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We have demonstrated previously that cord blood CD133+ cells isolated in the G0 phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133+G1 cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133+G0 cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133+G1 counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133+G0 population. In long-term BM cultures, the CD133+G0 cells generate significantly more progenitors than the CD34+G0 population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133+G0 versus CD133+G1 cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G0 cells. We conclude that CD133+ cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G1 counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.Bone Marrow Transplantation (2009) 43, 627–635; doi:10.1038/bmt.2008.368; published online 10 November 2008 [ABSTRACT FROM AUTHOR]

The efficacy of gene therapy for the treatment of inherited immunodeficiency has been highlighted in recent clinical trials, although in some cases complicated by insertional mutagenesis and silencing of vector genomes through methylation. To minimize these effects, we have evaluated the use of regulatory elements that confer reliability of gene expression, but also lack potent indiscriminate enhancer activity. The Vav1 proximal promoter is particularly attractive in this regard and may be useful in situations where high-level or complex regulation of gene expression is not necessary. X-linked severe combined immunodeficiency (SCID-X1) is a good candidate for such an approach, particularly as there may be additional disease-related intrinsic risks of leukemogenesis, and where safety is therefore a paramount concern. We have tested whether lentiviral vectors expressing the common cytokine receptor gamma chain under the control of the proximal Vav1 gene promoter are effective for correction of signaling defects and the disease phenotype. Despite low-level gene expression, we observed near-complete restoration of cytokine-mediated STAT5 phosphorylation in a model cell line. Furthermore, at low vector copy number, highly effective T- and B-lymphocyte reconstitution was achieved in vivo in a murine model of SCID-X1, in both primary and secondary graft recipients. This vector configuration deserves further evaluation and consideration for future clinical trials.

The relationship between the plasma concentration of valproic acid (VPA) and anticonvulsant or neurotoxic effects was studied in the rat. Anticonvulsant activity was assessed against; (1) maximal seizures induced either by electroshock or by intravenous injection of pentylenetetrazol; and (2) kindled amygdaloid epilepsy. Drug-induced neurotoxicity was determined by the rotarod test and by observation of behaviour. In the maximal seizure tests, tonic hindlimb extension was always abolished at plasma valproic acid concentrations of 225 microgram ml-1 and above. Tonic forelimb extension was not consistently blocked until the plasma drug concentration exceeded 530 microgram ml-1. In fully-kindled rats, plasma valproic acid concentrations of 300 microgram ml-1 and above markedly reduced the duration of amygdala afterdischarge activity and the intensity of behavioural seizures produced by amygdala stimulation. Analysis of the data from individual kindled rats revealed that there was a significant correlation between the estimated plasma concentration of valproic acid and the degree of seizure protection. Impairment of rotarod performance and marked ataxia occurred at plasma valproic acid concentrations above 510 microgram ml-1 and loss of righting reflex became evident at 970 microgram ml-1. From these results, it is concluded that the plasma concentration of valproic acid is closely correlated with the anticonvulsant and neurotoxic effects observed in individual rats after acute administration of sodium valproate.