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Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intratumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus demethylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single-cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus demethylation at diagnosis associate with suboptimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78). [Figure not available: see fulltext.].

The improvement of hematopoietic stem and progenitor cell (HSPC) engraftment remains a high-priority goal when limited cell doses are available, such as in cord blood (CB) transplantation and HSC gene therapy. We observed that monocytes are highly effective at improving the engraftment of both CB-CD34+ and lentivirus-transfected CD34+ cells in a xenogeneic model of HSC transplantation. Moreover, monocytes, in particular the CD14+CD16− classical subset, in co-culture systems increase survival and stemness of CB-CD34+ cells. Both soluble factors and direct-cell contact interactions, such as JAG/NOTCH and COX-2/PGE2 pathways, are critically involved in the HSC-monocyte crosstalk. Our results indicate that the infusion of monocytes improves engraftment when cell dose is a limiting factor.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

Aim: To determine the time needed to provide clinical pharmacy services to individual patient episodes for medical and surgical patients and the effect of patient presentation and complexity on the clinical pharmacy workload. Method: During a 5-month period in 2006 at two general hospitals, pharmacists recorded a defined range of activities that they provided for patients, including the actual times required for these tasks. A customised database linked to the two hospitals' patient administration systems stored the data according to the specific patient episode number. The influence of patient presentation and complexity on the clinical pharmacy activities provided was also examined. Results: The average time required by pharmacists to undertake a medication history interview and medication reconciliation was 9.6 (SD 4.9) minutes. Interventions required 5.7 (SD 4.6) minutes, clinical review of the medical record 5.5 (SD 4.0) minutes and medication order review 3.5 (SD 2.0) minutes. For all of these activities, the time required for medical patients was greater than for surgical patients and greater for 'complicated' patients. The average time required to perform all clinical pharmacy activities for 1071 completed patient episodes was 14.4 (SD 10.9) minutes and was greater for medical and 'complicated' patients. Conclusion: The time needed to provide clinical pharmacy services was affected by whether the patients were medical or surgical. The existence of comorbidities or complications affected these times. The times required to perform clinical pharmacy activities may not be consistent with recently proposed staff ratios for the provision of a basic clinical pharmacy service.

By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1 pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation. of unnatural amino acid moieties, in the following manner: WT → Cys(mutant) + H3CSO2SR → Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buffed residue in the S1' pocket of SBL, caused dramatic changes in k(cat)/K(M), of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter k(cat)/K(M), whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the β- carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor., References: Bone, R., Agard, D.A., (1991) Methods Enzymol., 202, pp. 643-671; Khosla, C., Caren, R., Kao, C.M., McDaniel, R., Wang, S.-W., (1996) Biotechnol. Bioeng., 52, pp. 122-128; Sears, P., Wong, C.-H., (1996) Biotechnol. Prog., 12, pp. 423-433; Shao, P., Arnold, F.H., (1996) Curr. Opin. Struct. Biol., 6, pp. 513-518; Perona, J.J., Craik, C.S., (1995) Protein Sci., 4, pp. 337-360; Ballinger, M.D., Tom, J., Wells, J.A., (1996) Biochemistry, 35, pp. 13579-13585; Russell, A.J., Thomas, P.G., Fersht, A.R., (1987) J. Mol. Biol., 193, pp. 803-813; Wells, J.A., Cunningham, B.C., Graycar, T.P., Estell, D.A., (1987) Proc. Natl. Acad. Sci. U.S.A., 84, pp. 5167-5171; Estell, D.A., Graycar, T.P., Miller, J.V., Powers, D.B., Burnier, J.P., Ng, P.G., Wells, J.A., (1986) Science, 233, pp. 659-663; Wangikar, P.P., Rich, J.O., Clark, D.S., Dordick, J.S., (1995) Biochemistry, 34, pp. 12302-12310; Cornish, V.W., Mendel, D., Schultz, P.G., (1995) Angew. Chem., Int. Ed. Engl., 34, pp. 621-633; Kuang, H., Brown, M.L., Davies, R.R., Young, E.C., Distefano, M.D., (1996) J. Am. Chem. Soc., 118, pp. 10702-10706; Peterson, E.B., Hilvert, D., (1995) Biochemistry, 34, pp. 6616-6620; Suckling, C.J., Zhu, L.-M., (1993) Bioorg. Med. Chem. Lett., 3 (4), pp. 531-534; Rokitas, S.E., Kaiser, E.T., (1986) J. Am. Chem. Soc., 108, pp. 4984-4987; Kukubo, T., Sassa, S., Kaiser, E.T., (1987) J. Am. Chem. Soc., 109, pp. 606-607; Radziejewski, C., Ballou, D.P., Kaiser, E.T., (1985) J. Am Chem. Soc., 107, pp. 3352-3354; Bech, L.M., Breddam, K., (1988) Carlsberg Res. Commun., 53, pp. 381-393; Bech, L.M., SÞrensen, S.B., Breddam, K., (1993) Biochemistry, 32, pp. 2845-2854; Gloss, L.M., Kirsch, J.F., (1995) Biochemistry, 34, pp. 12323-12332; Smith, H.B., Hartman, F.C., (1988) J. Biol. Chem., 263, pp. 4921-4925; Wynn, R., Harkins, P.C., Richards, F.M., Fox, R.O., (1996) Protein Sci., 5, pp. 1026-1031; Berglund, P., Stabile, M.R., Gold, M., Jones, J.B., Mitchinson, C., Bott, R.R., Graycar, T.P., (1996) Bio