目的 初步探索表皮生长因子受体(epidermal growth factor receptor,EGFR)在阴道念珠菌病发病过程中的作用及分子机制.方法 培养阴道上皮细胞(VK2/E6E7细胞),白念珠菌刺激后利用荧光定量PCR检测EGFR及可能相关的免疫通路因子的表达.构建EGFR-siRNA VK2/E6E7细胞模型,与白念珠菌共培养后,分别利用ELLISA及全自动细胞检测仪检测表皮生长因子受体敲除前后阴道上皮细胞分泌细胞因子的变化以及对白念珠菌感染防御能力的改变.构建阴道念珠菌感染小鼠模型,qPCR检测阴道组织表皮生长因子受体及免疫通路因子的表达;并检测EGFR磷酸化抑制剂阻断通路后,阴道组织局部的真菌载量和炎性细胞的变化.结果 qPCR检测显示,EGFR、STAT3、GM-CSF及IL-1β在VK2/E6E7细胞感染白念珠菌后表达升高,且具有统计学意义(P<0.05);ELLISA检测结果显示,EGFR-siRNA VK2/E6E7细胞感染白念珠菌后GM-CSF、IL-8、IL-1β、MIP-3α表达显著下降(P<0.05);全自动细胞检测仪检测结果显示,EGFR敲除细胞在感染白念珠菌30 h后的防御能力和细胞活性较正常细胞明显降低.qPCR检测显示小鼠阴道感染白念珠菌后EG-FR、HER2、STAT3、IL-8表达升高具有统计学意义(P<0.05);局部应用磷酸化抑制剂阻断EGFR通路后,小鼠阴道灌洗液菌载量较对照组明显增加,且在感染后第7日差异有统计学意义(P<0.05).结论 表皮生长因子受体及其通路因子在阴道念珠菌病发病过程中起到较为重要的作用.

Abstract. Objective:. Cutaneous and subcutaneous infectious granuloma (CSIG) is a broad group of inflammatory conditions that share important similarities in granulomatous reaction pattern and nonspecific clinical presentation. Here, we conducted the retrospective study to identify the clinical, pathological, and epidemiological correlations of CSIG cases at a signal center in China. Methods:. Data of patients diagnosed with CSIG between January 1, 2011 and December 31, 2015 were retrospectively collected, including socio-demographic information, pathogen diagnosis, clinical features, pathological results, treatment, and prognosis. Results:. This study included 256 patients (137 males and 119 females) with a mean age of 52 years. Infections were more common in those aged over 40 years old (76.17%). The most common pathogens were Mycobacterium leprae (26.56%), Sporothrix schenckii (23.83%), and Mycobacterium tuberculosis (15.63%). Mycobacterium marinum (8.98%) accounted for 51.11% of nontuberculous mycobacterial contagion. Lesions were most common in the distal extremities (32.03%). The predominant clinical forms were plaques (61/142, 42.96%) and nodules (41/142, 28.87%). Conclusions:. Various pathogens were responsible for the CSIG cases in this study, with M. leprae being the most common. CSIG should be considered as a likely diagnosis for patients with lesions on exposed parts of the body that present as plaques or nodules and has a history of trauma.

Objective To investigate the effect of ALS3 and SSA1 genes in Candida albicans on immune response of vaginal epithelial cells.Methods Wild strains and ALS3,SSA1 gene knockout strains(SC5314,Δals3,Δssa1)of Candida albicans were cultured and their morphology was determined.Human vaginal epithelial cell line VK2/E6E7was infected in a certain proportion.The damage of different strains to epithelial cells was evaluated by trypan blue staining and lactate dehydrogenase(LDH)activity detection.The co-culture of inflammatory cytokines and chemokines during infection was evaluated by enzyme-linked immunosorbent assay(ELISA).Results There was no significant difference in the effect of Δals3 on the germ tube length of Candida albicans,while theΔssa1 reduced the germ tube length by about 30％～40％ compared with the other two strains(P<0.001).Trypan blue staining and LDH assay showed that the cell damage ability of the three strains was proportional to the fungal load when infected with epithelial cells.Compared with wild strain,the cell damage ability ofΔssa1 was significantly reduced when infected with epithelial cells at the same MOI,and the difference was statistically significant(P<0.05),while the impact ofΔals3 mutant was weak or even slightly higher.Detection of inflammatory cytokines and chemokines showed that the ability of mutants to induce epithelial cells to produce pro-inflammatory factors and chemokines(GM-CSF,G-CSF,IL-1a,IL-8)was significantly weakened(P<0.05).Conclusion ALS3 and SSA1 gene expression played an important role in the local immune response of vaginal epithelial cells to Candida albicans infection,and SSA1 gene expression was more significant.

