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We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City, CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with r higher than 0.98 and a sensitivity of 0.1% proved reproducible.

Cells from the human leukemia cell line HL-60 undergo terminal differentiation when exposed to inducing agents. Differentiation of these cells is always accompanied by withdrawal from the cell cycle. Here we describe the isolation of a cDNA encoding a novel serine protease that is present in HL-60 cells and is down-regulated during induced differentiation of these cells. We have named this protease myeloblastin. Down-regulation of myeloblastin mRNA occurs with both monocytic and granulocytic inducers. Myeloblastin mRNA is undetectable in fully differentiated HL-60 cells as well as in human peripheral blood monocytes. We found that regulation of myeloblastin mRNA in HL-60 cells is serum dependent. Inhibition of myeloblastin expression by an antisense oligodeoxynucleotide inhibits proliferation and induces differentiation of promyelocyte-like leukemia cells.

References1 Huntly BJP, Reid AG, Bench AJ, Campbell LJ, Telford N, Shepherd P, Szer J, Prince HM, Turner P, Grace C, Nacheva EP, Green AR. Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia. is an auto-immune disease where the stroma usually functions normally, damage of the microenvironment has been observed in a subset of SAA patients. 5 We report here a 68-year-old woman patient with end stage SAA refractory to administration of growth factors, cyclosporine A, antithymocyte globuline and ineligible for an allogeneic hematopoietic stem cell transplantation (HSCT). This patient received two infusions of allogeneic MSC, in order to improve a possible defective BM stroma, to stimulate hypothetical residual HSC by local production of Correspondence: L Fouillard,

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In acute myeloid leukaemia (AML) initiating pre-leukaemic lesions can be identified through three major hallmarks: their early occurrence in the clone, their persistence at relapse and their ability to initiate multilineage haematopoietic repopulation and leukaemia in vivo. Here we analyse the clonal composition of a series of AML through these characteristics. We find that not only DNMT3A mutations, but also TET2, ASXL1 mutations, core-binding factor and MLL translocations, as well as del(20q) mostly fulfil these criteria. When not eradicated by AML treatments, pre-leukaemic cells with these lesions can re-initiate the leukaemic process at various stages until relapse, with a time-dependent increase in clonal variegation. Based on the nature, order and association of lesions, we delineate recurrent genetic hierarchies of AML. Our data indicate that first lesions, variegation and treatment selection pressure govern the expansion and adaptive behaviour of the malignant clone, shaping AML in a time-dependent manner.

The human GATA1, hGATA1 (previously called NF‐E1, GF‐1 or Eryf‐1), a major sequence‐specific DNA‐binding protein of the erythrocytic lineage, is a member of a zinc‐finger family of DNA‐binding proteins. We report here the cloning of a human cDNA for a new member of this family. This member, called hGATA3, has 85% amino acid homology with hGATA1 in the DNA‐binding domain and no homology elsewhere in the protein. Unlike hGATA1, hGATA3 is not localized on the X chromosome and we map it to the 10p15 band of the human genome. Northern blot analysis indicates that this factor is a T‐cell specific transcription factor, present before activation and up‐regulated during T‐cell activation. The encoded hGATA3 protein, made in an in vitro transcription‐translation assay, binds the WGATAR motif present in the human T‐cell receptor (TCR) delta gene enhancer and, by transfection in HeLa cells, we show that hGATA3 can transactivate this TCR delta gene enhancer. Interestingly this enhancer binds and is also transactivated by hGATA1. Conversely, the promoter of the human glycophorin B (GPB), which is erythroid‐specific and contains two WGATAR motifs, binds and is transactivated by hGATA1 and, to a lesser extent, by hGATA3. These results indicate that the activation of specific genes by hGATA1 or hGATA3 is partly governed by the lineage expression of these two factors during haematopoiesis and that, in the T‐cell lineage, hGATA3 binds the human TCR delta gene enhancer and is involved in its expression.

GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors.