Correlation between microsatellite discrepancy scores and transplant outcome after haemopoietic SCT for pediatric ALL.

Microsatellite analyses show that self-reported ethnicity often correlates poorly with true genetic ancestry. As unknown ancestral differences could potentially have an impact on transplant outcome, we developed an average allele length discrepancy (AALD) score to assess allele length discrepancy between donor/recipient (D/R) using microsatellites analysed routinely in post-transplant chimeric assessment. This was then compared with outcome in a homogeneously treated cohort of pediatric patients undergoing high-resolution sibling or matched unrelated donor transplantation for acute lymphoblastic leukemia (ALL). AALD scores formed a numeric continuum ranging from 0 to 1.4 (median 0.76) for sibling pairs and 0.8-2.17 (median 1.6) for high-resolution matched unrelated donor (HR-MUD) pairs. There was a trend for worse OS with increasing AALD score, which reached statistical significance above a threshold of 1.7 for OS. Patients whose transplants had an AALD score of ⩾1.8 had a risk of non-relapse mortality 4.9 times greater (P=0.025) and relapse risk three times greater (P=0.058) than those scoring <1.8. This approach will now be explored in a Centre International for Blood and Marrow Transplantation Research (CIBMTR) study of 750 D/R pairs across all disease groups; if confirmed, it has the potential to improve donor selection for patients with multiple prospective donors.

Improved survival in matched unrelated donor transplant for childhood ALL since the introduction of high-resolution matching at HLA class I and II.

We present the first detailed study analysing OS in BMT for paediatric ALL following the introduction of high-resolution (HR) HLA matching. A total of 356 consecutive paediatric ALL stem cell transplants performed between 1988 and 2007 were reviewed; 80 of them were performed following the introduction of HR HLA class I and class II matching to the transplant programme in 2002. Comparisons of matched unrelated donor (MUD) transplant outcomes before and after this period were made. Matching at the HR level for HLA-A, -B, -C, -DRB1 and -DQB1 (HR-MUD) correlated with a greater than 25% improvement in 2- and 5-year OS in paediatric ALL patients transplanted with MUDs (P=0.009, P=0.005, respectively). Two-year OS for contemporaneous HLA-matched sibling transplants (80.8%) and HR-MUD transplants (78.8%) was equivalent. At 6%, non-relapse mortality (NRM) in MUD transplants since 2002 was significantly reduced compared with previous epochs. Changes in treatment and epoch-dependent improvements in outcome were reviewed for possible confounders to the influence of HR typing using univariate and multivariate analysis.

Cardiac chloride channels: physiology, pharmacology and approaches for identifying novel modulators of activity.

Drugs that block cardiac cation channels have been marketed as the therapeutic answer to cardiac arrhythmia. However, such molecules have been only moderately successful at improving the survival of cardiac patients, and so new targets have been needed for future antiarrhythmic agents. This article outlines the properties and roles of Cl(-) channels, which are one of these new targets, and describes an approach for identifying novel CI(2) channel modulators.

Effects of urea and oxygen tension on K flux in sickle cells.

K influx and efflux (both ouabain- and bumetanide-resistant) in haemoglobin S-containing red cells (sickle cells) were markedly stimulated by urea (> 0.25 M). Stimulation was rapid and reversible. Volume-sensitive KCl cotransport in both HbA or HbS red cells is thought to be O2-dependent but we show here that urea-stimulated K fluxes in sickle cells were largely insensitive to O2 tension. Urea-stimulated K fluxes were not inhibited by lowering the external Ca concentration (with EGTA) but were abolished by Cl-substitution (with MeSO4 or NO3) or pretreatment of cells with the protein phosphatase inhibitor, calyculin A (0.1 muM). Results are consistent with a stimulatory action of urea on the KCl cotransporter, independent of oxygen tension, mediated via the phosphorylation cascade which regulates the transporter. The importance of this effect to the physiology and pathology of sickle cells is discussed.

Activation of a novel organic solute transporter in mammalian red blood cells.

1. Suspending human red blood cells in isotonic sucrose (low ionic strength, LIS) medium induces a significant increase in membrane transport of glutamine, glutamate, lactate, histidine, taurine, glycine, serine, choline and carnitine but not sorbitol or sucrose. 2. Progressive lowering of ionic strength by sucrose or NaCl replacement gave a similar activation profile for taurine influx as found earlier for residual K+(86Rb+) flux. 3. The induced taurine transport could be measured as enhanced influx and efflux. Influx was linear with external concentration up to 10 mM, largely insensitive to alteration in cell volume, and did not vary with red blood cell age. 4. Unlike previous results for residual K+ transport, altering transmembrane potential with gluconate or glucuronate media led to an increase in taurine influx similar to that observed in LIS media. Varying medium pH confirmed the effect was not due to alteration in pH. 5. The LIS-induced flux was sensitive to a variety of 'classical' anion transport inhibitors in the order of potency DNDS, DIDS, NPPB, DIOA, niflumic acid, furosemide (frusemide), glibenclamide, nitrendipine and bumetanide. 6. The taurine flux showed a temperature dependence similar to that of the LIS-induced residual K+ flux. High hydrostatic pressure (40 MPa), however, inhibited taurine flux but stimulated residual K+ influx in LIS media. 7. A significant enhanced taurine flux could be demonstrated in red blood cells of other species, including horse, cattle, pig and high and low potassium type sheep. 8. It is concluded that lowering ionic strength activates a transport pathway for organic molecules sharing some similarities with background Cl- channels and LIS-induced residual K+ fluxes. In the latter context, however, there are certain significant differences (effect of transmembrane potential; volume; pressure sensitivity; species distribution) which may be important, and the unequivocal identity of the two transport processes remains to be confirmed.

