No description available.

Allergic disease encompasses multiple complex syndromes including hayfever, food allergies, eczema and asthma. Atopy is the genetic predisposition towards an IgE-driven immune response in reaction to environmental stimuli, and often serves as a predictor for the development of allergies in the future. While disease etiology is not yet fully understood, many factors including genetics and the environment play a role in the development of allergic disease. Reliable methods for predicting atopic disease development are crucial in emerging therapeutic approaches, which aim to decrease allergic disease severity and clinical progression through early detection and preventative measures. While DCs are emerging as key players in the development of allergic disease, they are challenging to study in vivo due to their low numbers, and ex vivo methods remain relatively unstudied. In this project, receptor expression profiles of atopic-at-risk infants compared to not-atrisk infants were examined in DCs found in cord-blood at birth and CD34+-derived DCs cultured ex vivo. Atopic-at-risks exhibited a higher percentage of ex vivo pDCs expressing TSLPR when compared to not-at-risks. Additionally, an increase of FcεRI expression in atopic-at-risks was found approaching significance in in vivo mDCs. Furthermore, DC differentiation in culture from hematopoietic progenitors and the differences between in vivo and ex vivo DCs were studied. Results indicated a consistent 10-fold increase in the DC population after a 12-day culture compared to cord blood DC numbers. Additionally, a distinct DC population emerged as early as Day 3 with a substantial increase in the percentage of mDCs relative to pDCs. A trend of increased TSLP, CD80, CD86 receptor expression and decreased TLR-5, ST2, FcεRI receptor expression after culture in both mDCs and pDCs was also noted. / Thesis / Master of Science (MSc) / Allergic disease development typically begins in infancy, progressing classically in a series of stages from early life through adulthood. Currently, there is a lack of reliable predictive tests for the development of atopic sensitization and disease. This has slowed efforts to intercept and prevent allergy development at its earliest stages. Dendritic cells (DCs) link innate and adaptive immunity and are thought to be key players in the development of allergic disease. However, the low numbers of DCs in blood make them challenging to study. Methods such as inducing the differentiation of DCs from progenitors are often utilized to obtain a sufficient number of cells. This project investigates whether receptor expression of cord blood-derived DCs grown ex vivo are comparable to the profiles of in vivo DCs at birth. Furthermore, the expression of key receptors on DCs grown in vivo/ex vivo are compared in atopic at-risk, not-at-risk infants.

Diethylstilbestrol (DES) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been identified as immunotoxicants causing thymic atrophy, thymocyte hypocellularity, phenotypic changes detected by CD4 and CD8 surface antigens, and progenitor T-cell targeting in the fetal mouse. We hypothesized that gestational exposure to these two compounds may lead to comparable histologic and gene expression alterations in the fetal mouse thymus and liver. Treatment of pregnant C57Bl/6 mice with doses of 5 or 10 ug/kg TCDD or 48 ug/kg DES by oral gavage on gestation days (gd) 14 and 16 severely depressed day 18 thymic cellularity. Histologic evaluation of day 18 fetal thymuses showed disruption of normal cortico-medullary architecture after TCDD or DES. Decreased thymocyte density was noted primarily in cortical zones where pyknotic cells were increased by either TCDD or DES treatment. Using day 18 thymocyte suspensions and flow cytometry, 7-AAD showed decreases in viable thymocytes from TCDD- or DES-treated fetal mice, and concomitant increases in thymocytes in early apoptosis. When thymocytes were co-identified with CD4 and CD8 cell surface antigen expression, enhanced apoptosis occurred in CD4+CD8+ phenotype after TCDD treatment. After DES exposure, increased apoptosis occurred in CD4-CD8- and CD4-CD8+thymocytes. Both TCDD and DES increased liver to body weight ratios and decreased ratios of hematopoietic to hepatic cells present. Cytomegaly was seen in hepatocytes of TCDD and DES treated animals, and these cells had more variable features, such as increased cytoplasmic basophilia and more prominent nucleoli. Real time quantitative PCR demonstrated that DES decreased c-jun, Bcl-2, and PKCalpha mRNA expression. These results suggest a shift away from proliferative activity and may reflect alterations noted predominantly in the hematopoietic population. TCDD increased c-jun mRNA expression with modest decreases in PKCalpha, and marked decreases in p53 also noted. Decreases in p53 suggest a pro-proliferative status of hepatic cells, while decreases in PKCalpha may indicate decreases in phosphorylation of substrates required for normal cell cycle progression. The increased c-jun suggests that this gene may play a role in the hepatocyte hyperplasia, as well as the diminution of hematopoiesis. / Ph. D.

Background: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1(-/-)mice and Pim1(-/-)2(-/-)3(-/-) triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 (-/-) and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit S-35 methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Lau, Wai Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 141-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

O transplante de células-tronco hematopoéticas (CTHs) é o tipo mais bem-sucedido de terapia celular realizado até os dias atuais. No entanto, apesar do sucesso e da relevância clínica das CTHs isoladas a partir de fontes adultas, o uso destas células tem algumas limitações em relação à sua disponibilidade, compatibilidade imunológica e risco de contaminação. Desse modo, busca-se o desenvolvimento de soluções para as dificuldades apontadas para suprir a demanda de transplantes. Uma abordagem emergente para superar este problema é baseada na cultura e diferenciação de células-tronco embrionárias humanas (CTEhs). Estas são célulastronco pluripotentes e indiferenciadas com elevada capacidade de auto-renovação e diferenciação em todas as células derivadas dos três folhetos germinativos. No entanto, os métodos de diferenciação utilizados para a produção de CTHs a partir de células pluripotentes ainda não são eficientes. Os protocolos descritos até o momento têm gerado números variados e populações de células heterogêneas, e produz apenas CTHs muito primitivas e imaturas com baixa capacidade funcional in vivo. Parte desta dificuldade pode decorrer da ineficiência do microambiente de cultura para a diferenciação. Neste trabalho, nós demonstramos um eficiente protocolo de diferenciação hematopoética baseado em cocultivo de CTEhs com fibroblastos embrionários murinos com alto rendimento na geração de célulastronco/progenitoras hematopoéticas (CTPHs) que expressam os antígenos CD45, CD43, CD31 e CD34, e apresentam potencial clonogênico in vitro equivalente ao de células mononucleares isoladas de sangue de cordão umbilical. Nós fomos capazes de produzir todas as células das linhagens eritróide e mielóide em diferentes estágios de maturação, como também células positivas para marcadores linfóides. Demonstramos ainda que as células hematopoéticas surgem no sistema de cultura a partir de um endotélio-hemogênico constituído por células CD34+CD31+. No entanto, apesar das características maduras das CTPHs obtidas por tal método, os ensaios de reconstituição hematopoiética mostraram que estas células ainda possuem limitada capacidade funcional de enxertamento em camundongos imunocomprometidos quando transplantadas por via retro-orbital. / Hematopoietic stem cells (HSC) transplant is the most successful type of cell therapy carried out to date. However, despite the success and the clinical relevance of HSC isolated from adult sources, these cells have some limitations regarding its availability, immunological compatibility and risk of contamination. Thus, we seek to develop solutions to overcome these difficulties to supply the demand for transplants. An emerging approach to overcome this problem is based on human embryonic stem cells (hESCs) culture and differentiation. These are pluripotent and undifferentiated stem cells with high capacity for self-renewal and differentiation in all cells derived from the three embryonic germ layers. However, differentiation methods used for HSC production from pluripotent cells are not efficient yet. Protocols described so far have generated varying numbers and heterogeneous cell populations, and produce only very primitive and immature HSC with low in vivo functional capacity. Part of this difficulty may result from the inefficiency of the microenvironment of culture for differentiation. Here, we demonstrate an efficient protocol based on co-culture of hESCs with mouse embryonic fibroblasts for hematopoietic differentiation with high performance to generate in vitro hematopoietic stem/progenitor cells (HSPCs) that express CD45, CD43, CD31 and CD34 antigens with high purity of positive cells. We were able to produce all cells of erythroid and myeloid lineages at different stages of maturation. Lymphoid potential of hematopoietic cells was also evidenced. We demonstrated the primitive origin of hematopoietic cells through capillary-like structures constituted by hemogenic CD34+CD31+ cells. However, despite mature features of HSPCs obtained by our protocol, hematopoietic reconstitution assays showed that these cells have yet limited functional capacity for grafting into immunocompromised mice when exogenously transplanted by retro-orbital route.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains ix, 173 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 163-169).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Medicine. Title from pdf file main screen (viewed on April 27, 2010). Includes bibliographical references.