Browsing by Author "Gogal, Robert M."

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    An Assessment of the Relationship among Oxidative Stress, Adaptive Immunity and Genetic Variations in the Chicken, Gallus gallus
    Deng, Hui (Virginia Tech, 2010-09-01)
    Oxidative stress (OS) has been associated with aging and age-related diseases in humans, as well as with the decline in economic trait performance in poultry and other domesticated animals. However, the potential effects of OS on the poultry immune system are not well understood. In addition, the impact of bird genetic variation on redox balance remains to be elucidated. Thus, the central hypothesis of this dissertation is: The bird's adaptive immunocompetence is impacted by their OS level, which is not only influenced by environmental factors, but also related to genetic phenotype of either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). In the first phase of this study, White Leghorn chickens were provided ethanol at different concentrations in drinking water to induce OS. Biomarkers including malondialdehyde (MDA), glutathione (GSH), and plasma uric acid (PUA) were measured to assess OS before and after ethanol treatment. The adaptive immune response during an OS event was measured by plasma IgG and IgM levels, major lymphoid organ weights, CD4+/CD8+ cell ratio, and histopathological analysis of the immune organs. Results showed that when OS was induced by 10% ethanol, chicken adaptive immune responses decreased; however, when birds were exposed to 2% ethanol, there was an enhancement in antioxidant defense and immune response; These results would suggest a negative correlation between OS level and chicken adaptive immune response. In the second phase of the study, subsets of chickens were selected based on their high (H)- or low (L)-OS to assess for variations in their genetic phenotypes. Using MDA levels, 36 chickens were chosen to scan a 2734-bp region of mtDNA, but no definitive SNP was detected. In another experiment, 40 chickens were conversely selected according to three biomarkers for OS. Although no variation was found at eight SNP loci tested across the mitochondrial genome, mtDNA damage measured by 8-hydroxy-2′-deoxy-guanosine was shown to increase with time, and at higher levels in the high OS birds (p < 0.05). Thses results suggest that long-term high OS levels in chickens may increase the somatic mutation of their mtDNA. In the final phase of this dissertation, the effect of nDNA on OS, measured via a genome-wide association study was performed with 18 H and 18 L chickens using the latest chicken 60k SNP microarray for genotyping. Among 56,483 SNPs successfully genotyped, 13 SNPs across five independent loci were associated with OS at significance level of p ≤ 0.001, and another 144 SNPs were also associated with OS (p ≤ 0.01). These results indicate new loci and related genes for their genetic influence upon redox balance. In general, experiments carried out on White Leghorn chickens here have shown that adaptive immune response is tightly related to changes of OS. Further, genetic variance in nDNA is associated with the risk of high OS or the ability to better resist it.
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    Effects of Experimental Sarcocystis neurona-Induced Infection on Immunity in an Equine Model
    Lewis, S. Rochelle; Ellison, Siobhan; Dascanio, John J.; Lindsay, David S.; Gogal, Robert M.; Werre, Stephen R.; Surendran, Naveen; Breen, Meghan E.; Heid, Bettina; Andrews, Frank M.; Buechner-Maxwell, Virginia A.; Witonsky, Sharon G. (2014)
    Sarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM), affecting 0.5-1% horses in the United States during their lifetimes. The objective of this study was to evaluate the equine immune responses in an experimentally induced Sarcocystis neurona infection model. Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators. Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression. All experimentally infected horses displayed neurologic signs after infection. Neutrophils, monocytes, and lymphocytes from infected horses displayed significantly delayed apoptosis at some time points. Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls. Collectively, our results suggest that horses experimentally infected with S. neurona manifest impaired antigen specific response to S. neurona, which could be a function of altered antigen presentation, lack of antigen recognition, or both.
