Objective To explore the relationship between ERG4 gene overexpression and azole resistance in clinical Candida albicans strains. Methods The National Committee for Clinical Laboratory Standards (NCCLS)M27-A2 broth microdilution method was conducted to evaluate antifungal susceptibility of 34 clinical Candida albicans isolates in vitro. Total RNA was extracted from these Candida albicans strains and transcribed into cDNA. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of ERG4 gene. Statistical analysis was carried out by a two-sample t-test. Results The expression level of ERG4 mRNA was significantly higher in fluconazole-resistant than in -sensitive Candida albicans strains (4.20 ± 2.56 vs. 1.72 ± 1.33, t = 3.99, P < 0.05), higher in itraconazole-resistant than in -sensitive Candida albicans strains (3.60 ± 2.47 vs. 1.66 ± 1.61, t = 3.71, P < 0.05), and higher in voriconazole-resistant than in -sensitive Candida albicans strains (3.99 ± 2.72 vs. 2.07 ± 1.58, t = 2.91, P <0.05). Further more, increased ERG4 mRNA expression was also observed in isolates cross-resistant to all the three azole antifungal agents compared with those susceptible to all of them (4.49 ± 2.73 vs. 1.69 ± 1.82, t = 3.81, P < 0.05). Conclusions The overexpression of ERG4 gene may be associated with cross resistance to fluconazole, itraconazole and voriconazole in clinical Candida albicans strains, but its exact role is expected to be investigated through downregulation of the ERG4 gene.

Objective To investigate the effects of recombinant human pigment epithelium derived factor (rhPEDF)on in vitro proliferation of and expressions of interleukin 6(IL-6), IL-8 and vascular endothelial growth factor (VEGF)in cultured human HaCaT keratinocytes. Methods Some cultured HaCaT cells were treated with rhPEDF at various concentrations(25, 50, 100 μg/L)for different durations, and some treated with RPMI 1640 medium only served as the control group. Cell counting kit-8(CCK8)assay was performed to evaluate cell proliferation after 24-, 48- and 72-hour treatment, reverse transcription (RT)-PCR to measure the mRNA expressions of IL-6, IL-8 and VEGF in HaCaT cells after 24-hour treatment, and Western blot to detect the protein expressions of IL-6, IL-8 and VEGF in HaCaT cells after 48-hour treatment. Statistical analysis was carried out by two- and one-way analysis of variance, Student-Newman-Keuls(SNK)-q test and Pearson correlation analysis. Results After treatment with rhPEDF of 25-100 μg/L for 24 - 72 hours, the proliferation of HaCaT cells was significantly inhibited to different extents compared with the control group(all P < 0.05), and the inhibition rate significantly increased with the increase in treatment duration and concentrations of rhPEDF(F = 1115, 329.9, respectively, both P < 0.001). Moreover, there was a significant decrease in the expressions of IL-6, IL-8 and VEGF mRNAs(at 24 hours)and proteins(at 48 hours)in HaCaT cells after treatment with rhPEDF of 25 - 100 μg/L compared with the control group(all P < 0.05). The expression levels of VEGF mRNA as well as IL-6 and IL-8 proteins all significantly decreased with the increase of rhPEDF concentrations (all P < 0.05). The mRNA expressions of IL-6 and IL-8 were significantly lower in the 100-μg/L rhPEDF group than in the 25-μg/L rhPEDF group (both P < 0.05), and the protein expression of VEGF was significantly weaker in the 100-μg/L rhPEDF group than in 25-and 50-μg/L rhPEDF groups (both P < 0.05), but similar between the 25- and 50-μg/L rhPEDF groups (P > 0.05). Conclusions rhPEDF can inhibit the proliferation of HaCaT cells, and down-regulate the mRNA and protein expressions of IL-6, IL-8 and VEGF.

