The Coincidence of Cervical Ribs and Syringomyelia

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Measurement of Intracellular Fluorescence of Human Monocytes Relative to Oxidative Metabolism

Human monocytes (MN) produce O2â and H2O2 when stimulated by agonists. Dichlorofluorescin diacetate (DCFHâ DA) has been used as a substrate for measuring intracellular oxidant production in neutrophils. DCFHâ DA is hydrolyzed by esterases to dichlorofluorescin (DCFH), which is trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by action of cellular oxidants. DCFHâ DA can not be appreciably oxidized to a fluorescent state without prior hydrolysis. We have examined the utility of DCFHâ DA for the assessment of monocyte oxidative responses. The levels of intracellular fluorescence measured by flow cytometry were considerably less than expected from reported levels of O2â â production or chemiluminescence assays. Compared with neutrophils, monocytes produced minimal increases in DCF fluorescence after stimulation with phorbol myristate acetate as measured by flow cytometry, but both cell types showed increases in fluorescence when bulk cell suspensions were measured by spectrofluorometry. To determine the intracellular location of the DCFH, bulk fluorescence measurements were made on both whole and sonicated cell preparations. When intact mononuclear cells were preloaded with DCFHâ DA, then sonicated and oxidized with added excess H2O2, the increase in fluorescence was only 30% of the fluorescence of mononuclear cell sonicates to which DCFHâ DA was added and oxidized in a similar manner. These results suggest that a portion of the DCFHâ DA incorporated by intact cells, is not susceptible to oxidation by the added H2O2. Addition of NaOH to induce hydrolysis of any residual DCFHâ DA in the sonicates of DCFHâ DAâ loaded intact mononuclear cells resulted in a further increase in fluorescence upon addition of H2O2, suggesting that a significant portion of the DCFHâ DA was not hydrolyzed despite ample uptake of this dye by these cells. In contrast, no further increase in fluorescence was observed in sonicates of DCFHâ DAâ loaded intact neutrophils, suggesting complete hydrolysis of all incorporated DCFHâ DA to DCFH. When monocytes were allowed to phagocytose DCFHâ DAâ loaded Staphylococcus aureus, intracellular fluorescence was measurable by flow cytometry, indicating intracellular oxidation of the fluorochromes. We therefore propose that in monocytes the mechanism of intracellular processing of these fluorochromes differs from that in neutrophils owing to differences in intracellular localization of fluorochromes, site of oxidant production, and/or accessibility of the DCFHâ DA to esterolysis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141433/1/jlb0304.pd

Nervous and mental diseases /

Includes bibliographical references and indexes.Mode of access: Internet

Relationship of differential gene expression profiles in CD34+ myelodysplastic syndrome marrow cells to disease subtype and progression

Microarray analysis with 40 000 cDNA gene chip arrays determined differential gene expression profiles (GEPs) in CD34+ marrow cells from myelodysplastic syndrome (MDS) patients compared with healthy persons. Using focused bioinformatics analyses, we found 1175 genes significantly differentially expressed by MDS versus normal, requiring a minimum of 39 genes to separately classify these patients. Major GEP differences were demonstrated between healthy and MDS patients and between several MDS subgroups: (1) those whose disease remained stable and those who subsequently transformed (tMDS) to acute myeloid leukemia; (2) between del(5q) and other MDS patients. A 6-gene “poor risk” signature was defined, which was associated with acute myeloid leukemia transformation and provided additive prognostic information for International Prognostic Scoring System Intermediate-1 patients. Overexpression of genes generating ribosomal proteins and for other signaling pathways was demonstrated in the tMDS patients. Comparison of del(5q) with the remaining MDS patients showed 1924 differentially expressed genes, with underexpression of 1014 genes, 11 of which were within the 5q31-32 commonly deleted region. These data demonstrated (1) GEPs distinguishing MDS patients from healthy and between those with differing clinical outcomes (tMDS vs those whose disease remained stable) and cytogenetics [eg, del(5q)]; and (2) molecular criteria refining prognostic categorization and associated biologic processes in MDS