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    Experimental infection with Sarcocystis neurona alters the immune response: the effect on CD4+, CD8+, B-cell, monocyte and granulocyte populations in horses
    Lewis, Stephanie Rochelle (Virginia Tech, 2009-06-11)
    Previous studies have demonstrated differences in CD4+, CD8+ and B-cell populations between EPM affected and normal horses. The overall goal of our project was to further define the immune deficiencies associated with S. neurona infection. We hypothesized that PMA/I stimulated suppression in EPM horses is due to decreased proliferation of monocytes, CD4+ and CD8+ cells. Our objectives were 1) to determine whether S. neurona infection causes an increase in apoptosis of a particular immune subset, and 2) to determine whether S. neurona causes a decrease in the number of cellular divisions (proliferation) of a particular immune cell subset. For this study, nine S. neurona antibody negative, immunocompetent horses were obtained. Baseline neurologic examinations, SnSAG1 (S. neurona Surface Antigen 1) ELISAs on cerebrospinal fluid (CSF) and serum, and baseline immune function assays were performed. Horses were randomly divided into groups. Five horses were challenged for ten days via intravenous injection of autologous lymphocytes infected with S. neurona. Neurologic parameters of all horses were assessed for 70 days following infection. Immune function was based on proliferation responses to mitogens, as assessed through thymidine incorporation. Enumeration of cellular subsets, degree of apoptosis and number of cellular divisions were assessed through flow cytometry. SnSAG1 ELISA of serum and CSF samples performed post-infection confirmed infection and disease. All infected horses displayed moderate neurologic signs on clinical examination. Some significant differences in cellular activities were noted. Additionally, this is the first time the method using S. neurona infected lymphocytes has been reproduced successfully by different investigators.
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    Exposure to formaldehyde at therapeutic levels decreases peripheral blood lymphocytes and hematopoietic progenitors in the pronephros of tilapia Oreochromis niloticus
    Holladay, Steven D.; Smith, Bonnie J.; Gogal, Robert M. (Inter-Research, 2010)
    Formaldehyde (HCHO) was recently detected at concentrations above the cancer benchmark in 90% of 60 000 surveyed United States census tracks. Formaldehyde leaches into and mixes with water extremely well, exposing aquatic life. Further, formaldehyde is used therapeutically in aquaculture to remove external protozoa and other parasites from fish. The present study was undertaken to determine if sub-acute HCHO causes immunologic changes in tilapia Oreochromis niloticus. Fish were exposed to 0, 50, or 150 ppm of HCHO. Immune parameters examined included blood hematology, spleen/body weight ratio, spleen and pronephros total cellularity, leukocyte blastogenesis, and natural killer cell cytotoxicity. Organ/body weight ratios and total cellularity were not different from controls. Similarly, mitogen response and natural killer cell function were unchanged. Peripheral blood lymphocytes decreased as HCHO exposure increased. Formaldehyde exposure also decreased the number of progenitor cells in the fish pronephros. These observations suggest possible immunosuppressive effects of HCHO in fish.
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    Horses experimentally infected with sarcocystis neurona develop altered immune responses in vitro
    Witonsky, Sharon G.; Ellison, Siobhan; Yang, Jibing; Gogal, Robert M.; Lawler, Heather; Suzuki, Yasuhiro; Sriranganathan, Nammalwar; Andrews, Frank M.; Ward, Daniel; Lindsay, David S. (American Society of Parasitology, 2008-10)
    Equine protozoal myeloencephalitis (EPM) due to Sarcocystis neurona infection is I of the most common neurologic diseases in horses in the United States. The mechanisms by which most horses resist disease, as well as the possible mechanisms by which the immune system may be suppressed in horses that develop EPM, are not known. Therefore, the objectives of this study were to determine whether horses experimentally infected with S. neurona developed suppressed immune responses. Thirteen horses that were negative for S. neurona antibodies in serum and cerebrospinal fluid (CSF) were randomly assigned to control (n = 5) or infected (n = 8) treatment groups. Neurologic exams and cerebrospinal fluid analyses were performed prior to, and following, S. neurona infection. Prior to, and at multiple time points following infection, immune parameters were determined. All 8 S. neurona-infected horses developed clinical signs consistent with EPM, and had S. neurona antibodies in the serum and CSF Both infected and control horses had increased percentages (P < 0.05) of B cells at 28 clays postinfection. Infected horses had significantly decreased (P < 0.05) proliferation responses as measured by thymidine incorporation to nonspecific mitogens phorbol myristate acetate (PMA) and ionomycin (I) as soon as 2 days postinfection.
