Potent skin allograft survival prolongation using a committed progenitor fraction of bone marrow in mice.

BACKGROUND: The infusion of donor bone marrow (BM) into mice conditioned with antilymphocyte serum (ALS) and sirolimus (Sir) prolongs skin allograft survival and produces chimerism. This study identifies the BM cell(s) responsible for this effect and determines whether enrichment for these cells will improve efficacy. METHODS: Skin grafts from BALB/C mice were transplanted into C57BL/6 or C57BL/10 recipients by using ALS, Sir, and BM (or fractions). BM was fractionated by using immunomagnetic beads. Flow cytometry was used for phenotyping and detecting chimerism. RESULTS: The median graft survival in mice receiving 25 million BM cells was 61 days. Infusion of BM depleted of cells expressing CD19, CD3, CD11c, and c-kit had no effect on median graft survival, whereas infusion of fractions enriched for those cells resulted in median graft survival of 38, 48, 28, and 83 days, respectively. The administration of higher doses (4 x 10(6) and 8x10(6)) of fractions enriched for c-kit resulted in median graft survival of 124 and 197 days, respectively, without chimerism. This favorably compared with mice receiving 150 million BM cells that demonstrated transient mixed chimerism and a median graft survival of 190 days. The majority of cells in the c-kit+-enriched fraction expressed lineage markers. Removal of lineage positive cells from BM before infusion shortened median graft survival (90 days), indicating that the c-kit+ lin+ population is largely responsible for prolongation of graft survival. CONCLUSIONS: Cells enriched for C-kit+lin+ constitute approximately 5% of murine BM cells and are more potent than whole BM at prolonging skin allograft survival in mice treated with ALS and Sir.

Erythrocytic malaria growth or invasion inhibition assays with emphasis on suspension culture GIA.

Erythrocytic cycle malaria parasite growth or invasion inhibition assays (GIA) compare the effects of various test and control substances on malaria parasite growth in erythrocytes or invasion into erythrocytes in vitro. Although inhibitions by antimalarial drugs in vitro correlate well with drug protective levels required in vivo, as yet there are too few data to know how well inhibitions by antibodies in vitro correlate with the types and degrees of immune protection in vivo. Antibody-mediated GIA is frequently complicated by parasite strain-specific inhibitions, as well as nonspecific inhibitory factors generated in sera collected or stored under nonoptimal conditions. In this chapter, we describe methods for collecting and processing sera, for using different strains of parasite, and a simplified method for staining parasite DNA with Hoechst dye 33342 before quantitating parasites using ultraviolet (UV)-excited flow cytometry. We also describe a new type of GIA using suspension cultures in a 48-well plate. Critical to this method is enclosing the plate in a gassed, heat-sealed plastic bag, which, being low mass, can easily be rested at a 13.5 degrees angle on a rotor platform (114 rpm with 1-in. displacement) to produce gentle pulsatile waves of media in each well. The suspension GIA, which, relative to the static GIA, increased inhibition by one antibody and decreased inhibition by another (Table 1), may better simulate in vivo blood flow and may thus better predict in vivo efficacy.