OBJECTIVE:The immunological mechanisms behind different mucosa against candidiasis are largely unknown. In this study, we investigate the natural protective mechanisms and local cytokine responses of C. albicans-infected oral and vaginal epithelial cells.METHOD:The cell lines (Leuk-1 and VK2/E6E7) were cultured with C. albicans (SC5314, Δals3, and Δssa1) in indicated ratio, respectively. The morphological changes and colony growth of C. albicans were observed to evaluate the fungicidal ability of epithelial cells, and the cellular morphological changes and LDH activity measurements were used to assess cell damage. Further, we assess the production of cytokines and chemokines in co-culture supernatants using enzyme-linked immunosorbent assay (ELISA).RESULT:Our results show that the oral and vaginal epithelial cells use different strategies to combat this pathogen. Infected oral epithelial cells are adept at the production of cytokines (GM-CSF, IL-1α, and IL-1β) and chemokines (IL-8, MIP-3α, and RANTES), and yet, vaginal cells are more proficient at direct fungal killing. However, both epithelial cells play only a minor role in adaptive immunity to C. albicans. Further, C. albicans Als3p and Ssa1p genes also participate in local immune response since deletion of ALS3 or SSA1 causes reduction in cytokine and chemokine levels in both oral and vaginal cells. The dramatic decreases in both fungal % of cytotoxicity and the secretion of such cytokines as GM-CSF, MIP-3α, and RANTES in Δssa1-infected oral cells were consistent with a delayed germination process in that mutant.CONCLUSION:Human oral and vaginal epithelial cells performed different host response to C. albicans by fungal killing ability or secreting cytokines and chemokines.

In the last two decades, with the wide use of azoles, antifungal resistance among Candida parapsilosis has considered a matter of concern worldwide. The aim of this study is to evaluate the antifungal potentials of tetrandrine (TET) alone and in combination with fluconazole (FLC)/voriconazole (VRC) against C. parapsilosis. Susceptibility tests were performed by microdilution method, checkerboard assay, time-kill test, spot assay. Subsequently, rhodamine 6G efflux test and the expressions of transporter related genes, namely CDR1 and MDR1 for C. parapsilosis were analyzed by qRT-PCR. The susceptibility test showed that TET presented strong synergism with FLC and VRC with fractional inhibitory concentration index (FICI) in a range of 0.094–0.562. The susceptibility results were also confirmed by spot assay and time-kill studies. With TET treatment, a vast quantity of rhodamine 6G could not be pumped out from the cells as considerably intracellular red fluorescence was accumulated. Meanwhile, the expressions of efflux-associated genes presented varying degrees of inhibition. These results indicated that TET was a decent antifungal synergist to promote the antifungal efficacy of FLC/VRC, and the underlying antifungal mechanism might be associated with the inhibition of efflux pump and the elevation of intracellular drug content.

A 55-year-old male patient presented with plaques on the face for more than 20 years,and no immunodeficiency diseases were diagnosed.Skin examination showed large areas of pink plaques on the nose,bilateral cheeks and upper oral lips with slight desquamation,verrucous hyperplasia on the dorsal area of the nose,and a bean-sized verrucous protuberance on the tip of the nose.Histopathological examination of the skin lesions revealed pseudoepitheliomatous hyperplasia in the epidermis and hyphae-like structures in the stratum corneum.Moreover,there was diffuse infiltration of inflammatory cells in the dermis,which mainly included neutrophils,lymphocytes,histiocytes and multinucleated giant cells.Periodic acid-Schiff (PAS)-positive spore-like structures were observed in the multinucleated giant cells.Culture of the lesional tissues on Sabouraud dextrose agar (SDA) medium showed grey-brown villous colonies.Microculture on the potato dextrose agar (PDA) medium yielded dark septate hyphae and pycnidia filled with a large number of spores.Microsphaeropsis arundinis was identified by fungal molecular biological techniques.The patient was diagnosed with cutaneous phaeohyphomycosis caused by Microsphaeropsis arundinis.The patient was treated with CO2 laser for the removal of verrucous protuberance on the tip of the nose,and oral itraconazole capsules at a dose of 200 mg twice a day.After 3-month treatment,the skin lesions subsided and the drug was withdrew.During 6-month follow-up,no relapse occurred.

