ObjectiveTo study the expression of C3d in bullous pemphigoid lesions and its clinical significance.MethodsThe immunohistochemical SP method was used to detect the expression of C3d,IgG and IgA in tissue specimens from the lesions of 25 patients with bullous pemphigoid,10 patients with epidermolysis bullosa and normal skin of 10 human controls.The expression rates of these proteins were compared.Results The expression rates of C3d,IgG and IgA were 96％,72.0％ and 0 respectively in bullous pemphigoid tissue.Significant differences were found in the expression rates between C3d and IgG (x2 =4.17,P ＜ 0.05),as well as between C3d and IgA(x2 =22.04,P ＜ 0.01 ),in bullous pemphigoid tissue.No expression of C3d,IgG or IgA was observed in epidermolysis bullosa lesions or the control skin.ConslusionThe detection of C3d with immunohistochemistry in paraffin-embedded tissue sections may facilitate the diagnosis of bullous pemphigoid.

Objective To study the expressions of mucin (MUC) 1 and 2 in extramammary Paget's disease(EMPD) lesions. Methods Tissue specimens were obtained from the lesions of 19 patients with EMPD and normal skin of 19 human controls during cosmetic surgery. Streptavidin-perosidase (SP) technique was used to detect the expressions of MUC1 and MUC2 in these specimens. Results As haematoxylin-eosin (HE) staining showed, 3 cases were accompanied by poorly differentiated adenocarcinoma, 6 were invasive Paget's disease and 10 were intraepithelial EMPD. MUC1 was expressed in 2 cases accompanied by poorly differentiated adenocarcinoma, and in all the cases of invasive and intraepithelial EMPD; MUC2 was observed in all the cases of adenocarcinoma-complicated EMPD and invasive EMPD, but only in 2 of 10 cases of intraepithelial EMPD.Neither MUC1 nor MUC2 was observed in normal control specimens. A significant increase was observed in the expression of MUC1 in lesions of intraepithelial EMPD compared with invasive EMPD and adenocarcinoma-complicated EMPD (both P ＜ 0.05), and in the expression of MUC2 in lesions of invasive EMPD and adenocarcinoma-complicated EMPD compared with intraepithelial EMPD (both P ＜ 0.05). The expression of MUC1 was uncorrelated to that of MUC2 (r= -0.5, P＞ 0.05). Conclusions MUC1 is generally expressed in the lesions of EMPD, while MUC2 is expressed in those of adenocarcinoma-complicated EMPD and invasive EMPD.

Objective:To evaluate the effect of all-trans retinoic acid (ATRA) on the expression of epithelial-mesenchymal transition (EMT) -related molecules in human malignant melanoma A375 cells.Methods:Cultured A375 cells were divided into 4 groups: control-1 and -2 groups treated with Dulbecco′s modified Eagle medium (DMEM) for 24 and 48 hours respectively, and ATRA-1 and ATRA-2 groups treated with DMEM containing 10 μmol/L ATRA for 24 and 48 hours respectively. After the treatment, real-time quantitative PCR was performed to determine the mRNA expression of EMT-related genes E-cadherin, N-cadherin, vimentin and β-catenin in the above 4 groups, Western blot analysis to determine the relative expression of the above proteins, and direct immunofluorescence study to assess the fluorescence intensity of E-cadherin and vimentin in the ATRA-1, ATRA-2 and control-1 groups. Statistical analysis was carried out by using two-way analysis of variance, one-way analysis of variance and least significant difference- t test. Results:Real-time quantitative PCR showed that the E-cadherin mRNA expression was significantly higher in the ATRA-1 group than in the control -1 group ( F = 13.148, P < 0.05) , and higher in the ATRA-2 group than in the control-2 group ( F = 31.529, P < 0.05) ; the mRNA expression of N-cadherin, vimentin and β-catenin was significantly lower in the ATRA-1 group than in the control-1 group ( P < 0.05) , and lower in the ATRA-2 group than in the control-2 group ( P < 0.05) ; the ATRA-2 group showed significantly increased mRNA expression of E-cadherin ( F = 13.148, P < 0.05) , but significantly decreased mRNA expression of the other 3 proteins compared with the ATRA-1 group (all P < 0.05) ; there was no significant difference in the mRNA expression of the above molecules between the control-1 and -2 groups (all P > 0.05) . Western blot analysis showed that the protein expression of E-cadherin significantly increased, but the protein expression of N-cadherin, vimentin and β-catenin significantly decreased in the ATRA-1 and ATRA-2 groups compared with the control-1 group (all P < 0.05) ; compared with the ATRA-1 group, the ATRA-2 group showed significantly increased protein expression of E-cadherin ( P < 0.05) , but significantly decreased protein expression of N-cadherin, vimentin and β-catenin (all P < 0.05) . Direct immunofluorescence study showed that the fluorescence intensity of E-cadherin was significantly higher in the ATRA-1 group and ATRA-2 group (6.23 ± 0.08, 10.37 ± 0.13, respectively) than in the control-1 group (2.37 ± 0.14, both P < 0.05) , while the fluorescence intensity of vimentin was significantly lower in the ATRA-1 group and ATRA-2 group (15.17 ± 0.18, 10.29 ± 0.03, respectively) than in the control-1 group (50.16 ± 0.26, both P < 0.05) , and the cells in the ATRA-1 group and ATRA-2 group transformed from spindle- to cobble-stone-like in shape. Conclusion:ATRA can up-regulate the expression of E-cadherin, down-regulate the expression of N-cadherin, vimentin and β-catenin in A375 cells, and may inhibit the EMT of A375 cells.

