Chronic hepatitis C virus (HCV) infection exhibits persistent high viral load, inducing T cells differentiation and dysfunction in chronically infected individuals. Recent longitude studies in both HCV specific- and bulk T cells reveal that chronic immune stimulation is the driving force for the impaired T cell functions, however, the underlying mechanisms remain elusive. Here, we show that peripheral CD4+ T cells from chronically HCV-infected patients exhibit lymphopenia with the reduction of naïve population and expansion of effector memory T cells. CD4+ T cells from HCV patient also display elevated activation markers. including HLA-DR, GLUT1, Granzyme B, and short-lived effector marker CD127- KLRG1+, whereas stem cell-liked transcription factor TCF1 and telomere sheterin subunit TRF2 are significant reduced, comparing to age- and gender-matched healthy controls. Mechanistically, ex vivo T cell differentiation revealed that CD4+ T cells from HCV patients exhibit PI3K/Akt/mTOR signaling hyperactivation upon TCR stimulation, favoring pro-inflammatory effector differentiation with TRF2 downregulation, rendering telomere dysfunction induced foci (TIFs) accumulation, resulting in telomeric DNA damage and cellular apoptosis. Importantly, exacerbation of telomere deprotection by knockdown of TRF2 expression in healthy T cells resulted in an increase in telomeric DNA damage and T cell apoptosis; whereas overexpression of TRF2 in HCV-T cells led to an alleviation of telomeric DNA damage and T cell death. Additionally, inhibition of Akt signaling during T cell activation can preserve precursor memory population, while limiting inflammatory effector expansion, DNA damage, and cell death. Taken together, these results suggest that modulation of immune activation by inhibiting Akt signaling and protection of telomeres by enforcing TRF2 expression could open new therapeutic strategies to balance adaptive immune responses in the setting of chronic immune activation and inflammatory in vulnerable populations such as chronically viral infected individuals.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and is the most aggressive malignant primary brain tumor in adults. Without treatment, patients are expected to survive an average of three months. Conversely, current treatment regimens only extend survival to 12-14 months. Characteristically, GBM tumors are highly proliferative, invasive and stop responding to treatments relatively fast due to therapy resistance. Interestingly, GBM also exhibits high metabolic activity but manages to maintain a low level of reactive oxygen species (ROS). These ROS neutralization capabilities are sustained by system Xc–, a sodium-independent, electro neutral transporter that is found in the plasma membrane of GBM cells. System Xc– is composed of a regulatory heavy subunit (4F2hc) linked to a 12 transmembrane domain catalytic light chain subunit (xCT) that mediates the uptake of L-cystine into the cell, and L-glutamate out of the cell, at a 1:1 ratio. Imported cystine is quickly reduced to L- cysteine, the rate limiting substrate in glutathione (GSH) synthesis. Glutathione is a major antioxidant in the central nervous system that is responsible for maintaining intracellular redox homeostasis by neutralizing ROS by direct and indirect methods. The function of chemo and radiation therapy is to generate significant levels of ROS that tigger the cell to undergo apoptosis. High intracellular GSH levels in cancer cells are associated with drug resistance and detoxification of alkylating agents such as temozolomide (TMZ). Therefore, system Xc– represents a potential target to reduce glioma cell survival and reduce tumor progression. Sulfasalazine is an FDA approved drug in the treatment of arthritis and Crohne’s disease and has been shown to inhibit system Xc–. In vitro SASP studies demonstrated a strong antitumor potential in preclinical mouse models of malignant glioma. However, two clinical trials using sulfasalazine with standard chemo and radiation therapy to treat GBM patients were terminated due to off-target effects. Both results showed high toxicity and no change in the overall survival of patients. These studies demonstrate the need for a more effective inhibitor of system Xc–. To further elucidate the role of system Xc– in GBM survival, stable xCT knock-down and over-expressing U251 glioma cells were generated. These lines were characterized for survival, proliferation, apoptosis and resistance to oxidative and genotoxic insult. As expected xCT-knockdown cells exhibited lower GSH levels, increased intracellular ROS and markers for apoptosis after oxidative and genotoxic insult. The xCT-over-expressing cells displayed higher levels of GSH, increased resistance to hydrogen peroxide and various chemotherapy drugs including TMZ. An interesting unforeseen result of xCT over-expression in glioma cells was an increase in the metabolic activity as a result of increased mitochondria. Using xCT-modified glioma lines stably, we demonstrate for the first time that system XC– over-expression not only promotes survival under oxidative stress but may also decreases sensitivity to chemotherapy treatment and increase metabolic properties. Therefore, therapeutic manipulation of this transporter either alone or in combination with other treatments may improve clinical outcome in patients diagnosed with GBM.

