ETD Search Results: instcode:osu

85 matches in the database. These are records: 1 - 30.

[1] [2] [3]

1. Almgren, Colleen Marie D.V.M., Ph.D. The potential role of potent prolactin antagonists as chemotherapeutics for human cancers: an evaluation of select prolactin antagonists in human breast cancer cells.

Degree: PhD, Veterinary Biosciences, 2005, Ohio State University

► Prolactin stimulates growth and lactation in mammary epithelial cells. Recent evidence shows… (more)

Subjects: Health Sciences, General

Keywords: prolactin; prolactin antagonists; human breast cancer

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2. Al Wabel, Naser Ali. Acute induction of tracheo-bronchoconstriction in morphine/chloralose anesthetized dogs: physiological approach and principles of therapy.

Degree: PhD, Veterinary Biosciences, 2003, Ohio State University

► The respiratory system serves as a functional gas exchanger which, under neural… (more)

▼ The respiratory system serves as a functional gas exchanger which, under neural control, contributes greatly to homeostasis. This study was designed to evoke tracheo-bronchoconstriction by different stimuli and to study the respiratory and hemodynamic effects of three bronchodilators. Tracheo-bronchoconstriction was induced in morphine/chloralose-anesthetized dogs by 5% CO2, 10% O2 or intravenous bethanechol (0.5 mg/kg). Hypercapnia caused no significant (p>0.01) respiratory or hemodynamic effects. Hypoxia significantly (p<0.01) increased heart rate (HR), pulmonary artery pressure (Pap), cardiac output (CO), and peripheral vascular resistance (PVR). No significant changes were observed in left ventricular end-diastolic pressure (LVEDP), tracheal pressure (Tp), airway pressure (Paw), bronchial pressure (Brp), and pulmonary compliance (PC) due to hypoxia. Changes due to gas mixtures were transient and returned to baseline upon normal respiration. In contrast, bethanechol caused significant increases in Pap, LVEDP, PVR, Tp and Paw with no significant changes in HR, CO or Brp. Compared to gas mixtures, bethanechol produced greater respiratory and hemodynamic effects that may resemble asthma and chronic obstructive pulmonary disease. Four groups of dogs were used to compare the respiratory and hemodynamic effects of aminophylline, atropine, terbutaline, or saline control on bethanechol-induced bronchoconstriction. Both atropine (0.04 mg/kg) and aminophylline (20 mg/kg) increased HR significantly (p<0.006) compared to control. Atropine, starting at 0.02 mg/kg, significantly decreased the bethanechol-elevated Pap, with no changes in the other groups. Atropine (0.02 mg/kg) and aminophylline (20 mg/kg) significantly decreased LVEDP after bethanechol. Atropine decreased 65% of the elevated Tp compared to 47%, and 12.6% for terbutaline, and aminophylline, respectively. Only terbutaline significantly decreased Paw and increased PC. No significant changes were observed in CO, PVR, Brp, or in blood and end-tidal gases after any bronchodilator. The high efficacy of atropine in reversing bethanechol-induced tracheo-bronchial spasm suggests a dominant control of respiration by the parasympathetic system. With limitations, this model might be of benefit in evaluating the mechanism of other bronchodilators that alter autonomic nervous control, such as M3-selective antimuscarinics. Advisors/Committee Members: Hamlin, Dr. Robert L.

Keywords: Tracheo-bronchoconstriction; Bethanechol; Bronchodilators; Morphine/chloralose; Dog; Asthma; COPD; Hemodynamic

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3. Beamer, Gillian L. IMMUNOLOGIC MECHANISMS AND PREDICTORS OF SUSCEPTIBILITY TO MYCOBACTERIUM TUBERCULOSIS.

Degree: PhD, Veterinary Biosciences, 2009, Ohio State University

► It has been estimated that 2 billion people, equivalent to one-third of… (more)

▼ It has been estimated that 2 billion people, equivalent to one-third of the world’s population, are currently infected with Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB). M.tb is typically an aerosol infection that causes disease in the lungs. Most people develop protective immunity that effectively contains M.tb bacilli in a clinically silent form (latent TB) that is not contagious. However, in a proportion of individuals, latent TB progresses to contagious active disease (reactivation TB) due to complex environmental, genetic, and immunologic interactions that are incompletely defined. By accurately predicting TB disease outcome, at-risk patients could be specifically targeted for antibiotic and/or immunotherapeutic treatments to prevent active TB. This strategy is important because early aggressive therapies would prevent illness in predisposed individuals, and benefit society by eliminating those patients’ abilities to transmit M.tb to others. This work used comparative murine models to identify immunologic correlates and mechanisms of M.tb susceptibility and TB disease progression that may be applicable to humans. Following a low dose M.tb aerosol infection of susceptible and relatively resistant mice, we identified three immune mediators that were associated with M.tb susceptibility. These mediators, therefore, could predict the outcome and timing of TB disease progression: antigen specific interferon-γ (IFN-γ), interleukin-10 (IL-10), and chemokine C-C ligand 5 (CCL5). Persistently low antigen specific IFN-γ and low CCL5 were biomarkers of M.tb susceptibility and predicted TB disease progression four to five months prior to disease onset in mice. The studies using antigen specific IFN-γ are particularly relevant to man (where access to lung samples is limited) because blood antigen specific IFN-γ accurately predicted lung responses. In contrast to low IFN-γ and low CCL5, elevated levels of IL-10 in the lungs predicted reactivation TB approximately 4-8 weeks prior to disease onset in M.tb susceptible mice. Furthermore, IL-10 was shown to be a single cause of reactivation TB, as disease progression was delayed and protective immunity was enhanced by IL-10 blockade in vivo. Overall, these results indicate that single immune mediators may be used as biomarkers of TB disease progression in humans, and furthermore, that appropriately timed immunotherapeutic intervention may prevent the transition from controlled M.tb infection to active TB disease. Advisors/Committee Members: Turner, Joanne.

Subjects: Immunology

Keywords: mycobacterium tuberculosis; susceptibility; predictors; immunity

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4. Bowden, Nadine Yvonne. Structure and Function Studies of Human T-Lymphotrophic Virus Type 1 p30.

Degree: PhD, Veterinary Biosciences, 2011, Ohio State University

► The complex retrovirus human T-lymphotrophic virus type 1 (HTLV-1) is the causative… (more)

▼ The complex retrovirus human T-lymphotrophic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and a variety of inflammatory disorders. Only about 5% of infected individuals develop ATL and then only after a 30 to 50 year clinical latency period. Reasons for this are incompletely understood. Viral modulation of viral and cellular protein expression is thought to be a major contributor to the phenomenon. HTLV-1 Tax has a prominent role in modulation activities. Key Tax activities include altering cellular response to DNA damage, apoptosis signals, and cell cycle regulation. However, expression of Tax and some Tax modulated activities increase the probability of the virus being detected and eliminated by the immune system. The virus mediates this risk by employing other viral proteins to modulate the activities and effects of Tax. HTLV-1 p30 (p30), the focus of this thesis, is one of the viral proteins that provides a counter balance to the activities of Tax. p30 selectively retains tax mRNA in the nucleus, competes with Tax for limited transcriptional elements and transcriptionally regulates both HTLV-1 and cellular gene expression. Improving our understanding of how p30 accomplishes these activities will improve our ability to combat HTLV-1 infections. Critical to understanding the mechanisms of action of p30 is knowledge of which amino acid residues and post-translational modifications are required for the functions of p30. In chapter 2, we used bioinformatics analysis to predict motifs and post-translational modifications of p30. Our bioinformatics studies revealed 11 highly conserved serines, all predicted to be phosphorylated. To test the presence of phosphorylation we used P32 labeling and mass spectrometry. Mass spectrometry revealed two serines at positions 88 and 102 that were phosphorylated. In chapter 3 we explored the impact of p30 on expression of a subset of genes associated with cellular response to DNA damage. Two complementary, but different methods were utilized to assay mRNA expression; RT2 Profiler PCR Array and NanoString. We also evaluated the impact of p30 on gene expression in the presence and absence of DNA damage. Our data indicate that p30 selectively impacts gene expression patterns and furthermore the pattern of expression is altered by the presence of DNA damage. In chapter 4, we extended our NanoString based gene expression study from chapter 3. Our chapter 3 study showed expression levels of 5 genes in the DNA damage pathway, GML, XRCC6, ATRX, CDK7 and GTF2H2, were altered in a statistically significant fashion in the presence of p30 following induction of DNA damage. Using Ingenuity Pathway Analysis (IPA) we explored hypotheses to explain changes in expression of the group as a unit and of the individual genes. Our exploration led us to focus on XRCC6 because it is implicated in repression of the HTLV-1 LTR and the G2/M cell cycle checkpoint, two areas in which there are established roles for p30. Our data has introduced two viable venues to further explore the details of the functions of p30, the role of phosphorylation and the impact of decreased expression of XRCC6. Advisors/Committee Members: Lairmore, Michael.

Subjects: Molecular Biology; Virology

Keywords: HTLV-1 p30; phosphorylation; Nanostring; XRCC6; Ku70

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5. Brannick, Erin Marie. Assessing Informational Completeness in Veterinary Biopsy Submission Forms.

