Thesis (Ph. D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Mark T. Quinn. Includes bibliographical references (leaves 147-162).

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.S. )--University of Tennessee Health Science Center, 2009. / Title from title page screen (viewed on September 19, 2009). Research advisor: Jegdish Babu Ph.D. Document formatted into pages (x,36 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 32-36).

Magister Scientiae (Biodiversity and Conservation Biology) / Homopus areolatus is an endemic terrestrial tortoise that resides in a Mediterranean type of climate, which is characterised by winter rainfall and mild winter temperatures. Within ectotherms, such as H. areolatus, physiological changes are elicited by changes in the ambient temperature. These physiological changes are evident in the blood profile of reptiles. I described the morphology of immature and mature erythrocytes, leukocytes as well as thrombocytes of H. areolatus. Additionally, I evaluated erythrocytes, leukocytes and thrombocytes to assess the effects of season and cohort on these cells. Blood samples were collected in 2000 and 2001 at Elandsberg Nature Reserve in the Western Cape from H. areolatus cohorts (female, male, juvenile) in all seasons (spring, summer, autumn, winter). Blood smears were made and stained with modified Giemsa stain. SigmaStat was used for all statistical analysis. Immature erythrocyte types within H. areolatus included basophilic rubricytes, polychromatophilic rubricytes and polychromatophilic erythrocytes. Upon my evaluation, I encountered evidence to suggest that small and large immature erythrocytes possibly developed from two distinctive lineages. Further research is required to discern which lineage gave rise to which immature erythrocyte type. Cohort had no effect upon immature erythrocytes. Erythropoiesis was most prevalent during winter and spring within H. areolatus. Aberrant features of erythrocytes appeared to be more prevalent during autumn, which signified the driest season with limited food and water. Mature erythrocytes play a huge role in oxygen transport and metabolism in individuals. Factors such as size and shape are relevant since small, mature, ellipsoidal erythrocytes transport oxygen more efficiently than large, spherical erythrocytes. In H. areolatus small, mature, ellipsoidal erythrocytes appeared to be most prevalent during spring and summer. During winter however, large, spherical erythrocytes appeared to be most prevalent. Thrombocytes and seven types of leukocytes were observed within H. areolatus, namely heterophils, lymphocytes, eosinophils, basophils, monocytes, plasma cells and azurophils. Among cohort and season heterophils were most prevalent overall, followed by lymphocytes and eosinophils respectively. Basophils, monocytes, plasma cells and azurophils were present but overall, were relatively few. H. areolatus appeared to be healthy, and leukocyte counts as well as its dimensions appeared to be in accordance with other reptilian studies. This study serves as the first baseline haematological reference forH. areolatus. The study forms the second of its kind on South African tortoises, only one other haematological study has been done namely, P. geometricus which is a sympatric species to H. areolatus.

