Experiments using bone marrow cells from 10 of 10 separate normal marrow donors showed a similar pattern of results

Experiments using bone marrow cells from 10 of 10 separate normal marrow donors showed a similar pattern of results. Results Transduction of OCL precursors. and size and have increased numbers of nuclei per multinucleated cell, demonstrate increased resorption capacity, and are hypersensitive to 1 1,25-(OH)2D3, the active form Corynoxeine of vitamin D (2, 3). Immunocytochemical studies have shown that pagetic OCLs contain paramyxoviral-like nuclear inclusions that cross-react with antibodies to measles virus (MV), respiratory syncytial virus, and canine distemper virus nucleocapsid antigen (4C7). In situ hybridization studies have detected expression of MV nucleocapsid (transcripts (10). However, the role MV infection plays in the abnormal OCL phenotype in Pagets disease is unknown. We transduced MV genes into normal OCL precursors to determine their role in the abnormal OCL activity in Pagets disease. In particular, we constructed retroviral vectors expressing the and the MV matrix (MVM) genes, and transduced these constructs into normal marrow cells to determine whether the expression of a particular MV gene induced a phenotype in OCLs that was very similar to the phenotype of OCLs from Pagets patients. Results of these studies demonstrated that expression of the gene, but not the gene, in normal OCL precursors enhanced formation of OCLs that had many of the characteristics of pagetic OCLs. Methods Subjects and cell preparation. The Institutional Review Board of Corynoxeine Rabbit Polyclonal to CNGA2 the University of Texas Health Science Center at San Antonio approved these studies. Bone marrow cells were aspirated from the iliac crest of healthy normal donors on 10 separate occasions. Bone marrow mononuclear cells were separated on Ficoll gradients (density 1.077 g/mL) by centrifugation at 400 for 30 minutes, and then washed 3 times with -MEM, as described previously (11). Retroviral vector construction and viral supernatant preparation. and cDNA clones were generous gifts from Chris Richardson, University of Toronto, Toronto, Canada. A 1.6-kb cDNA fragment was excised from the pETL-NP#30 plasmid by digestion with mRNA expression under the control of 5 LTR viral promoter elements. Similarly, the MVM retroviral construct pILXB29M#2 was developed by subcloning a 1-kb DNA fragment encoding the gene into the pLXSN vector. The recombinant plasmid constructs were transfected into the PT67 amphotropic packaging cell line using the calcium phosphate method (12). Stable clonal cell lines PT67-NP#2 and PT67-M#2, producing MVNP and MVM Corynoxeine recombinant retrovirus at high titer (106 virus particles/mL), were established by selecting for resistance to neomycin (600 g/mL). Similarly, a PT67-EV control retrovirus producer cell line was established by transfecting the cells with the pLXSN empty vector (EV). Producer cell lines were maintained in DMEM containing 10% FBS, 100 U/mL each Corynoxeine of streptomycin and penicillin, Corynoxeine 4 mM L-glutamine, and high glucose (4.5 g/L). Retroviral supernatants from the producer cell cultures were collected and filtered (0.45 m pore diameter) for immediate use. The retrovirus stocks prepared were demonstrated to be helper-free by a marker assay (13). Viral titers present in the culture supernatants were determined by multiplicity of infection of NIH 3T3 fibroblast cells in serial dilutions, and scoring G418-resistant (0.5 mg/mL G418) CFU formed as described (14). Transduction of human bone marrow cells. Isolated human bone marrow cells were prestimulated for 1 day in -MEM containing 10 ng/mL each of IL-3, IL-6, and stem cell factor (R&D Systems Inc., Minneapolis, Minnesota, USA), and 10% FBS (GIBCO BRL, Grand Island, New York, USA). After prestimulation, the bone marrow cells were cultured for 96 hours at 37C in a humidified atmosphere of 5% CO2, at a density of 105 cells/mL to 2 105 cells/mL, with supernatant containing the vector. Cultures were supplemented with 4 g/mL of polybrene, 20 ng/mL of IL-3, 50 ng/mL of IL-6, and 100 ng/mL of stem cell factor. In preliminary experiments, we determined the cytokine combination that supported the highest transduction efficiency. After 24 hours, the cells were centrifuged and the old supernatant was removed. Freshly prepared viral supernatants supplemented with polybrene and growth factors were added, and cultures were continued for 24 hours. On day 3, cells were harvested for short-term CFU-GM clonogenic assays in methylcellulose (as described below). An aliquot of the.