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Background: The tissue-signaling cytokines IL-17 and IL-22 are critical to host defense against oral Candida albicans infection, by their induction of oral antimicrobial peptide expression and recruitment of neutrophils. Mucosal Th17 cells which produce these cytokines are preferentially depleted in HIV-infected patients. Here, we tested the hypothesis that defective IL-17- and IL-22-dependent host responses to C. albicans determine the phenotype of susceptibility to oropharyngeal candidiasis (OPC) in transgenic (Tg) mice expressing HIV-1., Results: Naïve CD4+ T-cells and the differentiated Th1, Th2, Th17, Th1Th17 and Treg lineages were all profoundly depleted in cervical lymph nodes (CLNs) of these Tg mice. However, naive CD4+ cells from Tg mice maintained the capacity to differentiate into these lineages in response to polarizing cytokines in vitro. Expression of Il17, Il22, S100a8 and Ccl20 was enhanced in oral mucosal tissue of non-Tg, but not of Tg mice, after oral infection with C. albicans. Treatment of infected Tg mice with the combination of IL-17 and IL-22, but not IL-17 or Il-22 alone, significantly reduced oral burdens of C. albicans and abundance of Candida hyphae in the epithelium of tongues of infected Tg mice, and restored the ability of the Tg mice to up-regulate expression of S100a8 and Ccl20 in response to C. albicans infection., Conclusions: These findings demonstrate that defective IL-17- and IL-22-dependent induction of innate mucosal immunity to C. albicans is central to the phenotype of susceptibility to OPC in these HIV transgenic mice.

Objectives: In this study, we developed a nanoparticulate nystatin formulation and performed a comparative evaluation against a commercial nystatin preparation of its in vitro and in vivo antifungal activities., Methods: A nystatin nanosuspension was prepared from a commercially available suspension by wet-media milling. The nanosuspension was characterized for particle size by laser diffraction and assayed for content by HPLC. Its in vitro activity was evaluated against Candida albicans strains SC5314 and LAM-1 (12.5-5000 μg/mL) using an agar plate assay and its in vivo efficacy was evaluated using a murine model of oral candidiasis. Briefly, DBA/2 mice were immunosuppressed with cortisone acetate, orally infected with C. albicans strain LAM-1, and treated for 14 days with conventional nystatin suspension, nystatin nanosuspension or saline control. Efficacy endpoints were oral fungal burden, mouse survival and organ histopathology. A single-dose pharmacokinetic study was also performed., Results: The median particle size of the nystatin suspension was reduced from 6577 to 137 nm. The HPLC assay demonstrated a nystatin content of 98.7% ± 0.8% of the label claim. In vitro activity was superior to that of the conventional nystatin suspension at 100-5000 μg/mL concentrations. Beginning on day 3 of treatment, lower oral burdens of C. albicans were found in the nanosuspension group compared with the suspension and control groups. Mouse survival was also superior in the nanosuspension group. No systemic absorption was observed., Conclusions: Taken together, these data reveal that nanonization of nystatin provides a novel approach to enhancing its efficacy in the treatment of oral candidiasis.

The critical impairments of innate and adaptive immunity that cause susceptibility to mucosal candidiasis in human immunodeficiency virus (HIV) infection have not been fully determined. We therefore conducted an analysis of macrophage-mediated responses to Candida albicans in transgenic (Tg) mice expressing Nef, Env, and Rev of HIV type 1 (HIV-1) in CD4(+) T cells, dendritic cells, and macrophages and developing an AIDS-like disease (CD4C/HIV(MutA) Tg mice). Macrophages were successfully recruited to the oral and gastric mucosae of these Tg mice in response to chronic carriage of C. albicans and displayed polarization toward an alternatively activated phenotype. Functionally, peritoneal macrophages from uninfected Tg mice exhibited increased phagocytosis of C. albicans and enhanced production of interleukin 6 and monocyte chemoattractant protein 1, demonstrating that the HIV-1 transgene independently activates selected macrophage functions. Production of H(2)O(2) by macrophages from Tg mice primed with gamma interferon and treated with phorbol 12-myristate 13-acetate or C. albicans was moderately reduced, but expression of the HIV-1 transgene did not alter production of nitric oxide or reduce killing of C. albicans. A knockout of the inducible nitric oxide synthase (NOS2) gene in these Tg mice did not augment oral or gastrointestinal burdens during chronic carriage of C. albicans or cause systemic dissemination, likely due to a redundancy provided by partially preserved production of H(2)O(2) and oxygen-independent candidacidal mechanisms. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine the susceptibility to mucosal candidiasis.

Controlled studies on the immunopathogenesis of mucosal candidiasis in HIV infection have been hampered by the lack of a relevant animal model. We have previously reported that oral Candida infection in CD4C/HIV transgenic mice expressing gene products of HIV-1 in immune cells and developing an AIDS-like disease closely mimics oropharyngeal candidiasis in human HIV infection. The role of defective dendritic cells and CD4+ T cells in impaired induction of protective immunity and in the phenotype of chronic oral carriage of C. albicans can now be investigated under controlled conditions in these transgenic mice.

