6A–F. As shown in Fig. 6A, Ficoll gradient 1.077 was used to demonstrate a lower percentage of monocytes (10.2%) compared with the results from Ficoll gradients 1119 and 1077 (17.4%, Fig.
6B). Fig. 6C shows the PBMC profile from the culture plates on the fifth day of culture, which had substantial cell debris and 79% lymphocytes. In contrast, in Fig. 6D, where the lymphocytes were passed again in double Ficoll (1.119 and 1.077), the levels of lymphocytes were higher (91.7%). The percentage of lymphocyte purity using anti-CD4 or anti-CD8 antibodies and the Epacadostat mw sorting using magnetic column demonstrated high levels for CD4 (91.7%, Fig. 6E) and CD8 (92.7%, Fig. 6F) T-cell purification. The immune response against Leishmania sp. is highly dependent on the microbicidal action of macrophages, which, although the host target cells of this protozoan, have full capacity for antigen presentation and establishment of an effective response against the parasite ( Pinelli et al., 1999). This methodology could be employed in immunogenic studies during testing of candidate vaccines against CVL. Thus, the microbicidal ability of antigen-specific CD4 or CD8 T cells co-cultured with Leishmania-infected
macrophages could be investigated in dogs during testing of vaccines or treatment strategies against CVL. Our results indicated that differentiated macrophages after 5 days of culture induced increases in both phagocytic and microbicidal activity (Fig. 1, Fig. 2 and Fig. 3). Moreover, only at this time point was it possible to selleck kinase inhibitor observe multinucleated giant cells and vacuolation of the cytoplasm. These results were encouraging for macrophages at this stage of maturation being satisfactory for application however in in vitro experiments using L. chagasi infection. Furthermore, the differentiation of peripheral blood monocytes into macrophages permits obtaining cells less invasively than puncture through the peritoneal compartment ( Zhang et al., 2008 and Sampaio et al., 2007). From the morphological point of view, the presence of multinucleated giant cells
from cell fusion in cultures of monocytes differentiated into macrophages is reported in humans. However, it is known that a variety of inflammatory conditions can generate these cells (Gerberding and Yoder, 1993). These cells were previously reported in canine macrophages in the 1970s, in the studies of Ho and Babiuk (1979), however, the cell fusion occurred only in cultures after 4 weeks. In addition, they proposed a virtually pure culture, after 10 days of culture, from which macrophages can be maintained for up to 2 months under in vitro conditions. However, it is noteworthy that the longer the duration of culture, the greater the chances of contamination by different microorganisms. Therefore, it would be helpful to standardize these cultures so that experiments could be performed more quickly. In this context, Goto-Koshino et al.