Further analysis by immunogold labeling provided qualitative information about patterns of virus-cell interactions that may be pertinent to the ability of the different AM subpopulations to support or restrict viral replication. In particular, immunolabeling within the phagolysosomes of hypodense AMs correlates with the granular staining pattern observed under light microscopy and is consistent with this subpopulation of cells preferentially inactivating the virus. In contrast, the distribution of gold particles free within the cytoplasm and in association with viral nucleocapsids may be related to the greater tendency of intermediate-density and high-density cells to support viral replication. Read the rest of this entry »

Figure 5 shows representative electron micrographs of RSV-exposed AMs immunolabeled with the pool of monoclonal anti-RSV antibodies. There were apparent differences between the three AM subpopulations in the patterns of cytoplasmic distribution of colloidal gold particles. Hypodense cells showed aggregates of colloidal gold particles within phagolysosomes (Figure 5, A), consistent with the granular pattern of RSV immunostaining observed by light microscopy. By contrast, intermediate- and high-density AMs (Fig 5, B and C) showed colloidal gold particles free within the cytoplasm, frequently in a perinuclear distribution that has been described for RSV, and in association with typical RSV nucleocapsids Examination of control sections (Fig 5, D) revealed rare single colloidal gold particles that did not show a particular distribution within cells. Read the rest of this entry »

Figure 2 shows typical cytoplasmic localization of RSV proteins within isolated AMs in the three subpopulations, as detected by immunocytochemis-try at 24 h postinfection. Positive RSV immunostaining within hypodense AMs showed a distinct granular pattern within the cytoplasm, whereas a less granular and more diffuse cytoplasmic immunostaining pattern was observed for intermediate-density and high-density cells. High-density AMs showed two-fold higher proportion of RSV-positive cells than hypodense AMs. As summarized in Figure 3, hypodense AMs showed the lowest percentage of RSV-positive cells in comparison to the other subpopulations of AMs and blood monocytes similarly exposed to RSV in vitro (p < 0.01). Read the rest of this entry »

After exposure to RSV, as described above, AMs were fixed in 2.5% glutaraldehyde-containing sodium cacodylate buffer (0.1 M, pH 7.4) for 1 h at 4°C, postfixed in osmium tetroxide for 1 h at room temperature, and embedded in epoxy resin following standard methods. Ultrathin sections were cut with an ultramicrotome (Reichert; Vienna, Austria) and collected onto Formvar vinyl alcohol-vinyl acetate formal copolymer-coated nickel grids (Ted Pella, Inc; Redding, CA). Following treatment with sodium metaperiodate to unmask antigenic sites and blocking nonspecific binding by incubation with normal rabbit serum (5% in PBS, 20 min), the sections were incubated with a cocktail of mouse anti-RSV monoclonal antibodies optimally diluted 1:100 as recommended by the manufacturer (NCL-RSV2; Novacastra Laboratories; Newcastle Upon Tyne, UK). Read the rest of this entry »

After aspirating the culture media, the cells were fixed in the wells for 10 min with 4% paraformaldehyde in phosphate-buffered saline solution (PBS), rinsed with PBS, and harvested by gently scraping with a sterile rubber policeman. Cytospin slides were then prepared from the collected cell suspensions. RSV proteins were detected by indirect immunoperoxidase staining using a primary rabbit polyclonal anti-RSV antibody and a secondary biotinylated goat antirabbit antibody (Dako A/S; Glostrup, Denmark) optimally diluted in TBS (0.05 M Tris-buffered saline solution, pH 7.6) containing 1% bovine serum albumin and 0.1% polysorbate 20 (Tween 20). Unless otherwise indicated, all incubations and washes occurred at room temperature. Read the rest of this entry »

Accordingly, we designated the subpopulation of AMs recovered at the interface 20 to 22% metrizamide as “high-density” AMs; cells recovered at the interface 18 to 20% metrizamide were called “intermediate-density” AMs; and those collected on top of the 18% metrizamide gradient were designated as “hypodense” AMs. The purity of AMs recovered from each fraction was 100% in hypodense, 96% in intermediate-density, and 72% in liigh-density cells. In the latter fraction, contaminating cells consisted most of eosinophils (24%). Morphologically, hypodense AMs were the largest cells, had vacuolated cytoplasm, and showed a low nuclear to cytoplasmic ratio; in contrast, liigh-density AMs were smaller and showed a high nuclear to cytoplasmic ratio. Read the rest of this entry »

Discontinuous gradients of metrizamide (Nycomed Pharma AS; Oslo, Norway) were prepared from 27% metrizamide stock solution diluted with 10 mM HEPES-buffered saline solution at a pH of 7.3 containing 0.1% each of glucose and gelatin. The method was derived from Fukuda et al, and Vadas et al, in which metrizamide-containing solutions are isotonic. Up to 10⁷ BAL cells were resuspended in 2 mL of HEPES-saline solution and layered on top of increasing concentrations of metrizamide gradients (3 mL at 27%; and 2 mL each at 22%, 20%, and 18% metrizamide) in 15-mL transparent polypropylene centrifugation tubes (Corning; Corning, NY). After centrifugation at 1,200 X g for 45 min at 18°C, the cells were collected from each interface, washed with Hanks’ balanced salt solution containing 2% FBS and were counted before being resuspended in MEM supplemented with 5% FBS. http://help-in-mens-health-problems.net/ fully The total cell recovery after density gradient centrifugation ranged from 70 to 75%. Read the rest of this entry »