The role of sex steroids on cellular events involved in vascular disease.

In this work we checked the hypothesis whether estrone, progesterone, and testosterone are able to modulate the interactions between platelets, monocytes, and endothelial cells either under basal or inflammatory conditions. Using adhesion assays we demonstrated that pretreatment of endothelial cells with estrone, progesterone, or testosterone prevented monocytes and platelets adhesion induced by the proinflammatory agent bacterial lipopolysaccharide. The hormones reduced the expression of mRNA of ICAM-1, VCAM-1, and P-selectin, endothelial surface proteins that mediate monocytes and platelets adhesion respectively. Integrins are the main leukocyte proteins that allow firm adhesion. Using flow cytometry we showed that estrone treatment of monocytes reduced CD11b and CD11c expression, either under basal or injury (lipopolysaccharide) conditions. The three steroids inhibited platelet aggregation in a nitric oxide dependent manner. Platelet function was not affected by the steroid treatment. The molecular mechanisms of action exerted by the steroids included the participation of the intracellular signaling pathways PKC, MAPK, and PI3K, which selectively and differentially mediate the stimulation of nitric oxide release. We evidence that estrone, progesterone, and testosterone modulate monocyte and platelet adhesion to endothelial cells, events that play a major role in the initiation and progression of vascular lesions. The steroid action was evidenced under basal or inflammatory conditions. The mechanisms of action exerted by the steroids included stimulation of nitric oxide production and the participation of PKC, MAPK, and PI3K systems.

Leukocyte perturbation associated with Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism due to the deficient activity of the enzyme alpha-galactosidase A. The non-degraded substrate, mainly globotriaosylceramide (Galα1-4Galß1-4Glcß1-1Cer; Gb(3)) accumulates progressively in the lysosome of various cells. The aim of this work was to analyse changes in leukocyte subpopulations and surface markers and to determine whether Gb(3) is increased in leukocytes of patients with untreated and treated Fabry disease. Blood samples obtained from 22 male Fabry patients (11 untreated and 11 on enzyme replacement therapy) and 22 normal controls were subjected to flow cytometric analysis of Gb(3) intracellular content, leukocyte subpopulations and cell markers. Based on the fluorescence intensity of bound monoclonal antibody, and relative to normal control leukocytes, Gb(3) appeared significantly increased in lymphocytes (but not in monocytes or granulocytes) from patients with Fabry disease. A significantly higher percentage of lymphocytes and CD19(+) cells and a reduced proportion of monocytes, CD8(+) cells and myeloid dendritic cells were detected in samples from Fabry patients compared with normal controls. CD1d expression was significantly lower and MHC class II surface expression was significantly higher in monocytes from Fabry patients than in normal controls. As previously observed for other adhesion molecules, the expression of CD31 (PECAM) was higher in leukocytes from Fabry patients. In conclusion, the differences recorded in this study reveal a leukocyte perturbation associated with the disease state in Fabry patients, whereas some abnormalities are less marked in treated patients.

Intracellular expression of CD69 in endometrial and peripheral T cells represents a useful marker in women with recurrent miscarriage: modulation after allogeneic leukocyte immunotherapy.

PROBLEM: To characterize in fertile women and women with recurrent spontaneous abortions (RSA) the expression and functional status of T cells expressing the CD69 molecule. METHOD OF STUDY: We analyzed by flow cytometry in peripheral blood and endometrium from fertile and RSA women, the surface and cytoplasmic expression of CD69 on gated T cells. In addition, we investigated by three-color flow cytometry the expression of cytokines, and subsets of memory T cells. RESULTS: In T cells, CD69 was restricted to the intracellular compartment with a higher frequency in RSA than in fertile women (68.2 +/- 12% versus 23.7 +/- 22%, P < 0.001, and 20 +/- 9.5% versus 2.1 +/- 3.8%, P < 0.005, in endometrium and peripheral blood, respectively). In contrast, the number of interferon-gamma+ (IFN-gamma+) secreting cells was higher (16 +/- 5% versus 6 +/- 1%) in fertile women. All 11 RSA women alloimmunized with parental leukocytes reached values of CD3 +/- CD69+ cells similar to those observed in fertile women. CONCLUSIONS: CD69 might represent a useful marker in the diagnosis and the follow up of RSA patients.

Modulation of CD44 in acute lymphoblastic leukemia identifies functional and phenotypic differences of human B cell precursors.

CD44 expression and other B cell markers were analyzed in 38 samples of B cell precursors (BCP) from patients with acute lymphoblastic leukemia (ALL). According to the expression of CD10 and CD44, we established the following five stages of BCP-ALL phenotypes that may represent different forms of interaction between BCP-ALL and bone marrow-adherent cells: stage 1, CD19+, CD44bright, CD10-; stage 2, CD19+, CD44bright, CD10dim/bright; stage 3, CD19+, CD44dim, CD10bright, CD20-/+; stage 4, CD19+, CD44dim, CD10dim, CD20+; and stage 5, CD19+, CD44bright, CD10-, CD20+. Next, we analyzed the modulation of CD44 according to the expression of the different BCP-ALL phenotypes by incubating the samples under different culture conditions, including addition of stromal cells and interleukin (IL)-7. In culture, the samples in stages 1 and 2 maintained high expression of CD44 and re-expressed this molecule when cultured after trypsin treatment, indicating ongoing synthesis of CD44. Similarly, the stage 3 samples cultured in the presence of stromal cells, IL-7, or both also upregulated CD44 expression in culture. In contrast, the low expression of CD44 on the presumably more mature stage 4 samples was not modified by the addition of stromal cells or IL-7 or when cultured after trypsin treatment, suggesting that those cells had arrested CD44 synthesis. We concluded that down-modulation of CD44 occurred in association with differentiation to phenotype stages 3 and 4 and we hypothesized that this down-modulation might be associated with the exit of BCP-ALL from the bone marrow.

Is the paternal mononuclear cells' immunization a successful treatment for recurrent spontaneous abortion?

PROBLEM: Alloimmunization as a treatment for recurrent spontaneous abortion (RSA) is still controversial due to the lack of enough controls to evaluate its effectiveness. The present study was conducted to compare the live birth rate in the presence or absence of immunotherapy. METHOD OF STUDY: Ninety-two women with RSA (79 primary [PA] and 13 secondary aborters[SA]) received immunotherapy. Thirty-seven RSA couples not receiving paternal alloimmunization, constituted the "control" group. RESULTS: The pregnancy rate in alloimmunized was 58 vs 46% in the control group. The live birth increased from 71% in the controls to 88% after immunotherapy. The alloimmunization induced mixed lymphocyte reaction blocking factors (MLR BFs) in 79% of women. However, they were also present in 83% of immunized women experiencing a new abortion. CONCLUSION: These results indicate that alloimmunization may be useful in the treatment of RSA.