Allogeneic endometrial regenerative cells: An "Off the shelf solution" for critical limb ischemia?

Critical limb ischemia (CLI) is an advanced form of peripheral artery disease which is responsible for approximately 100,000 amputations per year in the US. Trials to date have reported clinical improvement and reduced need for amputation in CLI patients receiving autologous bone marrow or mobilized peripheral blood stem cells for stimulation of angiogenesis. While such treatments are currently entering Phase III trials, practical and scientific pitfalls will limit widespread implementation if efficacy is proven. Hurdles to be overcome include: a) reduced angiogenic potential of autologous cells in aged patients with cardiovascular risk factors; b) invasiveness/adverse effects of bone marrow extraction and G-CSF mobilization, respectively; and c) need for on-site cellular manipulation. The Endometrial Regenerative Cell (ERC) is a mesenchymal-like stem cell derived from the menstrual blood that is believed to be associated with endometrial angiogenesis. We discuss the possibility of using allogeneic ERCs as an "off the shelf" treatment for CLI based on the following properties: a) High levels of growth factors and matrix metalloprotease production; b) Ability to inhibits inflammatory responses and lack of immunogenicity; and c) Expandability to great quantities without loss of differentiation ability or karyotypic abnormalities

Regulation of LFA-1 (CD11a/CD18) function

In the immune system, the complex cellular interactions that are necessary for the effective surveillance of the body against pathogens, and for launching an appropriate immune response to eliminate these pathogens, are mediated by adhesion molecules. The proper function of the immune system relies on the strict regulation of adhesive interactions between immune cells. The leukocyte integrin LFA-1 and its major ligand ICAM-1 constitute one pair of adhesion molecules that play critical roles in immune responses. This thesis presents the results of experiments designed to elucidate the mechanisms regulating the binding of LFA-1 to ICAM- 1. Murine recombinant soluble ICAM-1 was immobilized on polystyrene microspheres with a view to using the beads to probe the distribution of high-avidity LFA-1 on various cell lines. These microspheres bound specifically to high-avidity LFA-1 in a cytoskeleton-independent manner as treatment with cytochalasin B had no effect. Furthermore, the beads displayed a highly localized distribution on some cell types, whereas fluorescence staining indicated that LFA-1 was uniformly distributed on the cell surface. Thus, the cell surface distribution of highavidity LFA-1 can be different from that of LFA-1 in general, and is potentially significant in regulating LFA-1-mediated adhesion. The role of LFA-1 cytoplasmic domains in binding to ICAM-1 and in post-adhesion events was also investigated. Various truncated forms of LFA-1 α (CD 11 a) and β (CD 18) chains were generated by PCR and co-transfected into murine fibroblast TNR-2 cells in various combinations. The transfected fibroblasts were tested for their ability to adhere to ICAM-1 immobilized on plastic, and to spread out following this adhesion. The results indicated that both LFA-1 cytoplasmic domains are important for adhesion to ICAM-1, but that they play different roles. Furthermore, both cytoplasmic domains are required for post-receptor spreading. Fluorescent staining of these cells indicated no significant variation in the distribution of LFA-1 on the cell surface. As a further probe into the roles of LFA-1 cytoplasmic domains in regulating adhesion, we overexpressed chimeras consisting of the LFA-1 subunit cytoplasmic domains attached to the extracellular portion of murine CD4 in a B cell line as well as in a T cell line, and examined the effect on adhesion to ICAM-1 and fibronectin. The CD4/18 chimera drastically inhibited adhesion of both lines to ICAM-1 and fibronectin, whereas the CD4/11 chimera and a truncated form of CD4 lacking most of the cytoplasmic domain had no effect. Cell spreading after binding to ICAM-1 was also inhibited by the CD4/18 chimera but not the CD4/11 chimera. These results suggest that the CD 18 cytoplasmic domain interacts with intracellular molecules that regulate not only LFA-1 but also other integrins.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Functional organization of the chloroplast in the diatom Phaeodactylum tricornutum

The technique of protein A-gold immunoelectron microscopy was used to determine the distributions of two photosynthetic complexes in the thylakoid membranes of the diatom Phaeodactylum tricornutum. The fucoxanthin-chlorophyll a/c light-harvesting complex, believed to be associated with photosystem II, was found to be equally distributed among appressed and unappressed membranes, whereas photosystem I was slightly more concentrated in the latter. These results suggest that in diatoms, the two photosystems and their associated light-harvesting complexes are essentially equally distributed on the two types of membranes, in marked contrast to the lateral heterogeneity observed in higher plants and green algae. Furthermore, it was expected that in P. tricornutum, the nuclear-coded proteins of the fucoxanthin-chlorophyll a/c light-harvesting complex would be present in the vesicles of the periplastidal reticulum, which were postulated to be involved in protein transport into chloroplasts. Immunolabelling results, however, indicated that these proteins were absent from the periplastidal vesicles