Ellen van der Schoot

Copy number variation at the FCGR locus includes FCGR3A, FCGR2C and FCGR3B but not FCGR2A and FCGR2B.

Human Fcã receptors (FcãRs) are glycoproteins that bind the Fc region of IgG. The genes encoding the low‐affinity FcãRs are located on chromosome 1q23‐24. Beside single nucleotide polymorphisms (SNPs), gene copy number variation (CNV) is now being recognized as an important indicator for inter‐individual differences. Recent studies on identifying CNV in the human genome suggest large areas at chromosome 1q23‐24 to be involved, and CNV in this region has been associated with manifestations of systemic autoimmune disease. To study both SNPs and CNV of the low‐affinity FcãRs in one assay, we have developed a Multiplex Ligation‐dependent Probe Amplification (MLPA) assay. A novel CNV for FCGR3A was observed. Similar to FCGR3B and FCGR2C, a gene‐dosage effect of FCGR3A was found, that seemed to correlate nicely with the FcãRIIIa expression on NK cells. Next, we delineated the approximate boundaries of CNV at the FCGR locus. Variation in co‐segregation of neighboring FCGR genes was limited to four variants, with patterns of Mendelian inheritance. No CNV of the FCGR2A and FCGR2B genes was observed in over 600 individuals. In conclusion, we report a novel CNV of the FCGR3A gene that correlates with FcãRIIIa expression and function on NK cells. Only FCGR3A, FCGR2C and FCGR3B show CNV, in contrast to FCGR2A and FCGR2B.

The BloodGen project: toward mass-scale comprehensive genotyping of blood donors in the European Union and beyond.

Neil D. Avent, Antonio Martinez, Willy A. Flegel, Martin L. Olsson, Marion L. Scott, Núria Nogués, Martin Písǎcka, Geoff Daniels, Ellen van der Schoot, Eduardo Muñiz-Diaz, Tracey E. Madgett, Jill R. Storry, Sigrid H. Beiboer, Petra A. Maaskant-van Wijk, Inge von Zabern, Elisa Jiménez, Diego Tejedor, Mónica López, Emma Camacho, Goedele Cheroutre, Anita Hacker, Pavel Jinoch, Irena Svobodova, and Masja de Haas

SDF-1–induced actin polymerization and migration in human hematopoietic progenitor cells

OBJECTIVE The capacity of hematopoietic progenitor cells (HPCs; CD34(+) cells) to respond to chemotactic stimulation is essential for their homing efficiency, e.g., during stem cell transplantation. Previous studies established that stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 play an important role in the homing of HPCs. The aim of the present study was to analyze SDF-1-induced actin polymerization and migration of HL-60 cells and primary human CD34(+) cells. MATERIALS AND METHODS SDF-1-induced migration of CD34(+) cells from cord blood (CB) and peripheral blood (PB) across fibronectin-coated filters was measured in a Transwell assay. Actin polymerization was detected using fluorescent phalloidin and analyzed by confocal microscopy and FACS analysis. RESULTS SDF-1 induced a rapid and transient increase in actin polymerization and in polarization of the actin cytoskeleton in primary CD34(+) cells and HL-60 cells. SDF-1 was found to induce significantly more actin polymerization in CB CD34(+) cells that show fast migration in vitro compared to slow migrating PB CD34(+) cells. Moreover, CB CD34(+) cells that had migrated toward SDF-1 showed an elevated and prolonged rise in F-actin upon second exposure to SDF-1 compared to nonmigrated cells, although both cell types expressed equal levels of the SDF-1 receptor CXCR-4. CONCLUSIONS The relatively high migratory capacity of CB-derived human HPCs is not related to cellular polarization or high expression of the SDF-1 receptor but is largely determined by their capacity to efficiently polymerize F-actin in response to SDF-1.

Successful Therapy Reduction and Intensification for Childhood Acute Lymphoblastic Leukemia Based on Minimal Residual Disease Monitoring: Study ALL10 From the Dutch Childhood Oncology Group

PURPOSE Outcome of childhood acute lymphoblastic leukemia (ALL) improved greatly by intensifying chemotherapy for all patients. Minimal residual disease (MRD) levels during the first months predict outcome and may select patients for therapy reduction or intensification. METHODS Patients 1 to 18 years old with ALL were stratified on the basis of MRD levels after the first and second course of chemotherapy. Thereafter, therapy was substantially reduced in patients with undetectable MRD (standard risk) and intensified in patients with intermediate (medium risk) and high (high risk) levels of MRD. Seven hundred seventy-eight consecutive patients were enrolled. The method of analysis was intention-to-treat. Outcome was compared with historical controls. RESULTS In MRD-based standard-risk patients, the 5-year event-free survival (EFS) rate was 93% (SE 2%), the 5-year survival rate was 99% (SE 1%), and the 5-year cumulative incidence of relapse rate was 6% (SE 2%). The safety upper limit of number of observation years was reached and therapy reduction was declared safe.MRD-based medium-risk patients had a significantly higher 5-year EFS rate (88%, SE 2%) with therapy intensification (including 30 weeks of asparaginase exposure and dexamethasone/vincristine pulses) compared with historical controls (76%, SE 6%). Intensive chemotherapy and stem cell transplantation in MRD-based high-risk patients resulted in a significantly better 5-year EFS rate (78%, SE 8% v 16%, SE 8% in controls). Overall outcome improved significantly (5-year EFS rate 87%, 5-year survival rate 92%, and 5-year cumulative incidence of relapse rate 8%) compared with preceding Dutch Childhood Oncology Group protocols. CONCLUSION Chemotherapy was substantially reduced safely in one-quarter of children with ALL who were selected on the basis of undetectable MRD levels, without jeopardizing the survival rate. Outcomes of patients with intermediate and high levels of MRD improved with therapy intensification.

