Browsing by Author "Prater, Mary R."

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    Birth Defect Amelioration and Placental Cytokine Expression in Mnu-Exposed Dams Treated With Ifn-Gamma
    Laudermilch, Chelsea Lee (Virginia Tech, 2007-01-16)
    Each year, 7.9 million babies are born with birth defects. Seventy percent of those could be prevented, ameliorated, or repaired; yet 3.2 million children still die by the age of three (March of Dimes Global Report 2006). We have found that non-specific maternal immune stimulation with the cytokine interferon-gamma (IFN-gamma) can successfully ameliorate some of these defects in the C57BL/6N mouse model. We have observed a reduction in the distal limb malformations syndactyly, polydactyly, and webbing by 47%, 100%, and 63% respectively when IFN-gamma is given 2 days prior to MNU administration. We have also observed that IFN-gamma works at the placental level to protect against MNU-induced damage. Trophoblast loss and associated cytokine alterations occur in gestation day (GD) 14 placenta following GD9 MNU exposure, showing that fetal-maternal communication can be hindered due to MNU. In the labyrinthine layer of the placenta, we observed multifocal fibrinous necrosis of endothelial cells due to MNU, however IFN-gamma almost completely protected the trophoblast and endothelial cells when given to the dam as an immune stimulant. To determine the genes participating in these processes, gene microarray studies were conducted. Hepatocyte growth factor (HGF), interleukin 1 beta (IL1Β), and insulin-like growth factor 2 (IGF2) were elucidated as genes that were significantly expressed in GD12 placenta. These genes are similar in that they are all connected to the Jak-Stat signaling pathway. These findings provide a possible mechanism for birth defect reduction by maternal immune stimulation with IFN-gamma in MNU-challenged mice.
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    Conjugated Linoleic Acid in the treatment of murine autoimmune glomerulonephritis
    Hammond, Sarah Elizabeth (Virginia Tech, 2015-10-15)
    Conjugated linoleic acid (CLA) has been shown to reduce inflammation via Peroxisome Proliferator-Activated Receptor (PPAR)-γ in inflammatory disorders such as Crohn's Disease and Inflammatory Bowel Disease. We sought to determine whether CLA isomers would reduce inflammation via PPAR-γ in cultured mesangial cells, and in murine models of anti-glomerular basement membrane (anti-GBM) glomerulonephritis and Systemic Lupus Erythematosus (SLE). SV40-transformed mouse mesangial cells (MES13) were cultured with pure CLA isomers (c9,t11 or t10,c12-CLA or a 50:50 mixture prior to immune stimulation with lipopolysaccharide and interferon-γ. Next, cultured mesangial cells were transfected with small interfering RNA (siRNA) targeting PPAR-γ and treated with CLA isomers prior to immune stimulation. ELISA, qPCR, Western blot, and Griess reaction were performed to measure cytokine production, mRNA expression, induced nitric oxide synthase (iNOS) and nitrite production, respectively. Next, myeloid-specific (LysM creR2+) PPAR-γ knockout mice were treated with CLA prior to the induction of anti-GBM glomerulonephritis and evaluated for disease. Finally, NZM2410/J mice (a natural model of SLE) were treated with c9,t11-CLA and evaluated for disease progression. Treatment with CLA reduced IL-6 production in cultured mesangial cells, but not in siRNA-treated mesangial cells, supporting a PPAR-γ-mediated mechanism. CLA treatment increased both Transforming Growth Factor (TGF-β) and Interleukin-1 Receptor Antagonist (IL-1RA) mRNA expression independent of PPAR--γ. While CLA treatment reduced nitrite production and iNOS production to some degree, this was an inconsistent finding. Conversely, in the induced anti-GBM mouse model, CLA treatment increased mesangial cell IL-6 mRNA expression, reduced TGF-β expression, and had no effect on IL-1RA. Moreover, NZM2410/J mice that were fed a c9,t11-CLA-supplemented diet had reduced survival times, increased renal inflammation and increased serum IgG2a relative to controls. Taken together, these studies indicate that the in vitro MES13 cell line does not translate to the in vivo mouse model of anti-GBM induced glomerulonephritis. Furthermore, while CLA may have beneficial effects in other mouse models, it worsens disease in NZM2410/J mice. Findings from these models should be interpreted with caution.
