Myelodysplastic syndrome is not merely "preleukemia".

Megakaryocytes (Mks) mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. As the sites for platelet release have higher levels of oxygen tension (pO2) than the core of the BM where stem and progenitor cells reside, we investigated whether pO2 influences Mk maturation. Mks were generated from CD34+ cells (from mobilized peripheral blood from cancer patients) under 5% and 20% O2. At day 15, CD41+ Mk expansion in 20% and 5% O2 cultures was 85-fold and 31-fold respectively. Twenty percent O2 cultures also had higher levels of high ploidy (≥ 8N, eightfold higher) and proplatelet-forming (fivefold higher) Mks. At day 21, 20% O2 cultures had a fivefold higher number of apoptotic Mks. In contrast, 5% O2 promoted Mk colony-forming unit (CFU-Mk) generation and maintenance. Similar results were observed in cultures initiated with CD41+ Mks, indicating that pO2 directly affects Mks. The change from 20% to 5% O2 on day 5 and day 7 delayed both maturation and apoptosis, suggesting that these two processes are closely linked. These results were confirmed in CD34+ cultures from normal BM samples. These data may provide insights into in vivo Mk maturation, such as an explanation for hypoxia-induced thrombocytopenia in animals.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2141.2000.02457.x

Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes

Mobilization of hematopoietic stem cells

Bacterial superinfections are an important cause of morbidity and mortality during influenza A virus (IAV) epidemics. We demonstrate that incubation with the combination of IAV and Streptococcus pneumoniae caused marked reductions in survival of neutrophils in vitro compared with treatment with control buffer or IAV or S. pneumoniae alone. This cooperative effect was in part mediated by acceleration of neutrophil apoptosis as evidenced by increases in annexin-V binding and caspase-3 activation. However, GM-CSF did not increase survival of neutrophils exposed to IAV and S. pneumoniae. IAV enhanced neutrophil uptake of S. pneumoniae significantly. Furthermore, the combination of IAV and S. pneumoniae caused significantly more hydrogen peroxide production than IAV or S. pneumoniae alone. This increased respiratory burst activity contributed to the diminished neutrophil survival caused by IAV and S. pneumoniae. The NADPH oxidase inhibitor, diphenyleneiodonium, significantly improved survival of neutrophils treated with IAV and S. pneumoniae. These findings may help to explain the increased susceptibility of IAV-infected patients to infections with S. pneumoniae.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1189/jlb.69.1.50

Neutrophil survival is markedly reduced by incubation with influenza virus and Streptococcus pneumoniae: role of respiratory burst.

BACKGROUND: To study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin-V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death. METHODS: Apoptosis was induced in Jurkat cells by gamma-irradiation, incubation with camptothecin (CPT), or cytosine beta-D-arabinofuranoside (Ara-C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze-thaw procedure. Various AnV/PI- CD34+ fractions were cultured in a single-cell single-well (SCSW) assay. RESULTS: Jurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI- cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80-90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI- fractions (overall range 23-62%). Within total AnV+ and AnV+/PI- populations, only a minority of CD34+ cells showed ISEL positivity (range 4-8% and 0.8-6%, respectively). Different fractions of AnV+/PI- CD34+ cells did have clonogenic capacity. CONCLUSIONS: PCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo-nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV-fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI- CD34+ cells in the SCSW assay.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.10028

The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique.

Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma

N. D. Mckelvie

Papers

Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma