Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest, as it autonomously generates the first adult type hematopoietic stem cells (HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, we tested whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Our explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells (as was previously suggested). Rather, it is due to instructive signaling from ventral tissues. Furthermore, we identify Hedgehog protein(s) as an HSC inducing signal.
delayed in SDS neutrophils. This is the first study revealing a molecular defect, namely defective F-actin cytoskeletal remodeling, which may contribute to impaired chemotaxis in SDS patients. Design and Methods Neutrophil isolation and cell cultureBlood was obtained after informed consent of healthy volunteers (26-43 years) and SDS patients (2-29 years; all with mutations 183-184 A>CT [K62X]/IVS2 258+2T>C [C84fs]). The study was approved by the Medical Ethics Committees of the AMC Hospital and the institutional review board and was in accordance with the Declaration of Helsinki. Neutrophils were isolated as described. 8 PLB-985 and GFP-Rac2 PLB-985 17 cells were cultured in RPMI/10% FCS/penicillin (200 µg/mL)/streptomycin (200 µg/mL)/ L-glutamine (4 mM). PLB-985 cells were differentiated with 0.5% dimethylformamide (DMF; Sigma-Aldrich) for 5-7 days. Cellular spreading and immunofluorescenceNeutrophils or PLB-985 cells in Hepes medium (132 mM NaCl, 20 mM Hepes, 6mM KCL, 1 mM MgSO4, 1.2 mM K2HPO4, 1 mM CaCl2, 5 mM Glucose, 2.5% human albumin (Cealb; Sanquin reagents)) were seeded on fibronectin-coated coverslips and allowed to adhere for 30 min. Cells were stimulated with 20nM fMLP, fixed with 4% paraformaldehyde/PBS and processed for immunofluorescence staining as described. 18 Antibodies used: SBDS (rabbit polyclonal), Phalloidin-Alexa488 or BODIPY conjugated. Pictures were taken with a Zeiss LSM510 microscope with a Zeiss 65x oil lens and processed with LSM 510 software. Neutrophils (2×10 6 /mL) in Hepes medium were stimulated with 100nM fMLP or 40nM C5a. At indicated time points 2×10 5 cells were harvested and fixed with buffer 1 (Intraprep Permeabilization Reagent Kit; Immunotech), permeabilized with buffer 2 and stained with Phalloidin-Alexa488 (Molecular Probes, Invitrogen). Cells were analyzed on LSRII (BD Biosciences) flow cytometer for F-actin content. F-actin polymerization and cellular polarizationTo determine F-actin polarization, neutrophils were embedded with Mowiol 4-88 and analyzed by confocal microscopy. For each experiment at least 50 cells per time-point were analyzed in a blind fashion. Results and Discussion SBDS co-localizes with F-actin and Rac2 in human neutrophilsTo investigate a possible link between SBDS and the actin cytoskeleton, we performed immunofluorescence stainings in neutrophils and PLB-985 cells, with an anti-SBDS antibody that we have generated and which specifically recognized SBDS (Online Supplementary Figure S1A/B). In resting human neutrophils we observed that SBDS is localized prominently to the nucleus and to a lesser extent to the cytoplasm (Online Supplementary Figure 1C). However, in these resting cells there is not much polymerized F-actin cytoskeleton detectable (data not shown) and therefore we set out to investigate SBDS in relation to the F-actin cytoskeleton in activated neutrophilic cells. Human peripheral blood neutrophils were allowed to adhere to fibronectin and were subsequently stimulated with fMLP. These activated cells displayed Factin enriched c...
During the follow-up period (mean = 22 months; range = 12-54 months) cholesteatoma recidivism was observed in 6% of the patients (four ears), mostly in young patients (three ears). An acceptably dry ear (Merchant grade 0-1) was achieved in 96% of all cases. The remaining 4% of cases scored a Merchant grade 2. Overall, both air conduction thresholds and air bone gap were slightly lowered when comparing post-operative values to pre-operative values and significantly in the case of ossicular reconstruction. In none of the patients (0%) did post-operative wound infections occur.
BackgroundShwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified.Methodology/Principal FindingsWe observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle.Conclusions/SignificanceSBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34+ hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained.
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