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    Current Thoughts on Maternal Nutrition and Fetal Programming of the Metabolic Syndrome
    Brenseke, Bonnie; Prater, Mary R.; Bahamonde, Javiera; Gutierrez, J. Claudio (Hindawi, 2013-02-14)
    Chronic diseases such as type 2 diabetes and cardiovascular disease are the leading cause of death and disability worldwide. Although the metabolic syndrome has been defined in various ways, the ultimate importance of recognizing this combination of disorders is that it helps identify individuals at high risk for both type 2 diabetes and cardiovascular disease. Evidence from observational and experimental studies links adverse exposures in early life, particularly relating to nutrition, to chronic disease susceptibility in adulthood. Such studies provide the foundation and framework for the relatively new field of developmental origins of health and disease (DOHaD). Although great strides have been made in identifying the putative concepts and mechanisms relating specific exposures in early life to the risk of developing chronic diseases in adulthood, a complete picture remains obscure. To date, the main focus of the field has been on perinatal undernutrition and specific nutrient deficiencies; however, the current global health crisis of overweight and obesity demands that perinatal overnutrition and specific nutrient excesses be examined. This paper assembles current thoughts on the concepts and mechanisms behind the DOHaD as they relate to maternal nutrition, and highlights specific contributions made by macro- and micronutrients.
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    Development and testing of recombinant B. abortus RB51 vaccine strains carrying M. tuberculosis protective antigens
    Al Qublan, Hamzeh (Virginia Tech, 2015-06-23)
    Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most prevalent infectious diseases inflicting humankind. The World Health Organization estimates that one third of the world's population, approximately 2.2 billion people, is infected with TB with a mortality of 1.7 million people annually. Currently, the WHO estimates that each year more than 9 million people develop TB. Bacille Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only licensed TB vaccine in the world. Clinical studies have shown childhood vaccination with BCG to be protective against disseminating and meningeal forms of TB. However, the efficacy of BCG against pulmonary TB in adults has been variable and inconsistent (0-80%). The objective of this study is to develop and test the efficacy of the B. abortus vaccine strain RB51 as a platform for expression of M. tuberculosis antigens (Ag85B, ESAT6 and Rv2660c) and induction of a protective immune response against M. tuberculosis and B. abortus challenge in mice. Here we report the construction of two recombinant strains of B. abortus vaccine strain RB51 capable of expressing mycobacterial antigens Ag85B, ESAT6 and Rv2660c. Our studies show that expression of mycobacterial antigens in strain RB51 lead to induction of antigen-specific immune responses characterized by secretion of IgG2a antibodies as well as of IFN- and TNF-α. Mice immunized with a combination of two strains of RB51 in equal numbers, one carrying Rv2660c-ESAT6 and another carrying Ag85B, led to a 0.90 log reduction in CFU burden with significance nearly reaching borderline (p = 0.052). However, when mice were primed with the same strains of RB51 and boosted with proteins Ag85B and ESAT6, a significant level of protection (1 log reduction) compared to the PBS vaccinated group was achieved. The protection levels conferred by this vaccination strategy was similar to that conferred by BCG vaccine. In conclusion, we have shown that recombinant RB51 strains expressing mycobacterial protective antigens result in stimulation of antigen specific immune response without altering the vaccine efficacy in protecting against the more virulent strain of B. abortus 2308. These recombinant vaccines could potentially be used to protect against M. tuberculosis infection.