We report a case of imported pulmonary coccidioidomycosis caused by Coccidioides posadasii in a patient who was misdiagnosed as tuberculosis and mistreated with antituberculosis medications for 18 months. The symptoms were not relieved until antifungal treatment was started. An extensive review of the coccidioidomycosis cases occurring in China reveals 38 cases, 16 of which had no associated history of travel to any traditional endemic areas. We speculate that some factors may drive Coccidioides spp. transference to China, which then causes those domestic infections. Moreover, we indicate the first, to the best of our knowledge, possible endemic areas in China.

Paeoniflorin (PF), a Chinese herbal medicine, has been widely used in clinical practice in China because of its dual immunoregulatory effects. A previous study found that PF inhibited the biofilm formation of Candida albicans (C. albicans) in vitro; however, whether PF plays an antifungal role in vivo is still unexplored. In this study, we sought to examine the effect of PF alone or in combination with an antifungal agent, fluconazole (FCZ), using a mouse model of systemic candidiasis. The results showed that the survival time of mice treated with PF alone or PF + FCZ decreased compared with the Infected alone and FCZ treated groups, respectively (8.20 ± 1.75 vs 10.40 ± 2.50 days, P < 0.05; 24.60 ± 6.55 vs 29.00 ± 3.16 days, P < 0.05). The fungal burden in the kidney of mice increased in the PF alone and PF + FCZ treated groups compared with the Infected alone or FCZ treated group. Furthermore, it was found that the PF and PF + FCZ treated groups showed significantly decreased levels of serum interferon gamma (IFN-γ), interleukin (IL)-17, and IL-22, and an increased level of serum IL-4; PF had no effect on the production of tumor necrosis factor alpha (TNF-α). PF alone or in combination with FCZ decreased the proliferation of Th1 (IFN-γ+CD4+) and Th17 cells (IL-17+CD4+) and increased the expression of Th2 cells (IL-4+CD4+). These results suggested that PF treatment could be detrimental to the host response to systemic C. albicans infection in mice. Thus, caution might be required for clinical use of PF in patients with fungal infection.

We report a case of subcutaneous infection caused by Exophiala oligosperma . Erythematous ulcerated plaque with exudate was major clinical features. Histopathological examination showed yeast-like cells and fungal hyphae. Mycological and molecular identification revealed E. oligosperma as etiologic agent. Local debridement and oral itraconazole were effective. To the best of our knowledge, this is the first report of phaeohyphomycosis caused by E. oligosperma in mainland China. This report highlights the potential role of E. oligosperma as an emerging cause of infection in immunocompetent patients.

Objective: To determine the effects of itraconazole on the migration of murine bone marrow de-rived dendritic cells (DCs) and secretion of MMPs and RANTES. Methods: DCs were cultivated for 8 days with rmGM-CSF. DCs were divided into control and itraconazole (0.25, 0.5, 1 μM) treated groups. The via-bility and migration of DCs were determined by CCK-8 assay and transwell assay respectively. The levels of MMP-2, MMP-3, MMP-8, MMP-12 and RANTES were detected by Luminex. The levels of MHCII, CD40, CD80, CD86 and CCR7 were measured by flow cytometry. Results: The cytotoxicity of itraconazole on DCs was time and dose-dependent. The migration rate and levels of MMP-2, MMP-3, MMP-12 and RAN-TES in the itraconazole group were lower than those in the control group (Ps<0.05). There was no significant difference of the level of CCR and MMP-8 in two groups (P>0.05). Conclusion: Itraconazole inhibited the DCs cell migration and secretion of MMPs and RANTES.

临床资料 病例1,女,73岁.因双手掌结节伴活动受限7月余来我院就诊.患者7月前无明显诱因双手掌出现皮下肿物,无明显自觉症状.肿物逐渐增大,双手手指不能伸直,以小指无名指为重,功能受限.患者有肝癌病史4年,治疗情况不详.皮肤科检查:双手掌多发深在质硬结节,皮色,大小约0.6 cm× 0.5 cm.表面光滑、不活动,与局部皮肤有粘连.掌部皮肤褶皱,手指屈曲,活动受限.左手拇指皮下组织萎缩(图1).组织病理:角化过度,表皮轻度增生,真皮浅层可见较多成纤维细胞,下方胶原组织增生(图2).免疫组化:Actin(-),Desmin(-),MyOD1 (-),SMA(-).诊断:掌部纤维瘤病.治疗:手术切除.目前随访中.

