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 »

Virus
The Long strain A of human RSV was obtained (American Type Culture Collection [ATCC]; Manassas, VA). RSV was propagated on HEp-2 cell monolayers (ATCC) in minimal essential medium (MEM) supplemented with 2% heat-inactivated fetal bovine serum (FBS) and 50 μg/mL gentamicin (Gibco; Grand Island, NY). When > 75% of cells exhibited cytopathic effect (formation of syncytia, cell rounding, and detachment), the culture flasks were shaken with autoclaved 3-mm glass beads and the resulting cell suspension was sonicated on ice for 1 min at the maximum output from a sonicator (Vibra Cell; Sonics & Materials Inc; Danbury, CT). Cellular debris was removed by centrifugation at 1,500 X g and 4°C for 15 min and the cleared supernatant containing virus was kept at — 70°C and used for infection immediately after determining the titer by plaque assay. Read the rest of this entry »

Respiratory syncytial virus (RSV) is a major cause of acute bronchiolitis and pneumonia in young children. The pathogenesis of acute bronchiolitis has generally been considered to involve RSV infection of airway epithelial cells,² but more recently, several groups have established that RSV proteins also localize to alveolar macrophages (AMs) in vivo during acute bronchiolitis. Using a guinea pig model of experimental RSV infection, we have also demonstrated viral protein within AMs during acute bronchiolitis. AMs are cells derived from blood monocytes that protect the lower respiratory tract against pathogens (including viruses) and inhaled particles and maintain sterile conditions in the lung. Flovent inhalers website Despite the role of AMs in host lung defense mechanisms, several studies have reported that RSV can cause productive infection of human AMs and blood monocytes in vitro. However, only a minority of these cells are permissive to RSV infection, even with exposures to large amounts of virus, and the reasons for this limited permissiveness of AMs to RSV infection are poorly understood. Moreover, the compartmentalization of RSV proteins within infected AMs has not been evaluated by electron microscopy, such that our knowledge of virus-cell interactions at the ultrastructural level is limited. Read the rest of this entry »