Degree: MS, Veterinary Biosciences, 2010, Ohio State University

► Despite regulation and research in human medicine over the last two decades… (more)

▼ Despite regulation and research in human medicine over the last two decades related to content and informational completeness in laboratory test requisition forms, such as those submitted with biopsy specimens, no corollary regulation or research has occurred in veterinary medicine. Thus, the prevalence and potential causes of deficient or inadequate information in veterinary biopsy are largely unknown. Among, and even within, veterinary diagnostic laboratories, biopsy submission forms vary significantly in form composition and content. However, two general types of forms are in use: 1) Open-type (“open”) forms, characterized by predominately open-ended prompts such as “History:” followed by abundant space for independent clinician response, and 2) closed-type (“closed”) forms, characterized by predominately closed-ended prompts (i.e. checklists of predetermined responses) and limited space for independent clinician response. We hypothesized that open-type biopsy submission forms elicit quantitatively and qualitatively more complete case information than closed-type forms, especially regarding clinical history, lesion description, and lesion location. In this study, 225 open and 300 closed biopsy submission forms, randomly-selected from among all submissions to a single pathologist engaged in routine biopsy service for 3 laboratories over 3 years, were retrospectively evaluated for the amount (quantitative completeness) and value (qualitative completeness) of case information provided by clinicians. Four key concept areas- patient signalment, clinical history, lesion description, and lesion location- were assessed using a de novo grading scheme based upon 14 informational elements, which together recreate these clinical concepts for the pathologist analyzing the biopsy specimen. An overall deficiency rate (i.e. forms lacking all case information beyond patient signalment) of 3% was identified which is comparable to that observed for routine human biopsy submissions. Furthermore, the majority of cases had inadequate responses for one or more of the key concepts, with signalment information being most consistently reported and lesion description least consistently reported. Open-type forms were found to elicit longer responses from clinicians with more, and higher quality, information than closed-type forms in general and, specifically, in the areas of clinical history and lesion description. Both form type and prompts were also significantly associated with clinician reporting of key case information. Thus, deficient and inadequate submissions do occur in veterinary biopsy and the amount and quality of information provided by a clinician through the submission form is influenced both by the type (open versus closed) of submission form and the prompts presented on the form. Advisors/Committee Members: Stromberg, Paul.

Subjects: Veterinary Services

Keywords: biopsy; submission form; veterinary; informational completeness; requisition

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6. Buccellato, Matthew Allan. Host-mediated Alteration of Measles Virus Polymerase Activity: Consequences for the Outcome of Infection.

Degree: PhD, Veterinary Biosciences, 2008, Ohio State University

► Measles Virus (MeV) has a negative-sense, single-stranded RNA genome that is encapsidated… (more)

▼ Measles Virus (MeV) has a negative-sense, single-stranded RNA genome that is encapsidated by the viral nucleocapsid (N) protein. This ribonucleoprotein complex (RNP) forms the template for virus transcription and genome replication. The virus encoded RNA-dependent RNA polymerase (RdRp) complex is composed of the monomeric viral large (L) protein and the tetrameric polymerase co-factor (P) protein. Interaction between the RNP and the RdRp depends on transient contacts between P and the C-terminus of the N protein (NTAIL). Cyclic binding and release between P and N allow the polymerase complex to advance along the RNP template. The critical factor that should thus determine the rate of polymerase elongation, either during transcription or replication, is the binding affinity between N and P. This work addresses host-cell related factors that alter this affinity and thus influence the outcome of infection. Chapter 1 of this work describes how hsp72, the highly-inducible member of the 70-kDa heat shock protein family, along with other heat shock proteins, are expressed in the central nervous system and how these protein chaperones act to maintain cellular homeostasis, protect cells from insult, and preserve the specialized function of nervous tissue (i.e., to initiate and propagate the electrical impulses that dictate the actions of the rest of the body). These same heat shock proteins can also enhance the gene expression of neurotropic viruses (like MeV), and yet that stimulation of viral gene expression can enhance viral clearance by overcoming the host-restricted low levels of viral gene expression that otherwise confound adaptive antiviral immune responses. Thus, the stimulatory effect of hsp72 on viral gene expression remains consistent with its host protective functions – provided that the host is immune competent. Chapter 2 focuses on the role of hsp72 on MeV gene expression, specifically its ability to alter the activity of the viral polymerase, either during transcription or genome replication, or both. Using a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approach to monitor the kinetics of MeV transcription and genome replication, it was determined that the effect of hsp72 is to maintain nucleocapsid in a transcriptionally competent state during the late stages of infection. Chapter 3 describes a possible mechanism by which post-translational modification of Box-3 may alter the outcome of MeV infection in ways that mimic hsp72/Box-3 interaction. Here, the effect of tyrosine phosphorylation within NTAIL on polymerase activity and infection phenotype is examined. Information in this chapter provides a link between tyrosine phosphorylation and altered polymerase activity that is capable of promoting a persistent infection state. Finally, chapter 4 describes an examination of the contribution of co-chaperone molecules to the high binding affinity between hsp72 and NTAIL. Binding studies in this work show that HSP40 co-chaperones are necessary for the well-described high affinity binding between hsp72 and NTAIL, making these co-chaperones an additional host factor capable of modulating viral gene expression. Collectively, these studies delineate some of the effects that intracellular host-factors have on MeV infection. These findings help clarify the complex interactions that gives rise to the diverse outcomes of MeV infection. Advisors/Committee Members: Oglesbee, Michael.

Subjects: Microbiology; Virology

Keywords: measles virus; hsp72; hsp40; nucleocapsid; tyrosine phosphorylation

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7. Buck, Wayne R. Neuropathogenic mechanisms of feline immunodeficiency virus infection.

Degree: PhD, Veterinary Biosciences, 2004, Ohio State University

► We examined virological, neuropathological, and neurochemical parameters of cats infected at 3… (more)

▼ We examined virological, neuropathological, and neurochemical parameters of cats infected at 3 days and 2 months of age. Following intravenous infection of neonatal cats with 1000 tissue culture infectious doses-50% of the Maryland strain of FIV, plasma viral loads were 6,600 to 1,200,000 copies per ml. Plasma loads did not correlate with leukocyte proviral loads; latencies of auditory, visual, and sensory evoked potentials; or brain viral and proviral loads. Of 16 cats tested, viral RNA was undetectable in frontal cortex of 13 and of caudate nucleus 14. Brain proviral load was detected in all cats tested. Infected cats in this study did not develop clinical neurological signs or AIDS. Cerebral subcortical white matter volume was reduced in cats infected as neonates and killed 3 months post inoculation (MPI), but was normal 6 MPI. Caudate nucleus volume was reduced 19% in cats infected as juveniles and killed 6 and 24 MPI compared to 6 month old controls . Cerebral neocortical gray matter volume was decreased 14% in cats infected as juveniles and killed 24 MPI compared. Histopathological lesions of cats infected as juveniles and adults (6 months old at infection) consisted of minimal lymphocytic infiltrates. No neuronal loss was observed by either necrotic or apoptotic mechanisms using fluoro-jade and stereological techniques. Neurochemical analysis of the caudate nucleus of cats infected as neonates revealed increased dopamine content at 3 and 18 MPI . Tyrosine hydroxylase immunoreactivity was increased 6 and 18 MPI . Tyrosine hydroxylase enzyme activity was increased only at 3 MPI. In the caudate nucleus, glutamate transporter (GLT1) mRNA was decreased 3 MPI and Glutamate was decreased 6 MPI. In the frontal cortex, glutamine synthetase activity was decreased 3 and 6 MPI, but glutamine was elevated at 3 MPI. In summary, lack of correlation between viral load and electrodiagnostics suggests host responses to infection perpetuate functional disturbances. Neocortical atrophy without neuronal loss might be reversible. Neurochemical abnormalities indicate altered dopamine synthesis and turnover in the caudate nucleus during infection. Reductions in GLT1 mRNA glutamine synthetase activity suggest astrocytic injury occurs. Advisors/Committee Members: Mathes, Lawrence E.

Keywords: feline immunodeficiency virus; neuroAIDS; proviral load; viral load; evoked potential; neuropathology; stereology; neurochemistry; dopamine; tyrosine hydroxylase; glutamate; glutamine; glutamine synthetase; GLT1; glutamate transporter; brain

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8. Carlson, Tracy Karin. The Effects of Pulmonary Surfactant Protein-D on Innate Immune Cells and Tuberculosis Pathogenesis.