Early in infection, HIV crosses the blood-brain barrier and induces neuropathology. Viral presence in the CNS coupled with secretion of neurotoxic proteins causes neuroinflammation, glial dysfunction, excitotoxicity, and neuronal death. Despite advances in combined antiretroviral therapy, HIV-infected patients present with a spectrum of cognitive and psychomotor deficits collectively referred to as HIV-associated neurological disorders (HAND). A subset of HAND patients abuses drugs such as opiates like heroin and morphine show an exacerbation and rapid progression of HIV neuropathology; however, the mechanisms of this synergy are not well understood. Autophagy is a lysosomal degradative process which eliminates and recycles cytosolic components and is implicated in facilitating HIV-1 replication in the CNS and periphery, and in Tat-induced neurodegeneration. When a key initiator of autophagy Beclin 1 was silenced using siRNAs, there was a marked reduction of HIV-1 replication in human microglia and astrocytes and the corresponding inflammatory response. As such, the goal of the current study is to determine if diminished Beclin 1 is neuroprotective against Tat and morphine-induced neurodegeneration using heterozygous Beclin 1 (Becn1+/-) mice. Examination of Tat and morphine-induced inflammatory molecule secretion revealed that Becn1+/- mixed astrocyte and microglia (glia) exhibited attenuated secretion of cytokine IL-6 and chemokines RANTES and MCP-1 compared to control (C57BL/6J) glia, an effect mediated through the μ-opioid receptor. Dysregulation of autophagy-related gene expression and excessive intracellular calcium accumulation were limited in Becn1+/- glia. When determining the effects of Tat-and morphine co-exposure on neuronal survival in vitro, we found Becn1+/- neurons were particularly sensitive to injury, excitotoxicity, and toxic exposures; however, when C57BL/6J neurons were exposed to conditioned media of C57BL/6J and Becn1+/- glia treated with Tat and morphine, neurons treated with Becn1+/- supernatant had better outcomes than those treated with C57BL/6J conditioned media. Furthermore, despite minimal difference between strains in locomotor assessment, we observed significantly greater striatal neuron losses in adult C57BL/6J mice exposed to intrastriatal Tat-and systemic morphine compared to Becn1+/- mice. Our studies demonstrate the potential of targeting Beclin 1 in glia for the prevention of Tat and opiate-induced CNS dysfunction.

Glioblastoma is the most commonly diagnosed central nervous system primary malignancy and it is considered a terminal diagnosis with few treatment options available. Glioblastoma tumors frequently develop treatment resistance due in part to their highly heterogenic nature. The heterogeneity of glioblastoma is partially attributed to the presence of glioma stem-like cells (GSC), which are highly invasive and resistant to chemotherapy and irradiation treatments. Signaling of the receptor tyrosine kinase MET is a known regulator of GSC. Glioblastoma patients have an increasingly poor prognosis that corresponds with increasing MET expression. Both GSC and MET are known to contribute to treatment resistance in glioblastoma and several MET alterations have been observed in glioblastoma. In these studies, we investigated MET alterations that are commonly found in glioblastoma. Using patient-derived xenograft (PDX) lines, the MET alterations were characterized and confirmed to be MET positive, MET amplified, or harbor a PTPRZ1-MET fusion. We also included a MET null glioblastoma PDX line. The PDX lines demonstrated markers for GSC potential with all showing neurosphere formation, the ability to initiate tumor growth in immune-compromised mice, and expression of GSC markers GFAP, Sox2, and nestin. The MET alterations were further examined by examining tyrosine kinase inhibitors' effect on viability and MET signaling. Oncogene addiction through MET amplification was found to have the best response to inhibition. The MET fusion bearing line demonstrated less sensitivity to inhibition than has been shown in other studies, indicating a need for further research into co-mutations that increase sensitivity to MET inhibition. We also investigated the efficacy of novel MET-targeting chimeric antigen receptor T cells (MET.CART cells). The MET.CART cells were able to specifically target and successfully kill MET-expressing glioblastoma cells. Together these results imply the need for more personalized treatment of glioblastoma based on the molecular biology of the tumor.