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    Developmental Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Induced and Exacerbated Autoimmunity in Adulthood
    Mustafa, Amjad Issa (Virginia Tech, 2008-12-18)
    Developmental 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure can permanently alter immune system ontogeny, resulting in the dysregulation of a number of vital immune pathways. We hypothesized that developmental exposure to TCDD may also impair the establishment of self-tolerance, resulting in an increased risk of autoimmunity. For example, we observed that a single prenatal TCDD exposure given to non-autoimmune-prone high affinity aryl hydrocarbon receptor (AhR) C57BL/6 mice resulted in an immune complex-mediated autoimmune disease during the adult stage. Further using a similar TCDD exposure protocol, autoimmune-prone low affinity AhR SNF1 mice exhibited acceleration and exacerbation of lupus-like nephritis in adulthood. Examination of these mice showed that perinatal TCDD exposure adversely affected both primary immune organs of the adaptive immune system. In the thymic compartment, prenatal TCDD affected thymocyte cellularity, differentiation and maturation as well as central tolerance as indicated by high levels of autoreactive Vβ TCR T cells in the periphery. Prenatal TCDD also altered bone marrow B lymphopoiesis and B cell maturation and differentiation in the spleen. Functionally, these B cell changes resulted in high serum autoantibodies titers to dsDNA, ssDNA and cardiolipin suggesting a loss in central B cell tolerance. The functional assessment of T cells, via cytokine production showed that prenatal TCDD mice altered Th1/Th2 levels. As a result, significant changes were detected in the kidney characterized by increased immune complex deposition in the glomeruli, lymphocytic infiltration and general pathologic changes. This would suggest that multiple immune pathways are affected by prenatal TCDD and work either independently or synergistically to display immune-mediated disease during aging. Importantly, this study has also shown that the sex of an individual appears to influence both the type of immune pathways affected by TCDD as well as the progression and severity of the autoimmunity. In summary, these studies clearly demonstrate that postnatal immune system impairment due to prenatal TCDD exposure is not limited to immunosuppression but also can include inappropriate immune activation manifested as a hypersensitivity that can lead to the onset of autoimmune disease.
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    Effects of Quaternary Ammonium Disinfectants on Mouse Reproductive Function
    Melin, Vanessa Estella (Virginia Tech, 2015-07-25)
    Quaternary ammonium compounds (QACs) are antimicrobial disinfectants commonly used in commercial and household settings. While these compounds have been used for decades, reproductive toxicity has not been thoroughly evaluated. Extensive use of QACs results in ubiquitous human exposure to potentially toxic compounds. Reproductive toxicity of two common QACs, alkyl dimethyl benzyl ammonium chloride (ADBAC) and didecyl dimethyl ammonium chloride (DDAC), was investigated to determine gender-specific toxicity with an emphasis on male reproductive function. Breeding pairs of mice exposed for six months to ADBAC+DDAC exhibited decreases in fertility and fecundity, with fewer pregnancies and decreased numbers of pups over a six month period. Females proceeded through significantly fewer estrus cycles, and both ovulation and implantation rates were reduced. Males exhibited declines in both sperm concentration and motility. Male reproductive toxicity was further assessed in a series of in-vitro and in-vivo experiments. ADBAC+DDAC were cytotoxic to testicular Sertoli cells in culture at concentrations greater than or equal to 0.0005%. Changes in blood-testis-barrier integrity (BTB) were observed at 0.01% ADBAC+DDAC using a two-compartment culture system that measures transepithelial electrical resistance (TER). Sertoli cell cytotoxicity correlated with decreased TER at ADBAC+DDAC concentrations above 0.001%. In-vitro fertilization capacity of epididymal sperm was reduced in males given a 10-day rest period following ADBAC+DDAC exposure. Multigenerational changes in sperm parameters and in mRNA expression of enzymes involved with epigenetic modifications were evaluated across three generations. Sperm concentration and motility were reduced in F0 males exposed directly to ADBAC+DDAC. In F1 males, sperm concentration was increased and motility decreased, while there was no change in the F2 progeny. Genes involved in epigenetic modifications were altered in the exposed F0, with upregulation of two histone acetyltransferases (Hat1 and Kat2b) and downregulation of one lysine-specific demethylase (Kdm6b). F1 and F2 generations were not different from controls except for downregulation of the methyltransferase Dnmt1 in F1 progeny. The reproductive toxicity of ADBAC+DDAC identified in these studies, particularly to the male, compels further investigation into the potential effects that these compounds may have on human reproduction.
