DNA-Dependent Protein Kinase

After several rinses in PBS, labeled cells were analyzed in a flow cytometer at 488 nm

After several rinses in PBS, labeled cells were analyzed in a flow cytometer at 488 nm. Quantitative Analysis To quantify the graft in monkeys grafted with Teijin compound 1 test, defining statistical significance as value less than 0.05. AR in all monkeys. This increase was variable in intensity, and preceded, coincided or followed the histological evidence of AR (focal accumulations of lymphocytes) and/or the loss of myofibers of donor origin, and remained until the end of the follow-up (up to 8 weeks after tacrolimus withdrawal). Conclusions Flow cytometry detection of de novo circulating antibodies against the donors cells was consistently associated with AR. A clear increase in this antibody detection indicated current or recent AR. Smaller increases in comparison to the preimmunosuppression values were not associated with AR. Transplantation of myogenic cells (that is, mononuclear cells with the capacity to form multinucleated myofibers) has potential applications in the treatment of skeletal S1PR4 muscle diseases.1-4 Excluding autologous transplantation, graft viability depends on the control of acute rejection (AR). Because the permanent control of AR is not fully guaranteed in clinical practice due to the limits imposed by the toxicity of the immunosuppression drugs, monitoring of AR is essential to treat it if it occurs, so as to preserve the graft. We previously defined in nonhuman primates the histological features of AR in muscle biopsies after allotransplantation of myogenic cells.5 However, an aspect that until now has not deserved specific studies is the humoral response in this context. Flow cytometry detection of circulating antidonor cell antibodies was used since the early studies of myogenic cell transplantation in mice,6-9 dogs,10 monkeys,11-13 and human patients14-17 to determine the existence or not of AR. However, we lack elements to affirm that there is a relationship between AR and the detection of circulating antidonor cell antibodies in this context. In the present study, we wanted to contribute in understanding the value of circulating antidonor cell antibodies in the diagnosis of AR of the myofibers formed by the allotransplantation of myogenic cells, using nonhuman primates.18,19 To induce rejection of myofibers, we immunosuppressed monkeys of the genus with optimal levels of tacrolimus for 4 weeks (to allow complete myofiber formation by the grafted cells) and then we discontinued tacrolimus administration Teijin compound 1 to trigger AR. To monitor the graft by histology in some monkeys, we labeled the cells with a reporter gene. To confirm Teijin compound 1 that the immune findings were due to the allogeneic context and not to the expression of ?-galactosidase (?-Gal), we grafted in other monkeys cells with no genetic modification. To monitor the graft in this case, we transplanted cells from male donors into females and we detected the Y chromosome in the cell-grafted muscles by polymerase chain reaction (PCR). Cells used for transplantations were the only ones that so far proved to be myogenic in nonhuman primates and clinical trials, that is, satellite cell-derived myoblasts.3 For sake of simplicity, the word muscle in the rest of the article will be used as equivalent of skeletal muscle. MATERIALS AND METHODS Animals Seven cynomolgus monkeys (reporter gene and previously obtained in our laboratory from a cynomolgus monkey. Another cell line was proliferated without genetic manipulation from a muscle biopsy performed in one of the male monkeys included in the study (Table ?(Table1,1, monkey 3). In both cases, muscle samples were minced with fine scissors into fragments of less than 1 mm3 and then dissociated with 0.2% collagenase (Sigma, St. Louis, MO) in Hank balanced salt solution (HBSS) (Gibco, Grand Island, NY) for 1 hour, followed by another dissociation in 0.125% trypsin (Gibco) in HBSS for 45 minutes. The cells were subcultured in molecular, cellular, and developmental biology-120 culture medium20 with 15% fetal bovine serum (FBS) (Hyclone, Logan, UT), 10 ng/mL basic fibroblast growth factor (Feldan, St Laurent, Canada), 0.5 mg/ml bovine serum albumin (Sigma), 1.0 M dexamethasone (Sigma), and 5 g/mL human insulin (Sigma). ?-Gal + cells were produced by in vitro infection with a replication-defective retroviral vector LNPoZC721 (gift from Dr. Constance Cepko, Harvard University, Boston, MA), encoding.