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Fig 4. Effect of RhSP-D on IAV binding to neutrophils as assessed by fluorescent microscopy. Neutrophils were allowed to adhere to plastic coverslips followed by incubation of the cells with either FITC-labeled Bangkok 79 IAV alone (top panels) or IAV that had been preincubatec with either 1.6 (middle panels) or 3.2 4g/mL (bottom panels) of RhSP-D multimers for 15 minutes at 4°C. After incubation, the coverslips were washed, fixed with 1% paraformaldehyde, and mounted. The left panels show phase contrast microscopy and the right panels show fluores: cence microscopy of the same fields. Results depicted are representative of more than three separate experiments.

Figure 4 Effect of RhSP-D on IAV binding to neutrophils as assessed by fluorescent microscopy. Neutrophils were allowed to adhere to plastic coverslips followed by incubation of the cells with either FITC-labeled Bangkok 79 IAV alone (top panels) or IAV that had been preincubatec with either 1.6 (middle panels) or 3.2 4g/mL (bottom panels) of RhSP-D multimers for 15 minutes at 4°C. After incubation, the coverslips were washed, fixed with 1% paraformaldehyde, and mounted. The left panels show phase contrast microscopy and the right panels show fluores: cence microscopy of the same fields. Results depicted are representative of more than three separate experiments.

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set of experiments). These results contrast with those ob- tained with IAV opsonized with anti-HA MoAbs (Table 1 and Fig 8).
set of experiments). These results contrast with those ob- tained with IAV opsonized with anti-HA MoAbs (Table 1 and Fig 8).
Results shown are the mean + SEM (n = 3 to 4 experiments) neutro- phil HO, response (in nanomoles per minute per 4 x 10° cells) to stimulation with either Texas 77 or Bangkok 79 IAV alone or to aliquots of these strains of |IAV that had been preincubated with 800 or 400 g/mL, respectively, of the 81/4 or 73/1 antiviral MoAbs. Preincubation with MoAbs was performed as described in Fig 1 C. Where indicated (ie, IAV + MoAb/Prot A), MoAbs complexed with Staph protein A (as described in the Materials and Methods) before incubation with the virus. An equivalent concentration of Staph A to that used in these experiments had no effect on neutrophil HO. responses to IAV in the absence of MoAbs (eg, H.O, response to Texas 77 IAV in presence of Staph A was 0.86 + 0.16 nmol/min/4 x 10° cells). Where indicated, neutrophils were either preincubated for 90 minutes in control buffer or buffer containing neuraminidase (final concentration, 0.128 U/mL) before stimulation with IAV preparations.
Results shown are the mean + SEM (n = 3 to 4 experiments) neutro- phil HO, response (in nanomoles per minute per 4 x 10° cells) to stimulation with either Texas 77 or Bangkok 79 IAV alone or to aliquots of these strains of |IAV that had been preincubated with 800 or 400 g/mL, respectively, of the 81/4 or 73/1 antiviral MoAbs. Preincubation with MoAbs was performed as described in Fig 1 C. Where indicated (ie, IAV + MoAb/Prot A), MoAbs complexed with Staph protein A (as described in the Materials and Methods) before incubation with the virus. An equivalent concentration of Staph A to that used in these experiments had no effect on neutrophil HO. responses to IAV in the absence of MoAbs (eg, H.O, response to Texas 77 IAV in presence of Staph A was 0.86 + 0.16 nmol/min/4 x 10° cells). Where indicated, neutrophils were either preincubated for 90 minutes in control buffer or buffer containing neuraminidase (final concentration, 0.128 U/mL) before stimulation with IAV preparations.
Fig 8. Effect of pretreating neutrophils with neuraminidase on the ability of the cells to generate H.0. in response to IAV opsonized with RhSP-D or anti-HA MoAbs. Neutrophils were pretreated with neuraminidase (tracings B and D) or contro! buffer (tracings A and C) as described in Table 1, followed by the addition of IAV that had been preincubated with either RhSP-D (6.4 4g/mL RhSP-D multimers; left panel) or anti-HA MoAb (400 g/mL Bangkok 79 MoAb 73/1; right panel), as indicated by arrows. Scopoletin fluorescence tracings representative of 3 similar experiments are shown. Whereas neur- aminidase significantly reduced response to [AV opsonized with RhSP-D (tracing B), it did not reduce response elicited by [AV opso- nized with the MoAbs (tracing D).
Fig 8. Effect of pretreating neutrophils with neuraminidase on the ability of the cells to generate H.0. in response to IAV opsonized with RhSP-D or anti-HA MoAbs. Neutrophils were pretreated with neuraminidase (tracings B and D) or contro! buffer (tracings A and C) as described in Table 1, followed by the addition of IAV that had been preincubated with either RhSP-D (6.4 4g/mL RhSP-D multimers; left panel) or anti-HA MoAb (400 g/mL Bangkok 79 MoAb 73/1; right panel), as indicated by arrows. Scopoletin fluorescence tracings representative of 3 similar experiments are shown. Whereas neur- aminidase significantly reduced response to [AV opsonized with RhSP-D (tracing B), it did not reduce response elicited by [AV opso- nized with the MoAbs (tracing D).
Fig 7. Comparative ability of RhSP-D multimers and RhSP-D sin- gle arms to enhance IAV-stimulated neutrophil H,O. responses. Texas 77 IAV was preincubated with the indicated concentrations of either RhSP-D multimers or RhSP-D single arms. Aliquots of these samples were then added to neutrophils and the resulting H,O, re- sponses were measured using the scopoletin method as described. The mean + SEM of 4 experiments is shown. Whereas both RhSP- D preparations caused significant enhancement (*P < .05) of HO, responses as compared with those elicited by unopsonized IAV, RhSP-D multimers enhanced responses to a significantly greater de- gree than did RhSP-D single arms at all concentrations tested (P < 05). RhSP-D multimers caused a similar degree of enhancement of H,0, responses to Bangkok 79 IAV strain (data not shown).
Fig 7. Comparative ability of RhSP-D multimers and RhSP-D sin- gle arms to enhance IAV-stimulated neutrophil H,O. responses. Texas 77 IAV was preincubated with the indicated concentrations of either RhSP-D multimers or RhSP-D single arms. Aliquots of these samples were then added to neutrophils and the resulting H,O, re- sponses were measured using the scopoletin method as described. The mean + SEM of 4 experiments is shown. Whereas both RhSP- D preparations caused significant enhancement (*P < .05) of HO, responses as compared with those elicited by unopsonized IAV, RhSP-D multimers enhanced responses to a significantly greater de- gree than did RhSP-D single arms at all concentrations tested (P < 05). RhSP-D multimers caused a similar degree of enhancement of H,0, responses to Bangkok 79 IAV strain (data not shown).
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