Objective To explore the therapeutic mechanism of narrow-band ultraviolet B (NB-UVB)in psoriasis. Methods Forty-two patients with psoriasis vulgaris and 20 healthy controls were enrolled into this study. All the patients received 20 sessions of NB-UVB radiation. Psoriasis area severity index (PASI)was used to evaluate the severity of psoriasis. Blood samples were collected from all the patients before and 15 cured patients after the treatment as well as from 20 healthy controls, and skin samples from 10 patients before and after the treatment as well as from 10 healthy controls. Enzyme-linked immunosorbent assay (ELISA)was performed to determine the serum levels of IL-17 and IL-22, and real-time fluorescence-based quantitative PCR to measure the mRNA expressions of IL-17 and IL-22 in skin specimens. Statistical analysis was carried out by using the two-sample t-test, paired t-test and Pearson correlation analysis. Results After 20 sessions of NB-UVB radiation, 15 out of the 42 patients were cured with a significant decrease in PASI. Compared with the healthy controls, the 15 cured patients showed a significant elevation in the levels of IL-17and IL-22 proteins(IL-17: 34.26 ± 10.05 ng/L vs. 16.34 ± 4.73 ng/L, t = 7.016, P < 0.01; IL-22: 13.72 ± 4.45 ng/L vs. 5.03 ± 1.84 ng/L, t = 8.282, P < 0.01)and mRNAs (IL-17: 13.43 ± 2.12 vs. 5.26 ± 0.87, t = 6.312, P < 0.01; IL-22:16.53 ± 2.65 vs. 7.72 ± 2.13, t = 6.823, P < 0.01)before the treatment. The PASI score was positively correlated with the levels of IL-17 and IL-22 proteins in sera (r = 0.76, 0.70, respectively, both P < 0.05)and their mRNAs in skin lesions (r = 0.65, 0.68, respectively, both P < 0.05)in these patients. The serum levels and mRNA expressions of IL-17 and IL-22 all significantly reduced in the cured patients after the treatment compared with those before the treatment(all P < 0.05). Conclusion NB-UVB may treat psoriasis by downregulating the levels of IL-17 and IL-22 in peripheral blood and skin lesions in patients with psoriasis.

Objective To observe ultrastructural changes in a clinical isolate of Penicillium marneffei(PM) before and after treatment with amphotericin B or voriconazole by using scanning electron microscopy (SEM)and transmission electron microscopy (TEM). Methods A microdilution method was performed to determine the minimum inhibitory concentration (MIC)of amphotericin B and voriconazole against a clinical isolate of PM. Then, the PM isolate was treated with amphotericin B or voriconazole at their MICs and 10-fold MICs for 24, 48 and 72 hours. The ultrastructural changes in this isolate before and after the treatment were observed by using SEM and TEM. Results After the treatment with amphotericin B, SEM showed that the conidia or yeast cells of the PM isolate were gradually damaged, and their outer layers experienced detachment, shrinkage, breakage and adhesion with the increase in treatment duration and concentrations of amphotericin B; TEM also showed degenerated mitochondria, broken nuclei and cell walls, and shrunken cytoplasmic membrane with disappearance of cytoplasmic organelles. Similarly, the damage, shrinkage, shriveling and collapse of PM cells were seen by using SEM, and TEM showed many high-density electron-dense granules in cytoplasm, degeneration of mitochondria, roughening of cell wall surface, damage and shrinkage of cytoplasmic membrane, and disappearance of cytoplasmic organelles after voriconazole treatment. Conclusions Amphotericin B and voriconazole both had a strong antifungal effect on PM, and could induce evident ultrastructural changes, which were positively associated with treatment duration and concentrations. Moreover, amphotericin B caused more severe damage to PM compared with voriconazole.

Objective To evaluate the effects of alpha-lipoic acid (α-LA)on autophagy in human skin fibroblasts (HSFs). Methods HSFs at passage 3 - 5 were divided into several groups to be cultured with α-LA at final concentrations of 0, 0.01, 0.05, 0.10, 0.15, 0.20 and 0.50 mmol/L for 4, 12 and 24 hours, respectively. Methyl thiazolyl tetrazolium(MTT)assay was performed to evaluate cellular proliferative activity, monodansylcadaverin(MDC)staining to determine autophagy levels, and Western blot to measure the expression of the microtubule-associated protein 1 light chain-3B(LC3-B). Results After incubation for 24 hours, there was a significant difference in the proliferative activity of HSFs among all the groups (F = 10.41, P < 0.05), while no significant differences were observed after incubation for either 4 or 12 hours (F = 2.85, 1.34, respectively, both P > 0.05). MDC staining also showed a significant difference in the percentage of autophagosome-positive cells among all the groups after 24-hour incubation (F = 8.03, P < 0.05), but no significant difference after either 4- or 12-hour incubation (F = 0.11, 0.10, respectively, both P > 0.05). Western blot revealed that the degree of conversion from LC3-Ⅰ to LC3-Ⅱ(LC3-Ⅱ/LC3-Ⅰratio)was significantly different among all the groups after 24-hour incubation (F = 37.49, P < 0.05), but similar after 4- and 12-hour incubation (F = 3.38, 2.13, respectively, both P > 0.05). Conclusion α-LA may inhibit basal autophagy in HSFs.

Objective To explore the anti-inflammatory and antipruritic effects of Chushizhiyang ointment in a mouse model. Methods A total of 40 male 8-week-old BALB/c mice were included in this study, and randomly and equally divided into 4 groups. A mouse model of atopic dermatitis (AD)was established in three groups of mice by repeated application of 2,4-dinitroflurobenzene (DNFB)to shaved abdominal skin for sensitization and to shaved dorsal skin for stimulation. After establishment of the AD model, the three groups were topically treated with sodium chloride physiological solution (model group), hydrocortisone cream (hydrocortisone group)and Chushizhiyang ointment (Chushizhiyang group)respectively for 14 consecutive days. The remaining group receiving no sensitization or treatment served as the normal control group. All the mice were sacrificed 12 hours after the final treatment, and the dorsal skin of mice was resected followed by the determination of skin thickness and weight as well as hematoxylin-eosin(HE)staining and toluidine blue staining for the counting of leukocytes and mast cells respectively. Moreover, enzyme-linked immunosorbent assay(ELISA)was performed to measure the levels of interferon-gamma(IFN-γ), tumor necrosis factor alpha(TNF-α), interleukin 4(IL-4)and IL-5 in dorsal skin lesions. In addition, a local skin itching model was induced by histamine phosphate in Hartley guinea pigs, which was used to explore the effect of Chushizhiyang ointment on itch thresholds. Results Compared with the model group, both the Chushizhiyang group and hydrocortisone group showed reduced thickness and weight of dorsal skin in mice (all P < 0.01), numbers of infiltrating lymphocytes and mast cells (all P < 0.01)and levels of IFN-γ, TNF-α, IL-4 and IL-5 in skin lesions (P < 0.05 or 0.01)on day 15 after the start of treatment. The thickness and weight of dorsal skin in mice were significantly decreased in the hydrocortisone group (P <0.01), but experienced no significant changes in the Chushizhiyang group compared with the normal control group. Additionally, Chushizhiyang ointment could significantly increase itch thresholds in guinea pigs induced by histamine phosphate(P < 0.01). Conclusions Chushizhiyang ointment can significantly inhibit DNFB-induced AD in mice, likely by restoring the balance between Th1 and Th2 type cytokines. Moreover, Chushizhiyang ointment could markedly relieve itching induced by histamine phosphate in guinea pigs.