Degree: PhD, Veterinary Biosciences, 2011, Ohio State University

► Mycobacterium tuberculosis (M.tb), the causative agent of the disease tuberculosis (TB), is… (more)

▼ Mycobacterium tuberculosis (M.tb), the causative agent of the disease tuberculosis (TB), is among the leading causes of death due to a single infectious disease agent. M.tb is capable of surviving within the major pulmonary defense cell, the alveolar macrophage (AM), during respiratory infection. Normally, AMs engulf (phagocytose) and destroy potentially damaging particulate matter and bacteria, however, M.tb has evolved mechanisms to survive within AMs resulting in the establishment of infection and dissemination. AMs are constantly bathed in surfactant, a complex fluid containing multiple components of the soluble innate immune system including small amounts of the immunomodulatory protein, surfactant protein D (SP-D). SP-D has antimicrobial properties against M.tb including decreasing phagocytosis, thereby preventing the bacterium from gaining access to its intracellular niche, and decreasing intracellular bacterial survival through altered bacterial trafficking. SP-D binds to pathogenic M.tb and its major cell envelope lipoglycan, mannose-capped lipoarabinomannan (ManLAM), but only minimally to the nonpathogenic Mycobacterium smegmatis and its major cell envelope phosphatidylinositol-capped lipoarabinomannan (PILAM). Here we show that mutations to the SP-D protein, specifically, to the carbohydrate binding portion of the protein, enhance the interaction of SP-D with M.tb and its surface lipoglycoconjugates (ManLAM, lipomannan and phosphatidyl-myo-inositol mannosides). We hypothesized that increased binding to M.tb would enhance the antimicrobial effects of the protein and identified the R343V mutation as a particularly high mannose binding SP-D mutant. Structurally, SP-D consists of a carbohydrate recognition domain (CRD) which performs the carbohydrate binding function of the protein, an α-helical coiled-coil neck domain, a collagen-like region and an N terminus. With a truncated and mutated protein consisting of the neck and CRD (NCRD) of SP-D there is a corresponding lack of the macrophage antimicrobial effects of SP-D against M.tb even with the high binding R343V NCRD mutant. This illustrates the importance of the collagen-like and N terminal domains in proper interaction of SP-D with innate immune cells such as macrophages. Within the lung, SP-D is found in low concentration and beneath a lipid monolayer at the air interface. Therefore, it is both scarce and disadvantageously placed to interact with inhaled pathogens. We hypothesized that exogenous SP-D would decrease lesion formation and bacterial survival in a mouse model low dose aerosol challenge with M.tb. Surprisingly, SP-D therapy did not protect the lung although it appeared to decrease systemic spread of infection suggesting an unexpected role for SP-D in protection against TB. SP-D is highly conserved across species, but modifications to its amino acid sequence are associated with both structural and functional variation, particularly in interactions with macrophages. Therefore, we hypothesized that SP-D and cells from different species would not interact the same way as conspecific SP-D:cell combinations. Our data suggest that human SP-D protein enhances intracellular survival of bacteria on murine macrophages while decreasing intracellular survival in human macrophages. Therefore, different species’ SP-Ds are not interchangeable which should be taken into account in future experimentation with this protein. Our results have identified important new interactions of SP-D with macrophages and exciting new possibilities for SP-D immunotherapy in TB infection Advisors/Committee Members: Schlesinger, Larry.

Subjects: Biochemistry; Cellular Biology; Immunology

Keywords: Tuberculosis; Innate Immunity; Surfactant; SP-D; Macrophages

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9. Carsillo, Mary Elizabeth. Mechanisms of Measles Virus-Induced Immune Suppression in the Cotton Rat Model.

Degree: PhD, Veterinary Biosciences, 2009, Ohio State University

► The World Health Organization estimates that approximately 200,000 people, the majority of… (more)

▼ The World Health Organization estimates that approximately 200,000 people, the majority of them children, died in 2007 following acute measles. Bacterial pneumonia following virus-induced immune suppression is one of the leading fatal complications of acute Measles virus (MV) infection. Despite its clinical importance, the underlying mechanisms of MV-induced immunosuppression remain unresolved. To study MV-induced immune suppression we use the cotton rat, because it is the only rodent shown to replicate MV in the respiratory tract after intranasal infection and to develop T-cell proliferation inhibition, a hallmark of MV-induced immune suppression. Enhanced pulmonary susceptibility to secondary bacterial infection in MV-infected patients is well established. This might indicate a defect in the macrophage response, as these cells play an important role in immune responses against bacterial infections through phagocytosis and microbicidal activity as well as through cytokine production and the stimulation and modulation of T helper cell responses. To test the effect of MV on macrophages in vitro, we have established a method to culture cotton rat macrophages. Cotton rat bone marrow-derived macrophages are phenotypically and functionally more similar to human than rodent macrophages because they secrete little nitric oxide. After infection with measles virus, macrophages produce less IL12 than uninfected macrophages. Based on studies in humans it has been hypothesized that the lack of IL12 might lead to a T helper type 2 (TH2) response which might be mechanistically linked to immune suppression. In cotton rats, IL12 secretion was suppressed after infection with both wildtype and vaccine virus. After stimulation of spleen or lymph node cells with MV antigen, only IFNγ was detected, indicative of a TH1 response. Cytokines compatible with a mixed TH0 response, IFN and IL4, were detectable in supernatants after stimulation with PMA/ionomycin. A recombinant vaccine virus which secretes cotton rat IL4 was used to investigate the contribution of a TH2 response to immune suppression. This virus enhanced IL4 secretion but did not increase T-cell proliferation inhibition. These data show that measles virus infection leads to a decrease in IL12 secretion and an increase in IL4 secretion but this does not seem to correlate with development of a TH2 response and immune suppression. Inhibition of T-cell proliferation following MV infection results from down regulation of AKT kinase activity. AKT is also a key signalling molecule in a number of pathways for primary macrophage functions. In cotton rats, wildtype and vaccine MV infection lead to a downregulation of AKT activity in macrophages concurrent with decreased phagocytosis and bacterial killing, and increased susceptibility to S. aureus pneumonia in vivo. Contrary to MV, many viruses activate the PI3K-AKT signaling pathway as a strategy to slow down apoptosis and increase virus replication. Our data indicate that an increased level of active AKT kinase does not significantly effect MV transcription, replication and progeny release. This leads us to conclude that MV growth is independent of AKT kinase activity and that MV-induced pAKT suppression may be purely a strategy to contain immune responses. Advisors/Committee Members: Niewiesk, Stefan.

Subjects: Immunology; Virology

Keywords: measles virus; immune suppression; cotton rat; macrophage; T helper 1; T helper 2; nitric oxide; interleukin 12; AKT

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10. Chandler, Heather Lynn. Epithelial-mesenchymal transition in the anterior segment of the eye.

Degree: PhD, Veterinary Biosciences, 2006, Ohio State University

► Epithelial-mesenchymal transition (EMT) occurs as part of development, tissue repair, and tumor… (more)

▼ Epithelial-mesenchymal transition (EMT) occurs as part of development, tissue repair, and tumor progression. Complete EMT is characterized by transformation of closely associated immobile epithelial cells into individual motile fibroblast-like cells. This phenotypic change is characterized by disruption of all cell-cell junctions and increased protease production. EMT is stimulated by growth factors, and in all cases thus far studied, EMT appears to be controlled by transcription factors belonging to the Snail family. We demonstrate that Slug is required for corneal epithelial cell migration from murine corneal explants in vitro, and confirm the importance of Slug during EMT in the cornea by demonstrating the strong association between Slug expression and successful corneal wound healing in canine eyes in vivo. Slug is transiently expressed at the margins of healing corneal wounds and decreased Slug expression at corneal wound margins is accompanied by failure of EMT in non-healing corneal erosions of dogs. Finally, enhanced expression of Slug in corneal explants due to adenoviral transfection or tetracycline treatment increases the rate of epithelial cell migration. UVR exposure of the cornea leads to increased expression of markers of EMT, including Snail and Slug and MMPs. Canine chronic superficial keratitis (CSK) is an inflammatory disease associated with UVR exposure. Our findings suggest that overexpression of MMPs due to UVR exposure may be linked to changes in the cornea during CSK that allow an influx of inflammatory cells and neovascularization. We also demonstrate that EMT occurs in lens epithelial cells (LEC) during the repopulation of the lens capsule following cataract surgery with the subsequent development of posterior capsular opacification (PCO). In addition, there is increased expression of cyclooxygenase-2 (COX-2) in clinical samples of canine cataracts and PCO. Inhibiting the enzymatic activity of COX-2 effectively prevented EMT of LEC in our ex vivo model of PCO. The findings reported here demonstrate that partial EMT occurs in a variety of situations in differentiated epithelial cells of the adult anterior eye. Understanding the molecular events that initiate partial EMT in CEC and LEC will provide important insights into the pathology of ocular diseases and ways to control the process. Advisors/Committee Members: Kusewitt, Donna F.

Keywords: EMT; Slug; Cornea; Lens

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11. Chang, Hsiang-Lin. Keratinocyte growth factor as a survival factor in human breast cancer.

Degree: PhD, Veterinary Biosciences, 2005, Ohio State University

► Estrogens are considered to play a central role in human breast carcinogenesis,… (more)

▼ Estrogens are considered to play a central role in human breast carcinogenesis, among the endocrine factors associated with breast cancer. In estrogen-sensitive breast cancer cells, locally and distally produced growth factors engage intracellular signaling pathways and facilitate tumor cell growth. The inappropriate activation of growth factor signaling cascades can promote anti-hormone failure in breast cancer cells by stimulating cell growth and limiting apoptosis. KGF is a stromal origin growth factor and appears to act specifically on epithelial cells. Our group has shown that KGF stimulates breast cancer cell growth independently without estrogens. However, the mechanisms underlying the regulation of breast cancer by KGF is not well defined. In our study, we found that KGF enhanced breast cancer cell survival through the down-regulation of ER-α expression. Our results demonstrated that KGF could decrease ER without changing the mRNA expression of progesterone receptor (PR) and pS2. Tamoxifen (Tam) could induce MCF-7 cell death which could be blocked by KGF treatment. KGF alone did not stimulate MCF-7 cell growth. These findings lead to the hypothesis that KGF can promote anti-hormone failure through the regulation of apoptosis and KGF-induced the signaling pathways. Our experiments further proved that that the regulation of ER-α by KGF in MCF-7 cells is via PI3K/Akt pathway. KGF increased Akt phosphorylation and decreased ER mRNA expression which could be blocked by LY294002, a patent inhibitor of Akt pathway. KGF treatment also induced anti-apoptosis via the increase of the Bcl-2 and Bcl-xL proteins and the decrease of the active-form caspase-9 protein whereas LY294002 blocked the KGF-induced effects. KGF maintains the MCF-7 cell survival in the presence of 4OH-Tam. Our data suggested that KGF may function as a potential tumor promoter in regulating the process of tumor growth in human breast. Collectively, these studies provide insight into the role of KGF in the anti-estrogenic resistance of human breast and the underlying signaling pathway in the promotion of human breast carcinomas. The investigations not only further our understanding of human breast cancer biology but also provide rationales for the development of therapeutic approaches targeting KGF/KGFR signaling as a breast cancer treatment. Advisors/Committee Members: Lin, Young C.

