The origin of human dendritic cells (DCs) has long been debated. DCs are a subset of innate immune cells that are essential for initiating adaptive immune responses. Determining their ontogeny is critical for vaccine development and for unveiling the molecular mechanism of DC insufficiency, which underlies many primary immune deficiency disorders and leukemia. Like all blood cells, human DCs develop from hematopoietic stem cells through a sequence of increasingly restricted progenitors. Initially it was assumed that DCs should derive exclusively from myeloid progenitors. However, during the past few decades, a number of myeloid and lymphoid progenitors have been described and shown to have DC potential, instigating the debate of the myeloid vs. lymphoid origin of DCs. Hindering the resolution of this debate, human DC-restricted progenitors had not been identified. Further, the potential of known progenitors could not be interrogated due to the lack of a culture system that supports simultaneous differentiation of all human DCs subsets, in conjunction with other myeloid and lymphoid cells. In this work, we establish a culture system that supports the development of the three major subsets of DCs (plasmacytoid DCs or pDCs, and the two classical DCs, cDCs) as well as granulocytes, monocytes and lymphocytes. This system combines mitomycin C-treated murine stromal cell lines, MS5 and OP9, together with human recombinant cytokines, FLT3L, SCF and GM-CSF, and supports the differentiation of progenitor cells at a population and single cell level. Using this culture in combination with a phenotypic characterization of CD34+ progenitor cells, we identify four consecutive DC progenitors with increasing degree of commitment to DCs and describe their anatomical location of development. We show that DCs develop from a granulocyte-monocyte-DC progenitor (GMDP), which produces granulocytes and a monocyte-DC progenitor (MDP), which then generates monocytes and a common DC progenitor (CDP), which produces pDCs and a cDC precursor (pre-cDC), which finally produces cDCs only. Lastly, we establish a staining panel that allows the phenotypic identification and separation of newly found DC progenitors as well as all previously described myeloid and lymphoid progenitors. We investigate their inter-developmental relationship and DC potential at the single cell level. We show that each progenitor population with homogeneous phenotype is heterogeneous in developmental potential and prove that cell surface marker expression cannot be directly equated to a specific developmental potential. In order to resolve the unreliability of phenotype to draw developmental pathways, we propose to use the quantitative clonal output in order to delineate the DC developmental pathway. In summary, our studies provide a new tool to determine DC potential in vitro, identify new stages of DC development, and propose a new method for tracing the developmental pathway for DC lineage. This will generate a new model of dendritic cell hematopoiesis that can explain and reconcile the conflicts of data on DC origin and development.

No description available.

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The formation of germ cells in Drosophila melanogaster is dependent on the presence of ribonucleoprotein complexes called polar granules. A key component of these complexes is Oskar, a novel protein which has been shown to nucleate the granules. To investigate whether Oskar plays a further role in polar granule formation, I cloned the oskar gene from D. immigrans flies (osk[superscript imm]) and introduced it into D. melanogaster flies using P-element transformation. I found that osk[superscript imm] was able to rescue both the posterior patterning and germ cell formation defects of embryos from oskar mutant mothers. In addition, I found that the polar granules of embryos containing only Osk[superscript imm] as a source of Oskar protein resemble those found in D. immigrans embryos, indicating a new role for Oskar in determining the morphology of the polar granules. Germ cell formation in Drosophila is succeeded by migration of the germ cells to the site of gonad formation. A second line of research presented in this dissertation describes analysis of a novel protein important for both germ cell formation and migration, Bluestreak (Blue). Embryos from either heterozygous or homozygous Blue-mothers display defects in germ cell number and shape. I found that the ovaries of Blue-females have defects in the localization of Staufen and Oskar, sufficient to cause a reduction in pole cell number in embryos. In addition, genetic analysis of the interaction between Bluestreak and mutants which affect pole cell migration implicates Bluestreak in this process. Finally, I found that Blue localizes to centrosomes along with [gamma]-tubulin throughout the embryo, and to the nuclear membrane in pole cells. My findings introduce the possibility that Bluestreak may act to regulate germ cell migration in Drosophila.

A two by six factorial design was used to investigate the effect of zinc and all-trans retinoic acid (RA) on the growth of two human breast cancer cell lines differing in their expression of metallothionein (MT) and of estrogen receptors; MCF7 cells express estrogen receptors, BT-20 cells do not. Cells were treated with zinc to induce MT then treated with six concentrations of RA. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. / BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly induced by zinc treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. High RA concentrations increased lipid peroxidation. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by zinc in BT-20 cells but not in MCF7 cells. Glutathione did not appear to be a significant factor. / Induction of metallothionein by zinc may modulate the growth inhibitory effects of all-trans retinoic acid in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation.

A hybrid vector, pANIC1, was to be constructed which was capable of regulating expression of cloned genes in both Escherichia coli and Anacystis nidulans R2 by controlling the iron content of the growth medium. Plasmid pANIC1 would have origins of replication for E. coli and A. nidulans R2, and a marker gene conferring ampicillin resistance. It would also contain the promoter for the irpA gene which is active only in low iron growth conditions.The first two stages of the construction were successfully completed, but unfortunately the final construction proved to be unstable. Recent information has shown that operator sequences upstream from the irpA gene's promoter result in an unstable message. This may be interfering with the normal functioning of the host cell, resulting in an unstable construction. In future experiments it may be neccessary to alter the growth conditions or remove the upstream sequences in order to stablize the construction. / Department of Biology

No description available.

Bovine fibroblasts and epithelial cells were isolated from surgically biopsied mammary tissue. Characterization of population doubling time, cytoskeletal intermediate filaments, cryopreservation survival, and viability were performed on all fibroblast and epithelial cells. Several clonal fibroblast cell lines were cotransfected with a plasmid bearing the SV-40 Large-T-antigen, and the pSV-2 neo plasmid. Transfected cells were subsequently selected with G418 sulfate and cloned. / MAC-T cells and non-clonal primary bovine mammary epithelial cells proliferated in response to IGF-I, insulin, serum and serum albumin. MAC-T cells did not proliferate when cultured in EGF, estrogen, progesterone, estrogen+progesterone, growth hormone, prolactin, and only modest proliferation was obtained after TGF-$ alpha$ treatment. Subsequent experiments used serum, insulin or IGF-I (and its analogues) to stimulate cellular proliferation. Serum albumin was not added to serum-free media preparations since it stimulated cellular proliferation. / TGF-$ beta$ receptors were characterized in MAC-T cells and normal fibroblasts. Affinity labelling studies revealed that MAC-T and MF-2 cells contained type I, II, and III autoregulatable receptors. Fibroblast proliferation, was inhibited 50% by TGF-$ beta$. TGF-$ beta$ inhibited MAC-T cellular proliferation at concentrations among the lowest ever reported, ED$ sb{ rm 50}$ = 4 pm. TGF-$ beta$ was not cytotoxic at concentrations 1000-fold higher. / Retinoic acid (RA) also inhibited proliferation of MAC-T cells. Inhibition of proliferation did not occur when cells were growth stimulated by IGF-I analogues that do not bind IGFBPs. Unlike TGF-$ beta$, RA treatment increased IGFBP-2 and decreased IGFBP-3 protein expression by cells into media and on the cell's membrane. RA was cytotoxic at concentrations 10-fold higher than ED$ sb{100}$. / Fibroblasts and epithelial cells expressed the gap junction (GJ) protein, connexin43, with transformed fibroblasts expressing significantly less connexin43. Perinuclear and cell surface connexin43 was immunodetected in epithelial and fibroblasts cells. TGF-$ beta$, RA or cAMP, increased connexin43 protein expression, especially phosphorylated species. Only cAMP noticeably altered immunolocalization patterns of connexin43, causing a shift from perinuclear pools to the cell surface. None of the growth inhibitors affected GJ communication as measured by dye transfer. Therefore, mammary epithelial cells are growth inhibited by TGF-$ beta$ and RA by distinct mechanisms and both growth inhibitors significantly enhance the gap junction protein, connexin43, without increasing GJ communication.

Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves [109]-132. Also available on the Internet.