Additive in vitro effects of anti-sickling drugs.

To study the effect of anti-sickling drugs on cellular dehydration induced by entry of Ca, sickle cells were subjected to cyclical oxygenation-deoxygenation for 15 h in Ca-containing buffer. The consequential loss of cation (K) via the Ca-dependent K efflux (Gardos) channel caused cell dehydration and loss of deformability. Inhibition of a specific fraction of Ca entry by verapamil had no rheologically protective effect, whereas inhibition of the Gardos channel by clotrimazole or nitrendipine had a marked protective effect. When Gardos channel inhibition (by either clotrimazole or nitrendipine) was combined with stabilization of the oxy-conformation of sickle haemoglobin (by the substituted benzaldehyde 12C79), an additive protective rheological effect was achieved with 60-78% reduction in clogging rate of 5 microns diameter pores when compared with no drug. Therapeutic use of anti-sickling compounds in combination may achieve increased efficacy with lower toxicity.

Specific inhibition of Ca-activated K channels in red cells by selected dihydropyridine derivatives.

1. Thirty two dihydropyridine derivatives were screened as potential inhibitors of the Ca-activated K-channel in human red cells. 2. Three derivatives (26, 29, 32 see Tables 1 and 2) with high activity were then characterized in detail, and also tested against the smooth muscle Ca-channel and shown to have varying potencies. 3. One of the more potent derivatives (32) and nitrendipine were also tested on the Ca-activated K-channel, Maxi-K channel, from mouse pancreatic beta-cells. 4. We conclude from our results that it may be possible to develop selective Gardos-channel inhibitors based on these molecules, which may be of benefit in the treatment of sickle cell disease.

Volume-sensitive KCl co-transport and taurine fluxes in horse red blood cells.

Potassium (using 86Rb+ as a tracer), amino acid and taurine fluxes were measured in horse red blood cells (RBCs). No volume-sensitive component of alanine and glycine transport was observed, and although volume-sensitive taurine fluxes were observed in most animals, their absolute magnitudes were small. K+ fluxes, however, were shown to be particularly volume sensitive; they were stimulated by cell swelling and inhibited by cell shrinkage. Sizeable fluxes were present at normal cell volumes. The volume-sensitive K+ flux was Cl- dependent and was abolished by Cl- replacement with methylsulphate. The Cl(-)-dependent K+ fluxes in horse red blood cells were stimulated by lowering in external pH to 6.9 and by treatment with the sulphydryl-reacting agent, N-ethylmaleimide. They were inhibited by the potent K(+)-Cl- co-transport inhibitor, DIOA, ([(dihydroindenyl)oxy]alkanoic acid) but were insensitive to the Na(+)-K(+)-Cl- co-transport inhibitors, frusemide and bumetanide. A Cl- channel inhibitor, 5-nitro-2-(phenylpropyl-amino)-benzoate (NPPB), produced partial inhibition. These results suggest that regulatory volume decrease in horse red blood cells is achieved predominantly by volume-sensitive K+ efflux mediated via a K(+)-Cl- co-transport system with similar properties to those observed in the red blood cells of other species. The significance of these findings and their rheological consequences are discussed.

Mode of action and comparative efficacy of pharmacological agents that inhibit calcium-dependent dehydration of sickle cells.

1. Selected Ca-channel antagonists were tested at 20 microM as inhibitors of Ca(2+)-uptake in human sickle red cells. Nitrendipine, fendiline, and bepridil (and its stereoisomers), were found to be as effective as methoxyverapamil (D-600) in inhibiting a fraction (25%) of Ca(2+)-uptake. In contrast cetiedil and Org 30701 were ineffective. 2. The drugs were subsequently tested as inhibitors of Ca(2+)-induced K+ efflux (Gardos) from sickle cells. They all showed inhibitory activity, with the order of efficacy nitrendipine greater than fendiline greater than bepridil greater than cetiedil greater than Org 30701. 3. With a 15 h programme of deoxygenation/reoxygenation cycles in a gas exchanger, it was shown that the inhibitors protected against cellular dehydration and loss of filterability in the order nitrendipine greater than fendiline greater than bepridil greater than cetiedil greater than Org 30701. However, significant stomatocytosis occurred at high concentrations of cetiedil, and bepridil (including its stereoisomers and analogues) impairing cell deformability. 4. It is concluded that Ca-antagonists may partially block both Ca(2+)-uptake and Ca(2+)-induced K+ efflux. The latter pathway is significant in contributing to sickle cell dehydration and nitrendipine is the most effective inhibitor of this route.

Nitrendipine is a potent inhibitor of the Ca(2+)-activated K+ channel of human erythrocytes.

Nitrendipine, a classical blocker of L-type Ca2+ channels, is shown to be a potent inhibitor of the Ca(2+)-activated K+ channel of human erythrocytes. In erythrocytes suspended in a solution with physiological Na+ and K+ concentrations and in which the channel was activated using the Ca2+ ionophore ionomycin, nitrendipine inhibited K+(86Rb+) influx with an I50 of around 130 nM. Similar results were obtained for K+(86Rb+) efflux, and for K+(86Rb+) influx into cells suspended in a high-K+ medium.