Abstract There are eight foods that contribute to food allergies in the western world and peanut is the most common. Currently, there are no medical treatments that can cure an individual of food allergy, so avoidance of the allergic food is the only option. In the United States, there are three immunodominant allergic proteins accountable for patient sensitization to peanut, Arachis hypogea 1, 2, and 3 (Ara h 1, Ara h 2, Ara h 3). Therefore, research into why peanuts are more allergic than other foods that have homologous proteins is critical and may be obtained by studying the structural and allergenic properties of individual allergens and the changes that occur due to food processing. In this study, the basic and acidic subunits of Ara h 3 were cloned, expressed, and purified, and compared with each other and with the native Ara h 3 purified from peanut for differences in binding to IgE from peanut allergic individuals. Also, an in vitro Maillard reaction was performed on purified native raw Ara h 3 and patient serum IgE western blots were performed. This study concluded that an in vitro Maillard reaction enhanced IgE binding to Ara h 3, IgE binding to native Ara h 3 was in most cases higher than to the recombinant Ara h 3 subunits, and recognition of the acidic subunit was much higher than the and basic subunits in both the recombinant and native forms of the protein were investigated. Keywords:

A growing body of evidence supports the observation that marine disease outbreaks, especially those caused by opportunistic pathogens, are increasing in frequency and severity. One genus of such pathogens, Labyrinthula, has been identified as the causative agent of seagrass wasting disease, an epidemic that has historically plagued seagrass beds around the world. It is suspected that pathogenicity is intimately linked to the ability of the host to initiate defense responses, but a lack of compelling evidence prevents any meaningful application of preliminary observations. This body of work investigated the roles of host genotype, host immune status, and environmental stressors in dictating the susceptibility of Thalassia testudinum (turtlegrass) to seagrass wasting disease, through two investigational studies. The first, a lab-based study, addressed the deficit in empirical methods through the development of techniques that measured: 1) Labyrinthula loading in host tissue through a novel qPCR-based assay and 2) immune status in the seagrass host via four immune biomarker assays, measuring peroxidase (POX), exochitinase (EXOC), polyphenol oxidase (PPO), and lysozyme (LYS) activity. These methods were used to analyze turtlegrass individuals exposed to 1) abiotic stressors alone or 2) abiotic stressors followed by pathogen-challenge, in a controlled laboratory setting. The qPCR assay successfully quantified pathogen loading in seagrass tissue with high specificity. All four biomarkers were constitutively active in host tissue, but expression was largely unaffected by the chosen abiotic stressors. There were significant positive relationships between pathogen loading and two of the four biomarkers (POX and EXOC), regardless of abiotic stress treatment. Finally, despite the widely variable response among individuals, regardless of treatment, we identify a potential trade-off mediated immune response in T. testudinum, when faced with pathogen invasion. The second investigation was a field study conducted in Florida Bay, a shallow, subtropical estuary characterized by many spatiotemporally unique basins, where T. testudinum dominates. Samples collected from 15 representative sites were analyzed using the methods developed in the first study as well as historical monitoring databases, in an effort to identify ecologically significant trends that existed in patterns between: 1) pathogen loading and immune status; 2) pathogen loading and geographic site; 3) pathogen loading and morphometric characteristics; 4) pathogen loading and water quality data; 5) immune status and geographic site; 6) immune status and morphometric characteristics; and 7) immune status and water quality data. The results revealed that both pathogen loading and immune status varied as a function of location in Florida Bay. Furthermore, based on the trends observed among and between sites with regards to pathogen loading, immune status, leaf morphology and water quality, a mechanism in which all four of these parameter sets interact is proposed as a potential explanation for the differences observed in Labyrinthula prevalence and severity within the bay. The results of both investigations address whether wasting disease susceptibility is driven primarily by variability in the environment or in the host species, and provide valuable insight regarding the extent to which seagrasses possess the capacity for resilience against marine pathogens.