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    Evaluation of a Paratuberculosis Quantitative Polymerase Chain Reaction Assay with Microscopic Correlation
    Tyler, Ronald Dale Jr. (Virginia Tech, 2012-05-01)
    Paratuberculosis is an intestinal condition in ruminants infected with Mycobacterium avium subspecies paratuberculosis (MAP) and precedes Johne's disease, a chronic enteric disorder in ruminants caused by MAP infection. Necropsy with histopathology provides definitive diagnosis of Johne's disease and positive culture of MAP from tissues provides definitive diagnosis of paratuberculosis. To determine assay sensitivity, 85 formalin-fixed paraffin-embedded (FFPE) tissues from ruminants diagnosed with Johne's disease were tested with a commercial paratuberculosis quantitative polymerase chain reaction (qPCR) assay and had a sensitivity of 92%. To determine assay specificity, 21 FFPE tissues from animals without gastrointestinal disease combined with 13 FFPE tissues from non-ruminant animals (frog, dove, turtle, dog, and 2 cats) with non-paratuberculosis mycobacterial diseases were tested with the commercial qPCR assay and had a specificity of 100%. Slides prepared from the FFPE tissue blocks were stained with hematoxylin & eosin (H&E) and Ziehl-Neelsen's (acid fast stain), then examined for granulomatous inflammation and scored on a scale from 0-4 based on the quantity of acid fast bacteria (AFB). Digital microscopy and morphometric software were used to compute an acid fast bacteria area index (AFBAI) to evaluate a more precise correlation with the qPCR results. The quantity of AFB in tissue slides showed medium to strong correlation with the appropriate qPCR results. The results indicate that the commercial qPCR assay can be used on FFPE tissues with good results and the qPCR results have medium-strong correlation with quantitative acid fast histopathology.
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    High Saturated Fat Diet Induces Gestational Diabetes, Perinatal Skeletal Malformation and Adult-Onset Chronic Diseases
    Liang, Chengya (Virginia Tech, 2009-04-03)
    Adult exposure to high fat diet (HFD) has been linked to increased risk of musculoskeletal, cardiovascular, and metabolic diseases; however, the contribution of gestational HFD to elevated oxidative stress (OS), perinatal cardiovascular, skeletal, and metabolic dysfunction as well as long-term effects on adult offspring are incompletely understood. Pathophysiologic mechanisms linking gestational HFD, OS, and insulin resistance to perinatal development and adult-onset chronic diseases are explored in the present study, and maternal antioxidant (quercetin) is offered as a potential preventive dietary supplement to reduce fetal and maternal sequelae of HFD. Female C57BL/6 mice were fed "cafeteria-style" HFD (including 32.1% saturated fat to mimic a typical fast food menu) with or without quercetin for one month prior to conception, and throughout gestation. HFD dams developed gestational diabetes with significantly increased placental OS and vasculopathy. Neonates were smaller at birth than age-matched controls, and surviving offspring developed type 2 diabetes, hypertension and osteoporosis during adulthood, despite having been fed healthy diet throughout their postnatal life. Additional measures of bone using three-dimensionally reconstructed computed tomographic image analysis (microCT) revealed microarchitectural changes of bone at birth, and at 6 and 12 months postnatally. Fetuses from HFD dams displayed diminished bone mineral density (BMD) and disrupted endochondral and intramembranous ossification with significantly shortened distal limb lengths, as compared to offspring of standard rodent chow dams. Skeletal malformation persisted into adulthood despite the fact that both control and HFD offspring were fed conventional rodent chow throughout postnatal life. The offspring gestationally exposed to HFD showed significant decreased femoral BMD at 6 months of age and dysregulation of distal femoral trabecular architecture at 12 months of age, indicating development of osteoporosis. We were able to reduce incidence of placental vasculopathy, fetal maldevelopment and adult-onset type 2 diabetes, hypertension and osteoporosis with concurrent maternal quercetin supplementation during pregnancy. Collectively, these data suggested that maternal HFD increases placental OS and vascular damage during pregnancy, which are associated with fetal malformation and elevated adult-onset multisystemic chronic diseases. Maternal quercetin supplementation must be further explored as a potential dietary intervention for improved placental integrity, fetal development and lifelong health.
