Our results from neutropenic mice

Our results from neutropenic mice infected with both fungal strains revealed that the inflammatory process was more severe 72hours post-infection (Fig. 4). Angioinvasion was mainly observed 48hours after infection for both strains. Transformation of A. fumigatus conidia into hyphae in immunocompetent mice infected with the 699 strain was not observed, while intense filamentation after 48hours was observed in neutropenic mice infected with both strains (Fig. 5). Weigert staining highlighted vascular disruption in neutropenic mice (Fig. 6). Immunocompetent mice maintained vascular wall integrity and discrete bronchial wall rupturing. Disruption of elastic fibers in the vascular walls has been correlated to the virulence of A. fumigatus[27] but our findings from strain 699 (lower production of elastase) and 1753 (higher production of elastase) suggest that this enzyme is not a critical virulence factor but contributes to the pathogenesis of lesions observed in experimental IPA. Quantification of fungal burden by CFU count consists of plating serial dilutions of a tissue suspension and assuming that a single colony results from a unique viable cell [28]. This classic methodology, widely used because of its low cost, has generated controversial results. Filamentous molds such as A. fumigatus have challenged the efficacy of this method [26], [29]. It is believed that these microorganisms form cell clumps in tissue through continuous filamentation of hyphae during the infection; these Vincristine sulfate are not separated enough to form a single cell. Furthermore, the fragmentation of these structures by inadequate mechanical grinding of the tissue can reduce fungal viability, resulting in a CFU count that is not consistent with the real impairment of the infected tissue [28]. A research conduct by Sheppard et al. on 2006 [30] compared the fungal burden (by CFU counting) in the lungs from cortisone and/or cyclophosphamide-treated mice, which were infected by inhalation of conidia from A. fumigatus. The CFU were counted in lung tissues from mice sacrificed on the day of infection, ensuring that only conidia were present in the tissue. Mice were sacrificed on days 3, 5 and 7 post-infection. The CFU counts conducted on the day of infection with A. fumigatus conidia, which are small and easily scattered at this time-point, resulted in a more reliable count. The authors observed that the fungal burden decreased significantly 3 days after infection and remained stable in subsequent periods (5 and 7 days). Bowman et al. on 2006 [29], using quantification by PCR showed an inverse behavior. A gradual increase in fungal genetic material was observed on days 3, 5 and 7 post-infection, but this methodology does not allow for the assessment of fungal viability in tissue. This result can be explained by the fact that hyphae, unlike conidia, are multicellular structures and, therefore, a major template for the PCR reaction. In agreement with our results (Fig. 7), Duong et al. (1998) [23] also observed that CFU counts progressively decreased as infection duration increased. However, they observed that these counts were significantly higher in immunosuppressed mice when compared to immunocompetent mice at all time-points, which was unlike our findings comparing these groups. It should be emphasized that the presence of conidia was observed in the lung tissues of immunocompetent mice, even in 72hours post-infection (Fig. 5). Considering that phagocytic cells (from neutropenic or immunocompetent animals) suffered no functional changes we suggest that other leukocytes, such as macrophages, act in a compensatory manner to decrease the fungal burden in the absence of neutrophils. Finally, our results confirm that neutrophils have an essential role in the defense against A. fumigatus because their absence results in fungal invasion and major lesions in the lung parenchyma. However, we must consider that the mere presence of the mold in immunocompetent mice induced the migration of neutrophils to the focus of infection. The exacerbation of the inflammatory response following infection aims to prevent fungal invasion, but this phenomenon also causes tissue injury that may or not lead to death. In this study, the production of elastase by A. fumigatus strains was not crucial for its virulence, which suggests that the pathophysiology of experimental IPA could be multifactorial and depends mainly on host\'s immune conditions.