Taurine antibodies may have been elicited by GFP released from transduced cells, but were unable to produce a cytolytic reaction against intracellular GFP within viable blood and marrow cells. It is also possible that there was insufficient expression of GFP in the cells that persisted due to transcriptional silencing, and to allow recognition by antibodies or T cells. We were unable to detect GFP expression in PBMCs from the recipient with the highest levels of GFP gene marking by either flow cytometry or reverse trancription PCR. Higher levels of GFP expressingcells could lead to more vigorous immune responses that do lead to elimination of cells expressing the foreign transgene. In conclusion, these studies begin to establish a novel clinically phospholipases acceptable method to achieve engraftment of transduced HSC and persistence of cells expressing a new gene product. Further studies with higher dosages of fludarabine and, ideally, higher levels of transduced HSC, may allow exploration of the potential to achieve immune tolerance with a nonmyeloablative, but immune ablative conditioning regimen.At 2 months postnatal age monkeys were sedated with telazol for marrow collection.
The iliac crest was shaved, lidocaine infused, and the site was aseptically prepared using standard protocols.38 Approximately 10 ml of bone marrow was berberine collected into heparinized syringes using sterile technique. CD34 cells were isolated and cryopreserved using a controlled rate cryopreservation protocol as described below. At 3 months postnatal age, monkeys were sedated with telazol and supplemented with ketamine for busulfan infusions. Busulfan was administered i.v. in a 20 ml volume over a 2 hour infusion period at each of the dosages studied. The dosages were calculated based on surface area by utilizing body weight and crown rump length and a published surface area formula.39 Preinfusion peripheral blood samples were collected, and an indwelling i.v. catheter was placed. After administering a prophylactic dose of phenytoin, the busulfan infusion was initiated using a Baxter FLO GARD 6200 Volumetric Infusion Pump. At the end of the 2 hour everolimus busulfan infusion period, the indwelling catheter was removed, a postinfusion dose of dilantin administered, and blood samples were collected from a peripheral vessel at 0.5, 1, 3, and 4 hours postinfusion to determine busulfan levels.
Plasma was collected, frozen at 80, and then shipped frozen for analysis at the Clinical Chemistry Laboratory, Department of Pathology, Childrens Hospital Los Angeles. Plasma busulfan concentrations were determined as previously described11 and the AUC was calculated using trapezoidal estimation. Typically, the first busulfan infusion was performed on Monday and the second infusion performed on Wednesday. Fludarabine was reconstituted in 1 ml of sterile water and diluted in sterile saline to obtain 75, 87.5, and 100 mg/m2 doses. For animals that received fludarabine and busulfan, an i.v. injection of fludarabine was given prior to busulfan infusion on the first and third days, and fludarabine was administered alone on the second day under ketamine. CD34 cell isolation, transduction, and transplantation. CD34 marrow cells were isolated using the mini MACS immunomagnetic separation system.