Enhancing the immunity conferred by dendritic cells (DCs) to fungal infection represents a promising strategy in the number of immunocompromised individuals. In a previous study, we demonstrated that suppressor of cytokine signaling 1 (SOCS1) silencing can promote the maturation of DCs and induce an immune response against Candida albicans (C. albicans) in vitro. Herein, the effectiveness of SOCS1 suppression administered by SOCS1-siRNA-treated DCs is further evaluated in systemic candidiasis mouse model. The SOCS1-silenced DCs increase mouse survival and significantly decrease fungal colonization in the kidneys. We confirm that the serum IFN-γ levels in SOCS1-siRNA-treated mice are higher than in all other infected groups at the early stages of infection, which correlates with a higher differentiation of IFN-γ+CD4+ T cells (Th1) in the spleen. Meanwhile, the differentiation of IL-4-producing CD4+ T (Th2) or IL-17-producing CD4+ T cells (Th17 cells) remain unaffected under the same treatment, suggesting that SOCS1-silenced DCs significantly affect the IFN-γ-producing CD4+ T cells (Th1). However, at the late stages of infection when the differentiation of Th1, Th2 and Th17 cells decreases in SOCS1-silenced-DCs-treated mice, all the serum cytokines (IFN-γ, IL-4 and IL-17) are also reduced. In summary, treatment of mice with SOCS1-silenced DCs can protect mice from systemic infection during the early stages and thereby increase overall survival. We conclude that the increase in Th1 response in early stages avoids the cascade inflammatory response in later stages that is known to place such a large fungal load on the kidneys and cause subsequent death.

本文研究中药卷柏中炔多酚类成分卷柏素与氟康唑、酮康唑联用对念珠菌的体外抗真菌效果,并初步探讨对氟康唑增效的分子机制.采用棋盘法测定两类药物联用对念珠菌体外抗真菌活性,以FICI法评价结果.实验结果表明,卷柏素与氟康唑、酮康唑合用对白念珠菌、近平滑念珠菌表现协同作用(FICI≤0.5);对克柔念珠菌表现无关作用(FICI=1).qRT-PCR检测卷柏紊与氟康唑联用对白念株菌相关基因表达的影响显示,卷柏素能显著地逆转氟康唑诱导白念珠菌ERG5、ERG11、CDR1、CDR2、MDR1、FLU1和SIR2表达上调.Western-blot检测结果亦表明,卷柏素逆转组蛋白去乙酰化酶Sir2的上调.卷柏素显示出对氟康唑、酮康唑的抗念珠菌活性的体外增效作用.

We report a case of primary cutaneous mucormycosis caused by Mucor irregularis . A 66-year-old man was presented to our hospital with a history of gradually enlarging plaque on the right leg for about a year. The identification of pathogen based on the fungus morphology and DNA sequencing revealed M . irregularis as the responsible fungus for skin lesion. The lesion was removed incidentally by a surgery procedure, and no recrudescence was seen during a follow-up of 24-month observation.

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C.albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells served as the target cells,and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group).THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group.After transfection,the THP-1 macrophage-like cells in the above 2 groups were cocultured with heat-killed C.albicans separately.And then,fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C.albicans,and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR)-123 fluorescent cells.Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software.Results After transfection with siRNA-Dectin-1,the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t =26.163,P ＜ 0.001).After 1-,2-,4-hour co-culture of THP-1 macrophagelike cells with C.albicans,fluorescence microscopy showed that the phagocytosis rates of C.albicans by THP -1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5％ vs.22.1％,18.6％ vs.24.3％,39.2％ vs.59.1％,respectively,all P ＜ 0.05),so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C.albicans cells (2.2％ vs.4.7％,2.5％ vs.5.4％,5.1％ vs.8.3％,respectively,all P ＜ 0.05).After 30-minute,1-,2-and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C.albicans,flow cytometry showed that the MFI of C.albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs.45.7,54.3 vs.62.4,72.1 vs.84.9,93.6 vs.116.7,respectively,all P ＜ 0.05).Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C.albicans by macrophages.