Subjects: Health Sciences, Oncology

Keywords: Breast cancer; Keratinocyte growth factor; PI3K/Akt pathway; Apoptosis; Anti-hormone resistance

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12. Cheng, Zhihui. Transcriptional regulators of Ehrlichia chaffeensis during intracellular development and the roles of OmpA in the bacterial infection and survival.

Degree: PhD, Veterinary Biosciences, 2008, Ohio State University

► Ehrlichia chaffeensis, an obligatory intracellular bacterium of human monocytes-macrophages, causes human monocytic… (more)

▼ Ehrlichia chaffeensis, an obligatory intracellular bacterium of human monocytes-macrophages, causes human monocytic ehrlichiosis (HME). The bacterium has a biphasic developmental cycle in mammalian cells that alternates between a small ‘dense-cored cell’ (DC) and a large ‘reticulate cell’ (RC), as defined by morphological features, differential surface protein expression, and infectivity. However, how E. chaffeensis intracellular development is regulated is poorly understood. The type IV secretion (T4S) system is critical for the virulence of several pathogens. E. chaffeensis T4S apparatus, virB/D genes are split into two operons: virB3–virB6 (preceded by sodB) and virB8–virD4. Between these two operons, there are duplications of virB4, virB8, and virB9. We found that the transcription of all five loci was down-regulated prior to the release of E. chaffeensis from host THP-1 cells and up-regulated at the initiation of the exponential growth. An E. chaffeensis 12.3-kDa hypothetical protein, EcxR, specifically bound to the promoter regions upstream of virB/D loci. EcxR also activated transcription of all five virB/D loci in lacZ reporter constructs. The expression of ecxR was positively auto-regulated by EcxR. These results suggest that the five virB/D loci are coordinately regulated by EcxR to allow developmental stage–specific expression of the T4S system in E. chaffeensis. The two-component regulatory system (TCS) composed of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through the activation or repression of specific genes. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host-cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells one day post infection with closantel cleared the infection in dose-dependent manner. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were down-regulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in the infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes. Caulobacter crescentus CtrA (cell cycle transcription regulator A) is a transcriptional regulator that allows the coordination of cell cycle progression and morphogenesis by controlling the expression levels of ~100 genes. In E. chaffeensis, CtrA mRNA and protein levels were down-regulated within 6 h post infection in synchronously infected cell culture, and significantly up-regulated at the late stage of infection prior to the bacterial release. C. crescentus CtrA-binding motif (CtrA box) was predicted in the upstream regions of more than 30 E. chaffeensis genes. Temporal mRNA expressions of these genes were divided into four patterns: 1) up-regulated at the late exponential phase, similar to CtrA, 2) up-regulated at the lag phase and down-regulated during the exponential phase, 3) up-regulated at the lag phase but not down-regulated during the exponential phase, 4) constitutively expressed. E. chaffeensis CtrA specifically bound to the promoter regions of E. chaffeensis ctrA and surE, and rCtrA transactivated surE promoter-lacZ reporter constructs. These results suggest that CtrA is a global regulator controlling the intracellular development of E. chaffeensis. In E. chaffeensis, lipoproteins are required for bacterial infection to the host cells. OmpA (outer membrane protein A, also known as a peptidoglycan-associated lipoprotein) was E. chaffeensis surface exposed. E. chaffeensis binding and infection to the host cells were inhibited by rabbit anti-OmpA IgG. OmpA mRNA and protein expressions were down-regulated during the lag phase and the early exponential phase, but highly up-regulated at the late exponential phase. CtrA-binding site was predicted in the promoter region of ompA gene. CtrA bound to this region and activated OmpA expression. The results suggest that CtrA regulates ompA expression, consequently, E. chaffeensis infection of host cells. Advisors/Committee Members: Rikihisa, Yasuko.

Subjects: Biology; Cellular biology; Molecular biology; Pathology

Keywords: Ehrlichia Chaffeensis; type IV secretion system; two component regulatory system; CtrA; OmpA; lipoprotein

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13. Cooper, Megan Anne. Cytokine Regulation of Natural Killer Cell Activation and Homeostasis.

Degree: PhD, Veterinary Biosciences, 2002, Ohio State University

► Through their early production of cytokines and chemokines and their ability to… (more)

▼ Through their early production of cytokines and chemokines and their ability to lyse target cells without prior sensitization, natural killer (NK) cells are crucial components of the innate immune system. Human NK cells can be divided into two subsets based on their cell-surface density of the CD56 marker, CD56bright and CD56dim , each with distinct phenotypic and functional properties. Regulation of NK cell activation and homeostasis is regulated to a large degree by cytokines, and NK cells constitutively express a variety of cytokine receptors. During the early, innate immune response to infection, monocyte-derived cytokines (monokines), stimulate NK cells to produce immunoregulatory cytokines that are important to the host's early defense. Interleukin-1beta is one such monokine produced early in the immune response to various infections, but its role in promoting human NK cell cytokine production is unknown. Stimulation with this cytokine was found to induce abundant levels of CD56bright NK cell-derived IFN-gamma, a cytokine that is critical for the elimination of obligate intracellular pathogens. Further studies demonstrated that in response to various combinations of additional monokines or when co-cultured with activated monocytes, the CD56bright NK cell subset produces significantly greater levels of multiple cytokines than do CD56dim NK cells. These results suggest that human CD56bright NK cells have a unique functional role in the innate immune response as the primary source of NK cell-derived immunoregulatory cytokines. In order for NK cells to be maintained and available to fight infection in vivo, the homeostasis of these lymphocytes must be carefully regulated. However, very little is known about the factors controlling the survival of mature NK cells. We hypothesized that interleukin (IL)-15 may be a survival factor for NK cells. Based on results from two separate in vivo models, we have conclusively demonstrated that IL-15 is requisite for the survival of mature NK cells in vivo. Collectively, these studies provide new insight into the role of cytokines in the activation and homeostasis of NK cells and evidence for distinct functions of human NK cell subsets during the immune response. Advisors/Committee Members: Caligiuri, Michael A.

Subjects: Health Sciences, Immunology

Keywords: Immunology; Natural Killer Cell; Innate Immunity; Cytokines

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14. da Cunha, Daise Nunes Queiroz. Properties of Flow Through the Ascending Aorta in Boxer Dogs with Mild Aortic Stenosis: Momentum, Energy, Reynolds Number, Womersley’s, Unsteadiness Parameter, Vortex Shedding, and Transfer Function of Oscillations from Aorta to Thoracic Wall.

Degree: PhD, Veterinary Biosciences, 2009, Ohio State University

► Ejection murmurs of subaortic and aortic stenosis occur commonly in the mammalian… (more)

▼ Ejection murmurs of subaortic and aortic stenosis occur commonly in the mammalian population. Boxer-dogs have a high prevalence of systolic ejection murmurs (50 to 80%). The origin of these murmurs is a subject of discussion, especially in those cases that anatomical lesions are not evident, as with many cases of mild aortic stenosis. It has been speculated that turbulence is one of the most likely genesis of these ejection murmurs. Boxer-dogs with soft murmurs provide useful model to evaluate physical factors of aortic flow that may produce murmur.A total of 15 Boxer-dogs were evaluated with physical examination, electrocardiography, and a complete echocardiographic exam. For studies conducted in both the catheterization and NMR laboratories, 7 Boxers were induced to anesthesia for evaluation of the fluid mechanical parameters. Sounds were recorded from the ascending aorta and the torso surface in the cath-lab. S2 was used to calculate transfer function between oscillations in the heart and oscillations on the body surface. The images to study aortic blood flow were acquired on a 1.5T Siemens MRI system for 5 locations of the ascending aorta, 3 physiological states, and 7 dogs. Mean and peak velocity, area, Reynolds number, Womersly parameter, energy, flow, momentum, and vorticity were calculated from the velocity-encoded images using MRI. Reynolds numbers were above the critical values indicated in the literature. Re’s correlated well with momentum (r2 =0.54) and flow (r2 =0.86). Rotational velocity in CCW direction were greatest at the arch (p<0.05), clockwise vortices were greater at the root and Valsalva-sinus (p<0.05). Ten variables indicated that turbulence may have occurred at the proximal regions of the aorta. The murmur of mild aortic stenosis was originated in the proximal regions of the ascending aorta, demonstrated by the most “violent” fluid-mechanical activities in these regions. There was a mathematical relationship between intensity of oscillations within the heart generating sound and the intensity of oscillation detected on the thoracic wall as heart sound. Advisors/Committee Members: Hamlin, Robert.

Subjects: Acoustics; Anatomy and physiology; Animals; Fluid dynamics; Radiology

Keywords: Boxer dogs; systolic ejection murmur; mild aortic stenois; fluid mechanics; Reynolds number; Womersley parameter; Vorticies; Kinetic energy; momentum; Dobutamine; aucultation; catheterization; acoustics; Transfer function; heart sounds frequency and amplit

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15. Dorrance, Adrienne M. The Partial Tandem Duplication of the MLL (MLL PTD)in Leukemogenesis.

Degree: PhD, Veterinary Biosciences, 2008, Ohio State University

► Although many advances have been made in the treatment of cancer in… (more)

Subjects: Molecular biology

Keywords: leukemia; MLL; Hox

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16. Fossey, Stacey L. Characterization of STAT3 Activation in Osteosarcoma.

Degree: PhD, Veterinary Biosciences, 2010, Ohio State University

► Osteosarcoma (OSA) is the most common malignant bone tumor in both children… (more)

▼ Osteosarcoma (OSA) is the most common malignant bone tumor in both children and dogs. Treatment for both involves removal of the primary tumor followed by adjuvant chemotherapy. Unfortunately, 30% of children and over 90% of dogs will die of disease post-treatment. Recent evidence suggests that the molecular biology of OSA in dogs is similar to that in children including the presence of p53 mutations, aberrant Met expression, and similar transcriptional profiles. Given the similarities in biology and clinical behavior between canine and pediatric OSA, canine OSA represents a relevant animal model in which to investigate the molecular biology of this disease. While investigating signaling pathways in OSA cell lines, we identified the presence of constitutive STAT3 activation in all lines tested. The purpose of this work was to evaluate the potential utility of STAT3 as a target for therapeutic intervention in OSA. The first objective was to determine whether STAT3 was activated in human and canine OSA cell lines and canine OSA tissues and investigate how this activation is driven by upstream tyrosine kinases. OSA cell lines and tissues had activated STAT3 and this was found to occur through activation by upstream Src tyrosine kinase. Abrogation of STAT3 signaling by small interfering RNA or small molecule Src and STAT3 inhibitors led to apoptosis and loss of expression of targets such as VEGF and MMP2. This supported a role for STAT3 in enhancing tumor cell invasion and survival. The second objective was to investigate the effect of oncostatin M (OSM), a cytokine that activates the Janus kinase (JAK) family in enhancing STAT3 activation. The JAK family of kinases is known to activate STAT3 in cancer. OSM and members of its signaling pathway were found to be upregulated in human and canine OSA cell lines and canine tumor tissues. OSM stimulation led to STAT3, Src, and JAK2 activation with concurrent increases in MMP2 and VEGF expression. Enhanced invasion of OSA cells occurred with OSM stimulation, suggesting a role for this pathway in tumor progression and metastasis. The final objective was to characterize the efficacy and activity of a novel curcumin analog FLLL32 when applied to canine and human OSA cells. Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities however it does not attain sufficient blood levels to do so when ingested. This new analog was generated to have improved biochemical properties and specificity for STAT3. Treatment with FLLL32 led to decreases in STAT3 DNA binding activity followed by losses of viability, increased apoptosis, and decreased expression of VEGF, survivin, and MMP2, all STAT3 transcriptional targets. These studies provide evidence supporting the targeting of STAT3 in the treatment of canine and human OSA. Given the role of STAT3 in enhancing MMP2 and VEGF expression in OSA, this protein may prove to be an important part of targeted therapy for metastatic disease. Advisors/Committee Members: London, Cheryl.

Subjects: Molecular Biology

Keywords: STAT3; osteosarcoma

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17. Fuller, Geraldine Anne. ALTERATIONS IN MYOSIN AND MYOCYTE STRUCTURE IN AN EXTREMLY LONG TERM PACING MODEL OF CANINE DILATED CARDIOMYOPATHY.

Degree: PhD, Veterinary Biosciences, 2002, Ohio State University

► Canine dilated cardiomyopathy (DCM) induced by rapid pacing produces low cardiac output… (more)

▼ Canine dilated cardiomyopathy (DCM) induced by rapid pacing produces low cardiac output failure, mimicking naturally occurring human DCM. We studied long-range (>6 months) rapid right ventricular pacing to evaluate similarities to end-stage human DCM and determine differences from previously reported canine pacing studies. Samples were obtained from the right and left auricles (RAU, LAU), right and left atria (RA, LA), right and left ventricles (RV, LV), and LV subauricular and subatrial papillary muscles (PM) from 12 pacing and 11 control dogs. Since both beagle and mongrel hounds commonly are used in models of human DCM, we also investigated levels of cardiac myosin heavy chain (MHC) isoforms from 6 beagle and 6 mongrel controls. Distinct differences were observed, compared to previously reported, shorter-range studies, in numbers of apoptotic myocytes, cross-sectional myocyte size, and Bcl-2 protein levels. Myocyte degeneration and mitochondrial degradation were apparently more severe than previous reports. Rod body formation, indicative of severe myocyte degeneration, was observed. In addition, lower numbers of apoptotic myocytes, increased cross-sectional area with apparent wider size distribution and comparable levels of both Bcl-2 and Bcl-xL on western blot were observed in this study, different from previous pacing studies, but similar to end stage human heart failure. Differences in amounts of apoptosis and type II myocyte nuclei were observed based on sampling location, suggesting variation in cellular response to injury. Significant increases in levels of Beta MHC were observed in the RAU, RA, LAU, LA, and RV of pacing dogs compared to control. In addition, significant levels of ventricular light chain 2 (VLC2) were observed in the RA of pacing dogs, suggesting a compensatory switch in response to prolonged elevated RA pressure. Distinct differences were observed based on breed with levels of Beta MHC approximately 2 fold higher in the RAU, RA, LAU and LA of mongrels. The results of our study suggest that extremely long range rapid ventricular pacing is a relevant model for studying end stage human heart failure. In addition, breed and sampling sites appear to be factors that should be considered when canine models are used for studying human heart failure. Advisors/Committee Members: Yamaguchi, Mamoru.

Keywords: dilated cardiomyopathy; long-range right ventricular pacing myosin heavy chain isoforms myosin light chain isoforms mitochondrial degradation myocyte size apoptosis bcl-2 TUNEL type II myocyte nuclei

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18. Ge, Yan. Inhibitory mechanism of human neutrophil apoptosis by anaplasma phagocytophilum and indentification of novel surface proteins of A. Phagocytophilum and ehrlichia chaffeensis.

Degree: PhD, Veterinary Biosciences, 2007, Ohio State University

► The inhibition of neutrophil apoptosis plays a central role in human granulocytic… (more)

▼ The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signaling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. In this paper, it was found that the decrease of bfl-1 mRNA expression and activation of caspase 3 during spontaneous neutrophil apoptosis are inhibited by A. phagocytophilum infection. It was observed that most uninfected neutrophils lost mitochondrial membrane potential in contrast with high membrane potential in infected cells. Next, we studied the molecular signaling of the extrinsic apoptotic pathway (death receptor pathway) and the interaction between the intrinsic and extrinsic pathways in the inhibition of spontaneous human neutrophil apoptosis by A. phagocytophilum. Cell surface Fas clustering during spontaneous neutrophil apoptosis was significantly blocked by infection. The activation of caspase 8 and pro-apoptotic Bid were inhibited by A. phagocytophilum infection. These data together point to a novel mechanism induced by A. phagocytophilum involving both extrinsic and intrinsic pathways to ensure to delay the apoptosis of host neutrophils. Including inhibiting neutrophil apoptosis, the surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. To globally identify the major surface proteins of A. phagocytophilum, a membrane-impermeable biotin reagent was used to label the intact bacteria. Among the major proteins captured by biotin-streptavidin affinity purification, were five A. phagocytophilum proteins i.e., OMP85, two hypothetical proteins, P44 family proteins and OMP-1A. Except P44s, all of them were newly identified as major surface-exposed proteins. Ehrlichia chaffeensis belongs to the same family of Anaplasmataceae as A. phagocytophilum. So far, only P28, gp47 and gp120 have been shown as surface-exposed proteins. By using the affinity purification and proteomic methods, in addition to P28 and gp47, 31other proteins were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. The identification of E. chaffeensis surface-exposed proteins provides novel insights about E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interaction and vaccine development. Advisors/Committee Members: Rikihisa, Yasuko.

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19. Gibson, Kathryn Elizabeth. Neorickettsia spp.: Molecular Classification of a Vector and Roles of Bacterial Surface Proteins in Pathogenesis.

Degree: PhD, Veterinary Biosciences, 2011, Ohio State University

► Neorickettsia spp. are Gram-negative, obligate intracellular bacteria of the family Anaplasmataceae. Given… (more)

▼ Neorickettsia spp. are Gram-negative, obligate intracellular bacteria of the family Anaplasmataceae. Given their prevalence throughout the world, their propensity to cause human disease and deadly animal diseases, and the continuing discovery of new Neorickettsia spp., they are significant pathogens requiring better comprehension. The overall objective of this dissertation is to determine the host-bacterium relationships of Neorickettsia. Chapter 1 details background on Neorickettsia spp., with emphasis on Neorickettsia risticii and Neorickettsia sennetsu. The objective of Chapter 2 was to demonstrate the lineage of all N. risticii-infected trematode life stages. The study established the molecular identification of the N. risticii adult trematode host and its immature life stages, demonstrating as hypothesized that all life stages harboring N. risticii belong to the same clade. The objective of Chapter 3 was to determine the major surface proteins of N. sennetsu involved in host-pathogen interaction and to determine the roles of the major surface proteins. Four proteins: the 51-kDa antigen (P51), Neorickettsia surface proteins 2 (Nsp2) and 3 (Nsp3), and heat-shock protein 60 (GroEL), were found to have the highest surface expression. It was hypothesized that the two major β-barrel proteins, P51 and Nsp3 function as porins. The outer membrane fraction of N. sennetsu, as well as native P51 and Nsp3 were incorporated into proteoliposomes and tested by porin-swelling assays, and it was confirmed that P51 is a large porin. The objective of Chapter 4 was to determine levels of variation within predicted surface-exposed proteins of N. risticii, with the hypothesis being that geographic and temporal variation would occur among strains of N. risticii. Variation in P51 demonstrated geographic separation of N. risticii strains. Nsp2, Nsp3, and strain-specific antigen 3 (Ssa3) demonstrated temporal variation. Variety within the β-barrel proteins P51, Nsp2, and Nsp3 occurred mainly within regions predicted as external loops. Ssa3 variation mainly occurred in an N-terminal localized repeat region and consisted of changes in the number of 52-aa repeats. The objective of Chapter 5 was to determine causes of cytokine and chemokine induction in N. sennetsu infection, thus potential reasons for disease symptoms. The studies in this chapter first validated the mouse model of Sennetsu neorickettsiosis in immunocompetent BALB/c mice and then demonstrated cytokine and chemokine production by quantitative reverse-transcriptase polymerase chain reaction within spleens of infected mice during disease. Cytokine and chemokine production in the whole mouse was replicated in vitro within splenocyte and bone marrow-derived macrophage (BMDM) cultures, with significant induction of IL-1β, CXCL2, and IL-12A (p35) mRNAs occurring within whole bacteria and the Sarkosyl-purified bacterial outer membrane fraction of N. sennetsu. It was hypothesized that P51 is a major cause of cytokine induction, is recognized by Toll-like receptor 2, and that cytokine/chemokine induction is MyD88 dependent. Preliminary studies showed that cytokine/chemokine levels were reduced in MyD88-knockout mouse BMDMs and TLR2-knockout mouse splenocytes incubated with whole bacteria and in TLR2-knockout mouse BMDMs incubated with whole bacteria and bacterial outer membrane fraction. In conclusion, these studies demonstrated important host-pathogen relationships during Neorickettsial infection and disease useful for further understanding of these bacteria. Advisors/Committee Members: Rikihisa, Yasuko.

Subjects: Microbiology

Keywords: Neorickettsia risticii; Neorickettsia sennetsu; P51; surface proteins

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20. Grieves, Jessica Louise. Respiratory Syncytial Virus: Rodent Models and Vaccine Development.

Degree: PhD, Veterinary Biosciences, 2012, Ohio State University

► Respiratory syncytial virus (RSV) is the leading cause of lower airway disease… (more)

Subjects: Immunology

Keywords: respiratory syncytial virus; cotton rat; chinchilla; rodent models; mucosal vaccination

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21. Hamberg, Alexander David. Studies of circular single stranded DNA viruses of swine.

Degree: PhD, Veterinary Biosciences, 2009, Ohio State University

► Porcine circovirus 2 (PCV2) and porcine torque teno virus (TTV) are single… (more)

▼ Porcine circovirus 2 (PCV2) and porcine torque teno virus (TTV) are single stranded DNA (ssDNA) viruses that have the ability to cause disease in conjunction with other pathogens or sources of immunomodulation and are emerging pathogens of swine. PCV2 is the necessary infectious cause for the production of post-weaning multisystemic wasting syndrome (PMWS), and the sudden global emergence of epidemic PMWS remains unexplained. Immunohistochemical and in vitro culture-based methodologies have suggested that macrophages are likely sites of viral replication; however the tissues and cells involved in PCV2 replication have not been fully delineated. Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from non-replicating virion particles in vivo. In Chapter 1, a combination of IHC with commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNAs, presumably due to the lack of protein cross-linking in the former. These data demonstrate that conventional IHC detects PCV2 DNA forms in mononuclear cells of experimentally infected, PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the harsh conditions needed for ISH. In chapter 2, we investigated a PCV2 reference pathogenic isolate engineered to contain a unique genotype identified in archival porcine clinical samples collected 26 years prior to the first reports of epidemic PMWS. This three amino acid threonine-glycine-asparagine to alanine-threonine-alanine (Thr-Gly-Asn to Ala-Thr-Ala) sequence in the C-terminus region of the nucleocapsid protein distinguishes archival PCV2 from contemporary PCV2. Groups of gnotobiotic piglets inoculated with each virus demonstrated that this region of the PCV2 genome was critical for the spread of PCV2 beyond lymphoid tissues and suggest that genetic mutations in avirulent PCV2 may account for the emergence of virulent PCV2 and PCVDs including epidemic PMWS. The pathogenic ability of TTV has recently been demonstrated in gnotobiotic swine and is supported by experimental and epidemiologic data from conventional swine. In chapter 3 we used quantitative PCR to examine the course of TTV progression through tissues in vivo and assessed viral copy numbers of both PCV2 and genogroup 1 TTV (g1-TTV) DNAs with respect to clinical disease outcomes and co-infection with both viruses. While bone marrow was an important source of TTV DNA during early infection, spleen, liver and lung contained the highest levels of TTV during the late infection time periods. Currently little is known about the intra-cellular effects of TTV infection. In chapter 4 we examined a variety of g1-TTV-infected tissues from gnotobiotic piglets to explain the ultrastructural basis for histological lesions characteristic of g1-TTV. Transmission electron micrographs of porcine tissues infected with g1-TTV identified 35nm diameter electron-dense structures in serum and the cytoplasm of mononuclear cells in the bone marrow, lung and inguinal lymph nodes. The viral inclusion bodies observed by light microscopy did not contain virus-like particles. Advisors/Committee Members: Krakowka, George.

Subjects: Animal diseases; Livestock; Microbiology; Molecular biology; Pathology; Virology

Keywords: Porcine circovirus type 2; PCV2; porcine torque teno virus; TTV; qPCR; electron microscopy

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22. Haynes, Rashade Ameir Hakim II. Studies of the early immunological and virological events following Human T Lymphotropic Virus Type 1 infection in the rabbit model.

Degree: PhD, Veterinary Biosciences, 2009, Ohio State University

► Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell… (more)

▼ Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is associated with a variety of lymphocyte-mediated disorders. Establishment of persistent HTLV-1 infection is determined by a balance between host immune responses and virus spread. Early HTLV-1 specific cell-mediated immune responses that allow establishment of the infection are difficult to study without effective animal models. In addition the route of virus exposure is believed to be a key determinant of virus-associated spread, antiviral immune responses, and ultimately disease outcomes. The lack of knowledge of early events of HTLV-1 spread in vivo hinders a more complete understanding of the immunopathogenesis of HTLV-1 infections. Data in this thesis provides a better understanding of the early immunological and virological events following blood borne exposure to HTLV-1. Our data has provided important knowledge of the immune response against HTLV-1 infection during early infection. Serologic and proviral copy number analysis indicated that rabbits with higher proviral loads had significant CTL activity against HTLV-1 SU as early as 2 weeks post infection, while both low and high proviral load groups had minimal Tax-specific CTL activity throughout the study. Herein, we then used an established animal model of HTLV-1 infection to study early spatial and temporal events of the viral infection. Our data indicates that intravenous infection with cell-associated HTLV-1 targets lymphocytes located both in primary lymphoid and gut-associated lymphoid compartments. A transient lymphocytosis correlated with peak virus expression parameters by two weeks post infection before returning to baseline levels. Lastly, we examined the effects of Cyclosporine A (CsA)-induced immune suppression during early infection of HTLV-1 in an established rabbit model. Our data indicated that CsA-treatment prior to HTLV-1 infection enhanced early viral expression compared to untreated HTLV-1-infected rabbits, but did not alter longer term viral expression parameters. However, CsA treatment 1 week post infection diminished HTLV-1 expression throughout the 10 week study course. Collectively, data presented in this thesis indicates that HTLV-1 infection following a blood borne exposure induces a viral-associated lymphocytosis which acts as a way for the virus to spread rapidly before the HTLV-1 specific cell-mediated immune responses are formed. Advisors/Committee Members: Lairmore, Michael.

Subjects: Immunology; Virology

Keywords: HTLV-1; immunology; retrovirology; animal models; immune suppression

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23. Hinchcliff, Kenneth William. Modification of the physiologic responses to sustained submaximal exertion in horses by phenylbutazone and furosemide.

Degree: PhD, Veterinary Biosciences, 1990, Ohio State University

Advisors/Committee Members: Muir, W.W.

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24. Hiraragi, Hajime. Study of lentiviral vector for in utero gene transfer and functional analysis of human T-lymphotropic virus type p13(II).

Degree: PhD, Veterinary Biosciences, 2005, Ohio State University

► The role of human immunodeficiency virus-1 (HIV-1) accessory proteins in vectors is… (more)

▼ The role of human immunodeficiency virus-1 (HIV-1) accessory proteins in vectors is poorly understood. The impact of HIV-1 accessory proteins was analyzed in a murine in utero gene transfer model. PCR analysis revealed comparable levels of gene transfer by vectors, prepared in the presence or absence of HIV-1 accessory proteins, in multiple organs. Analysis of bone marrow-derived hematopoietic progenitor cells revealed successful gene transfer in both vector groups. However, two months after gene transfer, the percentage of transduced hematopoietic progenitor colonies was significantly reduced in animals that received vectors without accessory proteins. These findings suggested that in utero gene transfer to multiple organs were independent of HIV-1 accessory proteins, but required them for sustained gene transfer to hematopoietic progenitor cells. Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia. HTLV-1 encodes accessory proteins, including an open reading frame II product p13II. p13II localizes to mitochondria and reduce cell growth in vitro and tumorgenicity in mice, but its function in lymphocytes remains undetermined. Herein, we analyzed functional properties of Jurkat T cells expressing p13II. Our data indicated that p13II expressing Jurkat T cells were sensitive to ceramide- and FasL-induced apoptosis. Furthermore, pre-incubation of the p13II expressing cell with a farnesyl transferase inhibitor, chemical inhibitor of Ras, markedly reduced FasL induced apoptosis, indicating participation of Ras pathway in survival of p13II-expressing lymphocytes. Our data is the first to demonstrate that p13II alters Ras-mediated apoptosis in T-lymphocytes.HTLV-1 p13II’s role in the virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13II in HTLV-1 infection, using a cell line that expresses an infectious molecular clone of HTLV-1 with a selective ablation of p13II expression (ACH.p13). Infection of rabbits with ACH.p13 failed to establish HTLV-1 infection, as measured by antibody responses, viral antigen production from peripheral blood mononuclear cells, and provirus by PCR assay. Our data support the tenet that HTLV-1 p13II is essential for the virus to establish infection in vivo and suggests an essential role in the pathogenesis of HTLV-1 associated disease. Advisors/Committee Members: Lairmore, Michael.

Subjects: Biology, Microbiology

Keywords: HTLV-1; p13II; human T-cell leukemia; VIRUS TYPE; cell leukemia; VIRUS; leukemia

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25. Huang, Haibin. Analysis of lipoproteins, outer membrane proteins, and genetic diversities of Ehrlichia and Anaplasma species.

Degree: PhD, Veterinary Biosciences, 2006, Ohio State University

► Ehrlichia chaffeensis, Anaplasma phagocytophilum and Anaplasma platys are obligatory intracellular Gram-negative bacteria… (more)

▼ Ehrlichia chaffeensis, Anaplasma phagocytophilum and Anaplasma platys are obligatory intracellular Gram-negative bacteria belong to the family Anaplasmataceae. E. chaffeensis had lost lipopolysaccharide and peptidoglycan which are pathogen-associated molecular patterns (PAMPs) but kept other PAMPs, such as lipoproteins. In my dissertation research, using the specific inhibitor in lipoprotein processing, I showed that lipoproteins were essential for ehrlichial survival in vitro. The majority of putative lipoproteins can be detected expressed in cell culture. An E. chaffeensis infected dog developed antibodies against selected three lipoproteins and a strong delayed type hypersensitivity (DTH) to more than half of the putative lipoproteins. The lipid moieties of lipoproteins are strong adjuvants for both humoral and cellular immunity. E. chaffeensis infected dogs developed strong cellular immunity to lipoproteins indicated by DTH. Selected lipoproteins with strong DTH reactions in E. chaffeensis infected dogs were used to immunize dogs in a DNA vaccine format. After immunization, vaccination group dogs developed humoral immunity and cellular immunity. Upon challenge with E. chaffeensis, immunized group dogs were transiently infected with E. chaffeensis at the same levels as the placebo immunized dogs. Thus, the lipoprotein DNA vaccine constructs was not protective against challenge. P44s are A. phagocytophilum major outer membrane proteins encoded by a multi-gene family. They are always expressed in various systems, although different species of P44s are expressed. P44s are rich in â-strand structure. They have heat modifiability, a common feature of porin and other outer membrane proteins. The outer membrane of A. phagocytophilum has porin activity determined by proteoliposome swelling assay. The purified P44 protein has pore-forming activity. Very little was known about prevalence of A. platys in endemic area, Venezuela and diversity around the world. By 16S rRNA PCR screening we found that around 16% of dogs samples tested was A. platys positive. By comparing 16S rRNA and groEL sequences of A. platys in Venezuela with other available sequences from all over the world, we conclude that divergence of A. platys around the world is quite small. Advisors/Committee Members: Rikihisa, Yasuko.

Subjects: Biology, Veterinary Science

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26. Huang, Yi-Wen. Chemeopreventive activity of (-)-gossypol in prostate cancer.

Degree: PhD, Veterinary Biosciences, 2006, Ohio State University

► Prostate cancer is the most commonly diagnosed cancer except skin cancer in… (more)

▼ Prostate cancer is the most commonly diagnosed cancer except skin cancer in the United Stares. Gossypol, a natural polyphenolic compound present in cottonseeds, possesses anti-proliferative and pro-apoptotic effects in various cancer cells. The (-)-gossypol enantiomer is a more potent inhibitor of cancer cell growth than (+)- or (±)-gossypol. In this dissertation, we hypothesize that (-)-gossypol serves as a chemopreventive agent through suppressing cell growth, inducing apoptosis and reducing metastasis in prostate cancer cells. Firstly, we demonstrated that (-)-gossypol reduced cell viability in human primary cultured prostate cancer epithelial cells, as well as human and rat prostate cancer cell lines. The growth inhibition caused by (-)-gossypol treatment was associate with down-regulation of cyclin-D1 and Rb, but with up-regulation of p21 and TGF-β1 expression at mRNA and/or protein levels. Secondly, prostate cancer cells treated with (-)-gossypol induced apoptosis measured by DNA fragmentation. (-)-Gossypol treatment inhibited the anti-apoptotic Bcl-2 and Bcl-xL protein expression, but increased the pro-apoptotic Bax, and Bak protein expression. Consequently, the activations of caspase-3, -8 and -9 were observed, and the further activations of apoptotic downstream proteins, such as PARP and DFF 40, were found. The apoptotic effect was abolished by caspase inhibitors indicated a caspase-dependent pathway of (-)-gossypol-induced cell death. Thirdly, (-)-gossypol-induced apoptosis was associated the Bcl-xL inhibition through transcription repression and was independent of the level of Bcl-xL protein in cancer cells. Our data showed that (-)-gossypol treatment elevated the p53 protein, which subsequently up-regulated the expression of its downstream proteins p21 and PUMA, and p53 played a role, at least in part, in (-)-gossypol-induced apoptosis. Finally, (-)-gossypol was able to reduce cell invasiveness in MAT-LyLu cells, and MLL cells, a subline of the MAT-LyLu cells from the metastasized lungs of MAT-LyLu-bearing Copenhagen rats. (-)-Gossypol treatment induced the dose-dependence of growth inhibition, colony formation and in vitro invasive activity in both MLL and MAT-LyLu cells. Our data suggest that (-)-gossypol might serve as a chemotherapeutic and chemopreventive agent for prostate cancer. To develop (-)-gossypol-enriched foods such as cottonseed oil or cottonseed meal flour might have chemopreventive effect for healthy human individuals as well as prostate cancer patients. Advisors/Committee Members: Lin, Young C.

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27. Ishihara, Akikazu. Gene and Cell-Based BMP-2 and -6 Gene Therapy For Equine Bone Regeneration.

Degree: PhD, Veterinary Biosciences, 2009, Ohio State University

► Fracture is still a life-threatening disorder in horses, but current treatment options… (more)

▼ Fracture is still a life-threatening disorder in horses, but current treatment options have not provided satisfying outcomes for complicated and catastrophic fractures in horses. Cell-mediated and direct gene therapies have provided numerous promising results in recent years and may become as practical alternative solutions for the potential treatments of equine bone repair and regeneration. We demonstrated that sufficient gene transfer could be achieved by using an adenoviral (Ad) vector in equine cells. High vector dosages could be used in equine cells because of relative resistance to cytotoxicity in these cells compared to a human cell line. We evaluated the healing of equine metatarsal osteotomies and ostectomies in response to delayed percutaneous injection of adenoviral bone morphogenetic protein-2 (Ad-BMP2), Ad-BMP6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic (qCT) assessment of bone formation indicated greater and earlier mineralized callus in the bone defects injected with Ad-BMP2 or Ad-BMP6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP2 than Ad-BMP6, and both Ad-BMP-2 and Ad-BMP6 treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. These results demonstrated a greater relative potency of Ad-BMP2 over Ad-BMP6 in accelerating osteotomy healing. We also evaluated the healing of equine metacarpal/metatarsal osteotomies in response to the percutaneous injection of autologous dermal fibroblasts (DFb) genetically engineered to secrete BMP2 or demonstrate green fluorescent protein (GFP) gene expression administered 14 days after surgery. Radiographic assessment of bone formation indicated greater and earlier healing of bone defects treated with DFb with BMP2 gene augumentation. The qCT and biomechanical testing revealed greater mineralized callus and torsional strength of DFb-BMP2 treated bone defects. On the histologic evaluation, the bone defects with DFb-BMP2 implantation had greater formation of mature cartilage and bone nodules within the osteotomy gap and greater mineralization activity on osteotomy edges. In addition, we compared the DFb-mediated and direct adenoviral vector delivery of BMP2 for relative efficacy in bone regeneration. Equine rib drill defects were treated by percutaneous injection of either DFb-BMP2 or Ad-BMP2 vector. At week 6, both of the DFb-BMP2- and Ad-BMP2-treated rib defects had greater bone filling volume and mineral density, with DFb-BMP2 inducing greater bone volume and maturity in cortical bone aspect of the defect than Ad-BMP2. The transplantation of DFb alone induced modest bone formation. Increased mineral density and bone turnover were evident in the cortical and cancellous bone directly adjacent to the healing drill defects treated with either DFb-BMP2 or Ad-BMP2. Additionally, we demonstrated the safety of BMP2 gene therapy for articular fracture, because the direct intra-articular administrations of Ad-BMP2 did not cause of mineralization or ossification of articular cartilage and synovium tissues. In concert, both cell-mediated and direct BMP2 gene therapy may be considered as a potential treatment for various types of fractures and bone defects. Advisors/Committee Members: Bertone, Alicia.

