Establishment of an athymic BALB/c mouse model with chronic infection due to Phialophora verrucosa / 中华皮肤科杂志

Objective To establish an athymic (nu/nu)BALB/c mouse model with chronic subcutaneous infection due to Phialophora verrucosa (P.verrucosa),and to explore the role of T lymphocytes in defensing against invasive infection due to P.verrucosa.Methods Six immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of P.verrucosa hyphae suspensions at a concentration of 5.0 × 108 colony-forming unit (CFU)/ml into one footpad,while another 6 immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of 5.0 × 108 CFU/ml P.verrucosa conidium suspensions into one footpad.The degree of footpad swelling was measured with a vernier caliper every 3 days.Histopathological characteristics of infected footpads were further analyzed.Biopsy tissues were also subjected to fungal culture to analyze the growth of P.verrucosa in infection foci in mice.Results After the treatment with hyphae or conidium suspensions,the BALB/c mice experienced transient footpad swelling within 12 days,and basically recovered after 50 days.However,the nu/nu BALB/c mice experienced persistent footpad swelling with recurrent ulceration and crusting.As pathological examination revealed,all the inoculation loci in BALB/c mice experienced local infection,and the morphology of P.verrucosa in the infected foci was not changed over time.However,invasive infections due to P.verrucosa hyphae alone or a mixture of P.verrucosa hyphae and sclerotic cells were observed in all the inoculation loci in nu/nu BALB/c mice.The fungal culture showed that P.verrucosa could not grow in the footpads of BALB/c mice after 21 days,while P.verrucosa could persistently grow in the footpads of nu/nu BALB/c mice.Conclusion An experimental model with subcutaneous phaeohyphomycosis due to P.verrucosa has been successfully established by using nu/nu BALB/c mice,and the nu/nu BALB/c mice are more susceptible to P.verrucosa infection compared with the immunocompetent BALB/c mice.

Establishment of an athymic BALB/c mouse model with chronic infection due to Phialophora verrucosa / 中华皮肤科杂志

Objective To establish an athymic (nu/nu)BALB/c mouse model with chronic subcutaneous infection due to Phialophora verrucosa (P.verrucosa),and to explore the role of T lymphocytes in defensing against invasive infection due to P.verrucosa.Methods Six immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of P.verrucosa hyphae suspensions at a concentration of 5.0 × 108 colony-forming unit (CFU)/ml into one footpad,while another 6 immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of 5.0 × 108 CFU/ml P.verrucosa conidium suspensions into one footpad.The degree of footpad swelling was measured with a vernier caliper every 3 days.Histopathological characteristics of infected footpads were further analyzed.Biopsy tissues were also subjected to fungal culture to analyze the growth of P.verrucosa in infection foci in mice.Results After the treatment with hyphae or conidium suspensions,the BALB/c mice experienced transient footpad swelling within 12 days,and basically recovered after 50 days.However,the nu/nu BALB/c mice experienced persistent footpad swelling with recurrent ulceration and crusting.As pathological examination revealed,all the inoculation loci in BALB/c mice experienced local infection,and the morphology of P.verrucosa in the infected foci was not changed over time.However,invasive infections due to P.verrucosa hyphae alone or a mixture of P.verrucosa hyphae and sclerotic cells were observed in all the inoculation loci in nu/nu BALB/c mice.The fungal culture showed that P.verrucosa could not grow in the footpads of BALB/c mice after 21 days,while P.verrucosa could persistently grow in the footpads of nu/nu BALB/c mice.Conclusion An experimental model with subcutaneous phaeohyphomycosis due to P.verrucosa has been successfully established by using nu/nu BALB/c mice,and the nu/nu BALB/c mice are more susceptible to P.verrucosa infection compared with the immunocompetent BALB/c mice.

"A case of lymphocutaneous sporotrichosis caused by Sporothrix schenckii sensu stricto finally diagnosed by molecular identification based on ""ITS"" and ""CAL"" coding regions in combination with phenotypic analysis" / 中华皮肤科杂志