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    Identification of a protein kinase substrate in Sulfolobus solfataricus P2
    Redbird, Ruth Ann (Virginia Tech, 2010-04-01)
    Living organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes. A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies.
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    Immunotoxicity of Dermal Permethrin and Cis-Urocanic Acid: Effects of Chemical Mixtures in Environmental Health
    Prater, Mary R. (Virginia Tech, 2002-03-08)
    The present study examined adverse effects of sunlight exposure (mimicked by intradermal cis-urocanic acid, cUCA) on local and systemic immune responses, with or without co-exposure to the immunotoxic insecticide permethrin. A single exposure to cUCA caused diminished splenic macrophage phagocytosis that was persistent up to 30 days post-exposure. Five-day exposure to cUCA subtly increased splenocyte proliferation in response to the T cell mitogen Concanavalin A. Four-week exposure to cUCA caused increased splenic lymphocyte cellularity, thymic hypocellularity, and enhanced hydrogen peroxide production by splenic leukocytes. Single exposure to topical permethrin resulted in decreased thymic and splenic weight and cellularity, and inhibited antibody production by splenic B cells. cUCA worsened the negative effect of permethrin on both thymic weight and cellularity, and depressed splenocyte blastogenesis, hydrogen peroxide production, and antibody production. Five-day exposure to either cUCA or permethrin also caused persistent decreased contact hypersensitivity responses, an effect that became more than additive when the chemicals were administered concurrently. Defects in antigen processing and presentation by cutaneous Langerhans cells were evaluated as possible contributing mechanisms to the cutaneous immunosuppression, using mice with deleted genes. Vehicle-exposed IFNg knockout mice displayed approximately a 22.1% depression in the ear swelling response as compared to control C57BL/6N mice, suggesting that this cytokine may be required for mounting a control-level hypersensitivity response. Ear swelling in cUCA-exposed IFNg knockout mice displayed a 21.4% depressed response as compared to cUCA-exposed wild-type C57BL/6N mice, again suggesting that IFNg is an important cytokine in the contact hypersensitivity (CH) response. TNFaR knockout mice exposed to cUCA displayed 33.9% greater ear swelling than cUCA-exposed wild-type C57BL/6N mice, suggesting that increased TNFa may be involved in inhibited CH by cUCA. TNFaR knockout mice exposed to permethrin displayed 33.9% greater ear swelling than permethrin-exposed C57BL/6N mice, suggesting that increased TNFa may also be involved in inhibited CH by permethrin. C57BL/6N mice exposed to cUCA + permethrin displayed severe reduction of the CH response to 8.7% of the control level. IFNg knockout mice exposed to permethrin + cUCA showed essentially identical depression of the CH response as IFNg knockout mice exposed to either permethrin or cUCA alone. These results suggest that IFNg is required for the greater than additive immunotoxic effect that occurred when these two agents were co-administered. TNFaR knockout mice exposed to cUCA + permethrin displayed 8.7 fold greater ear swelling than similarly exposed C57BL/6N mice, again suggesting that increased TNFa is involved in inhibited CH by both cUCA and permethrin.
