A sensitive rolling-circle amplification (RCA)-based method utilizing species-specific padlock probes targeted to the internal transcribed spacer 2 region of the fungal ribosomal DNA gene complex was developed. The assay was rapid (2 hours) and specific. Of 28 fungal isolates (16 of Candida, six of Aspergillus, and six of Scedosporium spp.), all were all identified correctly.Fungal pathogens cause life-threatening infections in critically ill and immunosuppressed patients. Contemporary epidemiological trends reveal a shift toward species of Candida and Aspergillus other than Candida albicans and Aspergillus fumigatus and a range of emerging fungi including Scedosporium spp. and the zygomycetes (6,19). Given the reduced susceptibility of many of these pathogens to antifungal agents, timely identification to species level is essential for clinical management. However, standard culture-based identification methods are insensitive and slow (15).To overcome both problems, PCR-based tools have been developed. In particular, the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the fungal ribosomal DNA gene complex have shown promise as targets for species identification in a variety of formats including multiplex and/or real-time PCR assays (9, 16), DNA sequence analysis (1, 2, 12), and probe-based techniques (5, 7). The latter range from Southern blotting (5, 7) and reverse line blot (RLB) hybridization methods (23) to sophisticated microarray formats (10,11,17).Recently, the utility of circularizable oligonucleotide (padlock) probes has been demonstrated for the detection of target nucleic acid sequences, including nucleotide polymorphisms that differ by only a few base pairs, with high sensitivity (4,13,20). Such probes comprise two sequences complementary to the 5Ј and 3Ј termini of the target sequence joined by a linker region (Fig. 1A). Upon hybridization to the target, the probe ends are joined by DNA ligase to form a closed molecule. The intensity of the probespecific signal is then increased exponentially by rolling-circle amplification (RCA) (13) (Fig. 1B). There are few data on the application of padlock probes in the detection of polymorphisms in fungi. We report on a sensitive, RCA-based method using real-time PCR for species identification of clinically important Candida, Aspergillus, and Scedosporium spp.Twenty-eight clinical isolates were studied: two of C. albicans, two of Candida glabrata, three of Candida krusei, three of Candida tropicalis, three of Candida dubliniensis, three of Candida guilliermondii, four of A. fumigatus, two of Aspergillus flavus, and three strains each of Scedosporium apiospermum and Scedosporium prolificans. Species identity was confirmed by standard laboratory methods (3, 21) and ITS sequence analysis (23). Isolates were stored in sterile water at 25°C until required. DNA extraction and amplification of the ITS (ITS1, 5.8S rRNA, and ITS2) region, using the primers ITS1 and ITS4 (22), in preparation for hybridization with padlock probes (see below) were performed a...

As one of the most serious types of psoriasis, pathogenesis of erythrodermic psoriasis (EP) is unclear so far. In this study, we aimed to detect the levels of Th1/Th2 cytokine-associated transcription factors and T-lymphocyte clone in peripheral blood mononuclear cells (PBMCs) derived from EP patients, and gene expression level of T-bet/GATA-3 in skin lesion. The potential role of Th1/Th2 reaction pattern played in the pathogenesis of EP was also discussed. Serum levels of IFN-γ, IL-2, IL-4 and IL-10 were quantified by ELISA among 16 EP patients, 20 psoriasis vulgaris (PV) patients and 15 healthy controls. The expression levels of T-bet/GATA-3 in the skin lesion and PBMCs were examined by real-time qPCR. The ratio of Th1/Th2 was measured by flow cytometry. The levels of IFN-γ, IL-2, IL-4 and IL-10 were higher in EP patients than in the healthy controls. The levels of IL-4 and IL-10 were 69.44±11.45 and 12.62±4.57 pg/mL, respectively, in EP patients, significantly higher than those in PV patients and healthy controls (P<0.05). Flow cytometry revealed the levels of both Th1 and Th2 in PBMCs from EP patients were higher than those in healthy controls, and the Th1/Th2 ratio was dramatically lower than in PV patients (P<0.01). The ratios of IFN-γ/IL-4 and T-bet/GATA-3 in EP patients were both less than 1.0, suggesting a reversal when compared with the other two groups. Our study indicated that the EP patients exerted a Th1/Th2 bidirectional response pattern, and the balance of Th cell subsets inclines to Th2, which might be one of the important mechanisms of EP pathogenesis.

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Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of β-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The β-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the β-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production.

Fonsecaea pedrosoi (F. pedrosoi), a major agent of chromoblastomycosis, has been shown to be recognized primarily by C-type lectin receptors (CLRs) in a murine model of chromoblastomycosis. Specifically, the β-glucan receptor, Dectin-1, mediates Th17 development and consequent recruitment of neutrophils, and is evidenced to have the capacity to bind to saprophytic hyphae of F. pedrosoi in vitro. However, when embedded in tissue, most etiological agents of chromoblastomycosis including F. pedrosoi will transform into the sclerotic cells, which are linked to the greatest survival of melanized fungi in tissue. In this study, using immunocompetent and athymic (nu/nu) murine models infected subcutaneously or intraperitoneally with F. pedrosoi, we demonstrated that T lymphocytes play an active role in the resolution of localized footpad infection, and there existed a significantly decreased expression of Th17-defining transcription factor Rorγt and inefficient recruitment of neutrophils in chronically infected spleen where the inoculated mycelium of F. pedrosoi transformed into the sclerotic cells. We also found that Dectin-1-expressing histocytes and neutrophils participated in the enclosure of transformed sclerotic cells in the infectious foci. Furthermore, we induced the formation of sclerotic cells in vitro, and evidenced a significantly decreased binding capacity of human or murine-derived Dectin-1 to the induced sclerotic cells in comparison with the saprophytic mycelial forms. Our analysis of β-glucans-masking components revealed that it is a chitin-like component, but not the mannose moiety on the sclerotic cells, that interferes with the binding of β-glucans by human or murine Dectin-1. Notably, we demonstrated that although Dectin-1 contributed to the development of IL-17A-producing CD3+CD4+ murine splenocytes upon in vitro-stimulation by saprophytic F. pedrosoi, the masking effect of chitin components partly inhibited Dectin-1-mediated Th17 development upon in vitro-stimulation by induced sclerotic cells. Therefore, these findings extend our understanding of the chronicity of chromoblastomycosis.