Mireille Redard

Platelet accumulation in brain microvessels in fatal pediatric cerebral malaria

The pathogenesis of fatal cerebral malaria (CM) is not well understood, in part because data from patients in whom a clinical diagnosis was established prior to death are rare. In a murine CM model, platelets accumulate in brain microvasculature, and antiplatelet therapy can improve outcome. We determined whether platelets are also found in cerebral vessels in human CM, and we performed immunohistopathology for platelet-specific glycoprotein, GPIIb-IIIa, on tissue from multiple brain sites in Malawian children whose fatal illness was severe malarial anemia, CM, or nonmalarial encephalopathy. Platelets were observed in 3 locations within microvessels: between malaria pigment and leukocytes, associated with malaria pigment, or alone. The mean surface area of platelet staining and the proportion of vessels showing platelet accumulation were significantly higher in patients with CM than in those without it. Platelet accumulation occurs in the microvasculature of patients with CM and may play a role in the pathogenesis of the disease.

Pathogenic role of P-selectin in experimental cerebral malaria: importance of the endothelial compartment.

P-selectin is a leukocyte adhesion receptor expressed on the surface of activated platelets and endothelial cells. Its role in the pathogenesis of cerebral malaria was explored in a murine model of cerebral malaria. Infection of mice with Plasmodium berghei ANKA led to P-selectin up-regulation in brain vessels of cerebral malaria-susceptible mice but not of cerebral malaria-resistant mice. Treatment of susceptible mice with anti-mouse P-selectin mAb failed to prevent the development of the neurological syndrome. However, P-selectin-deficient mice infected with Plasmodium berghei ANKA had a cumulative incidence of cerebral malaria which was significantly reduced compared to wild-type animals (4.5% versus 80%, respectively), despite identical levels of parasitemia, platelet and leukocyte accumulation. To determine whether P-selectin on platelets and/or endothelium was responsible for the microvascular pathology, cerebral malaria was assessed in chimeric mice deficient in platelet or endothelial P-selectin, which were generated by bone marrow transplantation. Mice deficient only in endothelial P-selectin did not show any sign of cerebral malaria (vascular plugging, hemorrhages, or edema), while mice lacking only platelet P-selectin showed signs of cerebral malaria similar to that seen in wild-type mice. These results indicate that endothelial P-selectin plays an important role in the pathogenesis of cerebral malaria.

Localization of alpha 2, alpha 5 and alpha 6 integrin subunits in human endometrium, decidua and trophoblast

Integrins are heterodimeric glycoproteins acting as membrane receptors for extracellular matrix components. The specificity of these receptors towards one particular matrix glycoprotein depends on the type of alpha and beta subunit combination. Since integrins are involved in the migratory behaviour of cells and since cytotrophoblastic cells are constitutively invasive, we undertook to immunolocalize the alpha 2, alpha 5 and alpha 6 integrin subunits in normal and hydatidiform molar trophoblast, in an implantation site as well as in decidualized and non-decidualized endometrium. alpha 6 positivity was confined to villous cytotrophoblast and was clearly polarized towards the basement membrane. Extravillous cytotrophoblastic cells were alpha 6-negative but became alpha 5-positive. In contrast to normal trophoblast, villous cytotrophoblast from hydatidiform molar tissue was alpha 5-positive. We conclude that the expression of a alpha 5 integrin subunit on cytotrophoblastic cell surfaces is correlated with the appearance of an invasive phenotype. alpha 6 and alpha 2 integrin subunits were both localized on the surface and glandular epithelium of the endometrium and their expression was increased during the secretory phase but became low or undetectable after decidualization. In contrast, alpha 5 subunit positivity was weak in the same epithelial during the first half of the cycle but disappeared after ovulation. Stromal cell alpha 5 positivity was present throughout the cycle but increased dramatically in decidualized endometria. We conclude that the alpha 5 integrin subunit which disappears from the epithelium at the end of the cycle might allow migration of the epithelial cells and repair of the endometrium after menses. We also wonder if alpha 5 positivity is part of a change in the stromal cell phenotype induced by decidualization.

