http://repub.eur.nl/res/aut/1907/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/34562/ 2011-12-01T00:00:00Z Introduction: Microscopic differential analysis of leukocytes is a time-consuming activity for routine diagnostic laboratories. The criteria used to decide whether a manual differential should be performed should therefore be as strict as possible. The goal of this investigation was to give recommendations for the use of the left shift (LS) 1+ flag, which signals the presence of band neutrophils. Methods: The LS1+ flag of the ADVIA 120 and 2120 hematology analyzers was evaluated in 6 peripheral hospital laboratories in the Netherlands. In 2683 samples with exclusively a LS1+ flag, the percentage of band neutrophils were determined microscopically. A set of photographs of neutrophils were used to examine the differences between laboratories in the assessment of band cells. Results: In 18% of all samples with only a LS1+ flag, 5% or more band neutrophils were found. However, this percentage differed greatly between laboratories, as did the proportion of samples that received a LS1+ flag. Several factors were found to influence the amount and accuracy of the LS1+ alarm, i.e. band neutrophil counting by microscopists, specificity of request for leukocyte differentials, percentage of general practitioners requesting a leukocyte differential, and sample storage. Based on these findings, a number of recommendations were formulated. Conclusion:; Critical control of the factors influencing the LS1+ flag can significantly decrease the number of microscopic samples to be reviewed and may be valuable for every laboratory performing routine differentials, using any type of hematology analyzer. http://repub.eur.nl/res/pub/24568/ 2009-04-24T00:00:00Z BCR-ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR-ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR-ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR-ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR-ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR-ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR-ABL immunobead assay detects all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within ∼4h, and can be run in parallel to routine immunophenotyping. http://repub.eur.nl/res/pub/19345/ 2009-01-15T00:00:00Z Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a-/- HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis. http://repub.eur.nl/res/pub/35185/ 2007-10-01T00:00:00Z http://repub.eur.nl/res/pub/36301/ 2007-04-01T00:00:00Z The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rγ), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rγ had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rγ may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth. http://repub.eur.nl/res/pub/13983/ 2006-02-28T00:00:00Z Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands. http://repub.eur.nl/res/pub/7126/ 2005-11-30T00:00:00Z The human body is under constant siege of pathogens - bacteria, viruses, fungi and parasites. We can only survive because these attackers are continuously fought off by our immune system. Important tasks within the immune system of vertebrates are performed by T lymphocytes, the executors of speci.c, cellular immunity. The speci.city of T lymphocytes lies in their T-cell receptor (TCR), through which they sense the presence of antigens in their environment. Each T cell expresses a TCR with a unique antigen-recognition site, so all T cells together can respond to an enormous variety of antigens. The highly diverse T-cell repertoire is generated by random recombination of discrete TCR gene segments. Via the TCR, T cells recognize peptide antigens that are displayed by antigen presenting cells (APCs), in the context of major histocompatibility complex (MHC) class I or class II molecules. Mature T cells carry out their function in cellular immunity as either CD8+ cytotoxic T cells or as CD4+ helper T cells. The humoral part of the speci.c immune system is supplied by B lymphocytes, which can secrete their antigen receptors in the form of antibodies. T cells develop from multipotent precursors via a highly ordered, but complex differentiation pathway. A number of critical events occur during this T-cell development process. Cells proliferate, adopt a T-cell fate, and produce a TCR molecule via a strictly ordered process of gene rearrangements. Stringent selection processes make sure that the produced TCR molecule is self-MHC restricted but not reactive to self-antigens. Finally, the selected T cells are allowed to mature into functional effector T cells. To become highly differentiated and thoroughly ‘educated’ cells, T cells need a specialized microenvironment for their development. The organ which has evolved to ful.ll this task and which also gave T cells their name, is the Thymus. The hematopoietic cells that undergo T-cell development in the thymus are called thymocytes. The importance of the thymus as essential microenvironment for T-cell development is illustrated by children af.icted by the DiGeorge syndrome, who sometimes completely lack a thymus. These children have severely reduced T cell numbers and suffer from recurrent life-threatening infections. DiGeorge syndrome also illustrates the fact that T cells are the only hematopoietic cell type that absolutely require the thymus for their development. Although human T-cell development is the main theme of this thesis, most of the published knowledge about T-cell differentiation has been acquired by studies in the mouse. http://repub.eur.nl/res/pub/8406/ 2005-01-01T00:00:00Z To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin- cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Ddelta2-Ddelta3 rearrangement occurs at the most immature CD34+CD38-CD1a- stage. TCRB rearrangement starts at the CD34+CD38+CD1a- stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTalpha) expression pattern show that human TCRbeta-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before. http://repub.eur.nl/res/pub/10281/ 2004-01-01T00:00:00Z The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor beta-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/beta-catenin/TCF pathway in the most immature (CD4-CD8-CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (approximately 30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/beta-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor pool. http://repub.eur.nl/res/pub/9637/ 2001-01-01T00:00:00Z Initiation of gene transcription by transcription factors (TFs) is an important regulatory step in many developmental processes. The differentiation of T cell progenitors in the thymus is tightly controlled by signaling molecules, ultimately activating nuclear TFs that regulate the expression of T lineage-specific genes. During the last 2 years, significant progress has been made in our understanding of the signaling routes and TFs operating during the earliest stages of thymic differentiation at the CD4(-)CD8(-) double negative stage. Here we will review the TF families that play an important role in differentiation of thymocytes, particularly focusing on recent new information with respect to the Tcf, bHLH, GATA, and CBF/HES TF families.