Streptococcus zooepidemicus (Sz) and Streptococcus equi (Se) share 98% DNA sequence homology, but display different pathogenic properties. Infection by one organism does not cross-protect against the other. To better understand pathogenic differences between these organisms and gain information about which proteins are expressed in horses infected experimentally with Se, intrauterine Sz or naturally with respiratory Sz we compared antibody specificities of convalescent sera using ELISA. These comparisons were based on sets of 8 and 14 immunoreactive recombinant proteins of Se strain CF32 and Sz strain NC78, respectively. Sera from donkeys that were previously naturally affected with strangles and later developed Sz pneumonia secondary to an experimental influenza challenge were also included. Serum antibody responses were quantitatively and qualitatively much greater following recovery from strangles than following respiratory Sz infection. Increased reactions to Se proteins IdeE2, Se75.3, Se46.8, Se18.9 and Se42.0 were observed for the majority of strangles sera but not for sera from respiratory Sz infection cases. Reactions of sera from Sz respiratory disease to Sz proteins varied greatly and were mostly to HylC and ScpC. Interestingly, sera of donkey recovered from Sz bronchopneumonia did not show increased antibody reaction to any of the proteins even though these donkeys had also recovered from clinical strangles 6 months previously. Only 1/5 mare with Sz placentitis presented increased serum antibody responses to MAP. In conclusion, adaptive immune responses to Se of horses with strangles are stronger and involve a greater number of proteins than adaptive immune responses to Sz infection of the lower respiratory tract. In an effort to develop an improved vaccine against Se, modified live strain of EHV-1, RacH was constructed to express three recombinant antigens of Se SeM, IdeE and Se18.9. Two groups of 10 and 2 ponies were vaccinated intramuscularly or intranasally, respectively. Another group (n=6) vaccinated with empty RacH served as controls. Sera from 2/3 ponies from each vaccination groups and 1/2 serum from IN vaccinated ponies showed increased serum neutralizing antibodies to EHV-1. ELISA detected no significant increase in antibodies to proteins. Only one IM and IN vaccinated pony showed serum bactericidal activity post vaccination.

Chlamydia trachomatis is the most common sexually transmitted bacterial disease agent worldwide, and, though frequently asymptomatic, can cause extreme pathology including infertility. Chlamydial species exhibit a unique biphasic developmental cycle. Once attached to a cell surface, infectious elementary bodies (EB) are internalized within an inclusion, the membrane-bound structure in which EB transform to noninfectious, replicable reticulate bodies (RB). After multiple rounds of division, RB condense to form EB, which are released and can infect new host cells. In culture, exposure to stressors, such as beta-lactam antibiotics, induce chlamydiae to reversibly detour from normal development into a noninfectious, viable state termed persistence. Cell culture data suggest that persistent forms are resistant to azithromycin (AZM), a front-line antibiotic, and are able to alter the host transcriptome. Though persistence has been described in culture for over 50 years, whether or not it: i) occurs in vivo; and ii) influences chlamydial pathogenesis, transmission and therapy has remained unresolved. To address these questions, we developed an animal model of persistent chlamydial infection using amoxicillin (AMX) treatment. AMX exposure decreased shedding of infectious chlamydiae in C. muridarum-infected mice without affecting chlamydial viability, demonstrating the presence of persistent chlamydiae. Shedding of infectious EB resumed following AMX cessation. Shedding data and microarray analyses suggested that host immunity might limit chlamydia’s exit from persistence in our model. Thus, we hypothesized that cyclophosphamide (CTX) treatment would increase the magnitude of chlamydial shedding observed after AMX-treatment cessation. CTX treatment increased post-AMX shedding by more than 10-fold compared to AMX-only controls. To determine whether persistent chlamydiae are resistant to antibiotic eradication in vivo, we induced persistence by administering AMX and treated mice with various AZM dosing regimes. Persistently infected mice demonstrated increased treatment failure following AZM therapy compared to productively infected controls. These data suggest that persistent chlamydiae are refractory to treatment in vivo and provide an explanation for the observation that treatment fails in some patients. In addition to creating the first fully characterized, experimentally tractable, in vivo model of chlamydial persistence, these experiments provide evidence that persistent/stressed chlamydial forms may serve as a long-term reservoir of infectious organisms in vivo.