Evaluation of estrogen receptors by immunocytochemistry on fine-needle aspiration biopsy specimens from breast tumors

The estrogen receptor (ER) content of 31 surgically removed breast tumors (26 duct carcinomas, one lobular carcinoma, one papillary carcinoma, one colloid carcinoma, one duct carcinoma in situ, and one atypical fibroadenoma) was determined by a commercially available immunocytochemical method (Abbott Laboratories, ER‐ICA) on cytologic material obtained by fine needle aspiration biopsy (FNAB) of surgical specimens. Immunocytochemical staining of cells by a peroxidase‐antiperoxidase technique was evaluated on the basis of the percentage of positive cells and the intensity of staining. An immuno‐staining score for cytologic (IS‐CYTO) and histologic (IS‐HISTO) material was defined and a threshold of positivity determined to facilitate the semi‐quantitation of results and the comparison of cases. The results of immunostaining of cytologic material were compared with the evaluation of ER in corresponding tissue samples as determined by the radioligand binding assay using the dextran‐coated charcoal procedure (ER‐DCC) and by ER‐ICA using cryostat sections of frozen tissue. The sensitivity, specificity, predictive value of a positive test, and test efficiency of ER‐ICA in cytologic material as compared to ER‐DCC was 96%, 83%, 96% and 93%, respectively. The IS‐CYTO was significantly correlated with the IS‐HISTO in corresponding histologic material (r = 0.72, P < 0.001). In conclusion, the combination of ER‐ICA with FNAB represents a useful new technique for the evaluation of ER which may be applied to small primary tumors, tumor recurrences, and metastases.

The Application of Immunocytochemical Techniques to Routinely-Fixed and Stained Cytologic Specimens: An Aid in the Differential Diagnosis of Undifferentiated Malignant Neoplasms

Routinely-fixed and Papanicolaou stained smears with the cytologic diagnosis of undifferentiated malignant neoplasm that had been prepared with cells obtained by fine needle aspiration biopsy (n = 7), pulmonary lavage (n = 5), or thoracentesis (n = 3) from 15 unselected patients were stained by an immunocytochemical technique to evaluate the presence of keratin proteins and the leukocyte common antigen (LCA). Commercially available, well-characterized monoclonal antibodies with specificities for keratin proteins and the leukocyte common antigen, and a streptavidin-biotin-horseradish peroxidase labelling method were used. Evaluation of the stained smears revealed the presence of one of the two antigens in material obtained from each patient, thus indicating the probable cell-lineage of the neoplastic cells. The specificity of the monoclonal antibody reagents used was further evaluated in routinely-fixed and stained cytologic material from 24 histologically confirmed carcinomas and 12 lymphomas. In conclusion, immunocytochemical techniques may be successfully applied to routinely processed archival cytologic smears to determine the antigenic profile of morphologically undifferentiated cells and therefore aid in the differential diagnosis of undifferentiated malignant neoplasms.

Culture of Human Endothelial Cells Derived from Capillaries of the Decidual Tissue

Abstract. Human endothelial cells derived from the capillary bed of endometrial decidua were studied in vitro using medium 199 supplemented with 20% fetal calf serum. Cells in primary and subcultures were identified in parallel by electron microscopy and indirect immunofluorescence microscopy. The abundance of microfilaments appearing in small bundles, the micropinocytotic vesicles in the cell periphery, the occurrence of characteristic Weibel‐Palade bodies and the intense and specific fluorescence after decoration with antibodies to Factor VIII protein all were criteria for a positive demonstration of the endothelial character in the cell cultures derived from the capillary bed of the human decidua. The proliferative activity of the in vitro system was studied by autoradiography of the endothelial cells after exposure to [3H]‐thymidine at various periods of incubation. A statistically highly significant difference (p>0.001) was found between periods of 4‐6 and 6‐8 days of incubation and between 6‐8 and 8‐10 days of incubation (p>0.01). No such difference was found between 8‐10 and 10‐14 days of incubation. When the medium was switched to complete M 199+ 10% fetal calf serum and 10% human plasma containing 76 pg/ml of estradiol the thymidine index was 5 to 4 times as high as in the control medium after 5 to 6 days of incubation.