Showing papers by "Larry V. McIntire published in 2003"

Automated Selection of DAB-labeled Tissue for Immunohistochemical Quantification

Abstract: The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.

Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion.

Abstract: The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.

In vitro studies of erythrocyte-vascular endothelium interactions.

Abstract: Abnormal interactions between red blood cells (RBCs) and vascular endothelial cells (ECs) are crucial factors in causing vascular pathology in several diseases, including vaso-occlusive crises in sickle cell anemia and the development of vascular complications in diabetes mellitus and malaria. A mechanistic understanding of the specific nature of RBC–EC interactions and ensuing functional consequences can provide insights into the pathophysiology of RBC-related vascular disorders and a rational basis for developing novel therapies. This review discusses in vitro experimental models that are commonly used for investigating RBC–EC interactions, and current knowledge of the molecular mechanisms of RBC–EC adhesion and EC functions modulated by RBCs. Because blood flow-induced mechanical forces and convective mass transfer play significant roles in regulating vascular events, it is necessary to develop advanced dynamic experimental models for elucidating RBC–EC interactions under well-controlled, physiologically relevant mechanical environments. © 2003 Biomedical Engineering Society. PAC2003: 8719Tt, 8719Xx, 8718Ed