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Figure 5 | Veterinary Research

Figure 5

From: The protective immune response produced in dogs after primary vaccination with the LiESP/QA-21 vaccine (CaniLeish®) remains effective against an experimental challenge one year later

Figure 5

CMLA assay: inhibition of the macrophage parasitic index, iNOS activity and production of NO derivatives. Week 58 (W58) was 1 year after primary vaccination, but before the intravenous challenge, and W90 was 32 weeks after the challenge. The macrophages were infected with L. infantum promastigotes and incubated alone or in the presence of autologous lymphocytes for 72 h. After this, the lymphocytes were removed, the cell-free supernatants were conserved for analysis and the macrophages were fixed in order to evaluate the leishmanial killing. Panel A is a comparison of the ability of the dogs’ macrophages to inhibit parasite multiplication after interaction with autologous lymphocytes. The leishmanicidal activity was determined by counting in triplicate the number of parasites per 100 cells in the macrophages co-cultured with the lymphocytes and contrasting this with the macrophages cultured without the lymphocytes. The difference between these results is the percentage inhibition of the parasite index and is expressed as the CMLA index. Panel B is a comparison of the rate of expression of iNOS in the macrophages after 3 days of exposure to autologous lymphocytes. Fixed macrophages were incubated with rabbit polyclonal anti-NOS antibodies. Antibody binding was revealed by labelled anti-rabbit IgG to determine the percentage of iNOS positive macrophages. Panel C is a comparison of the rate of production of NO derivatives from the macrophages during co-culture with autologous lymphocytes. NO2 was determined in the culture supernatants using the modified Griess technique, providing a correlation with the production of the short-lived NO radical. When the CMLA, iNOS and NO2 measurements are consistently increased, this provides evidence of a functional interaction between specific memory lymphocytes and infected macrophages and indicates an increase of NO-mediated parasite killing as a result of vaccination.

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