Indoximod opposes the immunosuppressive effects mediated by IDO and TDO via modulation of AhR function and activation of mTORC1.

Indoximod has shaped our understanding of the biology of IDO1 in the control of immune responses, though its mechanism of action has been poorly understood. Previous studies demonstrated that indoximod creates a tryptophan (Trp) sufficiency signal that reactivates mTOR in the context of low Trp concentrations, thus opposing the effects caused by IDO1. Here we extend the understanding of indoximod's mechanism of action by showing that it has pleiotropic effects on immune regulation. Indoximod can have a direct effect on T cells, increasing their proliferation as a result of mTOR reactivation. Further, indoximod modulates the differentiation of CD4+ T cells via the aryl hydrocarbon receptor (AhR), which controls transcription of several genes in response to different ligands including kynurenine (Kyn). Indoximod increases the transcription of RORC while inhibiting transcription of FOXP3, thus favoring differentiation to IL-17-producing helper T cells and inhibiting the differentiation of regulatory T cells. These indoximod-driven effects on CD8+ and CD4+ T cells were independent from the activity of IDO/TDO and from the presence of exogenous Kyn, though they do oppose the effects of Kyn produced by these Trp catabolizing enzymes. Indoximod can also downregulate expression of IDO protein in vivo in murine lymph node dendritic cells and in vitro in human monocyte-derived dendritic cells via a mechanism that involves signaling through the AhR. Together, these data improve the understanding of how indoximod influences the effects of IDO, beyond and distinct from direct enzymatic inhibition of the enzyme.

Enterococcus in surface waters from the Des Moines River (Iowa) watershed: location, persistence and vancomycin resistance.

The object of this study was to quantify vancomycin-resistant enterococci in surface water from Central Iowa obtained from April 2007 to August 2007. Water from established sampling sites in four watersheds was plated on bile-esculin agar. Presumptively identified enterococci were categorized as "above the level of concern" if the sample contained ≥ 107 CFU per 100 ml. Confirmation of isolates as enterococci was based on growth at elevated temperature in high salt and on Enterococcus agar. Isolates that grew on 6 µg/ml vancomycin agar were deemed resistant. PCR analysis of resistant strains characterized vancomycin resistance genes. 77.2% of surface water samples from Central Iowa contained enterococci. Among enterococcal isolates, 10.4% grew on media containing 6 µg/ml vancomycin. PCR analysis of resistance genes showed a preponderance of VanC2/C3 in the area studied and VanB was not detected. Vancomycin-resistant Enterococcus is present in Central Iowa surface waters but resistance rarely involved VanA genotypes. Nevertheless, the potential for community-acquired infections remains a risk.

Quantitative real time PCR detection of Clostridium difficile growth inhibition by probiotic organisms.

BACKGROUND: Probiotic microorganisms are potential treatments for Clostridium difficile diarrheal disease (CDD) but better methods are needed to determine the relative potency of probiotic microorganisms against pathogenic organisms in mixed cultures. AIM: Quantify C. difficile in the presence of putative probiotic organisms using molecular methods to determine relative probiotic potency. MATERIALS AND METHODS: C. difficile strains were cultivated anaerobically. Serial dilutions of Lactobacillus cultures or microbial mixtures from kefir were co-cultured with C. difficile for 48 hours. Bacterial DNA was extracted and qPCR was used to measure C. difficile toxin A gene, on the basis of cycle threshold (Ct) number. RESULTS: Strains of Lactobacillus (human and ATCC derived), and mixed cultures from commercial kefir were co-cultured with C. difficile. Lactobacillus and the microbial mixture from kefir were ranked in order of their potency in C. difficile growth inhibition. CONCLUSIONS: PCR allows facile quantification of C. difficle in the presence of other. The technique measures relative potency of over-the-counter probiotics and may predict human strains meriting probiotic status.

Sodium choleate (NaCho) effects on Candida albicans: implications for its role as a gastrointestinal tract inhabitant.