Subjects: Veterinary services

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28. Kaewsakhorn, Thattawan. Roles of calcitriol and its analog on canine transitional cell carcinoma in vitro and in vivo, and in normal canine prostate tissue explaints.

Degree: PhD, Veterinary Biosciences, 2007, Ohio State University

► Although increasing data indicates inhibitory roles of calcitriol on tumor growth in… (more)

▼ Although increasing data indicates inhibitory roles of calcitriol on tumor growth in humans, little is known about its effects on canine tumors. The objectives of this study are to investigate the effects of calcitriol and its analogs on canine transitional cell carcinoma and canine prostate tissue explants. First, we investigated effects of calcitriol, seocalcitol and medium-chain triglyceride (MCT) on a canine transitional cell carcinoma cell line (TCC). The effects of calcitriol and seocalcitol on cell growth, cell cycle, vitamin D receptor (VDR) and Bcl-2 expression were determined with/without MCT. Second, we established a canine TCC mouse-xenograft model and used this model to examine effects of calcitriol, seocalcitol, and piroxicam on tumor growth. Third, the effects of calcitriol and dihydrotestosterone (DHT) were evaluated on arginine esterase (AE) acitivity and VDR expression in normal canine prostate tissue explants. In summary, our results showed that the VDR is present in canine TCC tumor and in the canine prostate. Calcitriol and seocalcitol significantly inhibited cell growth and calcitriol caused cell cycle arrest. Bcl-2 expression was decreased in cells treated with these compounds, although no significant changes in VDR expression were observed. MCT enhanced the growth-inhibitory effects of both compounds. We developed and used a canine TCC-mouse xenograft model to evaluate and compare the inhibitory effects of calcitriol, seocalcitol and piroxicam. Results showed that only seocalcitol reduced tumor volume compared to controls. The inhibitory effect of seocalcitol on tumor growth was supported by data from a Ki-67 staining. Blood calcium was higher in both calcitriol-and seocalcitol-treated mice compared to controls. In summary, our findings suggest a potential use for calcitriol and seocalcitol for the treatment canine TCC. We demonstrated that DHT increased AE activity and VDR expression in canine prostate tissue explants; however, there was no increase in AE activity in calcitriol-treated explants and a decreased in VDR expression. These results indicated that canine prostate tissue explants are a valuable model for the study of prostate pathobiology and pharmaceutical interventions. They also provided a basis for further investigation of roles of calcitriol as a therapeutic/preventative agent in benign prostatic hyperplasia in both dogs and humans. Advisors/Committee Members: Inpanbutr, Nongnuch.

Keywords: calcitriol, seocalcitol, canine transitional cell carcinoma, canine prostate gland

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29. Kijtawornrat, Anusak. Potential mechanisms for drug-induced prolongation of QT interval and genesis of torsades de pointes evaluated in the failing rabbit heart.

Degree: PhD, Veterinary Biosciences, 2007, Ohio State University

► Torsades de pointes (TdP) is a polymorphic ventricular tachycardia characterized by a… (more)

▼ Torsades de pointes (TdP) is a polymorphic ventricular tachycardia characterized by a distinctive pattern of undulating QRS complexes that twist around the isoelectric line. TdP has been associated with QT interval prolongation of the electrocardiogram; therefore, the QT interval has come to be recognized as a surrogate marker for the risk of TdP. Currently preclinical in vitro and in vivo methods as well as biomarkers for proarrhythmias have been imperfect in predicting drug-induced TdP in humans. The goal of the present dissertation is to create rabbit with myocardial failing heart as an in vivo animal model to predict TdP in humans and to determine mechanism(s) underlying TdP in this model. Electrocardiograms were recorded from bipolar transthoracic leads in conscious healthy rabbits, and the algorithm for removing effect of heart rate on QT is QTc=QT/(RR)0.72. The rabbit with myocardial failure was created by coronary ligation and validated with drugs known to be torsadogenic or non-torsadogenic in humans. A greater percentage of rabbits with failing hearts developed TdP following intravenous infusion of known torsadogens than did normal rabbits exposed to the same drug protocol. None of the rabbits in either group developed TdP when exposed to known non-torsadogens. These results suggested that a rabbit with myocardial failure possesses specificity and sensitivity to assess drugs that tend to induce TdP when given to humans. The results from isolated myocytes of myocardial failure suggested that (1) the re-entrant circuits emanating from action potential durations of different duration, (2) triggered activity in the form of EADs, and (3) increased dispersion of repolarization are responsible for genesis of the TdP in the failing rabbit heart. The torsadogenic-modifying effects of verapamil, ryanodine, KB-R7943, W-7, KN-93, and H-8 on ventricular premature depolarizations (VPDs) and TdP were then evaluated in the conscious failing rabbit heart. Verapamil, ryanodine and H-8 significantly delayed onset of VPDs and suppressed the occurrence of TdP. These results demonstrated that calcium entry via ICaL channel, calcium overload by SR Ca2+ release, and blocking of PKA may play a major role in the appearance of TdP suggested by the preventive effect of verapamil, ryanodine and H-8. Advisors/Committee Members: Hamlin, Robert L.

Subjects: Biology, Veterinary Science

Keywords: Conscious, Myocardial failing heart, Prolongation, QT, QTc, Rabbit, Torsades de pointes

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30. Kim, Seung-jae. Studies of HTLV-1 p12(I) in calcineurin binding, calcium-mediated cell signalling and viral transmission.

Degree: PhD, Veterinary Biosciences, 2006, Ohio State University

► The HTLV-1 pX ORF I encoded protein p12I is required for productive… (more)

▼ The HTLV-1 pX ORF I encoded protein p12I is required for productive infection. p12I regulates calcium-mediated signaling and induces activation of NFAT and IL-2 production. We identified a calcineurin-binding PSLPI/LT motif in p12I. p12I bound calcineurin in both immunoprecipitation and calmodulin bead pull-down assays. In contrast, serial mutations of p12I lacking the PSLPI/LT motif or had alanine substitutions (p12IAxAxAA) exhibited abolished or decreased binding affinity. Furthermore, p12I competed with NFAT for calcineurin binding. p12IAxAxAA enhanced NFAT nuclear translocation compared to wild type p12I and increased NFAT activity two fold greater than wild type p12I. Thus the reduced binding of p12I to calcineurin allows enhanced nuclear translocation and transcription mediated by NFAT. We further tested the role of p12I on LFA-1-mediated T-cell adhesion. Abrogation of pX ORF I expression in HTLV-1 infected cells (ACH.p12I) resulted in reduction of LFA-1-mediated adhesion compared to wild-type ACH cells. Furthermore, expression of p12I in Jurkat T-cells using lentiviral vectors, enhanced LFA-1-mediated adhesion. Similar to the intracellular calcium mobilizer, thapsigargin, expression of p12I induced cell surface clustering of LFA-1 without changing integrin expression. Data indicated that p12I promotes cell-to-cell spread by inducing LFA-1 clustering. Lastly, we investigated the role of expression of pX ORF I in early HTLV-1 transmission. We compared the activation status between ACH cells and ACH.p12 cells, and tested role of pX ORF I in bystander activation of uninfected cells by performing proliferation and flow cytometric analysis. Abrogation of pX ORF I decreased CD69 expression in infected cells, but did not affect bystander activation. Furthermore, we performed real-time RT-PCR to detect pX ORF I and III/IV mRNA after coculture of both ACH and ACH.p12 cells with target cells. We compared viral infectivity of ACH to ACH.p12 cells and viral mRNA expression in newly infected target cells. Data indicated that expression of pX ORF I is required for efficient HTLV-1 transmission and identified differential expression of Tax/Rex and pX ORF I mRNA during early cell-to-cell transmission. In conclusion, p12I modulates calcium-mediated signaling, enhances HTLV-1 transmission through LFA-1 clustering and facilitates early establishment of infection by regulating viral gene expression, providing further evidence that p12I is critically required for early HTLV-1 infection. Advisors/Committee Members: Lairmore, Michael D.

Keywords: TLV-1,calcium-mediated signaling,p12I,calcineurin,LFA-1, early transmission

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