Permissiveness of Guinea Pig Alveolar Macrophage Subpopulations to Acute Respiratory Syncytial Virus Infection In Vitro: RSV Immunostaining

In: Respiratory

26 Jul 2014

Permissiveness of Guinea Pig Alveolar Macrophage Subpopulations to Acute Respiratory Syncytial Virus Infection In Vitro: RSV ImmunostainingFigure 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). The percentage of RSV-positive cells within the high-density AM subpopulation was approximately two-fold higher than the hypodense AM subpopulation (20.6 ± 2.9% vs 10.6 ± 1.5%, respectively, p < 0.001) in detail canadian-familypharmacy.com. The percentage of RSV-positive intermediate-density AMs (18.6 ± 2.1%) was similar to that of high-density AMs, but was significantly lower than that of blood monocytes (25.2 ± 4.5%, p < 0.01). In specimens of unfractionated BAL cells, the predominantly granular pattern of cytoplasmic immunostaining was observed within large vacuolated cells, while smaller cells showed a more diffuse staining pattern, similar to results observed with hypodense and high-density fractions, respectively.
Viral Plaque Assays
Figure 4 illustrates the amounts of intracellular virus isolated by sonication from the different AM subpopulations at 24 h postinfection. The amounts of isolated virus increased with the buoyant density of AMs (hypodense AMs < intermediate density AMs < high-density AMs, p < 0.001), with high-density cells showing 18-fold greater amounts of RSV in comparison to hypodense AMs (4,554 ± 1,680 vs 252 ± 61 plaque-forming units/ 106 cells, respectively). No statistically significant difference was observed between high-density AMs and blood monocytes in the amounts of virus isolated at 24 h post-RSV infection (p = 0.15).

Figure 2. Iimnunostsiining with rabbit anti-RSV antibody (brown color) at 24 h postinfection. Note the granular pattern of positive KSY staining (arrow) within the cytoplasm of hypodense AMs (top left, A), with comparably less granular and more diffuse cytoplasmic staining in intermediate-densitv (top ripjit, B) and high-density AMs (bottom left, C). Bottom right, D: control immunostaining of unfractionated AMs without primary antibody (ABC-peroxidase reaction; Mayer’s hemalun counterstaining; scale bar represents 50 |xm).

Figure 2. Iimnunostsiining with rabbit anti-RSV antibody (brown color) at 24 h postinfection. Note the granular pattern of positive KSY staining (arrow) within the cytoplasm of hypodense AMs (top left, A), with comparably less granular and more diffuse cytoplasmic staining in intermediate-densitv (top ripjit, B) and high-density AMs (bottom left, C). Bottom right, D: control immunostaining of unfractionated AMs without primary antibody (ABC-peroxidase reaction; Mayer’s hemalun counterstaining; scale bar represents 50 |xm).

Figure 3. Percentage of RSV-positive cells in AM subpopulations and blood monocytes at 24 h postinfection. Hypo = hypodense AMs; Inter = intermediate-density AMs; High = high-density AMs; Mono = blood monocytes. One asterisk: p < 0.01; two asterisks: p < 0.001; NS = not significant.

Figure 3. Percentage of RSV-positive cells in AM subpopulations and blood monocytes at 24 h postinfection. Hypo = hypodense AMs; Inter = intermediate-density AMs; High = high-density AMs; Mono = blood monocytes. One asterisk: p < 0.01; two asterisks: p < 0.001; NS = not significant.

Figure 4. Amounts of intracellular RSV isolated from AM subpopulations and blood monocytes at 24 h postinfection. Hypo = hypodense AMs; Inter — intermediate-density AMs; High = high-density AMs; Mono = blood monocytes. One asterisk: p < 0.01; NS = not significant

Figure 4. Amounts of intracellular RSV isolated from AM subpopulations and blood monocytes at 24 h postinfection. Hypo = hypodense AMs; Inter — intermediate-density AMs; High = high-density AMs; Mono = blood monocytes. One asterisk: p < 0.01; NS = not significant


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