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    Improved Late-gestation Cardiac Morphology in Fetuses of Diabetic Mothers After Maternal Immune Stimulation: Potential Role of Dysregulated Apoptosis
    Gutierrez, Juan Claudio (Virginia Tech, 2009-01-22)
    The incidence of malformed newborns is higher in human pregnancies complicated by diabetes mellitus, as compared to non-diabetic pregnancies. Neural tube and cardiac defects predominate among the fetal malformations induced by hyperglycemia. Non-specific maternal immune stimulation is protective in mice against birth malformations caused by chemical or physical teratogens, or by maternal diabetes mellitus. Insulin dependent diabetes was induced in ICR females to study the late gestation fetal heart by morphometric analysis. Diabetic females treated with Freund's compete adjuvant (FCA) or interferon-gamma (IFNγ) were also generated to elucidate potential positive effects of maternal immune stimulation during the diabetic pregnancy by morphometric analysis and pathologic scoring. Insulin-dependent CD1 females were generated to analyze late gestation fetal myocardial apoptosis by flow cytometric analysis and by real time-polymerase chain reaction (RT-PCR) analysis of a panel of 5 genes involved in apoptosis/proliferation (Bcl-2, P53, Caspase3, Caspase9 and PkC-e). The morphometric analysis of fetal hearts revealed visibly obvious dilation of ventricular chambers and outflow channel of the left ventricle, and reduction of total myocardial ventricular area in late gestation fetuses, as predominant changes seen in the offspring of diabetic dams. Pathologic scoring revealed that maternal immune stimulation, particularly with FCA, in part alleviated fetal heart changes of cavitary dilation and myocardial reduction. Increased rate of apoptosis/necrosis in the fetal myocardium in late gestation during the diabetic pregnancy was evidenced by flow cytometric analysis. Particularly there was a significant increase in percentage of early apoptotic cells in the fetal myocardium detected by cell markers annexin V and propidium iodide. There was also a significant increase in percentage of late apoptotic/necrotic fetal myocardial cells in the diabetic group compared to the control group. These results suggest that maternal treatment with FCA may in part protect the heart from high hyperglycemia by reducing the number of myocardial cells undergoing apoptosis and necrosis. The RT-PCR analysis revealed subtle changes in gene expression for all the genes except Bcl-2. A paradoxical and dramatic up-regulation of this anti-apoptotic gene was observed in late gestation fetal myocardium from the insulin-dependent hyperglycemic groups. Possibly, this could be a mechanism to protect the fetal myocardial cell from the chronic exposure to a severe hyperglycemic insult and consequent apoptosis. In conclusion, maternal insulin-dependent diabetes caused morphological changes in the late gestation fetal heart. Such changes were in part related to dysregulation of myocardial apoptosis. Maternal immune stimulation with FCA improved fetal heart morphology, by a mechanism that may in part relate to normalizing fetal myocardial apoptosis.
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    Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates from healthy university student athletes
    Champion, Anna E.; Goodwin, Thomas A.; Brolinson, P. Gunnar; Werre, Stephen R.; Prater, Mary R.; Inzana, Thomas J. (2014-08-02)
    Background The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has been increasing in the general population, and there is concern that close or physical contact, such as in professional and collegiate sports, may increase spread of MRSA. We sought to determine the prevalence of MRSA colonization of male and female athletes from 9 different sports at a major, Division I University during a 12-week period, and determine the USA and SCCmec type from select isolates. Methods Swabs for culture of MRSA were obtained from nasal, axillary, and inguinal sites from healthy, asymptomatic student athletes and support staff each week for 12 weeks. Select MRSA isolates were typed by pulsed field gel electrophoresis (PFGE), and the genes encoding for MecA, cassette chromosome recombinase (Ccr), and several toxins were determined by multiplex polymerase chain reaction (PCR). Discrepant results were clarified by multi-locus sequence typing (MLST) and spa typing. Results Thirty-five percent (78/223) of test subjects were positive for MRSA during the study period, resulting in isolation of 139 MRSA isolates. However, 47% (37/78) of MRSA-positive participants carried MRSA in axillary or inguinal sites, but not in the anterior nares. There was significant correlation between MRSA carriage and participation in wrestling (76%, 19/25; adjusted odds ratio 29.7, 95% CI 5.8-151.5) and baseball (44%, 17/39; adjusted odds ratio 4.4, 95% CI 1.1- 17.4), compared with a staff prevalence of 18.1% (4/22), but other factors were not examined. Multiplex PCR analysis indicated that of the 32 isolates examined 26 could be typed, and all of these carried the SCCmec type IV cassette. PFGE typing identified USA types 300, 400, 500, 700, and 800. However, one isolate was not a known USA type, but was identified as a novel ST951 by MLST, and as spa type t216. Of the strains typed from the same individual, there was consistency, but also variation and alternation of the SCCmec and spa types isolated from individual subjects. Various staphylococcal toxin genes were identified in 31 of the 32 isolates analyzed. Conclusions Colonization by MRSA was greater in some student athletes than the average carriage rate for the general population, and only 53% of MRSA carriers were identified by nasal cultures. Carriage of MRSA clones on the same individual and transmission to contacts could vary over time, indicating colonization can be a dynamic process that may be difficult to control.
