Retroviral vector-mediated gene transfer into umbilical cord blood-derived megakaryocyte and platelet progenitors.

Optimizing platelet engraftment following hematopoietic stem cell transplantation is essential for minimizing transplant-related morbidity, particularly following umbilical cord blood transplantation (UCBT), where platelet engraftment frequently takes >60 days. One strategy for optimizing platelet engraftment following UCBT is to study or alter the genetic program of megakaryocyte/platelet (Mk/plt) progenitors. Retroviral vector gene transfer has previously proven useful for studying the biology of hematopoietic stem cells; however, procedures for transducing UCB cells of the Mk/plt lineage with retroviral vectors have not been described. We report here that Mk/plt progenitors generated from UCB progenitors can be efficiently transduced with retroviral vectors. Transduced Mk/plt cells were identified and quantitated by expression of a vector transgene encoding a truncated version of the human nerve growth factor receptor (NGFR). Vector-mediated NGFR expression could be readily detected in Mk/plt progenitors defined by immunophenotypic, morphologic, and functional criteria. In addition, NGFR expression persisted in mature anucleate platelets generated from the transduced Mk/plt progenitors. These methods may be useful for introducing genetic elements into Mk/plt progenitors to study various aspects of platelet development and biology and for marking ex vivo expanded Mk/plt progenitors to determine their contribution to engraftment.

Erythrocyte autoantibodies in paediatric patients with sickle cell disease receiving transfusion therapy: frequency, characteristics and significance.

The formation of erythrocyte autoantibodies following transfusion therapy has been described in case reports and small series. To determine the frequency, serological characteristics, and clinical significance of this phenomenon in paediatric patients with sickle cell disease, we analysed the patient database at the Duke University Pediatric Hematology Clinic. We identified children who received multiple erythrocyte transfusions, then reviewed clinical records to identify children who developed erythrocyte autoantibodies in association with transfusions. Among 184 paediatric patients who received multiple erythrocyte transfusions, 14 children (7.6%) developed warm (IgG) erythrocyte autoantibodies. Median transfusion exposure at the time of autoantibody formation was 24 erythrocyte units, range 3-341 units. The autoantibody reacted as a panagglutinin in 11 cases but had anti-e specificity in three patients. Surface complement also was detected in five patients. Clinically significant haemolysis was documented in four patients, each of whom had both surface IgG and C3 detected. The development of erythrocyte autoantibodies was associated with the presence of erythrocyte alloantibodies. Formation of warm erythrocyte autoantibodies in association with transfusions is not rare in paediatric patients with sickle cell disease. Clinicians should be aware of this complication and recognize that the presence of surface C3 is often associated with significant haemolysis.

Relapsed acute lymphoblastic leukemia: similar outcomes for autologous and allogeneic marrow transplantation in selected children.

The therapy of choice for relapsed childhood acute lymphoblastic leukemia is controversial. We retrospectively compared the outcome of 57 patients who received autologous bone marrow transplantation (BMT) with 17 patients who underwent allogeneic BMT for B cell lineage acute lymphoblastic leukemia after at least one marrow relapse. The allogeneic BMT cohort included only those who would also have been eligible for autologous BMT had they not had a matched sibling donor. Specifically, patients who were not in complete remission, those with T cell positive leukemia, t(9;22) or those with only an extramedullary relapse were excluded from both groups. Conditioning regimens included total body irradiation and chemotherapy. Age, white blood count at diagnosis, and duration of first and longest complete remissions were comparable for the two groups. The median follow-up of the event-free survivors was 4.8 years for those who received an autologous BMT (n = 26) and 4.6 years for those who received an allogeneic BMT (n = 8). The relapse rate was higher in the autologous BMT group and the incidence of non-leukemic deaths higher in the allogeneic BMT group. Event-free survival at 3 years was comparable for the two groups (47% +/- 7 vs 53% +/- 12, autologous vs allogeneic, respectively; P = 0.77). Based upon these findings, we concluded that the outcome for autologous BMT was equivalent to allogeneic BMT for relapsed childhood B cell lineage acute lymphoblastic leukemia in selected clinical situations.

Flunarizine enhancement of melphalan activity against drug-sensitive/resistant rhabdomyosarcoma.

Flunarizine, a diphenylpiperazine calcium channel blocker, is known to increase tumor blood flow. It also interferes with calmodulin function, repair of DNA damage and drug resistance associated with P-glycoprotein. Flunarizine was tested for its ability to modulate either cyclophosphamide- or melphalan-induced growth delay for a drug-resistant rhabdomyosarcoma xenograft (TE-671 MR) and the drug-sensitive parent line (TE-671), in which P-glycoprotein is not involved in the mechanism of drug resistance. Tumour blood flow was increased by 30% after a flunarizine dose of 4 mg kg-1, but no modification in growth delay was induced by melphalan (12 mg kg-1). In contrast, a 60 mg kg-1 dose of flunarizine had no effect on tumour blood flow, but the same dose created significant enhancement in melphalan-induced tumour regrowth delay in both tumour lines. The dose-modifying factor for flunarizine as an adjuvant to melphalan was approximately 2 for both tumour lines. Although blood flow measurements were not performed with the combination of flunarizine and melphalan, the results from flunarizine alone suggested that augmentation of melphalan cytotoxicity is not mediated by changes in blood flow. In contrast, flunarizine did not affect drug sensitivity to cyclophosphamide in groups of animals bearing the drug-sensitive parent tumour line. These results suggest that the mechanism of drug sensitivity modification by flunarizine is not related to modification of tumour blood flow, but may be mediated by modification of transport mechanisms that are differentially responsible for cellular uptake and retention of melphalan as compared with cyclophosphamide.