Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures.

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.

Demonstration of HBsAg as the antigen component in circulating immune complexes detected by peg-solid phase test.

The development of an enzyme-linked immunosorbent assay to identify HBsAg as the antigen component within circulating immune complexes using immobilized polyethylene glycol (PEG) is described. The method utilizes, on one hand, the ability of PEG to bind stably to plastic supports and, on the other, to precipitate circulating macromolecules. This method is easily performed, very cheap, quick and, above all, it helps define the biological nature of the immune complexes. HBsAg can be revealed as the antigen component of HBsAg/anti-HBs soluble immune complexes at concentrations of at least 20 ng/ml and either in antigen or antibody excess. Our results indicate that HBsAg circulates in a complexed form in 47% of HBsAg chronic carriers and in 10.7% of patients with liver disease who are positive for serum antibody to hepatitis B surface antigen (anti-HBs) and to core antigen (anti-HBc). None of the other groups of patients in the study had circulating HBsAg in the complexed form.

Isolated osteoclasts in primary culture: first observations on structure and survival in culture media.

Osteoclasts were isolated mechanically from the medullary bone of laying hens kept 7 days on a low calcium diet. Osteoclast enrichment was achieved with 3-4 sedimentations of the cell suspension in test-tubes prepared by layering on the bottom with BSA 10% in MEM-HEPES or PBS, above which the cells were suspended in MEM-HEPES or PBS. The final suspension of osteoclasts was cultivated in MEM with 10% FCS for 3 weeks. The cultures were observed by phase-contrast and scanning electron microscopy (SEM). By the third day, the osteoclasts were completely spread onto the plastic dishes and a variety of morphologies and of intercellular contacts was established. Osteoclasts in culture do not lose their morphology; they survive for long periods and can be used in many experimental systems.

Autoantibody to albumin of type G and M in acute and chronic viral hepatitis.

Autoantibodies to albumin (AAA) were tested by an ELISA method in patients with A, B and NANB acute and chronic hepatitis, and in a control group. AAA-IgM had a different behaviour in acute hepatitis type A, in which we observed a high average titre as compared with B, and NANB hepatitis, in which we observed a decrease in the average titre. In the chronic phase, we noted a decrement of the average titre in all the types of hepatitis. For AAA-IgG, in the acute phase the average titre in hepatitis A, B and NANB was lower than in the control group. In the chronic stage, only NANB hepatitis showed a decrement of the average titre of the antibody. On the base of these results, we can say that the involvement of AAA seems to be different in hepatitis A from the other two types, in which the decrement of average titre may be explained by the formation of immunocomplexes which are not detected by this test.

Resorption of vital or devitalized bone by isolated osteoclasts in vitro. The role of lining cells.

The maintainance of resorptive capability towards vital or devitalized bone in osteoclasts isolated from the medullary bone of laying hens and cultured for five days in vitro has been investigated morphologically with the aid of light and transmission electron microscopy. Devitalized bone particles ranging in size from 50 to 100 microns, added to cultures of osteoclasts, were rapidly surrounded by the osteoclasts which, in transmission electron microscopy, showed ruffled borders and clear zones at the surfaces of contact with bone-features typical of resorptive activity. Alternatively osteoclasts were added onto the endosteal surfaces of vital or devitalized diaphyses of quail femurs after removal of the endosteal and periosteal cell layers. The results indicated that, when the vital or devitalized bone surfaces were devoid of cells, the osteoclasts adhered and resorbed bone (as confirmed by transmission electron microscopy). When vital bone of quail was cultured for 24 h before the addition of osteoclasts a new cell layer was formed; it enveloped all bone surfaces and precluded the access of osteoclasts to bone. The role of these lining cells, ultrastructurally indistinguishable from resting osteoblasts, is discussed.

[Microcinematographic study of in vitro fusion between blood monocytes and differentiated osteoclasts]. / Studio microcinematografico di fusione in vitro tra monociti ematici ed osteoclasti differenziati.

The cell cycle of osteoclasts is hard to define, because regularly new nuclei enter in, and old nuclei are extruded from these polynucleated elements. The "transit-time" of a single nucleus averages 9-11 days in dogs and the factors conditioning this renewal are unknown. With the aim of elucidating in vitro the conditions necessary for the entrance of new nuclei in the polycarions, cultures of purified osteoclasts from medullary bone of laying hens and monocytes from circulating blood of the same animals have been performed after a previous period of 5 days of separate cultures each one of these two cell types. Part of the monocytes added to the osteoclast cultures, after a variable period of time entered in the polycarions by an active process of membrane fusion. Some phases of the process have been visualized at phase contrast and confirmed by time-lapse microcinematography and scanning electron microscopy.

Monocytes from circulating blood fuse in vitro with purified osteoclasts in primary culture.

The origin of osteoclasts from mononuclear phagocytes and the addition of new nuclei to already differentiated osteoclasts have already been documented by several authors, but the factors controlling these events have not yet been elucidated. With the aim of investigating this problem, monocytes, isolated from circulating blood of laying hens and cultured previously for 5 days, were added to osteoclasts isolated from the medullary bone of the same hen and cultured at low density. The cultures were either fixed and observed under phase contrast at 24-h intervals for 5 days or filmed by time-lapse cinemicrography. With both techniques the formation of extensive areas of membrane contacts, generally followed by fusion between some monocytes and osteoclasts, was observed. The absence of added resorbing factors and the possible mechanisms by which osteoclast precursors are recruited in vivo are discussed.

Mature osteocytes behaviour in a repletion period: the occurrence of osteoplastic activity.

The occurrence of osteoplastic activity in adult osteocytes from the femoral cortex of laying hens previously kept on a hypocalcaemic diet was studied after return of the animals to a calcium-rich alimentation. The following techniques were used: 1) tetracycline labelling of new calcifying matrix; 2) osteoid staining with alcian blue at pH 1.9 or toluidine blue at pH 3.1; 3) electron microscopic observations; 4) autoradiographic evaluation of (3H) proline uptake by osteocytes. Quantitative evaluation of (3H) proline uptake by osteocytes. Quantitative evaluation of the preparations consistently showed indication of osteoplastic activity in at least 20% of the osteocytes with relatively higher values in the metaphysis than in the diaphysis.

Osteoclasts and monocytes have similar cytoskeletal structures and adhesion property in vitro.

The distribution of some cytoskeletal structures (microtubules, microfilaments, intermediate filaments) has been studied by indirect immunofluorescence microscopy and affinity purified antibodies in osteoclasts isolated from medullary bone of laying hens and in hen blood monocytes cultured in vitro. Both cell types show similar patterns of distribution of cytoskeletal structures and this further supports the concept that these cells are closely related. Osteoclasts and monocytes are also similar in their adhesion patterns, because they adhere to fibronectin-free areas and show closely comparable cell-to-substrate interactions when observed with interference reflection microscopy.