At present, the human adenoviruses (Ads) comprise 51 members, which have been classified into six species (A to F). In general, adenovirus (Ad) tissue tropism or disease patterns vary according to species, although adenoviruses from different species can sometimes cause the same symptoms. The current interest in adenoviruses is partly due to the aim of using them as vectors for gene therapy. Hematopoietic cells are attractive targets for gene therapy and the transductions can be performed ex vivo. However, the most commonly used adenovirus vectors, based on Ad2 or Ad5, are inefficient in their transduction of hematopoietic cells since they attach poorly to these cells. Most Ads, including Ad2 and Ad5, appear to use the coxsackie-adenovirus receptor (CAR) (a component of tight junctions), for attachment to host cells. However, species B Ads do not bind to CAR and several studies have indicated that species B-based vectors would be more suitable for hematopoietic cells. Species B Ads can be further divided into species B1 and B2, which display different tissue tropisms. Species B1 Ads mostly cause acute respiratory infections whereas species B2 Ads have been associated with persistent infections of the kidney and urinary tract. One of the key determinants of tropism is believed to be the initial high-affinity attachment of the virion to host cell fiber receptors. By reciprocal blocking experiments and different ways of characterizing the species B attachment receptors, we have shown that the species B2 serotypes Ad11p and Ad35 and the species B1 serotypes Ad3p and Ad7p also differ in receptor usage. There are at least two different Ad species B receptors. Since one of these receptors appeared to be used by all four serotypes, we designated this receptor sBAR (species B adenovirus receptor). The other receptor appeared to be used exclusively by the two species B2 serotypes and was therefore designated sB2AR (species B2 adenovirus receptor). Binding to sBAR can be abolished by EDTA and restored with Mn2+ or Ca2+, whereas binding (of Ad11p and Ad35) to sB2AR is independent of divalent cations. Furthermore, sBAR appears to be trypsin sensitive whereas sB2AR is not. We also identified CD46 as a receptor for Ad11p. Even so, CD46 does not appear to be a functional receptor for Ad7p. Both Ad7p and Ad11p attached to CD46-transfected Chinese hamster ovary (CHO) cells more efficiently than to control CHO cells. However, only Ad11p (selectively) infected CD46-transfected CHO cells. Anti-CD46 antibodies inhibited Ad7p and Ad11p from binding to, and Ad11p from infecting, CD46-transfected CHO cells. However, in human cells, anti-CD46 antibodies had an inhibitory effect only on Ad11p binding (~30%) but did not affect Ad7p binding. In binding experiments with EDTA, divalent cations and pretrypsinized cells, Ad11p and Ad7p showed the same pattern in their binding to CHO-CD46 cells as in the previous study. Since Ad7p interacted almost as efficiently with control CHO cells as with CHO-CD46 cells after addition of Mn2+, it seems that Ad7p mainly addressed an endogenously expressed hamster receptor on CHO-CD46, the properties of which resemble sBAR. In addition, Ad3p and Ad7p attach poorly to PBMCs and CD46 is expressed on all nucleated cells. Thus, CD46 appears to correspond to sB2AR rather than to sBAR. With these differences in receptor usage in mind, we studied the binding and infectious capacity of these species B Ads in various hematopoietic cells. We found that all species B serotypes bound efficiently to CD34+ hematopoietic stem cells (HSCs) and also productively infected HSCs. However, only the sB2AR binding Ad serotypes Ad11p and Ad35 could attach primary PBMCs efficiently. Our results regarding the subsequent steps in infection of PBMCs suggest that both Ad11p and Ad35 enter PBMCs and deliver viral DNA to the nuclei of most PBMC cell types. However, productive infections were only clearly detected in stimulated T-cells (most frequently) and monocytes, whereas Ad infection seemed eclipsed in unstimulated lymphocytes. Replication of Ad DNA seemed seriously impaired in at least T-cells, indicating limited production of infectious particles in PBMCs. The capacity of species C Ads to establish persistent infections in lymphatic tissues has been described previously. These Ads also persistently infect various transformed hematopoietic cell lines in vitro. Our studies indicate that replication of the species B2 Ads is also restricted in cells of hematopoietic origin (both in primary and transformed cells). Taken together, the results indicate that species B2 Ads (as compared to other Ads) seem to enter and infect most hematopoietic cells efficiently, which is in line with the persistent nature of these Ads. They would presumably act as suitable vectors for efficient transduction of most cells of hematopoietic origin, as has already been shown for e.g. HSCs and dendritic cells. The finding that replication of Ads in T-cells appears to depend on the level of T-cell activation, strengthens the hypothesis that T-cells may serve as a reservoir for human Ads and raises possible safety issues for usage of species B-based vectors in hematopoietic cells.

In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase β subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase β subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis. We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens. Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.

Hämatopoetische Stammzellen besitzen die Fähigkeit, die dauerhafte Erhaltung ihrer eigenen Population im Knochenmark zu gewährleisten und gleichzeitig zur Neubildung der verschiedenen Zelltypen des peripheren Blutes beizutragen. Die Sequenz von Entscheidungsprozessen, die den Übergang einer undifferenzierten Stammzelle in eine funktionale ausgereifte Zelle beschreibt, wird als Linienspezifikation bezeichnet. Obwohl viele Details zu den molekularen Mechanismen dieser Entscheidungsprozesse mittlerweile erforscht sind, bestehen noch immer große Unklarheiten, wie die komplexen phänotypischen Veränderungen hervorgerufen und reguliert werden. Im Rahmen dieser Dissertation wird ein geeignetes mathematisches Modell der Linienspezifikation hämatopoetischer Stammzellen entwickelt, welches dann in ein bestehendes Modell der hämatopoetischen Stammzellorganisation auf Gewebsebene integriert wird. Zur Verifizierung des theoretischen Modells werden Simulationsergebnisse mit verschiedenen experimentellen Daten verglichen. Der zweite Teil dieser Arbeit konzentriert sich auf die Beschreibung und Analyse der Entwick- lungsprozesse von Einzelzellen, die aus diesem integrierten Modell hervorgehen. Aufbauend auf den entsprechenden Modellsimulationen wird dazu eine topologische Charakterisierung der resultierenden zellulären Genealogien etabliert, welche durch verschiedener Maße für deren Quantifizierung ergänzt wird. Das vorgestellte mathematische Modell stellt eine neuartige Verknüpfung der intrazellulären Linienspezifikation mit der Beschreibung der hämatopoetischen Stammzellorganisation auf Populationsebene her. Dadurch wird das Stammzellm- odell von Röder und Löffler um die wichtige Dimension der Linienspezifikation ergänzt und damit in seinem Anwendungsbereich deutlich ausgedehnt. Durch die Analyse von Einzelzellverläufen trägt das Modell zu einem grundlegenden Verständnis der inhärenten Heterogenität hämatopoetischer Stammzellen bei.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 85-105).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-113).

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2009. / Title from title page screen (viewed on February 4, 2010). Research advisor: John M. Cunningham, M.D. Document formatted into pages (xiv, 115 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 89-103).

The principal isoform of Src homology (SH) 2-domain containing 5' inositol phosphatase protein 1 (SHIP) is a 145kDa protein primarily expressed by cells of the hematopoietic compartment. The enzymatic activity of SHIP is responsible for hydrolyzing the 5' phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), and thereby preventing the recruitment of pleckstrin homology domain containing effector proteins. Furthermore, SHIP contains protein-protein interaction domains, such as an SH2 domain, two NPXY and several proline-rich motifs. All of these different domains endow SHIP with the capacity to impact signaling pathways important for proliferation, survival, differentiation and activation. Therefore, we hypothesized that SHIP-deficiency could result in the loss of hematopoietic cell homeostasis and functionTo this verify this hypothesis, we first studied the effect of SHIP ablation on hematopoietic stem cell (HSC) proliferation, survival, function and hom ing. Most interestingly we observed that SHIP impacts HSC homeostasis and their ability to home appropriately to the bone marrow. Then, since SHIP was shown to be activated after engagement of the c-mpl receptor by its ligand, thrombopoietin, we studied the impact of SHIP deletion on the function of megakaryocytes, the major target cell of that cytokine. We found that SHIP is also important for homeostasis of the megakaryocyte compartment. Thirdly, we studied the role of SHIP in natural killer (NK) cells biology. We observed that F4 generation SHIP-/- mice have increased NK cells in their spleen and that these cells exhibit a disrupted receptor repertoire. We verified the hypothesis that SHIP helps shape the receptor repertoire of NK cells, mainly through regulation of cell survival and proliferation. Also included, is a study on the role of a SHIP isoform lacking the SH2-domain, called stem cell-SHIP (s-SHIP) in the biology of embryonic stem (ES) cells. To date, this isoform i s expressed by stem/progenitor cells and not by normal differentiated cells. Due to its specific expression pattern, s-SHIP has the potential to have an important role in stem cell biology.