A Novel Assay to Trace Proliferation History In Vivo Reveals that Enhanced Divisional Kinetics Accompany Loss of Hematopoietic Stem Cell Self-Renewal

BACKGROUND: The maintenance of lifelong blood cell production ultimately rests on rare hematopoietic stem cells (HSCs) that reside in the bone marrow microenvironment. HSCs are traditionally viewed as mitotically quiescent relative to their committed progeny. However, traditional techniques for assessing proliferation activity in vivo, such as measurement of BrdU uptake, are incompatible with preservation of cellular viability. Previous studies of HSC proliferation kinetics in vivo have therefore precluded direct functional evaluation of multi-potency and self-renewal, the hallmark properties of HSCs. METHODOLOGY/PRINCIPAL FINDINGS: We developed a non-invasive labeling technique that allowed us to identify and isolate candidate HSCs and early hematopoietic progenitor cells based on their differential in vivo proliferation kinetics. Such cells were functionally evaluated for their abilities to multi-lineage reconstitute myeloablated hosts. CONCLUSIONS: Although at least a few HSC divisions per se did not influence HSC function, enhanced kinetics of divisional activity in steady state preceded the phenotypic changes that accompanied loss of HSC self-renewal. Therefore, mitotic quiescence of HSCs, relative to their committed progeny, is key to maintain the unique functional and molecular properties of HSCs

Garlic Accelerates Red Blood Cell Turnover and Splenic Erythropoietic Gene Expression in Mice: Evidence for Erythropoietin-Independent Erythropoiesis

Garlic (Allium sativum) has been valued in many cultures both for its health effects and as a culinary flavor enhancer. Garlic's chemical complexity is widely thought to be the source of its many health benefits, which include, but are not limited to, anti-platelet, procirculatory, anti-inflammatory, anti-apoptotic, neuro-protective, and anti-cancer effects. While a growing body of scientific evidence strongly upholds the herb's broad and potent capacity to influence health, the common mechanisms underlying these diverse effects remain disjointed and relatively poorly understood. We adopted a phenotype-driven approach to investigate the effects of garlic in a mouse model. We examined RBC indices and morphologies, spleen histochemistry, RBC half-lives and gene expression profiles, followed up by qPCR and immunoblot validation. The RBCs of garlic-fed mice register shorter half-lives than the control. But they have normal blood chemistry and RBC indices. Their spleens manifest increased heme oxygenase 1, higher levels of iron and bilirubin, and presumably higher CO, a pleiotropic gasotransmitter. Heat shock genes and those critical for erythropoiesis are elevated in spleens but not in bone marrow. The garlic-fed mice have lower plasma erythropoietin than the controls, however. Chronic exposure to CO of mice on garlic-free diet was sufficient to cause increased RBC indices but again with a lower plasma erythropoietin level than air-treated controls. Furthermore, dietary garlic supplementation and CO treatment showed additive effects on reducing plasma erythropoietin levels in mice. Thus, garlic consumption not only causes increased energy demand from the faster RBC turnover but also increases the production of CO, which in turn stimulates splenic erythropoiesis by an erythropoietin-independent mechanism, thus completing the sequence of feedback regulation for RBC metabolism. Being a pleiotropic gasotransmitter, CO may be a second messenger for garlic's other physiological effects

From Prototyping to Allotyping. The invention of change of use and the crisis of building types

The chapter analyses the invention and the form of the discourse on building conversion as one particular instance of redefining what a technology is and how it operates. I describe a shift from expert defined closure to lay based openness and tinkering as a shift from prototyping to allotyping: Since the early 1970s, change of use and building conversion have become a central and fashionable discourse among architects and architectural theorists. Before the 1970s, buildings were understood as technologies, as ‘society made durable’. The notion of building type was central to link a building to a given use. A bank was a bank because architects applied existing templates, prototypes, to turn a building into a bank. In the 1970s, suddenly buildings became flexible – discursively, since building conversion always existed: ‘Building type’ no longer was a meaningful link between a building and its use. A bank should not stay a bank, but become a hotel, a theatre or a flat, in short: an allotype. The chapter elucidate this central shift in thinking about buildings and reflects on the special case of allotyping buildings and how it continues to vex thinking about buildings

Distribution of injected anti-thy-1 monoclonal antibodies in mouse lymphatic organs: evidence for penetration of the cortical blood-thymus barrier, and for intravascular antibody-binding onto lymphocytes.

The localization of monoclonal anti-Thy-1 binding in mouse thymus, spleen, and lymph nodes was studied at early intervals after intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) injection of a single dose of the monoclonal antibody (MAb). Five minutes after i.v. injection, anti-Thy-1 was bound to cortical thymocytes surrounding capillaries in the thymic cortex, to thymic cells beneath the thymic capsule and to medullary thymocytes around venules of the thymus medulla. When anti-Thy-1 was injected i.p. or s.c. the MAb was first deposited in capillary walls in the thymus cortex and did not appear on thymocytes outside of capillaries until 60 min after injection. These findings suggest that thymic cortical capillaries are permeable for anti-Thy-1 MAb contrary to the generally accepted principle of a blood thymus barrier to antigens in thymic cortex. Some cortex capillaries also became permeable for peroxidase when injected 15 min after anti-Thy-1 MAb. Anti-Thy-1 MAb penetration into spleen white pulp and lymph node paracortex occurs along the circulatory pathway of the vascular system in the spleen and of lymphatics in lymph nodes. But those lymphocytes with a strong anti-Thy-1 MAb loading always appeared along the pathways of lymphocyte circulation indicating that the most intense contact between anti-Thy-1 MAb and T-lymphocytes occurs not in the lymphatic organs but during the intravascular period of recirculation of lymphocytes

Combined prophylactic suppression of graft-versus-host and host-versus-graft reactions following treatment of prospective bone marrow recipients with rat IgG 2 b anti-mouse T cell antibodies.

A new approach to avoid typical complications from bone marrow transplantation into MHC different mice was studied. Rat monoclonal anti-Thy-1 antibodies of the IgG 2 b isotype were identified, which inhibit T lymphocytes in vivo so that transplanted donor T cells as well as residual T cells of the conditioned marrow recipient were suppressed. A single injection of these antibodies after irradiation and before marrow transplantation did not only prevent graft-versus-host mortality but suppressed also host-versus-graft reactivity so that the radiation dose necessary for engraftment of donor cells differing in H-2, IA (both haplotypes) major histocompatibility antigens could be reduced to 6.0 Gy. In addition an anti-T leukemic cell effect from the injected monoclonal T cell antibodies was observed

A mouse model for the preclinical evaluation of immunosuppressive effector functions of human isotypes. The human IgG1 isotype is superior to IgG3.

Antibodies against T cells are widely used as immunosuppressive agents in clinical therapy. As effector functions of chimeric or humanized anti-T cell antibodies cannot be predicted in vitro, we compared T cell-depleting effects of human isotypes in vivo with their immunosuppressive consequences in a mouse BMT model. This system is based on chimeric antibodies with a mouse pan T cell specificity and human constant regions. To secure optimal immunosuppression, the specificity for Thy-1.2--one of the best-characterized T cell antigens--was selected, as Thy-1.2-specific antibodies prevent graft-versus-host disease in fully mismatched mice. Chimeric mouse anti-Thy-1.2 antibody with the human IgG1 Fc part was found to be equally effective in preventing graft-versus-host disease mortality as the highly protective anti-Thy-1.2 mouse IgG2a isotype, while human IgG3 was far less effective. This was not predictable by measuring the degree of T cell depletion in peripheral blood. T cell depletion in lymph nodes, however, exactly reflected the results obtained in the BMT system. In addition, this system offers the advantage of assessing the influence of reduced antigen density by using heterozygous Thy-1.2 mice

<em>In vivo </em>immunosuppression by pan-T cell antibodies relates to their isotype and to their C1q uptake.

There is considerable interest in the use of monoclonal anti-T cell antibodies for immunosuppression during organ transplantation. However, the in vitro cytotoxic titers of these monoclonal reagents do not correlate with their immunosuppressive potency when injected in vivo. A relationship nevertheless seems to exist between immunosuppression and the isotype of anti-mouse Thy-1 antibodies, because among several anti-Thy-1 antibodies of mouse and rat origin, the only two found to cause immunosuppression in vivo belonged to the rat IgG2b and mouse IgG2a isotype. We show here that a quantitative positive correlation exists between an antibody-induced humoral effector mechanism and immunosuppression. We measured the uptake of the C1q complement subunit by polyclonal rabbit and rat anti-thymocyte globulin and also seven monoclonal anti-Thy-1 antibodies in an immunohistochemical assay or a radioimmunoassay. Immunosuppression was studied in a murine graft-vs-host and skin allograft model. Our results suggest strongly that a stable association between the C1 protein and a potential binding antibody is an essential prerequisite of antibody-dependent cell inhibition in vivo that suppresses the immunoresponse against strongly incompatible transplantation antigens