The Bloodgen Project of the European Union, 2003-2009

The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.

Characterization of myeloid leukemia by monoclonal antibodies, with an emphasis on antibodies against myeloperoxidase

Since the last workshop on human leukocyte differentiation antigens, there are 14 well defined cluster-designated (CD) antigens which characterize myelomonocytic cells. Of these, 5 are potentially useful for myeloid leukemia typing (i.e. CD13, CD14, CD15, CD33, CD36) because they are cell lineage-specific and also expressed on immature cells. However, the reactivity of monoclonal anti-CD antibodies, directed against these antigens, with myeloblastic leukemia cells was found to be quite low. We produced monoclonal antibodies against myeloperoxidase. These antibodies react also with promyeloperoxidase, synthesized in HL-60 cell line cells. Monoclonal antimyeloperoxidase was found to be the most sensitive reagent to diagnose acute myeloid leukemia, even more sensitive than cytochemical stains (Sudan black, myeloperoxidase).

Intranasal Administration of Antibody-Bound Respiratory Syncytial Virus Particles Efficiently Primes Virus-Specific Immune Responses in Mice

ABSTRACT Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to the currently licensed palivizumab. While clearly beneficial during primary infections, preexisting antibodies might affect the onset of adaptive immune responses and the ability to resist subsequent RSV infections. Therefore, we addressed the question of how virus neutralizing antibodies influence the priming of subsequent adaptive immune responses. To test a possible role of the neonatal Fc receptor (FcRn) in this process, we compared the responses in C57BL/6 wild-type (WT) and FcRn−/− mice. We observed substantial virus-specific T-cell priming and B-cell responses in mice primed with RSV IgG immune complexes resulting in predominantly Th1-type CD4+ T-cell and IgG2c antibody responses upon live-virus challenge. RSV-specific CD8+ T cells were primed as well. Activation of these adaptive immune responses was independent of FcRn. Thus, neutralizing antibodies that localize to the airways and prevent infection-related routes of antigen processing can still facilitate antigen presentation of neutralized virus particles and initiate adaptive immune responses against RSV.

Dynamin 3 participates in the growth and development of megakaryocytes

High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38lo cells and culture-derived megakaryocytes (MKs). As previously published, three replicate microarray data sets from three different sources of organ donor marrow were analyzed using the software program Rosetta Resolver. After setting a stringent p value of <or=0.001 with a fold change cutoff of three or more in expression level, dynamin 3 (DNM3) was identified to be differentially expressed during the course of MK development with a mean fold-change of 8.2+/-2.1 (mean+/-standard deviation). DNM3 is a member of a family of mechanochemical enzymes (DNM1, DNM2, and DNM3) known for their participation in membrane dynamics by hydrolyzing nucleotides to link cellular membranes to the actin cytoskeleton. Real-time quantitative polymerase chain reaction confirmed that DNM3 increased by 20.7-+/-3.4-fold (n=4, p=0.09) during megakaryocytopoiesis and Western blot analysis showed that DNM3 protein was expressed in human MKs. Confocal microscopy revealed that DNM3 was distributed diffusely throughout the cytoplasm of MKs with a punctate appearance in proplatelet processes. Immunogold electron microscopy also showed that DNM3 is widely distributed in the cytoplasm of MKs, with no apparent localization to specific organelles. The open reading frame of DNM3 was cloned from culture-derived human MKs and determined to be 100% identical to the protein encoded by the DNM3 transcript variant ENST00000367731 published in the Ensemble database. Overexpression of DNM3 in umbilical cord blood CD34+ cells resulted in an increase in total nucleated cells, an amplification of total colony-forming cells and colony-forming unit-megakaryocytes, and a concomitant increase in the expression of nuclear factor erythroid 2 (NF-E2) and beta-tubulin. Together these findings provide the first evidence that a member of the dynamin family of mechanochemical enzymes is present in human MKs and indicate that DNM3 is an excellent candidate for playing an important role in mediating cytoskeleton and membrane changes that occur during MK/platelet development.

CD antigens 2001

The process of categorizing the antigenic molecules and epitopes associated with human white cells, via the collaborative study of monoclonal antibodies, dates back to the early 1980s, when the first HLDA (Human Leukocyte Differentiation Antigen) Workshop was held in Paris. This initial meeting listed only 15 agreed molecular entities, but it created an internationally agreed basis for the nomenclature of leukocyte molecules (the CD scheme), and also provided a forum for reporting studies on their function and practical relevance. A further six HLDA meetings have been held since the first Paris meeting. The most recent of these (“HLDA7”) took place last year in Harrogate, GB, and the proceedings of the meeting will be published later this year (Leucocyte Typing VII, Oxford University Press).

Reference: CD Antigens 2002.

The Proceedings of the 7th Human Leukocyte Differentiation Antigen (HLDA) Workshop are about to be published, detailing more than 80 new CD specificities. The next Workshop, planned for 2004, will continue this process, and a number of candidate CD molecules in the literature, identified by antibody