<p>NB: I had another committee member, Dr. Mark Larché; and would like to have his name included in the document.</p> <p>Thank you.</p> / <p><strong>Introduction: </strong>Increasing incidence of chronic immune diseases are mirrored by changing disease risk factors, which include maternal stress during pregnancy. To date, no studies have investigated the impact of prenatal stress challenge (PNS) on the fetal immune system. Fetal liver and bone marrow represent major sources of hematopoietic stem cells (HSC) at mid gestation, which differentiate and mature in the thymus. Disturbance of immune development may cause immune impairment in later life. Further, progesterone is recognized as a critical part of feto-maternal interaction. This study aimed to determine if PNS interferes with normal fetal immune development in mice and the impact of progesterone supplementation on stress effects. <strong>Methods: </strong>DBA/2J-mated BALB/c dams were sorted into three groups: control, PNS (gestation days (GDs) 12.5 and 14.5) and PNS plus progesterone supplementation (DHD). Fetal tissue was collected on GDs 16.5 and 18.5. Flow cytometric analysis examined frequency and phenotype of fetal immune cell populations: HSC in fetal liver and bone marrow, and different stages of T cell maturation and regulatory T (Treg) cells in the thymus. Fetal tails were collected to determine fetal sex by PCR analysis. <strong>Results: </strong>PNS induced a decrease in organ size on GD16.5, which was not seen on GD18.5 and was reversed by DHD treatment. PNS altered the percentage and absolute number of HSC within the liver and bone marrow populations, on GD16.5 and 18.5. There was a significant lag in T cell maturation as demonstrated by the altered expression of CD3 and skewed CD3-:CD3+ ratio. There was a significant decrease in Treg cells within CD3+ thymic cells in response to PNS. PNS effects in the thymus were ameliorated by DHD treatment. There was no PNS-induced sex bias. <strong>Conclusions: </strong>These results indicate that PNS compromises the developing fetal immune system, which could account for impaired immune responses in adults with chronic immune disease, and provide evidence for a therapeutic role of progesterone supplementation.</p> / Master of Science (MSc)

Human body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, have been hematophagous ectoparasites of humans for thousands of years. Despite being ecotypes, only body lice are known to transmit bacterial diseases to humans, and it appears that lower humoral and cellular immune responses allow body lice to possess a higher vector competence. We previously observed that the transcription level of the defensin 1 gene was up-regulated only in head lice following oral challenge of Bartonella quintana, a causative agent of trench fever, and also that body lice excreted more viable B. quintana in their feces. In this study, we first investigated this differential immune response by performing RNAi to knockdown defensin 1 by dsRNA injection. B. quintana was orally infected 72 h after injection and proliferation was compared at 2 hours (day 0) and day 4 post-infection. At day 0, bacterial cell numbers increased 1.5-fold in defensin 1 (Def1(-)) knocked down head lice compared with non-knocked down, pQE30-dsRNA injected, head lice control. At day 4, Def1(-) knocked down head lice had 2.55-fold more bacterial cells than control head lice and 1.65-fold greater than body lice, indicating that defensin 1 was active in reducing B. quintana cell number in non-knocked down head lice. Second, the levels of cytotoxic reactive oxygen species (ROS) generated by the epithelial cells of the alimentary tract were measured using two general indictors of ROS in both body and head lice at day 1 and day 4 following B. quintana challenge. Challenged body lice showed a 42% and 34% increase in ROS, whereas head lice showed a 70% and 22% increase at day 1 using CM-H2DCFDA and HPF as general indicators, respectively. On day 4, all challenged lice showed similar ROS levels except for body lice which maintained their ROS levels (40% increase using CM-H2DCFDA). Head lice are likely to have multiple immune and/or non-immune factors that suppress B. quintana proliferation, and the production of sustained ROS levels and/or the single knockdown of Defensin 1 is not enough to increase B. quintana proliferation in head lice to that seen in body lice.