Objective To investigate the effects of ciprofloxacin on dermal collagen synthesis and profibrotic gene expressions in an experimental mouse model of scleroderma induced by bleomycin. Methods Experimental mouse models of scleroderma were established by subcutaneous injection of bleomycin into the dorsal skin of 15 BALB/c mice for 4 consecutive weeks. Then, the mouse models were randomly and equally divided into 3 groups to be topically treated with 1% ciprofloxacin cream (ciprofloxacin group), 2.5% asiaticoside cream (asiaticoside group)and cream vehicle (model group)respectively for 5 consecutive weeks. Five mice firstly injected with sterile phosphate buffered saline (PBS)for 4 weeks then topically treated with cream vehicle for 5 weeks served as the blank control group. After the 5-week topical treatment, all the mice were sacrificed, skin specimens were resected from the dorsal skin of them, and subjected to HE staining and Masson staining. Further more, an immunohistochemical assay was performed to measure the expressions of type I collagen (COL-1), matrix metalloproteinase-1 (MMP1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1), semi-quantitative reverse transcription PCR to quantify the expressions of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGFβ1)and Smad3 genes, and alkaline hydrolysis-spectrophotometry to determine the level of hydroxyproline in skin. Statistical analysis was carried out by one-way analysis of variance and the least significant difference(LSD)test with the SPSS 17.0 software. Results Compared with the blank control group, the model group showed increased dermal thickness at injection sites (432.76 ± 93.74 μm vs. 301.69 ± 79.47 μm, P < 0.01). Masson staining revealed thick and dense collagen bundles in an irregular arrangement in the dermis in the model group, which was consistent with dermal fibrosis in scleroderma. The total content of collagen and staining intensity of COL-1, MMP1 and TIMP1 were all significantly decreased in the ciprofloxacin group and asiaticoside group compared with the model group (F = 1628.54, 33.29, 84.82, 224.81, respectively, all P < 0.01), while no significant changes were observed in dermal thickness (both P > 0.05). Moreover, compared with cream vehicle, asiaticoside down-regulated the expressions of the three profibrotic genes(CTGF, TGFβ1 and Smad3)to different extents (all P < 0.05), while ciprofloxacin only inhibited the expressions of TGFβ1 and Smad3 genes (both P < 0.05)with no significant effect on CTGF gene expression (P > 0.05). Conclusion Ciprofloxacin may counteract dermal fibrosis by inhibiting the TGFβ1/Smad3 pathway and modulating the unbalanced expressions of MMP1 and TIMP1.

Objective To analyze clinical features of and hypertrophy-related factors in patients with hypertrophic port-wine stains (PWS). Methods Patients with PWS were enrolled into this study from Anhui Provincial Hospital and the First Affiliated Hospital of Anhui Medical University between January 2010 and August 2014. Clinical features of hypertrophic PWS were investigated. The factors related to hypertrophy in PWS were analyzed by univariate and multivariate unconditional logistic regression analyses. Results A total of 262 patients with PWS were enrolled, 82 (30 males and 52 females)of whom had hypertrophic PWS with a median age of 32.5 years (range, 18 - 54 years). Among the 82 patients, 66(80.48%)had plaque-like hypertrophic PWS, 9(10.98%)had papular or nodular type, and 7 (8.54%)had mixed type; 56.10% (46/82)were aged ≥ 30 years, 41.46% (34/82)varied from 11 to 30 cm2 in lesional area, and 85.36% (70/82)showed purple lesions. There was a significant difference between patients with hypertrophic PWS and those with flat PWS in the distribution of age, lesional area and color(χ2 = 25.559, 10.580, 90.630, respectively, all P < 0.05), while gender, Fitzpatrick′s skin type, lesional site and distribution were unrelated to hypertrophy in PWS (all P > 0.05). Multivariate unconditional logistic regression analysis revealed that an age ≥ 30 years(OR = 2.889, 95%CI: 1.459 - 5.721)and purple lesions(OR = 19.984, 95% CI: 5.704 - 70.023)were factors related to skin hypertrophy in PWS. Conclusion An age ≥ 30 years and purple lesions seem to be hypertrophy-related factors in PWS.

