The relative pathogenicities of three Candida albicans strains differing in the function of ADE2 (the gene encoding phosphoribosylaminoimidazole carboxylase) were evaluated in a murine candidiasis model. C. albicans strain CAI7 (ade2/ade2), previously constructed by site-specific recombination, was avirulent in immunosuppressed mice compared to the parent strain, CAF2-1, and a heterozygous ADE2/ade2 strain obtained by transforming CAI7 with a wild-type allele. The reduced virulence of CAI7 was correlated with the inability to proliferate in either synthetic medium or serum without the exogenous addition of >10 g of adenine/ml. The loss of virulence upon site-specific disruption of the ade2 locus, and the restoration of wild-type virulence with the repair of just one ade2 allele, confirmed that the ADE2 gene and de novo purine biosynthesis were required for Candida pathogenicity. The potential of the phosphoribosylaminoimidazole carboxylase enzyme as a novel target for antifungal drug discovery is discussed.Candida albicans is an opportunistic human pathogen causing mucosal and cutaneous infections as well as life-threatening systemic infections in immunosuppressed patients. The incidence of candidiasis has increased markedly over the past 20 years, coinciding with an increase in immunosuppressed individuals (6). This increase is primarily due to the expanded use of chemotherapy and organ transplantation and to the rapid increase in the numbers of AIDS patients. Candidiasis is currently treated with amphotericin B or with a number of drugs collectively referred to as azoles. Amphotericin B exerts its antifungal activity by binding to the fungal steroid ergosterol and disrupting membrane integrity (1), whereas the azoles directly inhibit the biosynthesis of ergosterol (9). The potential for widespread resistance to azole drugs is an increasing concern (16). The development of safe, efficacious antifungal drugs that exhibit a novel mechanism of action is an important challenge for medical research. A greater understanding of virulence mechanisms and genes required for C. albicans pathogenesis is needed to meet this challenge.Recent attention has focused on the identification of virulence factors that dictate the ability of C. albicans to invade tissues and circumvent host defense responses. Tissue adherence (17), cell wall mannan, serine proteases, cellular hydrolases (2), and phospholipases (8) are examples of virulence factors utilized by C. albicans for infection. Also, a number of C. albicans housekeeping and morphogenesis genes are essential for C. albicans pathogenesis. Although not classical virulence factors, these genes are critical for C. albicans survival in a host. For example, purine, pyrimidine, and heme biosynthesis, which are all necessary for cell growth and division in vitro, are required for C. albicans pathogenesis in experimental murine candidiasis (10). Similarly, myristoyl-coenzyme A:protein N-myristoyl transferase, which is involved in protein myristoylation (22), and the PHR1 gene, which fu...

A new in vitro accelerated biological model, the macrophage-FeCl2-stress system was used for the evaluation of dexamethasone (DEX)-polymer formulations. This model combines the effects of cells (macrophages), transition metal ions (Fe2+), and polymer stress to promote material biodegradation. The cell and material effects of DEX, either in solution or incorporated into a polyetherurethane matrix (DEX/PEU), were monitored. Cell morphology and hydroperoxide formation in the polymer during cell culturing were characterized. After a subsequent treatment with FeCl2 the development of environmental stress cracking in the polymer was evaluated. We attempted to duplicate the biodegradation of PEU in terms of environmental stress cracking (ESC). Our results support the direct involvement of macrophages in polyetherurethane oxidation, probably by inducing hydroperoxide formation in the polymer structure. Under the influence of stress or strain, polymers with sufficient hydroperoxides degrade in the presence of Fe2+ metal ions in a manner that closely resembles the stress cracking that is observed in vivo. By contrast, polymers treated with either agents that inhibit cell activation and/or the oxidative burst, or with cells with no oxidative burst did not show signs of the biodegradative process. We demonstrated a reduction in hydroperoxide formation and no later ESC development in macrophage-cultured PEU in the presence of DEX in solution or in DEX-loaded PEU. We believe the prevention of initial polymer oxidation by reducing the cell's potential to produce oxidative stress at the tissue-biomaterial interface can directly inhibit the ESC degradation of chronically implanted polymers. The in vitro macrophage-Fe-stress system is a valuable tool for reliable assessment and cost-effective evaluation of biomaterials.

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The Caco-2 cell culture model of human small intestinal absorptive cells was used to investigate transepithelial transport. Transport of permeability markers such as mannitol demonstrated that Caco-2 monolayers became less permeable with increasing age in culture. Cells were routinely used for transport studies between day 18 and day 32. A transport index was determined for each compound by calculating the ratio of transport of the molecules under investigation to transport of an internal standard such as the permeability marker mannitol. Comparison of transport rates at 4 and 37 degrees C was a simple approach for differentiating primary transport mechanisms (passive paracellular, passive transcellular, or transporter-mediated) but must be coupled with additional experimental manipulations for definitive determination of transport pathways. Compounds predicted to undergo predominantly paracellular transport (mannitol, FITC, PEG-900, and PEG-4000), transporter-mediated transcellular transport (glucose, biotin, spermidine, or alanine), or lipophilic transcellular transport (alprenolol, propranolol, clonidine, or diazepam) showed differential effects of temperature on rates of transport as well as the transport index.

Abstract:A new in vitro accelerated biological model, the macrophage-FeCl 2 -stress system was used for the evaluation of dexamethasone (DEX)-polymer formulations. This model combines the effects of cells (macrophages), transition metal ions (Fe 2+ ), and polymer stress to promote material biodegradation. The cell and material effects of DEX, either in solution or incorporated into a polyetherurethane matrix (DEX/PEU), were monitored. Cell morphology and hydroperoxide formation in the polymer during cell culturing were characterized. After a subsequent treatment with FeCl 2 the development of environmental stress cracking in the polymer was evaluated. We attempted to duplicate the biodegradation of PEU in terms of environmental stress cracking (ESC). Our results support the direct involvement of macrophages in polyetherurethane oxidation, probably by inducing hydroperoxide formation in the polymer structure. Under the influence of stress or strain, polymers with sufficient hydroperoxides degrade in the presence of Fe 2+ metal ions in a manner that closely resembles the stress cracking that is observed in vivo. By contrast, polymers treated with either agents that inhibit cell activation and/or the oxidative burst, or with cells with no oxidative burst did not show signs of the biodegradative process. We demonstrated a reduction in hydroperoxide formation and no later ESC development in macrophage-cultured PEU in the presence of DEX in solution or in DEX-loaded PEU. We believe the prevention of initial polymer oxidation by reducing the cell's potential to produce oxidative stress at the tissuebiomaterial interface can directly inhibit the ESC degradation of chronically implanted polymers. The in vitro macrophage-Fe-stress system is a valuable tool for reliable assessment and cost-effective evaluation of biomaterials.