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This study describes an improved immunohistochemical method for the sensitive and specific identification of estrogen receptors (ERs) in paraffin sections from formalin-fixed and routinely processed breast carcinoma tissues, using DNase pretreatment to expose nuclear antigenic sites and commercially available immunoreagents (including monoclonal antibody) in kit form. Results were compared with dextran-coated charcoal cytosolic assay (DCC) and with conventional immunohistochemistry on frozen sections. Sensitivity and specificity for determinations on paraffin sections were 88% and 86%, respectively, and statistical analysis showed very good agreement between DCC and paraffin sections (kappa = 0.805). The DNase technic on paraffin sections allows excellent correlation between histologic characteristics and ER status and reduces DCC sampling error resulting from stromal dilution and tumor variability. This method offers a reliable and reproducible alternative when tissue is not suitable or unavailable for DCC or frozen tissue analysis and can be used for retrospective studies on stored tissue blocks.

Thrombo-embolic events remain one of the most serious complications in patients with nephrotic syndrome (NS). A causal relationship between coagulation abnormalities and the occurrence of thrombosis in NS has been searched. Several studies have demonstrated multiples abnormalities in the coagulation factors and platelets; however those studies did not provide adequate explanations for the thrombotic tendency of NS. This thrombotic tendency could be related to deficiency in natural coagulation inhibitors. Plasma ATIII level has been reported to be decreased both in some adults and children. Protein C is found normal or high in NS whereas protein S is increased. Nevertheless a possible decrease in functional protein S cannot be excluded.

The effect of rapid daily variation of glycemia and labile HbA1 on both antithrombin III (ATIII) activity and plasma concentration in ten insulin dependent diabetics has been evaluated. The variations of both plasma glucose and labile HbA1 were inversely correlated to the alterations of ATIII activity (r = -0.71 and r = -0.73, respectively, p less than 0.001), while no change in ATIII plasma concentration was present. These data suggest a direct role of glucose in determining rapid alteration of ATIII biologic activity, in vivo, in diabetes, probably mediated by labile non-enzymatic glycation.

Intact vascular endothelium provides several anticoagulant mechanisms for the maintenance of blood fluidity and the prevention of thrombosis. High-affinity binding of proteolytic active thrombin to thrombomodulin at the cell surface effectively facilitates the activation of the potent anticoagulant protein C (PC). Rapid inactivation of cell-bound thrombin by antithrombin III (ATIII) accelerated by heparin-like structures represents another anticoagulant mechanism. In the present investigation the interference of these two events has been studied. Inhibition of thrombin bound to cultured bovine aortic endothelial cells (BAEC) by ATIII and the effect of the inhibitor on the activation of PC has been studied using purified components of bovine origin. Exposure of thrombin (45 nM) with prewashed confluent BAEC-monolayers for 10 min resulted in the binding of 12% thrombin. The subsequent incubation with various concentrations (0.3-2.4 microM) of ATIII revealed no acceleration of the inhibition of thrombin by ATIII at the endothelial cell surface when compared with the uncatalyzed fluid phase reaction. However, compared with the uncatalyzed fluid phase reaction. However, heparin added to the reaction mixture substantially increased the inactivation of cell-bound thrombin. Modified ATIII that did not possess heparin cofactor activity presented a comparable inactivation pattern for endothelial cell bound-thrombin as native ATIII indicating that heparin-like structures did not accelerate the interaction. When PC (32 nM) and ATIII (1.8 microM) competed for thrombin bound to BAEC, activation of PC was demonstrated within the initial 6 min of the incubation amounting to 62% of the activated PC formation in the absence of ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2+. On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.