We performed flow cytometric analysis of CD34+ cell apoptosis in 59 patients with myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) secondary to MDS (MDS‐AML) using annexin V‐FITC, which binds to exposed phosphatidylserine on apoptotic cells. Apoptosis was significantly increased in FAB subtypes RA, RARS and RAEB (<10% blasts) (56.5% (15.1–86.5%)) compared to normal controls (18.5% (3.4–33.4%), P < 0.0001) and RAEB‐t/MDS‐AML (16% (2.1–43.2%), P < 0.0001). There was no correlation between % apoptosis, Full blood count or cytogenetics in any disease category. Two‐colour cytometric analysis of permeabilized CD34+ cells stained with antibodies to Bcl‐2, Bcl‐X (anti‐apoptotic), Bax and Bad (pro‐apoptotic), demonstrated significantly higher ratios of pro‐ v anti‐apoptotic proteins in early MDS (2.47 (1.19–9.42) compared to advanced disease (1.14 (0.06–3.32), P = 0.0001). Moreover, using repeated measures of variants (ANOVA), we found that variations between individual Bcl‐2‐related proteins differed significantly according to disease subtype (P < 0.0005). Our results confirm that CD34+ cell apoptosis was significantly increased in MDS subtypes RA and RARS and fell with disease progression. Early MDS was also associated with a significantly higher CD34+ cell pro‐ v anti‐apoptotic Bcl‐2‐family‐protein ratio than advanced disease. Furthermore, patterns of expression of individual Bcl‐2 related proteins differed significantly between different disease categories. However, no correlation between pro‐ v anti‐apoptotic Bcl‐2‐family‐protein ratios and the degree of apoptosis was observed.
There is increasing interest in the role of blood polymorphonuclear leukocytes (PMNs) in the pathogenesis of sickle cell crisis. We studied the adherence of PMNs from 18 sickle cell patients in crisis, 25 out of crisis, and 43 healthy subjects (controls) to monolayers of human umbilical cord endothelium that were either untreated or pretreated with tumor necrosis factor α (TNFα). Overall, the PMNs from patients in crisis were more adherent than control PMNs to untreated endothelial monolayers (mean 53% increase; P < .001) and TNFα-treated monolayers (mean 41% increase; P < .002). Increased adhesiveness was not associated with an abnormal expression of CD11a, CD11b, CD11c, CD18, CD62L, or CD15. There was an increase in the number of PMNs expressing CD64 in patients in crisis (median value, 44%) compared with patients out of crisis (median, 21%; P = .025) and controls (median, 6.5%; P < .001). Sera from patients in crisis had normal levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, TNFα, interleukin-1 (IL-1), IL-6, or IL-8 and did not modify the adherence of PMNs or their expression of CD64. Only IFN-γ induced CD64 expression on PMNs, but this effect was not associated with enhanced binding to endothelium. Because PMNs bound to endothelial monolayers were CD64+ and CD64-enriched PMNs were 7 times more adherent to endothelial monolayers than CD64-depleted PMNs, it is likely that CD64 is a marker of adherent PMNs. Two of the three anti-CD64 antibodies used in our antibody blocking studies (clones 32.2 and 197) partially inhibited the binding of sickle cell PMNs to untreated endothelium (mean inhibitions of 33% [P = .01] and 21% [P = .03], respectively), whereas only one (clone 197) inhibited binding to TNFα-treated endothelium (mean inhibition, 29%; P = .004). In some patients with sickle cell disease, an enhanced PMN adhesion to vascular endothelium could contribute to the vascular occlusion that characterizes the acute crisis of the disease.
Progressive chronic lymphocytic leukaemia is characterized by the accumulation of neoplastic B-cells in the tissues and correlates with the expression of prognostic biomarkers, such as CD38, CD49d and matrix metalloproteinase-9 (MMP9), which are involved in migration and tissue invasion. In this study we investigated the physical relationship between these molecules and demonstrated that CD38, CD49d, MMP9 and CD44 were physically associated in a supramolecular cell surface complex. Our findings provide a molecular basis for the correlation between expression of these proteins and prognosis and, as the complex is not present in normal B-cells, suggest a novel leukaemia-specific therapeutic target.
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