Models to Analyse the Interaction with Immune Cells

Both innate and acquired immunity are essential for the development of resistance to pathogenic fungi (Romani, 2004). Phagocytic cells, such as neutrophils, DCs and macrophages represent a first line of defence against microorganisms. They recognize different PAMPs (pathogen-associated molecular patterns), exposed on the surface of several pathogens by different specific PRRs receptors (Pattern Recognition Receptors), expressed on the surface of the immune cell. PAMPs-PRRs interaction is considered a prerequisite for the innate system to be able to discriminate between fungal pathogens of different nature and to develop the appropriate adaptive immune response (Underhill & Gantner, 2004).

Neutrophils are important in early immunity stages to fungal infection. The quick arrival of these cells at the site of infection depends on their ability to respond to chemoattractants released by pathogens in situ. Their main function involves phagocytosis, mediated by complement and Fc receptors, and killing of ingested pathogens by oxidative-mediated mechanisms (Aderem & Underhill, 1999; Underhill & Ozinsky, 2002; Witko-Sarsat et al., 2000) but, unlike macrophages and DCs, they do not function as antigen presenting cells (APCs) to T cells (Mansour & Levitz, 2002).

DCs are bone marrow-derived cells of both lymphoid and myeloid origin present in all lymphoid organs. DCs are potent APCs with a unique ability to sense and respond to fungus-associated information by means of PAMPs-PRRs patterns that result in a qualitatively different adaptive T-helper cell (TH) immune responses (Bacci et al., 2002; d'Ostiani et al., 2000).

Macrophages are tissue specific phagocytes specialized as APCs and microbe-killing cells. These tissue-resident macrophages are found at a higher frequency than are DCs, making them more likely to encounter a pathogen upon initial infection.

Protocols for the isolation and purification of these immune cells from healthy human donors commonly making use of flow cytometry are described for neutrophils, monocytes, macrophages, and DCs (Drenth et al., 1995; Read et al., 1993). Generally, the identity of the immune cells is determined by fluorescent-activated cell sorter (FACS) analysis in which a cell type specific antibody is coupled to a second antibody that is linked to certain fluorochromes as FITC or phycoerythryne.

Isolated monocytes (CD14+) can differentiate into macrophages by incubation with 10% human serum or be transformed into DCs by incubation with IL-4 and GM-CSF and Langerhans cells (LC) can be obtained from cord blood CD34+ progenitors (Sallusto & Lanzavecchia, 1994; Serrano-Gomez et al., 2004). Neutrophils are also easily isolated either from blood of healthy donors or from the blood and peritoneal cavity of mice. Murine peritoneal macrophages can be recovered from mice after intraperitoneal injection of sterile 10% thioglycolate medium (Kaposzta et al., 1999; Taylor et al., 2002). Human alveolar macrophages (HAMs) can be obtained from bronchoalveolar wash (BAL) of human donors (Dubourdeau et al., 2006) or mice (Steele et al., 2005).

In addition to the isolation of immune cells from donors or animals, several immune cell lines are available. The most commonly used macrophage cell lines are the murine peritoneal macrophages-like cell line RAW264.7 (ATCC number TIB-71), which is differentiated with IFN-y and J774A.1 (ATCC TIB-67), a murine (BALB/c; haplotype H-2d) cell line derived from a reticulum sarcoma (Ralph et al., 1975) that display certain phenotypic characteristics similar to murine peritoneal macrophages. An alternative to the use of primary alveolar macrophages is the MH-S cell line of murine alveolar macrophages (Matsunaga et al., 2001). HL-60 is a myelomonocytic cell line which differentiates into granulocytes when the cells are grown in medium supplemented with dimethilformamide, dimethyl sulfoxide, or retinoic acid (Harris & Ralph, 1985; Mullick et al., 2004).

In addition to these standard lines, some others have been constructed to analyse specifically the role of certain receptors in the recognition of surface components of the fungal cell wall, such as the K562-DC209 (Relloso et al., 2002) and HEK293 (Gantner et al., 2005) that express the DC-SIGN and Dectin-1 receptors respectively. To determine the contribution of a given host cell receptor to the interaction with a pathogen, immune cells can be derived from mice devoid of the molecule of interest (Blander & Medzhitov, 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.

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