Objective To explore the effects of narrow-band ultraviolet B (NB-UVB)on the serum levels of thymic stromal lymphopoietin (TSLP)and interleukin-25 (IL-25), as well as on the expressions of TSLP receptor (TSLPR)and IL-25 receptor (IL-25R)mRNAs in peripheral blood mononuclear cells (PBMCs)from patients with atopic dermatitis(AD). Methods A total of 40 patients with AD and 30 healthy volunteers were enrolled in this study. All the patients were treated with NB-UVB at 0.3 - 2.5 J/cm2 thrice a week for 12 consecutive weeks. Venous blood samples were obtained from these patients before and after the treatment as well as from these healthy controls. Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA)was performed to detect serum levels of TSLP and IL-25, and reverse transcription PCR(RT-PCR)to determine the mRNA expression levels of TSLPR and IL-25R in PBMCs from these subjects. The scoring atopic dermatitis (SCORAD)system developed by the European Task Force on Atopic Dermatitis was used to estimate the severity of AD, and visual analogue scale (VAS)to evaluate the degree of itch. Statistical analysis was carried out by the two-independent-sample t-test for intergroup comparisons and paired t-test for comparisons between pre- and post-treatment samples from these patients. Results After the treatment with NB-UVB, the total response rate reached 75%(30/40)in these patients, with a significant decrease in SCORAD score from 55.26 ± 10.88 before the treatment to 20.36 ± 5.12 after the treatment (t = 10.29, P < 0.05)and in VAS score from 8.20 ± 1.37 to 3.05 ± 1.02(t = 8.16, P < 0.05). Before the treatment, the patients showed a significant increase in the serum levels of TSLP(198.24 ± 29.47 ng/L vs. 120.13 ± 19.65 ng/L, t = 29.70, P < 0.05)and IL-25(160.54 ± 34.89 ng/L vs. 120.41 ± 43.07 ng/L, t = 14.65, P < 0.05), as well as in the mRNA expression levels of TSLPR (8.57 ± 1.34 vs. 1.94 ± 0.39, t =7.07, P < 0.05)and IL-25R(6.81 ± 0.50 vs. 1.48 ± 0.47, t = 18.89, P < 0.05)compared with the healthy controls. With the improvement of conditions after the treatment, a significant decrease was observed in the serum levels of TSLP (151.87 ± 14.78 ng/L, t = 18.56, P < 0.05)and IL-25(130.52 ± 29.65 ng/L, t = 9.07, P < 0.05), as well as in the mRNA expression levels of TSLPR (2.89 ± 0.53, t = 5.21, P < 0.05)and IL-25R (3.90 ± 0.37, t = 7.35, P < 0.05)in PBMCs from these patients compared with those before the treatment, and differences disappeared in all of these parameters between the patients and controls (all P > 0.05). Conclusions TSLP and IL-25 may play important roles in the development of AD, and NB-UVB may treat AD by downregulating the expressions of them and their receptors.

Objective To evaluate the safety and efficacy of a domestic recombinant human tumor necrosis factor receptor type Ⅱ- IgG Fc fusion protein (rhTNFR-Fc)for the treatment of moderate to severe psoriasis vulgaris. Methods A multicenter, randomized, double blind, parallel-group, positive drug-controlled clinical trial was conducted. According to random numbers generated by a hierarchical segmentation method using the SAS 9.2 software, patients with moderate to severe psoriasis vulgaris were randomly divided into two groups to be injected with two kinds of domestic rhTNFR-Fc under the trade names of Anbainuo(test group)and Yisaipu(control group)respectively at a dose of 25 mg twice a week for 12 consecutive weeks. The primary endpoint was the proportion of patients achieving a 50%, 75% and 90% reduction in psoriasis area and severity index(PASI50, PASI75 and PASI90)at week 2, 6 and 12 after initiation of the treatment. Adverse reactions were also recorded. Statistical analysis was carried out by using the chi-square test, Fisher′s exact test, two-sample t-test, and noninferiority trials with the software SAS 9.2. Results A total of 180 patients were enrolled in this study from 5 centers, and 174 completed this trial, of whom, 88 were assigned to the test group and 86 to the control group. Analysis of the full analysis set (FAS)revealed no significant differences in PASI50(75.6%(68/90)vs. 82.2%(74/90), P > 0.05)or PASI75(51.1%(46/90)vs. 50.0%(45/90), P > 0.05) between the test group and control group, but a significant increase in PASI90 in the test group compared with the control group (30.0% (27/90)vs.16.7% (15/90), χ2 = 4.472, P < 0.05)at week 12. Drug-related adverse reactions included elevation of transaminases, leukopenia, upper respiratory infections, injection-site reactions, abnormalities in urine routine test and purified protein derivative (PPD)skin test results, etc. There was no significant difference between the two groups in the incidence rate of adverse reactions(χ2 = 0.188, P > 0.05), most of which were mild, and subsided spontaneously or after appropriate treatment. Conclusion The domestic rhTNFR-Fc (trade name:Anbainuo)25 mg twice a week for 12 weeks is effective and safe for the treatment of moderate to severe psoriasis——————————vulgaris.