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Levels of circulating red blood cell (RBC)-derived vesicles are increased in sickle cell anaemia (SCA) and thalassaemia intermedia (TI) but the mechanisms, effects and controlling factors may differ. This study found that levels of vesicles and intravascular haemolysis were linked as shown by the correlation between levels of vesicles and plasma Hb. Vesicle levels were 6-fold greater in SCA and 4-fold greater in TI than in controls. The proportion of plasma Hb within vesicles was increased in SCA and TI with a significantly higher proportion in TI. We examined whether subpopulations of RBC expressing phosphatidylserine (PS) were a source of PS(+) vesicles and observed a significant association. Thrombin generation was promoted by the vesicles in which 40-50% expressed PS. In TI, markers of thrombin generation were significantly related to PS(+) RBC. Splenectomy in TI had significant effects including greater increases in vesicle levels, plasma Hb, PS(+) RBCs and thrombin generation markers than in unsplenectomised patients. In hydroxycarbamide (HC)-treated SCA patients these measures were decreased compared with untreated controls. The relationship between vesicle levels and plasma Hb suggests a mechanism linking vesiculation to haemolysis and consequently nitric oxide (NO) bioavailability and suggests a means by which HC treatment improves NO bioavailability.

This report concerns congenitally Na(+)-K(+) leaky red cells of the 'hereditary stomatocytosis' class. Three new isolated cases and one new pedigree are described, and one previously reported case is expanded. In all cases, Western blotting of red cell membranes revealed a deficiency in the 32 kDa membrane protein, stomatin. All showed pronounced cation leaks at 37 degrees C with markedly abnormal intracellular Na(+) and K(+) concentrations, like all other such stomatin-deficient cases. Consistent with recent findings in two previously described British pedigrees, immunocytochemistry demonstrated that the deficiency of stomatin was not complete. On typical blood films, some red cells showed positive stomatin immunoreactivity, while most were negative, although in one case only a minority were negative. All platelets and neutrophils were stomatin positive. The cases differed markedly between themselves with regard to the temperature dependence of the passive leak to K(+). Three showed a simple monotonic temperature dependence, while two showed a minimum at around 20-25 degrees C, such that the cells were extremely leaky at 0 degrees C, giving the phenotype known as 'cryohydrocytosis'. These patients are the only two known cases of stomatin-deficient cryohydrocytosis. Both showed a congenital syndrome of mental retardation, seizures, cataracts and massive hepatosplenomegaly, probably defining a new haemato-neurological syndrome.

Summary. The migration of haemopoietic stem and progenitor cells across endothelium lining bone marrow sinuses is a critical first step in the homing and successful engraftment of these cells. We have previously shown that freshly isolated mobilized peripheral blood CD34+ cells adhere to the endothelial surface but do not transmigrate unless activated by growth factors. The aim of this work was to examine the relationship between cell cycle progression, cell division and migration across endothelium. We now show that the enhanced migration of cytokine-activated cells is selective for cells which are in G0G1 phase of the cell cycle. Thus, the transmigrated population of CD34+ cells was enriched for cells in G0G1 phase, and sorted cells in G0G1 migrated more efficiently than those in S+G2M. Conversely, cells in S+G2M were more adherent to endothelium, a finding that may explain their reduced migration. Using the cytoplasmic dye, carboxyfluorescein diacetate succinimidyl ester, to track the divisional kinetics of CD34+ cells, we found that migration occurred preferentially in non-divided cells. Thus, although CD34+ cells require cytokine activation in order to migrate, cell division is not required for transmigration, which occurs optimally before cells enter S phase. The superior migratory ability of CD34+ cells in G0G1 phase of the cell cycle may have important implications for the homing and engraftment of ex vivo expanded cells.

Summary. Fetal haemopoietic cells continually circulate and migrate into tissues, and thus may have specialized homing capabilities. In this study we investigated the in vitro features of haemopoietic cells in fetal blood and liver which are relevant to homing and engraftment. Fetal cells were examined for long-term culture-initiating cell (LTC-IC) and progenitor content, adhesion molecule expression, cell cycle behaviour and transendothelial migratory activity. The LTC-IC content of fetal CD34+ cells is similar to that of CD34+ cells from cord and adult mobilized blood. In contrast to adult and cord blood CD34+ cells, fetal CD34+ cells were actively cycling (11·0 ± 1·7% and 28 ± 1·1% of fetal blood and liver CD34+ cells, respectively, in S+G2M, P

Dendritic cells may be generated ex vivo from CD34+ progenitor cells or peripheral blood mononuclear cells. Initial reports suggested that monocyte-derived dendritic cells (MoDCs) arise from a proliferating precursor and several groups subsequently reported successful retroviral transduction of these cells, again implying that cell division occurs. As this is of importance in the development of immunotherapy protocols, we investigated whether monocytes proliferate as they differentiate into MoDCs and also their susceptibility to retroviral transduction. During MoDC differentiation, there was a 51 ± 12% reduction in cell number, 98% of cells were in G0/G1, no DNA synthesis was detectable and the cell cycle regulatory proteins pRb and p130 were in the hypophosphorylated forms observed in non-cycling cells. As expected from these results, MoDCs were refractory to transduction with a GALV1 pseudotyped Moloney murine leukaemia virus (MoMLV)-based retroviral vector. In contrast, generation of DCs from purified CD34 progenitors was accompanied by rapid entry into the cell cycle and a 41.1-fold cell expansion at the end of 14 d culture.

When cord blood is used as a source of haemopoietic stem cells for transplantation, fewer cells are required per kg of recipient. This greater engraftment efficiency of cord blood cells may relate to an increased ability to traverse sinusoidal endothelium, a crucial step in the homing of stem cells. We report that freshly isolated cord blood progenitors migrated more efficiently than mobilized adult cells. Cord blood progenitors responded rapidly to growth factor stimulation with an increase in migratory ability within 24 h whereas mobilized adult cells responded only after 72 h (P