Permissiveness of Guinea Pig Alveolar Macrophage Subpopulations to Acute Respiratory Syncytial Virus Infection In Vitro: Immunogold Electron Microscopy

In: Respiratory

28 Jul 2014

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.
Discussion
The overall objective of this study was to determine whether the permissiveness of normal guinea pigs’ AMs to acute RSV infection in vitro may be related to their degree of cellular maturation achieved in vivo. We prepared three populations of AMs based on previous reports that showed an inverse relationship between cellular maturity and buoyant density, in which high-density AMs had similar properties to blood monocytes from which AMs differentiate. Tadanafil so Our results confirmed these previous findings and extended them by showing that comparatively immature, high-density AMs had a significantly higher percentage of RSV-positive cells than more mature, hypodense AMs, with intermediate values obtained for intermediate-density AMs. In addition, die results of viral plaque assays revealed that the amounts of intracellular virus were significantly increased from hypodense to intermediate-density to high-density AMs, with high-density AMs and blood monocytes showing similar amounts. Importantly, high-density AMs had a two-fold greater percentage of RSV-positive cells than hypodense AMs by immunoey-tochemistry but showed 18-fold more replicating virus by plaque assays. Taken together, diese findings demonstrate that the permissiveness of AMs to acute RSV infection is inversely related to dieir degree of maturity and suggest that high-density AMs support RSV replication whereas hypodense AMs may restrict RSV replication after internalization of the vims.

Figure 5. Electron micrographs of hypodense (top left, A), intermediate-density (top light, B), and high-density AMs (bottom left, C) showing immnnogold labeling (arrows) with the pool of monoclonal anti-RSV antibodies at 24 h postinfection. Note the localization of immunoreactive RSV proteins within a membrane-bound phagolysosome (pi) in hypodense AMs, free cytoplasmic \iral proteins near the nucleus (N) in intermediate-density AMs, and immunolabeling of viral nucleoeapsids (asterisk) within the cytoplasm of high-density AMs. Bottom right, D: control section (high-density AMs) that was not incubated with primary antibody (scale bars represent 50 nm).

Figure 5. Electron micrographs of hypodense (top left, A), intermediate-density (top light, B), and high-density AMs (bottom left, C) showing immnnogold labeling (arrows) with the pool of monoclonal anti-RSV antibodies at 24 h postinfection. Note the localization of immunoreactive RSV proteins within a membrane-bound phagolysosome (pi) in hypodense AMs, free cytoplasmic \iral proteins near the nucleus (N) in intermediate-density AMs, and immunolabeling of viral nucleoeapsids (asterisk) within the cytoplasm of high-density AMs. Bottom right, D: control section (high-density AMs) that was not incubated with primary antibody (scale bars represent 50 nm).


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