A 54-year-old female farmer presented with a pea-sized red nodule on the left upper limb near the wrist for 15 days,which occurred after trauma,gradually became swollen and ruptured,and developed into multiple nodules arranged in a chain in 30 days.Skin examination revealed multiple hard purple-red nodules arranged in a line on her left upper limb,some of which were ruptured with a small amount of purulent exndate.Histopathological examination further revealed that the focus of infection manifested as pyogenic granuloma-like inflammation mainly infiltrated with mixed inflammatory cells.Periodic acid Schiff (PAS) staining showed no fungal structures,including fungal spores,hyphae and asteroid body.The biopsy tissue culture yielded the fungus.According to the morphological analysis of the cultures and results of molecular identification based on internal transcribed spacer (ITS) and calmodulin (CAL) coding regions,this case was finally diagnosed as lymphocutaneous sporotrichosis caused by Sporothrix schenckii sensu stricto.The patient was treated with oral potassium iodide 10％ solution in a dose of 10 ml thrice a day.After 2-month treatment,the patient felt that the lesions were obviously improved,but afterwards she was lost to follow-up.This research report suggests that phenotypic analysis combined with ITS/CAL-based molecular identification can accurately identify Sporothrix schenckii complex at the species level.

Molecular identification of Trichophyton mentagrophytes complex strains in Hubei province / 中华皮肤科杂志

Objective To profile the intraspecific type of Trichophyton mentagrophytes clinically isolated from different anatomical sites of patients, and to compare the performance of different target sites for the identification of Trichophyton mentagrophytes complex strains. Methods A total of 48 Trichophyton mentagrophytes strains, which were clinically isolated from Department of Dermatology, Wuhan No. 1 Hospital in the latest 3 years, were included in this study. The phenotypes of these Trichophyton mentagrophytes isolates were primarily determined by morphological observation and the urease test. PCR was performed to amplify the nuclear ribosomal internal transcribed spacer(ITS) region and the D1?D2 domains of the large?subunit ribosomal DNA(28S rDNA)followed by DNA sequencing. Then, Clustal X2 software and MEGA 6.0 software were used to analyze the ITS and D1?D2 sequences and to build phylogenetic trees by the maximum?likelihood method (bootstrap = 2000). Results As the ITS sequence?based phylogenetic tree showed, the probability that the 48 isolates were grouped into the Trichophyton interdigitale clade reached 92%. However, Trichophyton interdigitale could not be effectively differentiated from Trichophyton quinckeanum by the D1?D2 sequence?based phylogenetic tree. In addition, Trichophyton interdigitale showed various appearances, including woolen type, downy type, cream type, powdery type and granular type. Conclusions Trichophyton mentagrophytes can be identified to the species level based on the sequence of ITS region, which shows higher efficiency in identifying Trichophyton mentagrophytes complex than the D1?D2 domains. Morphological characteristics can not serve as the basis for intraspecific typing of Trichophyton mentagrophytes.

Erythromycin resistance in propionibacteria isolated from patients with acne in Wuhan city is associated with mutations in 23S rRNA gene and presence of erm genes / 中华皮肤科杂志

Objective To determine whether erythromycin-resistant propionibacteria isolated from patients with acne in Wuhan city harbor 23S rRNA gene mutations as well as the transposon Tn5432 carrying ermX genes.Methods Twenty-nine Propionibacterium strains isolated from outpatients with acne in Wuhan city were included in this study.The E-test method was used to determine the susceptibility of these strains to erythromycin and clindamycin.PCR was performed to amplify the 23S rRNA,ermX,ermX (cj),IS1249a and IS1249b genes from resistant strains followed by DNA sequencing and nucleotide alignment.Results Among the 29 Propionibacterium strains,19 were identified as P.acnes and 10 as P.avidum.All of these Propionibacterium strains were resistant to erythromycin (MIC ＞ 256 μg/ml) and clindamycin (MIC ＞ 256 μg/ml),except for 3 P.acnes strains sensitive to clindamycin.Seven P.acnes strains resistant to both antibiotics exhibited an A→G transition at a position cognate with Escherichia coli 23S rRNA 2058.An A→G transition at a position cognate with E.coli 23S rRNA 2059 was identified in one clindamycin-resitant and three clindamycin-sensitive P.acnes isolates.The ermX gene was found in the remaining 8 P.acnes isolates and 2 P.avidum isolates,with the sequence 100％ identical to the reference sequence of the ermX gene of P.acnes in Genbank.Meanwhile,the ermX (cj) gene was successfully amplified from the other 8 P.avidum isolates,which showed 99％ sequence homology with the ermX (cj) gene of Corynebacterium jeikeium,but 94％ homology with the ermX gene of P.acnes in Genbank.Both IS1249a and IS1249b genes were amplified in the 10 ermX gene-positive Propionibacterium strains,but not in the other ermX gene-negative strains.Conclusions The erythromycin resistance in Propionibacterium isolates from Wuhan city may be associated with the A→G transition at the E.coli equivalent bases 2058 and 2059 of the 23S rRNA gene,as well as the presence of the erm X (transferred through the transposon Tn5432) and ermX (c j) genes.

