Exposure to heat-inactivated Trichophyton rubrum resulting in a limited immune response of human keratinocytes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively.</p><p><b>METHODS</b>HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment.</p><p><b>RESULTS</b>HaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1.</p><p><b>CONCLUSION</b>The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.</p>

The immune response of human keratinocytes to Trichophyton rubrum conidia is partially mediated by toll-like receptor-2, 4, dectin-1 and cytokines / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of Trichophyton rubrum exposure on the expressions of toll-like receptor-2 (TLR-2), TLR-4 and dendritic cell associated C-type lectin-1 (Dectin-1) and cytokine secretions in human keratinocytes cell line HaCaT.</p><p><b>METHODS</b>The mRNA of TLR-2,4, and dectin-1 in the HaCaT co-cultured with the conidia of Trichophyton rubrum conidia for 24 h was measured with real-time PCR. The mean fluorescence intensity and the percentage of cells positive for TLR-2, 4, and dectin-1 was detected during the co-culture using flow cytometry. The cytokine secretion profiles in the cell culture supernatant was analyzed using a cytokine antibody array.</p><p><b>RESULTS</b>The TLR-2,4, and dectin-1 mRNA expressions, mean fluorescence intensity and percentage of positive cells for TLR-2,4, and dectin-1 all increased in HaCaT cells in response to Trichophyton rubrum conidia exposure. The results of cytokine antibody array demonstrated obviously increased secretions of IL-8, I-309, IFN-γ, IL-6, and IL-13 in the culture supernatant of HaCaT cells in response to Trichophyton rubrum exposure.</p><p><b>CONCLUSION</b>The immune responses and immunological recognition of human keratinocytes to Trichophyton rubrum conidia are partially mediated by up-regulating the expressions of TLR-2, TLR-4 and dectin-1 and secretions of multiple cytokines.</p>

Comparison of a glucose consumption based method with the CLSI M38-A method for testing antifungal susceptibility of Trichophyton rubrum and Trichophyton mentagrophytes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The prevalence of dermatophytoses and the development of new antifungal agents has focused interest on susceptibility tests of dermatophytes. The method used universally for susceptibility tests of dermatophytes was published as document (M38-A) in 2002 by the Clinical and Laboratory Standards Institute (CLSI), dealing with the standardization of susceptibility tests in filamentous fungi, though not including dermatophytes especially. However, it is not a very practical method for the clinical laboratory in routine susceptibility testing. In this test, we developed a novel rapid susceptibility assay-glucose consumption method (GCM) for dermatophytes.</p><p><b>METHODS</b>In this study, we investigated the antifungal susceptibilities of dermatophytes to itraconazole (ITC), voriconazole (VOC), econazole nitrate (ECN) and terbinafine (TBF) by glucose consumption method (GCM), in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A method. Twenty-eight dermatophyte isolates, including Trichophyton rubrum (T. rubrum) (n = 14) and Trichophyton mentagrophytes (T. mentagrophytes) (n = 14), were tested. In the GCM, the minimum inhibitory concentrations (MICs) were determined spectrophotometrically at 490 nm after addition of enzyme substrate color mix. For the CLSI method, the MICs were determined visually.</p><p><b>RESULTS</b>Comparison revealed best agreement for TBF against T. mentagrophytes and T. rubrum, since MIC range, MIC50, and MIC90 were identical from two methods. However, for ITC and VOC, GCM showed wider MIC ranges and higher MICs than CLSI methods in most isolates. For ECN against T. rubrum, high MICs were tested by GCM (0.125-16 microg/ml) but not M38-A method (0.5-1 microg/ml). The overall agreements for all isolates between the two methods within one dilution and two dilutions for ITC, VOC, ECN and TBF was 53.6% and 75.0%, 57.1% and 75.0%, 82.1% and 89.3%, and 85.7 and 85.7%, respectively.</p><p><b>CONCLUSION</b>Measurement of glucose uptake can predict the susceptibility of T. rubrum and T. mentagrophytes to ECN and TBF.</p>