MPD in the mouse BM transplant model. BM cells were collected from C57/B6 SJL mice 2 d after 5 fluorouracil treatment. Lin? cells were purified, cultured for 2 d with IL 3, SCF, IL 6, and TPO, infected twice with the viral particles containing empty vector, CSF3Rwt, or CSF3RT617N, AZD8330 ARRY-424704 and finally injected intravenously into lethally irradiated C57BL6 mice. After 5 wk, mice were sacrificed and the total number of cells or the percentage of cells including granulocytes, B lymphocytes, or T lymphocytes was measured in CD45. 2 donor mouse cells in the blood, BM, and spleen. Spleen from Migr engrafted, CSF3Rwt engrafted, and CSF3RT617N engrafted mice. The results are the means SD of four mice of each group from two independent experiments, P 0. 05 compared with Migr engrafted mice. JEM VOL.
206, August 3, 2009 1705 BRIEF DEFINITIVE REPORT but also induces a mobilization of hematopoietic progenitors. Thus, in contrast to the truncating mutations found in severe congenital neutropenia, the CSF3RT617N mutation does not confer a major HSC proliferative advantage but rather increases myeloid differentiation. CAY10505 In conclusion, the family described in this report is the first case of germline CSF3R mutation leading to a chronic neutrophilia and mobilization of hematopoietic progenitors, a phenotype similar to that induced by administration of rhG CSF. The T617N TM mutation induces both proliferation and granulocytic differentiation. Progression to a myelodysplastic syndrome malignant hemopathy was observed on 1 out of 12 affected patients.
The precise role of the acquired CSF3R mutations found in severe congenital neutropenia is not completely resolved. Nevertheless, their association with leukemic progression is increasingly strong. Therefore, more investigations are necessary to know if CSF3RT617N mutation predisposes to malignant myeloid hemopathies. MATERIALS AND METHODS Patient cells. Blood samples from patients, normal subjects, and G CSF mobilized patients were collected after informed consent was obtained. The study was approved by the Local Research Ethics Committee from the H?el Dieu and the Henri Mondor hospitals, and informed consent was obtained from each patient in accordance with the Declaration of Helsinki. Purification and in vitro differentiation of CD34 cells.
Mononuclear cells were separated over a Ficoll density gradient, and CD34 cells were purified by a double positive magnetic cell sorting system according to manufacturer,s recommendations. CD34 cells were either plated for 15 d in methylcellulose in the presence of 25 ng/ml SCF and G CSF, or amplified for 21 d in granulocytic conditions in serum free liquid medium containing IMDM with penicillin/streptomycin/glutamine, ? thioglycerol, BSA, a mixture of sonicated lipids, and insulin transferrin in the presence of recombinant human cytokines. Western blot analysis. After purification, CD34 cells were deprived in IMDM alone for 4 h in the presence or not of JAK2 inhibitor at 1 M. AZD1480 was a gift from AstraZeneca. Cells were stimulated by 20 ng/ml G CSF for different intervals, washed in PBS, and lysed in denaturing loading dye buffer. Western blot analysis was performed using conventional techniques using anti Jak2, anti STAT3, anti STAT5, anti ERK p42 p44, and anti AKT antib