14800-DvRappard

93 Donor macrophages and remyelination in metachromatic leukodystrophy 6 Five-μm-thick frozen tissue sections were stained with Oil red O and with antibodies against M1marker CD40 (1:500, Dako, ab13545) and CD64 (1:250, Abcam, ab104273) 11,12 , M2 marker mannose receptor (CD206, 1:500, Dako, ab125028)[10] and oligodendrocyte lineage-specificmarker Olig2 (1:100, Millipore, AB9610) as described. 13 Frozen tissue was also used for double staining of the M1 and M2 markers CD40 and CD206, respectively. CD40 immunoreactivity was visualized with an Envision+ systemHRP-labelled antibody and 3,3’-Diaminobenzidine (Dako, K4002, K3467), CD206 with liquid permanent red (1:100, Dako, K00640) after secondary incubation with biotinylated secondary antibody (1:100, Dako, E0432) followed by streptavidin with an alkaline phosphatase conjugate (1:100, Sigma-Aldrich, 11089161001). Sections were counterstained with haematoxylin. Fluorescence in situ hybridization (FISH) against chromosomes X and Y was performed using a XY CEP probe (Abbott, 05J10-051) and a FISH Accessory Kit (Dako, K5799). Image acquisition and analysis Pictures were taken with a Leica DM6000B microscope (Leica microsystems). The number of positive pixels was quantified using the colour deconvolution plugin for imaging software ImageJ. 14,15 Total cell number were counted with the ImageJ cell counter. Oligodendrocytes were classified as mature or precursor cells (OPC) based on Olig2 intensity as validated before. 16 Statistical analysis Statistical analysis was performed with GraphPad Prism v7.0a. Data are displayed as mean ± standard error of the mean. An unpaired t-test or the nonparametric Mann- Whitney U-test was performed to evaluate differences between non-transplanted and HCT-treated patients (considered significant when p <0.05). RESULTS Patients Patient 1 presented at age 2 years with peripheral demyelinating polyneuropathy and mildly delayed myelination on brain MRI (figure 1b). Absent ASA activity and mutations c.245C>T, p.(Pro82Leu) and c.1168C>T, p.(Arg390Trp) in ARSA confirmed the diagnosis of late-infantile MLD. Cognitive function was age-adequate. Because there was no clear CNS involvement, HCT from a mismatched (5 out of 6 matched) unrelated female cord blood donor was performed, unfortunately followed by disease progression albeit rapid successful engraftment and full donor chimerism. He died one year after HCT. Patient 2 was diagnosed at age 7 years with gait abnormalities due to mild spasticity and