Macrovascular Endothelium Models

A commonly used model for the macrovascular endothelium are primary human umbilical vein endothelial cells (HUVEC) because they can be isolated relatively easily (Jaffe et al., 1973; Marin et al., 2001). In the simplest forms of single cell type assays, HUVEC are seeded for in vitro infection on multi-well tissue culture dishes or directly on coverslips for microscopic observations. Usually, they are grown to confluence as monolayers on a fibronectin or collagen matrix in order to reproduce the interactive nature of the endothelial cell conjunction.

Adhesion to EC is the first step in most situations for pathogens to leave the bloodstream. To quantify the adhesion step, EC monolayers are incubated with fungal cell suspensions for a defined period of time after which non-adhered cells are washed off. For small inocula, attached cells can then be visualized microscopically and quantified directly. A method described by Ibrahim et al. (1995) made use of an agar-containing medium which was allowed to solidify on the rinsed EC layer. After incubation, the number of attached organism was determined by colony counting and adhesion expressed as a percentage of the initial inoculum. For inocula with high cell concentrations, the same authors applied a radiometric assay based on incorporation of L-(35S) methionine into the fungal cells (Mayer et al., 1992) to measure adherence indirectly.

Using this system for in vitro C. neoformans infection, the role of the capsule and its main component GMX for adherence to EC was investigated, which led to the conclusion that acapsular or poorly encapsulated forms of the fungus may be the form that crosses the epithelium to initiate invasive disease (Ibrahim et al., 1995). The contribution of the C. albicans ALS (agglutinin-like sequence) gene family to adhesion to EC and other host cells has been also extensively investigated (Fu et al., 1998; Hoyer, 2001; Zhao et al., 2005). Host niche-specific functions in adhesion could be assigned for single ALS members by a comparative study of different in vitro models of non-professional phagocytic cells (Zhao et al., 2004).

A different radiometric assay for quantification of adhesion was employed by Spreghini et al. (1999) to identify host cell structures and their corresponding C. albicans receptors that mediate this process. The multifunctional adhesive glyc-oprotein vitronectin (VN) is present in the ECM of endothelia and was known to be bound by C. albicans specifically (Jakab et al., 1993; Limper & Standing, 1994). 3H glucose-labelled yeast cells were bound to ECM proteins or the endothelial cell line EA.hy 926 and adhesion was calculated. It could have be shown that VN is directly involved in adhesion of C. albicans to EC by binding to integrin-like VN receptors expressed by the fungus and, in addition, that a p105 focal adhesion kinase (Fak)-like protein plays a role in controlling yeast cell adhesion (Santoni et al., 2002).

A non-static assay is described, which adds flow as a physiological constant to the macrovascular endothelial in vitro system. This assay (ProteoFlow; LigoCyte Pharmaceuticals, Inc., Bozeman, MT, USA) was originally used for studying the interaction of EC with leukocytes under simulated physiological shear (Bargatze et al., 1994) and was adopted to address the question of how cell surface hydrophobicity (CSH) influences the interaction of C. albicans with EC under physiological conditions (Glee et al., 2001). HUVEC were grown as monolayers on the luminal surface of glass capillary tubes and yeast cell adhesion under physiological flow rates monitored by real-time video capturing. Hydrophobic cells showed more adhesion to EC than hydrophilic cells and previous activation of the EC monolayer by the cytokine IL-1ß increased the binding of hydrophobic C. albicans, probably by induction of the integrin receptors ICAM-1 and ICAM-2 on EC. Treatment of hydrophobic cells with a monoclonal antibody against 6C5 inhibited binding events as it was shown in static assays for the binding on laminin and fibronectin before (Masuoka et al., 1999).

Invasion of EC can be addressed by several fluorescence-based assays which have been developed to quantify the uptake of fungal cells by EC. Differential fluorescence and immunostaining allow enable the identification of adhered fungal cells from those which are taken up by the endothelium monolayer directly (Levitz et al., 1987; Phan et al., 2000, 2005). To monitor unspecific particle uptake by EC, an assay with fluorescein-labeled biotinylated polystyrene beads can be included (Wasylnka & Moore, 2002). An alternative method to determine the invasion index is the nystatin-protection assay in which extracellular organisms are eliminated while intracellular fungal conidia are protected from the fungicide (Wasylnka & Moore, 2002).

In C. neoformans, this methodology has proved to be useful to show that acap-sular, but not encapsulated strains, were internalized by HUVEC (Ibrahim et al., 1995). The phagocytotic process was shown to be dependent upon functional microfilaments as well as on the presence of a heat-labile serum factor, probably complement. This is in contrast to the situation in C. albicans, where internalization occurred in a serum-independent manner (Filler et al., 1995). Therefore, it was concluded that adherence and phagocytosis of both organisms are mediated by different receptors. While C. albicans yeast cells adhere to EC but are poorly phago-cytosed (Phan et al., 2000), the hyphal form of the fungus both adheres avidly and induces its own endocytosis (Filler et al., 1995; Rotrosen et al., 1985). Recently, several lines of evidence have indicated that N-cadherin could be the host cell structure which serves as receptor that recognizes still unknown adhesins on C. albicans hyphae, mediating their endocytosis (Phan et al., 2005).

It is known, that EC also bind and internalize A. fumigatus conidia and hyphae (Paris et al., 1997). However, in contrast to C. albicans, it is the conidial form which is more avidly internalized in vitro (Lopes Bezerra & Filler, 2004). A distinct feature of the pathogenesis of this fungus is that it shows angioinvasion in the lung with subsequent thrombosis (Fraser, 1993). While quiescent endothelia express an anticoagulant (and antiadhesive) phenotype, coagulation is promoted by activated EC (Aird, 2005). Tissue factor is a component which acts at the initial steps of the coagulation cascade (Morrissey, 2001). Recently, evidence was found that internalized hyphae, but not internalized conidia, of A. fumigatus induce HUVEC to express tissue factor. In contrast, although C. albicans is known to stimulate various EC responses (Orozco et al., 2000), it did not stimulate tissue factor. Thus, the difference in the ability to stimulate prothrombotic states in EC reflects the histopa-thology which characterizes infections by the distinct fungi (Lopes Bezerra & Filler, 2004).

The physiological intact endothelium displays polarized functions and structures that distinguish the luminal from the abluminal side (Haller & Kubler, 1999). This polarity has to be taken into account since it may confer differences in the pathogenesis of locally invasive disease versus hematogenously disseminated infection. On the one hand, fungal pathogens enter into the bloodstream by crossing the endothelial cell line from the abluminal to the luminal surface. This is the case when A. fumigatus or C. neoformans invade the lining of the pulmonary vasculature in the lung or when C. albicans enters the blood system leaving its normal habitat in the intestine under host conditions that favour its switch from commensalism to pathogenicity. On the other hand, microorganisms leave the vascular compartment to colonize deeper tissues and organs in the opposite direction, i.e. they pass the endothelium from the luminal to the abluminal side. Recent EC models reflect the different modes of crossing the endothelial barrier (Kamai et al., 2006).

Endothelia from different sites of the body display different characteristics. Current research in vascular biology points to these differential phenotypes and aims to determine organ-related endothelial functions (Kallmann et al., 2002; Sana et al., 2005). Integration of this newly gained knowledge into the experimental setup for in vitro interactions of fungal pathogens with EC should lead to conclusions that are relevant in the situation found in vivo.

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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