Killing and Proliferation

Most of the studies of phagocyte-mediated killing are done with macrophages and neutrophils as they play a predominant role in the host defence against fungal infection. Although is has been proposed recently that DCs also play an important role, they are less potent in killing fungal pathogens than monocytes, macrophages, and neutrophils.

The classical method of determining phagocytosis and intracellular killing is the counting of the surviving fungal population (plate counting) after the incubation with host cells. Fungal cells are added to give appropriate infection ratios with host cells and the percentage of killing is estimated after lysis of the immune cells (Kullberg et al., 1993; Netea et al., 2004b; Vonk et al., 2002). As an alternative, an end-point survival assay can be used that covers a range of high multiplicities of infection. This system has proved to be useful to determine differences in the resistance to host phagocytes in mutants altered in signal transduction pathways (Marcil et al., 2002). Phagocytosis and killing assay with acapsular or poorly encapsulated strains in comparison to the capsulated C. neoformans strains established the role of the structure in these processes. It inhibits phagocytosis of the fungus by macrophages, DCs and neutrophils and also inhibits the killing inside the host cells (Kozel, 1977; Levitz et al., 1997).

H. capsulatum has adapted to survive within human macrophages. The fungus is able to inhibit phagosome-lysosome fusion by controlling the intraphagosomal pH and creating an intracellular environment which permits its proliferation (Eissenberg et al., 1988, 1993). In contrast, DCs seem to be efficient in killing of the pathogen. The mechanisms underlying this difference has been investigated using FITC-dextran labelled DCs (Gildea et al., 2005) while the intracellular growth of H. capsulatum could be quantified by measuring the incorporation of [3H]-leucine (Newman & Gootee, 1992). A comprehensive compilation of methods that allows the investigation of the interaction of this fungus with immune cells can be found in Newman (2005).

Killing of target fungal cells by immune effector cells can be addressed by the methods outlined above (see section 'Techniques to monitor cell viability and tissue integrity' in this chapter). Assays based on XTT or MTT have been used in C. albicans to determine the damage caused by immune cells to extracellular hyphae (Netea et al., 2004a; Vonk et al., 2002, 2005) and in A. fumigatus to test the antifungal activity of a new compound in combination with neutrophils, monocytes, and macrophages (Choi et al., 2004).

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

Get My Free Ebook


Post a comment