目的 了解中国大陆地区近30年暗色丝孢霉病的流行病学和诊治状况.方法 通过CNKI、万方、维普、Embase和Pubmed检索1987～2017年中国大陆地区暗色丝孢霉病相关文献,进行数据分析和总结.结果 共纳入文献106篇,共计有115例患者.感染类型以皮肤及皮下组织型为主,继发于皮肤屏障被破坏、特发性免疫功能异常最多.诊断主要依据真菌镜检、组织病理、真菌培养和分子鉴定.治疗以系统抗真菌药物为主,伊曲康唑为首选用药.107例患者中因该病死亡10例,占9.35％;其中以中枢神经系统型死亡率最高,达75％.结论 中国大陆地区暗色丝孢霉病感染数有明显上升趋势.临床医生应提高该病各型临床及病原诊断水平,并积极开展分子鉴定和体外药物敏感性试验,为临床治疗提供更为有效的依据.

Malignant melanoma is the deadliest form of all skin cancers. Itraconazole, a commonly used systemic antifungal drug, has been tested for its anti-tumor effects on basal cell carcinoma, prostate cancer, and non-small cell lung cancer. Whether itraconazole has any specific anti-tumor effect on melanoma remains unknown. However, the goal of this study is to investigate the effect of itraconazole on melanoma and to reveal some details of its underlying mechanism. In the in vivo xenograft mouse model, we find that itraconazole can inhibit melanoma growth and extend the survival of melanoma xenograft mice, compared to non-itraconazole-treated mice. Also, itraconazole can significantly inhibit cell proliferation, as demonstrated by Ki-67 staining in itraconazole-treated tumor tissues. In in vitro, we show that itraconazole inhibits the proliferation and colony formation of both SK-MEL-28 and A375 human melanoma cells. Moreover, we demonstrate that itraconazole significantly down-regulates Gli-1, Gli-2, Wnt3A, beta-catenin and cyclin D1, while it up-regulates Gli-3 and Axin-1, indicating potent inhibitory effects of itraconazole on Hedgehog (Hh) and Wnt signaling pathways. Furthermore, itraconazole significantly suppresses the PI3K/mTOR signaling pathway - indicated by the down-regulated phosphorylation of p70S6K, 4E-BP1 and AKT - but has no effect on the phosphorylation of MEK or ERK. Our data suggest that itraconazole inhibits melanoma growth through an interacting regulatory network that includes Hh, Wnt, and PI3K/mTOR signaling pathways. These results suggest that this agent has several potent anti-melanoma features and may be useful in the synergesis of other anti-cancer drugs via blockage of the Hh, Wnt and PI3K/mTOR signaling pathways.

Mucor irregularis is an emerging fungal pathogen that cause cutaneous infection and could cause death. However, little is known about its mechanism of pathogenesis. There is evidence suggesting virulence vary with mating types in fungi, including the Mucorales. Here, we characterized the mating type locus of M. irregularis and the mating type ratio of 17 clinical isolates in China. Genomic data indicated M. irregularis is heterothallic having two mating types - bearing either SexP or SexM allele. Also, we employed a mice model to study the inflammation and pathological effects of different mating types. The comparison of the inflammatory response, cytokine profiles and Th-1, Th-2 and Th-17 cells numbers in each mating type treated mice showed that the severity and disease progress were enhanced in (+) mating type treated mice. One (+/0) mutant strain, with multiple mutations at the mating locus, had defects in sexual mating ability but appeared to be more virulent than the (-) mating type. Although (+) mating type appeared to be more virulent, most of our clinical isolates presented belonged to (-) mating type. Our findings support the involvement of MAT genes in sexual fertility, and the influence of mating type on the severity of cutaneous infection.

The antifungal effects of ambroxol (Amb; the metabolite VIII of bromhexine) against Cryptococcus planktonic cells and mature biofilms were investigated in this study. Amb showed antifungal activity against planktonic cells and mature biofilms. Disk diffusion test similarly showed antifungal profile for planktonic cells. Furthermore, Amb was found to be synergetic with fluconazole against planktonic cells and reduced the adherence of cells to polystyrene. Our results suggest that Amb can inhibit cryptococcal cells and biofilms, indicating its potential role in the prevention and treatment of cryptococcosis.