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  • br Material and methods br Results br


    Material and methods
    Discussion A. fumigatus utilizes several virulence factors that facilitate its establishment in human hosts. Among these factors, the best known are hydrolytic enzymes such as proteases and phospholipases, toxins, melanine and catalases that neutralize the reactive oxygen intermediates [11], [12], [13]. Production of proteases such as elastases by A. fumigatus strains has been related to the virulence of this mold. Previous studies [11], [16] have demonstrated production of this enzyme in media containing elastin as the unique nitrogen source. Blanco et al. [17], observed that strains isolated from clinical samples with remarkable invasiveness had higher elastase activity, and strains isolated from local lesions had minimal or no elastase activity, like our observations. Kothary et al. [11] and Kolattukudy et al. [16] concluded that strains producing elastase caused extensive destruction of lung parenchyma in mice inoculated with these strains. These mice died within 72 to 96hours post-infection, while non-elastase-producing strains did not cause any relevant pulmonary injury, even in neutropenic animals. Unlike these observations, our findings about elastase producers strains showed survival curves overlapped in neutropenic mice within 72 to 120hours post-infection. In this way, our result does not allow us to correlate elastase activity with mortality or invasive potential in neutropenic or immunocompetent mice. Similarly, another study [18] could not establish a link between enzyme production and the development of invasive disease when they investigated the production of elastase in solid media using strains of A. fumigatus isolated from patients with IPA. The establishment of experimental IPA depends directly on the immunosuppressive regimen used [19] and the immunosuppressive schemes most often utilized include treatment with corticosteroids, and/or chemotherapy [3], [4], [20] or treatment with monoclonal TPMPA receptor against surface antigens of granulocytes [21]. Generally, glucocorticoids and cyclosporin A act preferentially on acquired immune cells such as circulating lymphocytes. Chemotherapeutic agents such as cyclophosphamide and vinblastine act on many types of leukocytes, both from the innate and acquired immune response. Finally, Gr-1/Ly-6G monoclonal antibodies act specifically by depleting granulocytes. We developed a neutropenia model that successfully facilitated the establishment of experimental IPA, by specific neutrophils depletion (Table 2). Our analysis of the fungus/host interaction by transmission electron microscopy (Fig. 3) showed that monocytes and neutrophils from both neutropenic and immunocompetent mice presented phagocytic activity when located near fungal structures. This activity was demonstrated by the formation of surface projections on these phagocytes. We also observed membrane structures involving conidia, a crucial step directly before digestion of the pathogen by the action of oxidant substances and lytic enzymes produced by the phagocytes. These observations are consistent with another findings [20], [22], which showed that the internalization of conidia involved the formation of cytoplasmatic projections by phagocytic cells. The mode of interaction was not affected by neutropenia, meaning that phagocytic cells (from neutropenic or immunocompetent animals) suffered no functional changes. Parameters such as mortality profile, histopathological analysis of affected organs and quantification of tissue fungal burden by CFU counts are widely studied and compared in pathophysiology studies of IPA. Results vary according to the immunosuppressive regimen adopted, the virulence of the pathogen and the animal model employed [19], [21], [23], [24], [25], [26]. The survival curve built from the mortality data from neutropenic mice depicted a very similar profile for mice infected with the A. fumigatus lower (699) and higher (1753) elastase producer strain (Fig. 2). Using an inoculum of 5×107 conidia, no mouse survived for more than 5 days post-infection. Conversely, all immunocompetent animals infected with the same strains survived until 7 days post-infection. These results may indicate that the virulence of A. fumigatus is not only linked to the production of this enzyme, but could be ascribed by other biological features and host's immune conditions.