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    Response of Peripheral Blood Lymphocytes from RAO-affected Horses to b2-Agonist Stimulation
    Werner Becker, Marianne Patricia (Virginia Tech, 2011-05-03)
    Recurrent airway obstruction (RAO) affects middle-age horses, inducing bronchoconstriction and airway inflammation. β2-agonists like salbutamol are used as treatment, promoting airway smooth muscle (ASM) relaxation and bronchodilation. In addition to ASM, inflammatory cells express the β2-adrenoreceptors (β2-AR). In other species, β2-agonists promote peripheral blood lymphocyte (PBL) cytokine expression towards a pro-inflammatory phenotype. RAO horses are a good model for evaluating chronic changes in human asthma. However, little is known about the effect of β2-agonist stimulation on equine PBL inflammatory response. The aims of this study were to develop an indirect method to evaluate the response of equine PBLs to β2-agonist stimulation, and to compare it between cells from RAO and non-affected horses. Isolated PBLs were activated with ConA and stimulated with salbutamol. Response to agonist binding was indirectly determined using flow cytometric methodology and verified by Western blot. Activated PBLs from RAO horses demonstrated a significant response to β2-agonist binding whereas cells from non-affected horses did not. Response of PBLs from RAO horses was attenuated when pre-treated with a β2-antagonist but unaffected following pre-treatment with a β1-antagonist, indicating that the response of PBLs from these horses to salbutamol binding was mainly through the β2-AR. Preliminary investigation of bronchoalveolar lavage (BAL) lymphocytes from RAO horses demonstrated that they also responded to β2-agonist binding, while cells from non-affected horses did not. These findings represent a novel tool for further investigation of the role of β2-agonist binding in diseases like asthma and RAO, and support the use of this model for future studies.
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    The Role of Maternal High Fat Diet in the Pathogenesis of Metabolic and Bone Disease in the Adult Offspring
    Brenseke, Bonnie Margaret (Virginia Tech, 2013-01-11)
    Chronic diseases such as osteoporosis, type 2 diabetes, and cardiovascular disease are diseases of long duration, slow progression, and are by far the leading cause of death worldwide. A growing body of evidence links adverse exposures in early development with an increased risk of chronic diseases in adult life. The studies presented in this dissertation sought to exploit this phenomenon to determine the extent to which gestational and lactational exposure to a high fat diet predisposes the offspring to certain diseases in later life and if the eating habits of adult offspring would be able to mitigate or exacerbate these conditions.  In the study presented in Chapter III, dams fed an atherogenic high fat diet prior to conception and throughout gestation and lactation experienced excess hepatic lipid accumulation and poor birth outcome as characterized by smaller litter sizes and higher post-delivery mortality. In the offspring, gestational and lactational exposure to such a diet resulted in growth restriction and skeletal aberrations indicative of osteoporosis, despite being fed a standard rodent diet post-weaning. We propose that dietary-induced hyperlipidemia, along with pregnancy-associated factors, resulted in fatty liver and subsequently reduced litter sizes and increased early mortality, and that the skeletal aberrations seen in the mature offspring represent dietary-induced inhibition of osteogenesis in favor of adipogenesis. In the study presented in Chapter IV, early exposure to a high fat diet resulted in central obesity, elevated lipid levels, hyperglycemia, and additional markers used in the diagnosis of the metabolic syndrome. Altering the diets of the mature offspring demonstrated that the eating habits of adulthood have the potential to mitigate or exacerbate certain metabolic parameters established earlier in life. Mechanisms contributing to the observed metabolic aberrations could include developmental plasticity and mismatch, catch-up growth, and altered programming of the appetite regulatory network. Collectively, this research suggests that early exposure to a fat-rich diet can lead to metabolic and skeletal aberrations in the adult offspring and adds support to the developmental origins of health and disease hypothesis by finding that adverse nutritional exposures in early life can play a role in the chronic diseases of adulthood.