Candida albicans at times resides in the intestinal tract, where it experiences exposure to bile salts suggesting a study of the effects of crude bile salts in the form of sodium choleate (NaCho) on C. albicans growth, expression of virulent phenotypes, and adaptation to physiological challenges in vitro. Growth and phenotype alteration was examined by challenging clinical isolates of C. albicans with a wide range of NaCho concentrations by using conventional microbial physiology methods. Our results showed that (1) NaCho did not inhibit growth of yeast cells, up to 40 mg/ml; (2) NaCho powerfully stimulated the hypha formation; (3) NaCho at 2.5 and 5 mg/ml significantly induced CDR1p and biofilm formation, but these effects decreased at higher NaCho concentrations; (4) loss of cell integrity with exposure to 56 degrees C for 15 min, was exacerbated by increasing levels of NaCho; (5) NaCho protected yeast from hydrogen peroxide damage in a dose-dependent manner; and (6) catalase activity was increased by NaCho exposure.

Antifungal mechanisms supporting boric acid therapy of Candida vaginitis.

BACKGROUND: Boric acid is a commonly cited treatment for recurrent and resistant yeast vaginitis, but data about the extent and mechanism of its antifungal activity are lacking. OBJECTIVES: The aim of this study was to use in vitro methods to understand the spectrum and mechanism of boric acid as a potential treatment for vaginal infection. METHODS: Yeast and bacterial isolates were tested by agar dilution to determine the intrinsic antimicrobial activity of boric acid. Established microbial physiology methods illuminated the mechanism of the action of boric acid against Candida albicans. RESULTS: C. albicans strains (including fluconazole-resistant strains) were inhibited at concentrations attainable intravaginally; as were bacteria. Broth dilution MICs were between 1563 and 6250 mg/L and boric acid proved fungistatic (also reflected by a decrease in CO(2) generation); prolonged culture at 50,000 mg/L was fungicidal. Several organic acids in yeast nitrogen broth yielded a lower pH than equimolar boric acid and sodium borate but were less inhibitory. Cold or anaerobic incubation protected yeast at high boric acid concentrations. Cells maintained integrity for 6 h in boric acid at 37 degrees C, but after 24 h modest intrusion of propidium iodide occurred; loss of plate count viability preceded uptake of vital stain. Growth at sub-MIC concentrations of boric acid decreased cellular ergosterol. The drug efflux pump CDR1 did not protect Candida as CDR1 expression was abrogated by boric acid. Boric acid interfered with the development of biofilm and hyphal transformation. CONCLUSIONS: Boric acid is fungistatic to fungicidal depending on concentration and temperature. Inhibition of oxidative metabolism appears to be a key antifungal mechanism, but inhibition of virulence probably contributes to therapeutic efficacy in vivo.

Tetracycline effects on Candida albicans virulence factors.

OBJECT: To determine if tetracycline, previously reported to increase the probability of developing symptomatic vaginal yeast infections, has a direct effect on Candida albicans growth or induction of virulent phenotypes. METHOD: In vitro, clinical isolates of yeast were cultivated with sublethal concentrations of tetracycline and yeast cell counts, hyphal formation, drug efflux pump activity, biofilm production, and hemolysin production were determined by previously reported methods. RESULTS: Tetracycline concentrations above 150 microg/mL inhibited Candida albicans, but at submicrogram/mL, a modest growth increase during the early hours of the growth curve was observed. Tetracycline did not inhibit hyphal formation at sublethal concentrations. Hypha formation appeared augmented by exposure to tetracycline in the presence of chemically defined medium and especially in the presence of human serum. Efflux pump CDR1 was upregulated and a nonsignificant trend toward increased biofilm formation was noted. CONCLUSION: Tetracycline appears to have a small growth enhancing effect and may influence virulence through augmentation of hypha formation, and a modest effect on drug efflux and biofilm formation, although tetracycline did not affect hemolysin. It is not clear if the magnitude of the effect is sufficient to attribute vaginitis following tetracycline treatment to direct action of tetracycline on yeast.

Effect of commonly used herbicides on the virulence factor CDR1 in Candida albicans.

The ubiquitous yeast Candida albicans becomes a troublesome pathogen by inducing virulence factors in response to environmental stimuli. Among these virulence factors is a drug-exporting transport protein, Cdrlp, which renders cells resistant to certain antifungal drugs. The expression of the CDR1 gene responds to a wide spectrum of stimuli, including drugs, heat shock, and steroid hormones. The aim of the present study is to characterize the effects of commonly used herbicides on the expression of CDR1. Following exposure of C. albicans cultures to varying doses of herbicides and azole drugs, CDR1 expression was quantified by flow cytometry using a reporter strain in which expression of a green fluorescent protein is under the control of the CDR1 promoter. Correlating CDR1 expression with cell growth and survival revealed that-similar to antifungal azole drugs-herbicides induce CDR1 expression only at inhibitory doses. It is concluded that none of the tested herbicides mimics the worrisome action of hormones, which increase virulence without reducing survival.

