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The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.

In this study, we introduce an application of flow cytometry for the concurrent detection of phagocytotic cells and surface molecules involved in the phagocytic process. E. coli expressing green fluorescent protein (GFP) were applied as the phagocytosable particles. Blood samples were incubated with E. coli expressing GFP, followed by indirect immunofluorescence using four candidate monoclonal antibodies (mAbs). Granulocytes that had phagocytosed E. coli exhibited high levels of GFP intensity, in contrast to the nonphagocytosed cells. By comparing the level of expression of molecules expressed on phagocytosed granulocytes with that of nonphagocytosed cells by flow cytometry, it enabled the determination of the expression and alteration of the cell surface molecules upon phogocytosis. Of the four mAbs used in this study, upon phagocytosis, molecules recognized by mAbs WK13, COSA5A and COSA33NL were up-regulated. However, CD15 recognized by mAb VIMD5 was downregulated. The proposed method will benefit the study of phagocytic mechanisms in the future.

Background and Objectives The expression of A Disintegrin and Metalloproteinase 8 ( ADAM8) is associated with several inflammatory diseases. Elevated ADAM8 levels have been shown in gingival crevicular fluid of patients with chronic periodontitis. The objective of this study was to investigate ADAM8 expression in chronic periodontitis tissues compared with that in normal tissues. ADAM8 expression and its inductive mechanism were examined in human gingival epithelial cells ( HGECs) and human gingival fibroblasts. Material and Methods Total RNA and protein were extracted from gingival biopsies of 33 patients with chronic periodontitis and those of 23 healthy volunteers. ADAM8 mRNA and protein expression was analyzed by real-time polymerase chain reaction, immunoblotting and immunohistochemistry. ADAM8 expression in control and stimulated cells in the presence or absence of specific inhibitors for mitogen-activated protein kinase pathways was assayed by real-time polymerase chain reaction, immunoblotting, flow cytometry and immunofluorescence. Results ADAM8 mRNA and protein expression in chronic periodontitis tissues was significantly greater than that in normal tissues ( p < 0.01). Significantly increased ADAM8 expression was detected in the gingival epithelium of chronic periodontitis tissues ( p < 0.001). ADAM8 mRNA expression in HGECs, but not in human gingival fibroblasts, was significantly induced by stimulation with Fusobacterium nucleatum ( p < 0.05), partially via the p44/42 mitogen-activated protein kinase pathway. ADAM8 expression in the cell lysates and on the surface of HGECs was induced by stimulation with F. nucleatum. Conclusion ADAM8 expression is increased in inflamed chronic periodontitis tissues and localized within gingival epithelium, consistent with an upregulation of ADAM8 expression in F. nucleatum-stimulated HGECs, suggesting a possible role of ADAM8 in innate immunity of periodontal tissue. [ABSTRACT FROM AUTHOR]

β3 subunit is described as one of the Na, K ATPase subunits. Recently, we generated a monoclonal antibody (mAb), termed P-3E10. This mAb was shown to react with the Na, K ATPase β3 subunit or CD298. By immunofluorescence analysis using mAb P-3E10, it was found that all peripheral blood leukocytes express Na, K ATPase β3. The presence of β3 subunit on leukocytes is not in a quantitative polymorphic manner. Upon phytohemagglutinin or phorbol myristate acetate activation, the expression level of the Na, K ATPase β3 subunit on activated peripheral blood mononuclear cells was not altered in comparison with those of unstimulated cells. Red blood cells (RBCs) of healthy donors showed negative reactivity with mAb P-3E10. However, more than 80% of thalassemic RBCs showed positive reactivity. By immunoprecipitation, moreover, a protein band of 55–65 kDa was precipitated from normal RBC membrane using mAb P-3E10. These results evidenced that the β3 subunit of Na, K ATPase is expressed on RBC membrane but the epitope recognized by mAb P-3E10 is hidden in normal RBCs. Furthermore, we showed the association of β3 subunit and α subunit of Na, K ATPase. This information is important for further understanding of the functional roles of this molecule. [ABSTRACT FROM AUTHOR]