Adhesion Internalization

The adhesion of pathogens or isolated surface components to immune cells - as well as phagocytosis - can be quantified by flow cytometry using specific fluorochromes when the assay is performed with immune cells that do not adhere to tissue culture plates. Normally, fungal cells are labelled with a fluorescent dye (such as FITC) and able to interact with target cells under defined conditions (Newman & Holly, 2001) analysing the size and intensity of the resulting population by flow cytome-try. The interaction between host cell receptors and fungal cells can be addressed by the specific inhibition of the binding using specific antibodies or compounds that are presumed or well-established ligands. For example, DC-SIGN recognizes a large array of pathogens in a mannan-dependent and Lewis oligosaccharide-dependent manner (Feinberg et al., 2001; Frison et al., 2003). Therefore, binding to the pathogen can be blocked by anti-DC-SIGN antibody or by galactomannans (Relloso et al., 2002).

Quantification of adhesion/phagocytosis is difficult given the necessity to differentiate between surface-bound particles and those cells internalized. Although cyto-plasmic cells can be observed by transmission electron microscopy quantitative assays are difficult to carry out since cells and particles are not easily counted (Ezekowitz et al., 1990). Different methods rely on the use of fluorescence microscopy in which yeast cells are pre-labelled by either staining chitin with diaethanol or specific antibodies against fungal surface components or lectines (Levitz et al., 1987; Parod et al., 1986). However, internalized pathogens within living immune cells cannot be studied using this methodology. Quenching fluorescence of surface-bound pre-labelled particles with FITC, crystal violet, or TB (Hed et al., 1985; Lundborg et al., 2006) has been used. The Giemsa stain can be used after tannic acid staining to generate different colours for internalized and surface-bound particles under brightfield illumination (Giaimis et al., 1992). Alternatively, yeast particles can be also stained with TB and distinguished by differential interference contrast microscopy, thereby bypassing the requirement of two different filters. Immune complexes attached to and ingested by neutrophils have been quantified by cytofluorometry using a fluorescence quenching assay which permits differentiation between attachment and ingestion as the fluorescence intensity decreases in the phagosolysosome as a result of the low pH (Sahlin et al., 1983).

Other authors have used calcofluor staining to assess the ability of viable and nonviable C. albicans cells to adhere to, and to be internalized by, host mammalian cells in vitro (Henry-Stanley et al., 2004). Fluorescence microscopy has also been used for C. neoformans phagocytosis assays where specific antibodies for capsule components are used, such as 18B7 (Casadevall et al., 1998). Internalization of the C. neoformans cells can be verified by immunofluorescence staining with Texas Red-conjugated antibody to mouse IgG (Fan et al., 2005). The need to have appropriate antibodies, as it is the case of A. fumigatus, can be bypassed by a combined biotin-calcofluor-staining (BCS) technique in which biotinylated conidia are incubated with BCS macrophages. Extracellular and intracellular conidia are then visualized by calcofluor but only extracellular conidia are stained in red using Cy3-labelled streptavidin (Luther et al., 2006).

In these experiments, the phagocytosis index is commonly ascertained by determining the number of unbound cells (which are removed by washing and quantified by plate counting). Thus phagocytosis can be expressed by subtracting the colony-forming units counted in the washing fluid from those obtained from dose suspension or as percentage of fungal yeast used for macrophages infection.

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.

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