Obesity and insulin resistance are characterized by elevated pro-inflammatory proteins in the blood and immune cell accumulation in the visceral adipose tissue. Resident leukocytes release tumor necrosis factor α (TNFα) and other inflammatory cytokines which stimulate adipocyte lipolysis, recruit leukocytes to adipose tissue, promote pro-inflammatory immune cell polarization, facilitate oxidative stress, and activate intracellular kinases which dull insulin signaling cascades in metabolic tissues. Immune cell mediated dysregulation of stromal and parenchymal cells has raised suspicion that insulin resistance is an immune disorder initiated by activated white blood cells with over-nutrition. Efforts to improve pathological metabolism by reducing inflammation have yielded mixed results in humans and animal models. The role of inflammation and immune cell accumulation in the visceral fat (VF) in the progression of insulin resistance remains presently debated. There is, however, a consensus that identifying the triggers for obesity and impaired insulin signaling is of the utmost importance. The goal of this report is to identify dietary fat absorption as a key initiator of inflammatory action and insulin desensitization which may be dampened by reducing immune cell accumulation in adipose tissue. To explore how lean, healthy organisms become obese and insulin resistant, we examined the inflammatory consequences of isocaloric but variable macronutrient loads in the VF of lean mice. Mice were administered single liquid meals composed of low-fat (10% fat) or high-fat (60% fat) diet and observed by intravital microscopy to quantify leukocyte-endothelium interactions in mesenteric postcapillary venules (MPCV) 1, 2, 3, and 4 hours after oral gavage. Leukocyte rolling and leukocyte adhesion were transiently elevated within 1 hour after feeding and returned to baseline levels 4 hours later. Endothelial cell surface expression of P-selectin (Psel), a rapidly activated cell adhesion molecule (CAM), confirmed that high-fat feeding induced Psel dependent leukocyte rolling through the VF microcirculation. Furthermore, leukocyte accumulation in the VF was modestly increased by a single high-fat meal (HFM). Repetitive high-fat diet (HFD) consumption for 24 hours prolonged elevated leukocyte-endothelium interactions and promoted neutrophil accumulation in the VF. The neutrophilic enzyme myeloperoxidase (MPO), a producer of the chlorinating agent hypochlorous acid, increased in abundance and activity in the VF of HFM fed mice. Elevated leukocyte-endothelium interactions, leukocyte infiltration, and MPO activity in VF were not observed in Psel deficient (Psel-/-) mice following lipid overload. To ascertain if MPO is required for sustained endothelial activation, leukocyte-endothelium interactions and leukocyte infiltration were monitored in high-fat fed MPO deficient (MPO-/-) mice. Similar to the Psel-/- mice, MPO-/- mice were protected from the inflammatory effects of high-fat feeding. Our data supports postprandial hyperlipemia as an inducer of transient and Psel dependent inflammatory reactions that are sustained by prolonged HFD consumption. To study whether early phase inflammatory interventions granted late phase metabolic improvements, wild-type (WT), Psel deficient (Psel-/-), and MPO deficient (MPO-/-) C57BL/6 mice were given ad libitum access to LFD (10% fat) or HFD (60% fat) for 12-16 weeks. All mouse groups given HFD became obese. Prolonged HFD consumption sustained elevated leukocyte-endothelium interactions in MPCVs and was accompanied by increased local and systemic TNFα in WT mice. High-fat fed WT mice were hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant compared to LFD fed controls. Psel-/- mice were protected from leukocyte-endothelium interactions as well as local and systemic TNFα accumulation despite extended HFD consumption. Surprisingly, high-fat fed Psel-/- mice were equally hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant as the inflamed, high-fat fed WT mice. MPO-/- mice were also protected from elevated systemic TNFα and gained slightly less weight than the other high-fat fed groups. While MPO-/- mice were hyperglycemic and glucose intolerant, they did have improved insulin stimulated glucose clearance. The data presented in this report demonstrates the pro-inflammatory nature of postprandial hyperlipemia and the insulin desensitizing nature of prolonged HFD consumption. Ablation of VF immune cell accumulation by Psel deletion is not sufficient for improving insulin signaling or glycemic control, which is consistent with prior reports. Deletion of MPO, however, did result in slightly less obesity and marginally improved insulin signaling. We conclude that while immune cell accumulation in the VF contributes to the progression of insulin resistance, it is not a prerequisite for metabolic pathology development. / Physiology

Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis. / Ph. D.

Equine neutrophils (PMN) play a critical role in inflammatory processes in horses. The objective of this study was to characterize equine PMN function ex vivo following stimulation with calcium ionophore A23187 (A23187) and phorbol myristate acetate (PMA). These stimulants trigger different branches of the PMN activation process that occurs in vivo. Equine PMN were isolated from the whole blood of six clinically normal geldings using a one-step discontinuous Percoll gradient technique. Neutrophil aggregation, degranulation, and superoxide anion production were evaluated in assay systems which had previously been established to guantitate PMN function. Dose-response curves for A23187 and PMA were derived for the three functions. Results indicate that equine PMN aggregation and superoxide anion production are more responsive to activation by PMA as the maximum change in percent transmittance and maximum nanomoles of superoxide anion produced following PMA stimulation (60.82% and 10.4nmols/10⁶cells, respectively) were greater than those values stimulated by A23187 (41.5% and 5.2nmols/10⁶cells, respectively). However, degranulation was found to be more responsive to A23187 stimulation (maximum percent degranulation= 56.1%) than to PMA stimulation (maximum percent degranulation= 30.7%). Dose-response curves following A23187 and PMA stimulation revealed that superoxide anion production had the lowest threshold concentration among the three functions. Degranulation had the highest threshold concentration among the three functions for both stimulants. Results indicate that equine PMN functions differ in their dependence on second messengers in the activation pathway. These functions also occur in a dose-dependent manner and differ in the threshold concentrations required for their stimulation. / Master of Science

Mortality and decreased weight gain resulting from infection and disease in dairy calves is a problem within the dairy industry. Colostrum is the sole source of maternal immunity for the calf, having a substantial impact on health and survival. To date, colostrum quality is determined by concentration of antibodies. Colostrum also contains proteins and cells, which may enhance immune development in the neonate. Our goals were to determine the impact of colostral immune cells on (1) immune status during the first month of life and (2) immune development over time. To determine the impact of adoptively transferred colostral immune cells, calves were fed either whole colostrum (WC) or cell-free colostrum (CFC) at birth. During the first month of life, calves fed CFC had decreased numbers of CD4+ T cells when compared to WC-fed calves. However, CFC-fed calves had a greater percentage of monocytes during the first month of life. To determine the influence of colostral immune cells on immune development, cellular blood parameters were measured in response to two series of vaccinations (A and B). After vaccination series A, CFC-fed calves had decreased numbers of B cells when compared to WC-fed calves. After vaccination series B, CFC-fed calves had decreased levels of interleukin-2 gene expression and numbers of CD4+ and gamma delta T cells when compared to WC-fed calves. This study demonstrates that colostral immune cells impact immune status and development in dairy calves. / Master of Science

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.