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    SBES Advanced Multi-scale CT Facility at Virginia Tech - From Multi-scale to Multi-energy and Multi-Parameter Imaging Capabilities
    Wang, Ge; Wyatt, Christopher Lee; Yu, Hengyong; Sharma, Kriti S.; Prater, Mary R.; Xiao, Shuhai; Markert, Chad; Saul, Justin; Fox, Edward A.; Lee, Seung W.; Feser, Michael; Lau, S. H.; Yun, Wenbing; Wang, Steve (2010-04-05)
    While clinical CT scanners are available at our medical school, for preclinical imaging we have a Scanco micro-CT scanner, an Xradia micro-CT scanner and an Xradia nano-CT scanner. With all these scanners, we can cover image resolution and sample size over six orders of magnitude. The Scanco scanner has resolution 16 µm and FOV 20-38 mm. The Xradia micro-CT scanner, purchased using an NIH SIG grant in 2008, is the highest resolution micro-CT system on the market. It produces 0.5 µm resolution and handle samples of up to 100 mm diameter. The Xradia nano-CT scanner, purchased using an NSF-MRI grant in 2009, has 50 nm resolution and represents the state-of-the-art. It allows tomographic imaging in either the attenuation or Zernike phase contrast mode. For the high-resolution performance of the micro-/nano-CT systems, special housing is vital to ensuring technical development and biomedical applications. We have a dedicated space for these systems in the Institute for Critical Technologies and Applied Sciences (ICTAS; http://www.ictas.vt.edu) Building A, adjacent to the Nanoscale Characterization and Fabrication Lab (NCFL; http://www.ictas.vt.edu/NCFL) at Virginia Tech, which hosts most other cutting-edge imaging systems under one roof.
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    Short- and Long-Term Effects of Commercially Available Gold Nanoparticles in Rodents
    Bahamonde Azcuy, Javiera del Pilar (Virginia Tech, 2014-01-24)
    Gold nanoparticles (GNPs) are currently being intensely investigated for their potential use in biomedical applications. Nanotoxicity studies are urgently needed to validate their safety in clinical practice. The objective of this research was to assess the acute, subacute, and chronic effects of a single intravenous exposure to commercially available GNPs in two in vivo models, mice and rats. Gold nanoparticles were purchased and independently characterized. Animals were exposed to either 1000 mg GNPs/kg body weight (GNP group) or an equivalent volume of phosphate buffered saline (PBS group) intravenously via the tail vein. Subsets of animals were euthanized 1, 7, 14, 21, 28 days (female BALB/c mice and female F344 rats) or 20 weeks (female and male C57BL/6 mice) post-exposure and samples were collected for biochemistry, histopathology, electron microscopy, and atomic absorption spectrometry analysis. Independent characterization demonstrated that the physicochemical properties of the purchased GNPs were in good agreement with the information provided by the supplier. Important differences in GNP-induced immune responses were identified when comparing mice and rats 1 to 28 days post-exposure. Gold nanoparticles stimulated the formation of liver microgranulomas in mice, along with transiently increased serum levels of the proinflammatory cytokine interleukin-18. No such alterations were found in rats. Species differences in GNP biodistribution and excretion were also detected, with higher relative accumulation of GNPs in spleen and longer fecal excretion in rats. In the long-term (20 weeks after dosing), exposure to GNPs incited chronic inflammation in mice, characterized by the persistence of microgranulomas in liver, spleen, and lymph nodes, as well as further increased serum levels of interleukin-18. Impairment of body weight gain was also observed in the GNP-exposed group. No sex differences were detected. In conclusion, GNPs are not innocuous and have the ability to incite a robust macrophage response in mice. However, considering the mildness of the toxic effects identified despite the high dose selected for the study, GNPs continue to have great potential for biomedical uses. Further studies are needed in order to determine specific mechanisms of toxicity and the role of chronic inflammation in the development of adverse effects after co- or post-exposures.