Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A . actinomycetemcomitans, H . aphrophilus, and H . paraphrophiius. A. actinomycetemcomitans was divided into two groups of strains. A . lignieresii fell outside or on the border of the A . actinobacillus class. A . ureae, H . injluenzae, H. parainjluenzae, P . haemolytica, and P. multocida fell outside the A . actinomycetemcomitans, H . aphrophilus, and H . paraphrophilus classes.Organisms belonging to the Actinobacillus-HaemophilusPasteurella group, which constitute the family Pasteurellaceae, have assumed increasing clinical importance in medicine and dentistry over the last few years (see references 30, 34, 38, 39). Unfortunately, generic separation in this family has been questioned, and with conventional biochemical tests it can be difficult to distinguish among the sometimes confusingly similar species of these genera. This particularly applies to Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. Therefore, additional criteria should be sought to assist in the taxonomic separation of organisms belonging to the Actinobacillus-Haemophilus-Pasteurella group.Like a number of other gram-negative rod-shaped bacteria, organisms belonging to the genera Actinobacillus, Haemophilus, and Pasteurella form budding extrusions (extensions of the outer membrane) which can be either attached to the bacterial cell surface or released to the environment during growth (16,19,23,25,35). These structures are referred to below as outer membrane vesicles or outer membrane fragments. They serve as vehicles for toxins and enzymes (for a review, see reference 24), and their small sizes (21 to 500 nm) permit them to cross epithelial barriers that are impermeable to whole cells.The aim of this study was to examine whether the enzymic contents of vesicles and fragments and the enzymic contents of whole cells, as determined by using the API ZYM micromethod, can be used for taxonomic distinction among members of the Actinobacillus-Haemophilus-Pasteurella group. Previously, the API ZYM method has been used to distinguish whole cells of A . actinomycetemcomitans, Actinobacillus lignieresii, and H. aphrophilus (29). Another purpose of this study was to determine whether the enzyme contents of vesicles and fragments differed from the enzyme contents of whole cells within the same species. * ...

Actinobacillus actinomycetemcomitans, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica and Pasteurella multocida strains were examined by transmission electron microscopy for the presence of bacteriophages. Phages were detected in serotype a (SUNY 75) and e (UOH 1705) and in the fresh clinical isolates UOH Q1243 and UOH Q1247 of A. actinomycetemcomitans. Phages were not found in serotype b, c and d strains of A. actinomycetemcomitans, in the fresh clinical isolate UOH Q1244 of this species or in old strains (including reference strains) of related species from the Actinobacillus-Haemophilus-Pasteurella group.

Johne. B., Myhrvold, V. & Mmland. B. Effects of endotoxins from Bucteroides intermedius and Esdicrichiu coli on cytotoxic and lysosomal activity in peritoneal macrophages from endotoxin responder and low responder mouse strains. Acta path. microbiol. immunol. scand. Sect. C, 95: 85-95, 1987. Peritoneal macrophages from normal mice strains (C3H/Tif and C57BL/6J) and from the endotoxin (lipopolysaccharide. LPS) low responder strain C3H/Hej were exposed to two structurally different endotoxins from Bucieroides interrnc~diics and Escherichia coli in vitro. lntracellular activity of a lysosomal enzyme (acid phosphatase) and macrophage mediated cytotoxic activity against a tumor cell line (L929) were tested. Both endotoxins caused increased levels of acid phosphatase activity in normal mice macrophages. No change was obtained in the C3H/Hej macrophages exposed to E. coli LPS; however, the B. inicwnc.diii.s LPS was able to strongly elevate intracellular enzyme level in the C3H/Hej low responder macrophages. Cytotoxic activities were investigated in macrophage supernatants and in co-cultures of stimulated macrophages and target cells. Cytotoxic activity evaluated by measuring release of radioactivity from "C-thymidine labelled tumor cells was increased with both endotoxins in normal mouse macrophages. but not in non-responder macrophages. When macrophage-mediated effects on tumor cells were tested by counting target cells left per culture, a reduction in target cell number was observed in endotoxin-treated low responder macrophage as well as in normal strain macrophage cultures more pronounced, however, in the normal strain. Cell contact between cytotoxic macrophages and target cells was 'verified by scanning electron microscopy. The results suggest that LPS effects on macrophages are dependent upon the functional parameters studied, and that the chemical composition of a particular LPS is important for its selective effects on macrophage functions.

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Human monocytes were isolated from peripheral blood by Lymphoprep density‐gradient centrifugation and adherence to fibronectin. The cells were loosened by ethylene‐diamino‐tetra‐acetate (EDTA), frozen by different freezing methods, thawed, washed and compared to unfrozen cells. After freezing, thawing and washing, cell recovery was calculated and found to vary with the freezing procedure. The best result was about 76% cell recovery. No morphological differences were observed between unfrozen and frozen cells. The experiments also showed that the percentage of cells that attached or phagocytized IgG‐opsonized erythrocytes (E‐IgG) via the Fc‐receptor was unaltered after freezing. Neither was there any difference between unfrozen and frozen monocytes with respect to their ability to phagocytize latex particles. There was no significant difference in reactivity between monocytes frozen for one day and those frozen for six weeks.