Application of BIOMED-2 primers in the detection of T cell receptor γ gene rearrangements in patients with mycosis fungoides / 中华皮肤科杂志

Objective To estimate the value of BIOMED-2 primers for the detection of T cell receptor γ (TCR-γ) gene rearrangements in different types of specimens from patients with mycosis fungoides (MF).Methods Totally,15 paraffin-embedded tissue specimens,14 fresh tissue specimens and 18 whole blood specimens were obtained from 28 patients with MF,and subjected to DNA extraction.BIOMED-2 multiplex PCR tubes TCRγ (A+B) were used for the analysis of TCRγgene rearrangements.Data were processed by SPSS 13.0 software,and statistical analysis was done by chi-square test and Fisher's exact probability test.Results TCR-γ gene rearrangements were detected in 3 paraffin-embedded tissue specimens,11 fresh tissue specimens and 12 blood specimens,with significant differences in the detection rate between the three samples (x2 =13.047,P ＜ 0.01).The fresh tissue samples showed a significantly higher detection rate than the paraffin-embedded tissue samples (X2 =12.523,P ＜ 0.01).The detection rate of TCRγgene rearrangements was 3/6 in paraffin-embedded tissue samples collected in 2011,significantly higher than that in the other 9 paraffin-embedded tissue samples collected before 2011 (Fisher's exact probability test,P =0.044),but similar to that in 14 fresh tissue specimens (12/14,Fisher's exact probability test,P =0.044).Decreased detection rate of TCRγ gene rearrangements was observed in blood samples compared with fresh tissue specimens,but no statistical difference was observed between the two types of specimens (x2 =2.358,P ＞ 0.05).Conclusions BIOMED-2 multiplex PCR tubes TCRγ(A+B) are suitable for the detection of clonal rearrangements of TCRγgene in different types of specimens,especially in fresh tissue specimens,from patients with MF.

Differential expression of reactive oxygen species in human neutrophils stimulated by the yeast phase of Sporothrix Schenckii and Candida albicans / 中华皮肤科杂志

Objective To compare the reactive oxygen species (ROS) expression in human neutrophils phagocytizing Sporothrix Schenckii and Candida albicans yeast cells,and to compare the fungicidal activity of human neutrophils against Sporothrix Schenckii and Candida albicans.Methods Human neutrophils were isolated from peripheral blood by using density gradient centrifugation method,and cultured with the presence of the yeast phase of a Sporothrix Schenckii clinical isolate and a standard strain of Candida albicans (ATCC 90028)at a multiplicity of infection of 10 or 1 for 60-210 minutes.Subsequently,flow cytometry with ROS probe (2',7'-dichlorofluorescein diacetate,DCFH-DA) was carried out to for the real time detection of intracellular ROS level,confocal laser scanning microscopy (CLSM) for the observation of ROS distribution.In addition,the fungicidal efficiency of neutrophils against Sporothrix Schenckii and Candida albicans was estimated by the number of colonies after additional culture of neutrophil lysates on brain-heart infusion agar (BHIA) medium.Statistical analysis was done by using univariate analysis of variance and LSD-t test.Results The intracellular ROS level peaked at 60 minutes in neutrophils incubated with Sporothrix Schenckii yeast cells,then decreased rapidly from 60 minutes to 210 minutes.Compared with the neutrophils incubated with Candida albicans yeast cells,those with Sporothrix Schenckii yeast cells showed a higher ROS level (expressed as mean fluorescence intensity) at 60minutes (159.67 ± 11.34 vs.112.22 ± 9.66,P＜ 0.01),but a lower ROS level at 120 minutes (89.01 ± 9.81 vs.110.25 ± 7.28,P＜ 0.05) and 180 minutes (57.63 ± 8.46 vs.109.98 ± 9.00,P＜ 0.01).CLSM revealed that ROS was mainly distributed in neutrophils with phagocytized fungal spores,and especially on the surface of phagocytized spores.Furthermore,the percentage of yeast cells killed by neutrophils was significantly lower for Sporothrix Schenckii than for Candida albicans at 180 minutes (19.21％ ± 3.68％ vs.26.63％ ± 4.97％,P ＜ 0.01).Conclusions Differential expression of intracellular ROS was observed in neutrophils after phagocytosis of Candida albicans and Sporothrix schenckii.Neutrophils exert a stronger fungicidal activity against Sporothrix Schenckii in comparison with Candida albicans,which may be associated with the rapid decrease of ROS level in neutrophils after phagocytosis.