Candida albicans causes oropharyngeal candidiasis (OPC) but rarely disseminates to deep organs in human immunodeficiency virus (HIV) infection. Here, we used a model of OPC in CD4C/HIV(Mut) transgenic (Tg) mice to investigate the role of polymorphonuclear leukocytes (PMNs) and CD8+ T cells in limiting candidiasis to the mucosa. Numbers of circulating PMNs and their oxidative burst were both augmented in CD4C/HIV(MutA) Tg mice expressing rev, env, and nef of HIV type 1 (HIV-1), while phagocytosis and killing of C. albicans were largely unimpaired compared to those in non-Tg mice. Depletion of PMNs in these Tg mice did not alter oral or gastrointestinal burdens of C. albicans or cause systemic dissemination. However, oral burdens of C. albicans were increased in CD4C/HIV(MutG) Tg mice expressing only the nef gene of HIV-1 and bred on a CD8 gene-deficient background (CD8-/-), compared to control or heterozygous CD8+/- CD4C/HIV(MutG) Tg mice. Thus, CD8+ T cells contribute to the host defense against oral candidiasis in vivo, specifically in the context of nef expression in a subset of immune cells.

Although the echinocandin caspofungin primarily inhibits the synthesis of cell wall 1,3-beta-D-glucan, its fungicidal activity could also potentially perturb the expression of virulence factors involved in the ability of Candida albicans to cause infection. Expression of the C. albicans secretory aspartyl proteinase (SAP) and phospholipase B (PLB) virulence genes was determined by reverse transcription-PCR after the addition of caspofungin to cells grown for 15 h in Sabouraud dextrose broth. In cells that remained viable, expression of SAP1 to SAP3, SAP7 to SAP9, and PLB1 was unaltered after exposure to fungicidal concentrations (4 to 16 micro g/ml) of caspofungin over a period of 7 h. However, expression of SAP5 increased steadily beginning 1 h after exposure to caspofungin. These results indicate that caspofungin is rapidly fungicidal against C. albicans, before any suppression of SAP or PLB1 gene expression can occur.

To comprehensively assess the in vivo expression of Candida albicans hydrolytic enzyme genes during oropharyngeal candidiasis (OPC), a controlled sequential analysis of the temporal expression of individual members of the SAP (secretory aspartyl proteinase) gene family and PLB1 (phospholipase B) in a murine model of OPC was conducted. Acute infections in intact C3H and DBA/2 mice were terminated by clearance of C. albicans within 7 days after oral inoculation, but transgenic (Tg) mice expressing human immunodeficiency virus type 1 were persistently colonized until a final outgrowth before death. In contrast to the sustained expression of other SAP genes and PLB1, SAP7 and SAP8 were conspicuously distinguished by their transient expression in both intact and Tg mice. SAP5 and SAP9 were most strongly expressed throughout the course of infection in the Tg mice. These findings indicate that expression of individual members of the C. albicans SAP gene family is differentially regulated during experimental OPC.

To elucidate the implications of secreted aspartyl proteinase (Sap)2p in the pathogenesis of Candida infections, the SAP2 gene was expressed in Saccharomyces cerevisiae and overexpressed in Candida albicans. The coding region of SAP2, including its signal sequence and propeptide, was amplified by PCR and cloned downstream of the S. cerevisiae or C. albicans ADH1 promoter. Plasmid expression of SAP2 in S. cerevisiae showed that the signal peptide was functional. Integrative transformation of S. cerevisiae and C. albicans was accomplished by homologous recombination within the URA3 locus for S. cerevisiae and the SAP2 locus for C. albicans. Negative control transformants carried plasmids either without the SAP2 insert or with mutated sap2. S. cerevisiae and C. albicans transformants showed similar growth rates to their parental strains or negative controls, when grown in medium containing amino acids. However, in medium with BSA as sole nitrogen source, constitutive expression of SAP2 enabled S. cerevisiae to grow and increased the growth rate of C. albicans. In both media, only S. cerevisiae transformants harbouring SAP2 secreted the enzyme, as confirmed by proteinase activity assays and immunoblotting. When C. albicans was grown in amino acids medium, the enzyme was detected exclusively in transformants constitutively expressing SAP2. However, in BSA medium these strains secreted enzyme earlier and secreted higher amounts of enzyme and total proteinase activity. In pathogenicity studies in intact mice, expression of Sap2p as a sole putative virulence factor did not cause S. cerevisiae to become virulent and constitutive overexpression of SAP2 did not augment virulence of C. albicans in experimental oral or systemic infection.

An increasingly diverse array of clinically relevant animal models of candidiasis have been established that mimic both the immune perturbations of the host and tissue-specific features of candidiasis in humans. Cause-and-effect analysis of Candida host–pathogen interactions using these animal models has made a quantum leap forward in the genomic era, with the concurrent construction of C. albicans mutants with targeted mutations of putative virulence factors, the application of microarrays and other emerging technologies to comprehensively assess C. albicans gene expression in vivo, and construction of transgenic and knockout mice to simulate specific host immunodeficiencies. The opportunity to combine these powerful tools will yield an unprecedented wealth of new information on the molecular and cellular pathogenesis of candidiasis. [Copyright &y& Elsevier]