Key physiological differences in Candida albicans CDR1 induction by steroid hormones and antifungal drugs.

The Candida albicans CDR1 gene, encoding an ABC transporter that functions as an efflux pump, is thought to be involved in pathogenic adaptation and uses mammalian hormones and other environmental cues to regulate its activity. Exposure of several clinical isolates of C. albicans to 1 x 10(-8) M 17beta-oestradiol increased CDR1 expression and the isolates showed a positive correlation between oestrogen induction of CDR1 and growth in the presence of oestrogen. A reporter strain carrying the GFP gene under the control of the CDR1 promoter was used to analyse the effect of steroid hormones and antifungal drugs on CDR1 expression by flow cytometry. We found that among the many hormones tested, only oestradiol and progesterone induce CDR1 expression. CDR1 induction requires hormone concentrations greater than 10(-8) M, a threshold reached in vivo only by progesterone. Using the GFP-reporter strain, we show CDR1 induction by female but not male human serum and demonstrate that exposure of C. albicans to physiological concentrations of progesterone measurably increases resistance to fluconazole, miconazole and 5-fluorouracil. Simultaneous exposure of C. albicans to hormones and antifungal drugs provided evidence that both agents induce CDR1 expression via different mechanisms with different saturation points.

Adherence and blocking of Candida albicans to cultured vaginal epithelial cells: treatments to decrease adherence.

BACKGROUND: Pathogenesis of mucosal microorganisms depends on adherence to the tissues they colonize and infect. For Candida albicans, cell surface hydrophobicity may play a significant role in tissue binding ability. METHODS: A continuous cell line of vaginal epithelial cells (VEC) was grown in keratinocyte serum-free medium (KSFM) with supplements and harvested by trypsinization. VEC were combined with yeast cells to evaluate adherence and inhibition of adherence. In this experimental setup, yeast stained with fluorescein isothiocyanate were allowed to attach to VEC and the resulting fluorescent VEC were detected by flow cytometry. RESULTS: VEC were cultured and examined daily after plating and showed morphology similar to basal epithelial cells. Culture media supplemented with estradiol showed increased VEC proliferation initially (first 24 h) but cell morphology was not altered. Fluorescinated Candida cells bound effectively to the cultured VEC. Using fresh cells exposed to various preparations of K-Y, we showed that all formulations of the product reduced Candida binding to VEC by 25% to 50%. While VEC were generally harvested for use in experiments when they were near confluent growth, we allowed some cultures to grow beyond that point and discovered that cells allowed to become overgrown or stressed appeared to bind yeast cells more effectively. CONCLUSION: Flow cytometry is a useful method for evaluating binding of stained yeast cells to cultured VEC and has demonstrated that commercially available products have the ability to interfere with the process of yeast adherence to epithelial cells.

Flow cytometry of Candida albicans for investigations of surface marker expression and phagocytosis.

Several cytoplasmic virulence factors of Candida albicans are altered in the presence of estrogen and this fact may imply the existence of a global virulence regulatory system in this organism. The response of virulence-associated surface markers to estrogen, however, has not been studied. We exploited flow cytometry methods for assessment of the iC3b receptor analog and mannoproteins on 2 clinical yeast strains selected for their different rates of growth in the presence of estradiol 17beta. Although, as expected, iC3b receptor analog expression increased in the presence of glucose, growth in the presence of estradiol did not increase the levels of iC3b receptor analog on either organism. Exposure to human serum caused massive conversion to mycelial growth, but cells examined by flow cytometry did not show increased levels of iC3b receptor analog expression, possibly due to inability of the flow cytometer to sample the mycelial forms of Candida. In contrast, estradiol increased expression of mannoproteins as evidenced by concanavalin A binding to yeast. This increase occurred in both yeast strains but was less pronounced with strain GT188, which also showed limited growth in estradiol compared to strain GT142. Effective phagocytosis by human neutrophils required exposure of yeast to human serum. Yeast grown in the presence of estradiol were ingested by human PMN but not at a significantly greater rate than yeast grown without estradiol. While flow cytometry appears to be useful in determining estrogen-enhanced concanavalin A binding to yeast, it probably does not reflect the surface markers on large mycelial masses. Consequently, the results of this study are applicable to Candida primarily in its yeast form.

Surface modifying substances that reduce apparent yeast cell hydrophobicity.

OBJECTIVE: To determine whether several topical compounds and other chemical entities are able to diminish the surface hydrophobicity of yeast cells. METHOD: Hydrophobicity of yeast cells was determined by binding styrene microspheres to the surface of untreated yeast or yeast pre-incubated with various substances with potential for cell surface modification. The degree of microsphere adherence to yeast cells was measured by flow cytometry. RESULTS: A significant reduction in cell surface hydrophobicity was observed when yeast was incubated in protein-containing media. Other compounds that effectively reduced microsphere binding were various formulations of K-Y and heparin. Divalent cations (Ca+ + , Mg+ + , Zn+ + , Cu + + ) were also potent inhibitors of microsphere adherence. It was possible to remove substances contributing to microsphere binding by chemical extraction of the yeast. Yeast having reduced microsphere binding activity also showed diminished binding of concanavalin A. CONCLUSIONS: Several commercially available compounds were able to block binding of styrene microspheres to yeast. Some of the binding activity appeared to be attributable to mannose-containing surface components. These findings have implications for formulating therapeutic products that might block yeast binding to tissues.

Evaluation of relative yeast cell surface hydrophobicity measured by flow cytometry.

OBJECTIVE: To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties. METHODS: Yeast isolates were suspended in phosphate-buffered saline and mixed with deep blue-dyed polystyrene microspheres. Flow cytometry was used to detect the degree of microsphere binding to yeast cells. Different strains of yeast were compared for intrinsic microsphere binding activity and changes in growth conditions were invoked to modify the relative surface hydrophobicity. RESULTS: Commercially available blue-dyed polystyrene microspheres showed strong fluorescence in the FL3 channel, whereas yeast cells did not show appreciable FL3 fluorescence. Microspheres and yeast were generally distinguishable on the basis of size revealed by forward light scatter. This method showed a wide variation in intrinsic cell surface hydrophobicity among Candida albicans strains. Likewise, variation in hydrophobicity of non-albicans yeast species was observed. Growth on solid media, incubation at 25 degrees C, or 250 mg/dl glucose concentration increased hydrophobicity compared with growth in liquid media, incubation at 37 degrees C, or 50 mg/dl glucose, respectively. Growth in 1 x 10(-9) M estradiol had no appreciable effect on hydrophobicity. CONCLUSIONS: Stained latex microspheres fluoresced in the FL3 channel of the flow cytometer and bound to yeast cells to an extent related to the surface hydrophobicity of the yeast. Binding detected by flow cytometry showed that clinical yeast isolates varied in intrinsic binding capacity and this binding ability was altered by different growth conditions. The implications for virulence regulation among yeast isolates are discussed.

Isolation and partial characterization of Hsp90 from Candida albicans.

Hsp90 is a stress-induced protein involved in many cellular processes including the regulation of signal transduction and steroid hormone response pathways in higher eukaryotic cells. Candida albicans hsp90 has a mass of 82 -Da and has previously been implicated as a virulence factor. A 47-kDa C-terminal fragment of Candida hsp90 is a target for an immune response to C. albicans infections. A C. albicans hsp90 specific polyclonal antibody was developed against a synthetic peptide containing a previously defined epitope of the 47-kDa fragment. This antibody was used to investigate the cellular localization and induction of hsp90 in the fungus. By means of cell surface protein extraction, hsp90 is shown to be localized on the cell surface as well as in the cytoplasm. On the cell surface, it appears only as an 82-kDa protein. In the cytoplasm, anti-hsp90 detected the 82-kDa protein as well as 72-kDa and 47-kDa bands on SDS-PAGE gels. The cytoplasmic protein bands were heat inducible and appeared to be estrogen induced as well, suggesting that C. albicans modulates hsp90 expression in response to environmental changes. Since the 82-kDa protein is also found on the surface of the cells, hsp90 may be directly involved in sensing environmental changes. It may also be important for recognition of its host or elements of the host immune system and antibody responses to the molecule and may therefore be useful for diagnostic or prognostic evaluation.