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    Immunopathologic effects associated with Sarcocystis neurona-infected interferon-gamma knockout mice
    Witonsky, Sharon G.; Gogal, Robert M.; Duncan, R. B.; Lindsay, David S. (American Society of Parasitology, 2003-10)
    Interferon-gamma knockout (IFN-gamma KO) mice were infected with Sarcocystis neurona merozoites to characterize the immunopathology associated with infection. By day 14 postinfection (PI), mice developed splenomegaly and lymphadenopathy, characterized by marked lymphoid hyperplasia with increased numbers of germinal centers. Additional histopathologic changes included increased extramedullary hematopoiesis, multifocal mixed inflammatory infiltrates in the liver, perivascular infiltrate of the liver and lung, and interstitial pneumonia. The total number of B-cell splenocytes (P < 0.05) and the percentage of B-cells increased on day 14 PI in the spleen and on day 28 PI in the lymph nodes (P < 0.05). By day 28 PI, the number of B-cell splenocytes decreased significantly. A non-subset-specific decrease in percentages of CD4 lymphocytes throughout all lymphoid organs was observed on day 14 PI. However, total CD4 and CD44/CD4 splenocytes increased significantly by day 28 PI. Early-activation CD8 lymphocytes were reduced in the blood and spleen, whereas memory CD8 lymphocyte percentages and total numbers were significantly increased. On the basis of the results, we propose that S. neurona-infected IFN-gamma KO mice are immunocompromised and unable to clear the infection. Thus, they develop B-cell exhaustion and a delayed, but sustained. increased number of memory CD4 and CD8 lymphocytes due to chronic antigen stimulation.
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    Protective immune response to experimental infection with Sarcocystis neurona in C57BL/6 mice
    Witonsky, Sharon G.; Gogal, Robert M.; Duncan, Robert B.; Lindsay, David S. (American Society of Parasitology, 2003-10)
    Immunocompetent C57BL/6 mice were infected with Sarcocystis neurona merozoites to assess the protective immune response to active infection. Using a direct agglutination test, all infected mice seroconverted to S. neurona merozoite antigens by day 14 postinfection (PI). Further, mice developed splenomegaly and bilateral symmetrical lymphadenopathy by day 14 PI, which appeared to be resolving by day 28 PI. Histologic analysis revealed a marked increase in germinal center formation in the spleen and lymph nodes by day 14 PI. Corresponding to gross and histopathological changes, the percentage of B-cells decreased significantly by day 14 PI but then increased significantly and persisted at day 28 PI in the blood, spleen, and multiple lymph nodes. There was a sharp nonspecific significant decrease in CD4 percentages by day 14 PI in the blood, spleen, and lymph nodes. Early-activation CD8 lymphocytes (CD62/CD8) were significantly down-regulated coinciding with a significant compensatory up-regulation of memory (CD44/CD8) lymphocytes in multiple organs. We propose that the protective cell-mediated immune response to S. neurona involves both CD4 and CD8 cells, with CD8 lymphocytes appearing to play a more critical role.
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    Signal Transducer and Activation of Transcription (STAT) 4 beta, a Shorter Isoform of Interleukin-12-Induced STAT4, Is Preferentially Activated by Estrogen
    Karpuzoglu, Ebru; Phillips, Rebecca A.; Dai, Rujuan; Graniello, Carmine; Gogal, Robert M.; Ahmed, Sattar Ansar (Endocrine Society, 2009-03)
    Estrogen, a natural immunomodulatory compound, has been shown to promote the induction of a prototype T helper 1 cytokine, interferon (IFN)-gamma, as well as to up-regulate IFN gamma-mediated pro-inflammatory molecules (nitric oxide, cyclooxygenase 2, monocyte chemoattractant protein 1). Because IL-12 is a major IFN gamma-inducing cytokine, in this study we investigated whether estrogen treatment of wild-type C57BL/6 mice alters IL-12-mediated signaling pathways. A recent study has shown that IL-12 activates two isoforms of signal transducer and activation of transcription (STAT) 4, a normal-sized (full-length STAT4 alpha) and a truncated form (STAT4 beta). Interestingly, we found that estrogen treatment preferentially up-regulates the phosphorylation of STAT4 beta in splenic lymphoid cells. Time kinetic data showed the differential activation of STAT4 beta in splenic lymphoid cells from estrogen-treated mice, but not in cells from placebo controls. The activation of STAT4 beta was mediated by IL-12 and not IFN gamma because deliberate addition or neutralization of IL-12, but not IFN gamma, affected the activation of STAT4 beta. In contrast to IL-12-induced activation of STAT4 beta in cells from estrogen-treated mice, STAT4 beta was not increased, rather it tended to be decreased. In this context, STAT4 alpha-induced p27(kip1) protein was decreased in concanavalin A + IL-12-activated lymphocytes from estrogen-treated mice only. By using the in vitro DNA binding assay, we confirmed the ability of pSTAT4 beta to bind to the IFN gamma-activated sites (IFN gamma activation sequences)/STAT4-binding sites in estrogen-treated mice. Our data are the first to show that estrogen apparently has selective effects on IL-12-mediated signaling by preferentially activating STAT4 beta. These novel findings are likely to provide new knowledge with regard to estrogen regulation of inflammation. (Endocrinology 150: 1310-1320, 2009)