Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Although nano-sized metal colloids are used in industrial and medicinal applications, little is known about the potential liver toxicity of these materials after occupational or intentional exposures. To begin to resolve some outstanding hepatotoxicity concerns, the inflammatory response of hepatocytes after exposure to metal colloids was assessed. Four ~30-nm-sized metal colloids, including silver (nano-Ag), copper (nano-Cu) and nickel (nano-Ni) were examined in an effort to understand the induced cytokine expression in a murine liver cell line (AML12). Here we also utilized another system, co-cultures of hepatocytes, Kupffer’s cells, and lymphocytes isolated from C57BL6 mice. Cells were exposed to the materials over dose-response (0.1mg/L to 1000mg/L) and time-dependent (4 h, 48 h, and 1-week) studies. Cytotoxicity was measured via metabolism of resazurin and validated via MTT assay and cell counts. Inflammatory response was determined by cytokine profiles (TNF-a and IL-6), as well as by mRNA and protein expression of heat shock protein (Hsp70). Results from cells exposed to nano-Ag to doses of up to 100mg/L exhibited no significant changes in cytotoxicity, IL-6, or TNF-a production, or Hsp70 expression. Both nano-Cu and nano-Ni exposed cells exhibited decreased metabolism, increased Hsp70 induction, and increased inflammatory responses (IL-6 and TNF-a). Dynamic light scattering and electron microscopy were used to characterize particle size and surface charge. All three metal colloidal systems demonstrated different particle size distributions, agglomerated sizes, and surface zeta potentials. Furthermore, each metal colloid system elicited different inflammatory biomarker responses and stress protein expression.

Thesis (M.S.)--University of Wyoming, 2008. / Title from PDF title page (viewed on Dec. 4, 2009). Includes bibliographical references (p. 75-88).

Reproduction de : Thèse doctorat : Médecine. Immunologie et biologie parasitaire : Reims : 2005. / Titre provenant de l'écran-titre. Bibliogr. p.121-141.

Thèse de doctorat : Chimie organique. Pharmacochimie : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 307-324.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 111-122).

published_or_final_version / Pathology / Master / Master of Medical Sciences

 Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts. Despite intensive chemotherapy and allogeneic bone marrow transplantation, the treatment outcome of AML remains unsatisfactory, with a cure rate of only about 30%. Therefore, novel therapeutic strategies targeting the pathogenetic pathways of leukemia initiation and progression are needed. Using intracellular phospho-flow analysis with normal bone marrow as reference, we detected an increase in phosphorylated-STAT5 (pSTAT5) in three leukemic cell lines (K562, KG-1 and ML-2) and 15 primary AML samples. Treatment with specific JAK2 inhibitor TG101209 and JAK2/3 inhibitor AG490 significantly reduced pSTAT5 level and leukemia cell growth associated with an increase in apoptosis and decrease in cellular proliferation. The clonogenic activities of these leukemia cell lines were also significantly reduced. Furthermore, treatment with these inhibitors in K562 and KG-1 also significantly reduced the WNT signaling activity, as enumerated by the TOP/FLASH luciferase assay. In addition, genes associated with oncogenic potential and anti-apoptosis were significantly reduced, consistent with the pathogenetic role of JAK-STAT pathway. In summary, the present study highlighted the importance of the JAK2-STAT5 signaling pathway in sustaining AML. The results may open up a new avenue whereby new therapeutic strategies targeting AML can be designed. / published_or_final_version / Medicine / Master / Master of Philosophy

Background and objectives: A lot of studies proved that adipocyte fatty acid binding protein (A-FABP), an adipokine mainly expressed in adipocytes and macrophages, is the key link between obesity and inflammation which is suggested to be a therapeutic target for obesity-related diseases. Loss-of-function study was employed by using A-FABP knockout (KO) mice generated by our group to investigate role of A-FABP in high fat high cholesterol (HFHC) diet-induced obesity. Key findings: 1. Our study confirmed that HFHC diet-induced A-FABP KO mice have a significantly increased body weight when compared to the wild-type (WT) control mice. 2. Higher adiposity was the major reason for the A-FABP KO mice to be heavier than the WT controls under HFHC diet induction. 3. The marked increase of the weight of subcutaneous fat and peri-renal fat contributed to the higher adiposity of the HFHC-diet induced A-FABP KO mice when compared to the WT controls. 4. The HFHC-diet induced A-FABP KO mice significantly consumed less oxygen and produced less carbon dioxide suggesting the reduced energy expenditure but had higher weekly energy intake when compared with the WT controls, leading to higher adiposity. 5. The A-FABP KO mice were protected against HFHC diet induced glucose intolerance, insulin resistance, hyperglycemia and hyperinsulinemia when compared with the WT controls. There was also a better insulin secretion in response to glucose stimulation in A-FABP KO mice under prolonged HFHC diet induction when compared with the WT controls. 6. The A-FABP KO mice were protected against the development of hypercholesterolemia and hypertriglycemia when compared the WT controls under HFHC diet induction. However, there was no significant difference in the fasting serum free fatty acids (FFA) level among A-FABP WT and KO mice fed with standard chow (STC) or HFHC diet. 7. A-FABP KO mice were protected against isolated systolic hypertension (ISH) under HFHC diet induction. 8. The A-FABP KO mice were protected against HFHC diet-induced liver injury as indicated by a lower serum ALT level suggesting a better liver function when compared with the WT controls. 9. Under HFHC diet induction, M1 macrophage polarization was dominant in fat tissues of A-FABP WT mice but M2 macrophage polarization was dominant in fat tissues of A-FABP KO mice, suggesting an improved inflammatory status in the adipose tissue of the A-FABP KO mice when compared with the WT controls. This may also be the reason for why HFHC diet-induced A-FABP KO mice have an increased body weight but are metabolically healthier compared to their WT controls. Conclusions: A-FABP KO mice had a significant higher body weight and higher adiposity due to the reduced energy expenditure and increased weekly food intake as indicated in the metabolic cage study and the reason for metabolic healthier is due to the alleviated HFHC diet induced M1 macrophage polarization in various adipose tissues suggesting an improved inflammatory status in A-FABP KO mice comparing to the WT controls. / published_or_final_version / Medicine / Master / Master of Philosophy

Although tremendous efforts have been made to search for other novel growth factors in promoting marrow recovery after irradiation or chemotherapy, there have not been any efficient and safe agents discovered so far. Danggui Buxue Tang (當歸補血湯) as a traditional Chinese herbal decoction, is commonly used for replenishing blood loss in menstruating women, or enhancing erythropoiesis and immune responses in various settings. Our previous study confirmed that Danggui Buxue Tang promotes haematopoiesis and thrombopoiesis both in vitro & in vivo. Recent studies also showed that parenteral Astragalus regulates haematopoiesis in myelosuppressed mice and has protection effect on UV irradiated human dermal fibroblasts. However, astragaloside IV, as the major component of Astragalus, the "Monarch" (君葯) in Danggui Buxue Tang, the bioactivity and its possible mechanism on haematopoiesis remains unclear. My studies showed that astragaloside IV had promoting effect on different lineages of haematopoietic CFUs forming including erythrocytes, granulocytes, monocytes and megakaryocytes both in normal and irradiated mice. In the K562 and CHRF apoptotic model, astragaloside IV exerted proliferation effect and induced K562 into megakaryocytic differentiation. Astragaloside IV up-regulated phosphorylation of ERK and it was abolished by PD98059. Meanwhile, astragaloside IV increased phosphorylated ERK migration into nuclei which enhanced cell survival and differentiation. EGFR inhibitor also attenuated the enhancing effect of astragaloside IV on ERK phosphorylation. It suggested that astragaloside IV is likely to function through EGFR with subsequent activation of ERK1/2 pathway. Furthermore, astragaloside IV also increased Bcl-2/Bax ratio by up-regulating Bcl-2 alone. Bone marrow derived mesenchymal stromal cells are the major supporting cells involved in the haematopoietic microenvironment. My studies demonstrated that astragaloside IV also indirectly enhanced haematopoiesis by stimulating cytokine release from MSCs, especially IL-6, IL-8, MCP-1 and GRO1. I also found that matured and activated population of neutrophils was increased after cultured with mesenchymal stromal cells conditional medium stimulated by astragaloside IV. This finding further supported why there was a significant increment of CFU-GM in vitro culture with murine bone marrow collected from mouse model after astragaloside IV treatment, where MSCs serve as the feeder layer in such system in mice. In conclusion, my studies explored the directly and indirectly dynamic and multiple targeted function of astragaloside IV on haematopoiesis. In addition to activating haematopoietic cells, astragaloside IV also stimulated mesenchymal stromal cells to secret cytokines that could modulate haematopoiesis and up-regulated neutrophil production and maturation. It provided a holistic view on how astragaloside IV induced synergistic effect on haematopoietic cells and mesenchymal stromal cells in the marrow microenvironment. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy