was developed for studying the endothelial injury and activation by these stimuli in vitro (Section I). The effects of LPS and IFN, in the absence and presence of hypercholesterolemia, a traditional risk factor for atherosclerosis, on endothelial function and pro-inflammatory gene expression in vivo were also studied (Section II). We found that LPS and IFN synergistically activated MAEC by upregulating pro-inflammatory genes, such as inducible nitric oxide synthase and vascular cell adhesion molecule-1. Several signal transduction pathways, such as the Janus kinase/signal transducer and activator of transducer-1 (STAT1), the p38 mitogen-activated protein kinase, and the protein kinase A pathways, were involved in this regulation. Other effects of LPS included inducing the production of superoxide anion and hydrogen peroxide by MAEC. These reactive oxygen species, especially oxidized low-density lipoprotein cholesterol, caused MAEC injury, DNA breakage and gene expression. The in vivo effects of inflammation in endothelial dysfunction and atherogenesis were studied using a mouse model (C57BL/6) on an atherogenic diet. A single low dose of LPS (1.0 mg/kg, intraperitoneal injection) caused an impairment of endothelial-dependent vasorelaxation in response to acetylcholine in these mice but not their normal diet fed littermates. Endothelial-independent vasorelaxation remained unaffected. Chronic treatment with LPS (once a week for 12 weeks) or 16-week of the atherogenic diet each caused endothelial dysfunction. A more significant effect was observed with the combi (open full item for complete abstract)

Committee: Dale Hoyt (Advisor) Subjects: Health Sciences, Pharmacy

Master of Science (MS), Ohio University, 2006, Chemical Engineering (Engineering)

Toll-like receptors (TLRs) are a family of proteins (e.g. TLR4) widely acknowledged as the body's first line of defense against invading microbes. TLR4 induction can take place both under pro- as well as pseudo-inflammatory stimuli. This TLR4 induction has been implicated in the genesis of inflammation, which subsequently can lead to the pathogenesis of atherosclerosis and autoimmune diseases. Investigating what brings about TLR4 expression during inflammation is of fundamental importance. Hence, we sought to determine the effects of cytokines on TLR4 expression in human endothelial cells. For the limited range of conditions tested, our studies suggest that TLR4 expression is unaffected when endothelial cells are treated with pro-inflammatory Lipopolysaccharide, Interferon beta and Interferon gamma. Further studies are needed to determine if these results are valid for a wider range of conditions.

Committee: Douglas Goetz (Advisor) Subjects: