Robert Wadley

Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches

Hematopoietic stem cell (HSC) niches have been reported at the endosteum or adjacent to bone marrow (BM) vasculature. To investigate functional attributes of these niches, mice were perfused with Hoechst 33342 (Ho) in vivo before BM cell collection in presence of pump inhibitors and antibody stained. We report that the position of phenotypic HSCs, multipotent and myeloid progenitors relative to blood flow, follows a hierarchy reflecting differentiation stage, whereas mesenchymal stromal cells are perivascular. Furthermore, during granulocyte colony-stimulating factor-induced mobilization, HSCs migrated closer to blood flow, whereas stromal cells did not. Interestingly, phenotypic Lin(-)Sca1(+)KIT(+)CD41(-)CD48(-)CD150(+) HSCs segregated into 2 groups (Ho(neg) or Ho(med)), based on degree of blood/Ho perfusion of their niche. HSCs capable of serial transplantation and long-term bromodeoxyuridine label retention were enriched in Ho(neg) HSCs, whereas Ho(med) HSCs cycled more frequently and only reconstituted a single host. This suggests that the most potent HSC niches are enriched in locally secreted factors and low oxygen tension due to negligible blood flow. Importantly, blood perfusion of niches correlates better with HSC function than absolute distance from vasculature. This technique enables prospective isolation of serially reconstituting HSCs distinct from other less potent HSCs of the same phenotype, based on the in vivo niche in which they reside.

The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity

Trophozoites of the microaerophilic flagellate parasitic protozoon Giardia intestinalis have only a limited capacity to detoxify O(2). Thus, when exposed to controlled concentrations of dissolved O(2) >8 microM, they gradually lose their ability to scavenge O(2). In a washed cell suspension stirred under 10% air in N(2) (equivalent to 25 microM O(2)), inactivation of the O(2)-consuming system was complete after 3.5 h; during this period accumulation of H(2)O(2) (3 micromol per 10(6) organisms) and oxidation of cellular thiols to 16% of their initial level occurred. Under 20% air (50 microM O(2)), respiratory inactivation was complete after 1.5 h, and under air (258 microM O(2)), after 50 min. Loss of O(2)-consuming capacity was accompanied by loss of motility. Use of the fluorogen 2, 7-dichlorodihydrofluorescein acetate indicated that intracellular H(2)O(2) is produced at extranuclear sites. Flow cytometric estimation of the plasma membrane electrochemical potentials using bis(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC(4)(3), showed that values declined from -134 mV to -20 mV after 4.5 h aeration. Incubation of organisms with 60 microM H(2)O(2) for 10 min gave partial collapse of plasma membrane potential and complete loss of O(2) uptake capacity; motility and viability as assessed by DiBAC(4)(3) exclusion were completely lost after 1 h. Inactivation of the O(2)-consuming system and loss of viability were also observed on exposure to singlet oxygen photochemically generated from rose bengal or toluidine blue.

IL-16 Regulation of Human Mast Cells/Basophils and Their Susceptibility to HIV-1

AIDS patients often contain HIV-1-infected mast cells (MCs)/basophils in their peripheral blood, and in vivo-differentiated MCs/basophils have been isolated from the blood of asthma patients that are HIV-1 susceptible ex vivo due to their surface expression of CD4 and varied chemokine receptors. Because IL-16 is a ligand for CD4 and/or an undefined CD4-associated protein, the ability of this multifunctional cytokine to regulate the development of human MCs/basophils from nongranulated progenitors residing in cord or peripheral blood was evaluated. After 3 wk of culture in the presence of c-kit ligand, IL-16 induced the progenitors residing in the blood of normal individuals to increase their expression of chymase and tryptase about 20-fold. As assessed immunohistochemically, >80% of these tryptase+ and/or chymase+ cells expressed CD4. The resulting cells responded to IL-16 in an in vitro chemotaxis assay, and this biologic response could be blocked by anti-IL-16 and anti-CD4 Abs as well as by a competitive peptide inhibitor corresponding to a sequence in the C-terminal domain of IL-16. The additional finding that IL-16 induces calcium mobilization in the HMC-1 cell line indicates that IL-16 acts directly on MCs and their committed progenitors. IL-16-treated MCs/basophils also are less susceptible to infection by an M/R5-tropic strain of HIV-1. Thus, IL-16 regulates MCs/basophils at a number of levels, including their vulnerability to retroviral infection.

T Cell Transfer Model of Colitis: A Great Tool to Assess the Contribution of T Cells in Chronic Intestinal Inflammation

Inflammatory bowel diseases (IBD) consist of Crohns disease (CD) and ulcerative colitis (UC) affecting about 0.1% of the western population. These two chronic gut diseases affect youth at their prime of life causing diarrhoea, intestinal bleeding, and severe gut discomfort. Mouse models of colitis have been major tools in understanding the pathogenesis of IBD. A number of mouse models are available to assess the contribution of T cells in the pathogenesis of CD and UC. Among these, the T cell transfer model of colitis is the most widely used model to dissect the initiation, induction, and regulation of immunopathology in chronic colitis mediated by T cells. The methodology below describes the classification of various animal models and explains the T cell transfer model in detail, including flow cytometry-based isolation of naïve T cells that are used in the transfer, immunological concepts, detailed immune-pathological assessment, shortcomings of the model, and the latest improvements to this colitis model. A special focus is paid to the utilisation of the T cell transfer model in delineating the immunopathology in a primary epithelial defect model of colitis, namely Winnie.

Flow Cytometry Measurement of Bone Marrow Perfusion in the Mouse and Sorting of Progenitors and Stems Cells According to Position Relative to Blood Flow In Vivo

Identification of the precise location, where hematopoietic stem cells (HSCs) reside in the bone marrow, has made a great leap forward with the advance of live time-lapse video 2-photon fluorescent microscopy. These studies have shown that HSCs preferentially resides in the endosteal region of the BM, at an average of two cell diameters from osteoblasts covering endosteal bone surfaces. However, this equipment is very sophisticated and only a very few laboratories can perform these studies. To investigate functional attributes of these niches, we have developed a flow cytometry technique in which mice are perfused with the cell-permeable fluorescent dye Hoechst33342 in vivo before bone marrow cells are collected and antibody stained. This method enables to position phenotypic HSC, multipotent and myeloid progenitors, as well as BM nonhematopoietic stromal cells relative to blood flow in vivo. This technique enables prospective isolation of HSCs based on the in vivo perfusion of the niches in which they reside.