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Blood, Vol. 106, Issue 7, 2259-2268, October 1, 2005 This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology Blood Sano et al. 106: 2259 Supplemental materials for: Sano et al, Vol 106, Issue 7, 2259-2268 A typical feature of soluble lysosomal enzyme precursor is to be secreted into the extracellular space and then internalized by neighboring or distant cells via mannose-6-phosphate– or mannose-receptor–mediated endocytosis. The precursor is correctly compartmentalized and processed in lysosomes. Transduced BMCs expressed sustained levels of the precursor and mature forms of -gal (Figure S1) and had a 10-fold increase in enzymatic activity compared with nontransduced cells. Enzyme activity was 140.27 nmol/h/mg protein for non transduced BMCs and 1113.40 nmol/h/mg protein for MSCV--gal-GFP–transduced BMCs. The activity in the conditioned medium used as a source of the corrective enzyme was 95 nmol/h/mL. The enzyme precursor secreted into the medium was effectively internalized by GLB1–/– human fibroblasts (target cells), and it restored -gal activity to nearly normal values (from 7.31 nmol/h/mg to 290 nmol/h/mg protein). Thus, transduced BMCs, particularly HSCs, have the potential to become a continuous source of therapeutic enzyme once engrafted into GLB1–/– recipients. Files in this Data Supplement: Table S1. Groups of mice transplanted with either the MSCV–β-gal–GFP vector or the MSCV-GFP vector (n=60) (JPG, 29.5 KB) Figure S1. β-gal uptake and secretion studies (JPG, 8.75 KB) - (A) Western blot analyses of nontransduced (1) and MSCV--gal-GFP–transduced (2) BMCs show efficient expression of the precursor (*85 kDa) and mature (**64 kDa and ***24-kDa C-terminal peptide) forms of the enzyme in transduced cells. (B) Western blot analysis showed that the secreted -gal precursor was taken up by deficient human fibroblasts after 5 days of exposure to conditioned medium from MSCV--gal-GFP–transduced BMCs. -gal assay also demonstrated efficient processing of the precursor form into the mature and catalytic active enzyme (290 nmol/h/mg protein). Figure S2. Immunohistochemical analyses in systemic organs of mice that received MSCV-β-gal-GFP–transduced BMCs (JPG, 66.3 KB) - Several -gal+ and GFP+ cells were detected in the liver and intestine (shown as examples) of treated mice 3 months after BMT. Scale bar equals 50 µm. Figure S3. Immunostaining of different brain regions with β-gal antibody (JPG, 40.5 KB) - Anti–-gal immunostaining of brain sections revealed numerous -gal+ cells in the cortex, pons, and hippocampus of Glb1–/– mice that received transplants (Glb1–/– BMTGlb1–/–) compared with Glb1–/– mice that received mock transplants (Glb1–/– BMTmock). Scale bar equals 25 µm. Figure S4. Immunohistochemical analyses of SDF-1β+ cells in mice receiving transplants (JPG, 91.3 KB) - Overlay of SDF-1 staining with the astrocyte marker GFAP demonstrated colocalization of the two markers. This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef

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