G-test positive serum inhibits ROS-dependent killing of Candida albicans by interfering with internal- ized expression of neutrophil Dectin-1 / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans ( C.albicans ) mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans (MOI=10) which was pretreated with β-1, 3-D-glucanase (10 U/ml) or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans (MOI=10) for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans ( MOI=10 ) in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans (LSD-t test, P<0.01).Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner (Dectin-1,R2=0.702,P<0.01;ROS,R2=0.588,P<0.01 ).Conclusion Taken together, these results demonstrated that the soluble β-1, 3-D-glucan in G-test positive serum may play a role in inhibiting the ROS-dependent killing of C.albicans by interfering with internalized expression of neutrophil Dectin-1.

Intracellular expression of Dectin-1 mediates the killing of Candida albicans by human neutrophils in a manner dependent on ROS production / 中华微生物学和免疫学杂志

Objective To investigate the mechanism hy which Dectin-1 mediates the killing of Candida albicans(C.albicans) by human neutrophils in a manner dependent on the production of reactive oxygen species (ROS).Methods After stimulation with FITC-C.albicans at a multiplicity of infection (MOI) of 10 for 30 or 60 min,PE-anti-human Dectin-1 monoclonal antibody (2.5 μg/106 cells) was used to detect the expression of Dectin-1 in human neutrophils by flow cytometry.For Dectin-1 inhibition test and ROS assay,human neutrophils (2×106/ml) were respectively pre-incubated with different concentrations of blocking antibody (0.5,1,2.5 and 5 μg/ml) for 60 min at 4℃,and then with 25 μmol/L 2′,7′-Dichlorofluorescein diacetate for another 20 min at room temperature.Afterwards,under stimulation with live C.albicans at a MOI of 10,the rate of intracellular ROS production over time in blocking and control groups was measured continuously at 10 min intervals for up to 120 min.In addition,localization of Dectin-1 and ROS in human neutrophils was observed by confocal/two-photon laser scanning microscopy after stimulation with live C.albicans.For the detection of candicidal activity,after pre-treatment with different concentrations of Dectin-1 blocking antibody as mentioned above,neutrophils were stimulated with live C.albicans (MOI=1) for 60 min,serial dilutions of cell lysate were plated onto yeast agar,and CFU were enumerated after incubation at 37℃ for 48 h.The candicidal activity was represented as [1-(CFUblocking group/CFUbuffer)] × 100％.Results Under stimulation with FITC-C.albicans at a MOI of 10 for 30 and 60 min,positive percentage of intracellular Dectin-1-expressing neutrophils increased significantly when compared with initial level (0 min,8.32％ ; 30 min,16.82％ ; 60 min,23.88％) (versus 0 min,P＜0.01).However,positive percentage of cell-surface Dectin-1-expressing neutrophils remained almost unchanged after stimulation for 30 and 60 min (versus 0 min,P＞0.05).Upon blockage of Dectin-1,the stimulated ROS generation (R2=0.306,P＜0.01) and candicidal activity (R2=0.251,P＜0.01) of neutrophils were partly and irreversibly inhibited in a dose-dependent manner when compared with control group.In addition,the intracellular Dectin-1 is recruited and co-distributed with ROS on the surface of phagocytized C.albicans as observed by confocal microscopy.Conclusion Taken together,these results demonstrated that an internalized expression pattern of human Dectin-1 might contribute to the ROS-dependent killing of serum-opsonzied C.albicans which was phagocytized by human neutrophils.

Two cases of cutaneous lymphadenoma: a histological and immunohistological study / 中华皮肤科杂志

The histological and immunohistological features of two cases of cutaneous lymphadenoma was studied. A single, erythematous nodule with smooth surface developed on the face of both patients. The lesion slowly progressed. Histology revealed irregular epithelial lobules in the dermis which showed a peripheral palisaded border of basaioid-like cells as well as a center composed of clear cells. Some epithelial lobules and surrounding stroma were infiltrated by numerous small lymphocytes. Immunohistological study showed that the lymphocytic infiltration was predominantly composed of T cells (CD3 positive) along with a small number of B cells (CD20 positive). Within epithelial lobules and surrounded stroma, there were numerous dendritic cells that were positive for S-100 and CDia but negative for cytokeratin 7, cytokeratin 20 or carcino-embryonic antigen. In the center of epithelial lobules in one case, a few cells positive for epithe-lial membrane antigen and CD30 was observed. The diagnosis of cutaneous lymphadenoma was made according to the pathological and immunohistochemical findings, and the infiltration was predominated by CD3-positive lymphocytes in this uncommon epithelial neoplasm.