Objective To explore the changes and clinical significance of regulatory T cell/T help cell 17 (Th17) in the peripheral blood from patients with dermatomyositis (DM).Methods Thirty-six patients with DM were recruited and thirty-six healthy individuals were enrolled as normal controls.The percentage of regulatory T cell/Th17 cell was assessed by flow cytometry and the mRNA levels of Foxp3/RORγt were detected by real-time quantitative reverse wanscription polymerase chain reaction (RT-PCR).The plasma levels of TGF-β1/IL-17 were measured by enzyme linked immunosorbent assay (ELISA).T-test was used for statistical analysis and Pearson's linear analysis were used for correlation analysis.Results The percentage of CD4+ Foxp3+ T cell,the mRNA expression of Foxp3 and the plasma levels of TGF-β1 decreased significantly in patients with DM than in normal controls [(7.2±2.3)％ vs (9.1±3.2)％,(0.44±0.19) vs (0.62±0.25),(37 400±4814) pg/ml vs (41 200±6348) pg/ml,P＜0.05].The percentage of Th17 cell,the mRNA expression of RORγt and the plasma levels of IL-17 increased in patients with DM compared with normal controls [(1.22±0.41)％ vs (0.83±0.14)％,(0.85±0.34) vs (0.61±0.21),(77±32) pg/ml vs (58±20) pg/ml,P＜0.05].There was negative correlation between the percentage of regulatory T cell,the mRNA levels of Foxp3,the plasma levels of TGF-β1 and the serum levels of CK in patients with DM (r=-0.573,-0.593,-0.618,P＜0.05).There was positive correhtion between the percentage of Thl7 cell,the mRNA levels of RORγt,the plasma levels of IL-17 and the serum levels of CK in patients with DM (r=0.582,0.609,0.623,P＜0.05).Conclusion The imbalance of regulatory T cell/Th17 cell may exist in the peripheral blood of patients with DM.Regulatory T cell/Th17 cell may be associated with the pathology of DM.

Objective To evaluate the effect of transforming growth factor-β (TGF-β) on the expressions of epithelial-to-mesenchymal transition (EMT)-related molecules in human A431 epidermal squamous cell carcinoma cells.Methods Cultured A431 cells were classified into two groups:TGF-β group treated with TGF-β of 7.5 μg/L for 72 hours,and control group remaining untreated.Subsequently,cell morphology was observed under an inverted microscope,and real time-PCR and Western blot were performed to quantify the mRNA and protein expressions of EMT-related molecules including E-cadherin,N-cadherin,vimentin and β-catenin respectively.Results After TGF-β treatment,the cells became dispersed and spindle in shape.The mRNA expression levels of E-cadherin,Ncadherin,vimentin,and β-catenin in TGF-β group were 0.317,2.475,11.340 and 2.615 folds those in the control group respectively.Western blot showed a marked increase in the expression of N-cadherin,vimentin,and β-catenin proteins but a notable decrease in that of E-cadherin in the TGF-β group compared with the control group.Conclusions TGF-β can induce EMT in A431 cells,and increased expression of TGF-β may contribute to the invasion and metastasis of SCC.

Objective To study the effect of NS398, an inhibitor of cyclooxygenase 2 (COX2), on the growth and apoptosis of human squamous cell carcinoma cell line Tca8113. Methods Cultured Tca8113 cells were incubated with NS398 (0, 6.25, 12.5, 25, 50, 100 μmol/L) for 24, 48 and 72 hours, respectively. Thereafter, MTT method, flow cytometry and transmission electron microscopy were applied to detect the proliferation, cell cycle and apoptosis of Tca8113 cells, respectively. Results The proliferation of Tca8113 cells was inhibited by NS398 in a dose- and time-dependent manner (both P<0.05). FCM analysis showed the appearance of a typical hypodiploid apoptotic (Sub-G1) peak, an increase in the percentage of cells at G0/G1 phase and a decrease in that at S and G2/M phases in NS398 ( 100 μmol/L) -treated Tca8113 cells. Moreover, the cell proliferation index was significantly downregulated by NS398 of 100 μmol/L from 41.03 to 24.33 (P<0.05). Under an electron microscope, morphological changes characteristic of apoptosis were observed in NS398-treated Tca8113 cells. Conclusion NS398, an inhibitor of COX2, could effectively inhibit the growth of Tca8113 cells in vitro by induction of apoptosis.

Objective To evaluate the inhibitory effect of photocatalytic titanium dioxide (TiO2)on the growth of a human epidermal squamous cell carcinoma cell line A431 and its mechanism.Methods Cultured A431 cells were classified into various groups to remain untreated (blank control group),be treated with different concentrations (100,200,300,400,500,600 mg/L) of TiO2 nanoparticles alone or in combination with ultraviolet (UV,main wavelength 253.7 nm,power 30 W,distance 30 cm,exposure duration 15 min) irradiation.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell growth,annexin V-fluorescein isothiocyanate/propidium iodide (PI) double staining to observe cell apoptosis,and Rho123 staining to determine mitochondrial transmembrane potential.Statistical analysis was carried out using SPSS 13.0 software.Analysis of variance (AOV),t test and Student-Newman-Keuls (SNK) test were performed to assess the differences in these parameters between these groups.Results The growth of A431 cells was inhibited by pretreatment with TiO2 nanoparticles followed by UV irradiation,and the inhibitory effect was enhanced as the dose of TiO2 nanoparticles increased.As AOV and SNK test showed,there were significant differences in the growth inhibition rate among A431 cells treated with different concentrations of TiO2 nanoparticles at the three time points (24,48 and 72 hours) after UV irradiation (n =6,F =21.54,77.56,20.27,respectively,all P ＜ 0.05).No statistical inhibition was observed in the growth of A431 cells treated with TiO2 nanoparticles alone compared with untreated A431 cells (all P ＞ 0.05).Photocatalytic TiO2 nanoparticles also induced the apoptosis but decreased the mitochondrial transmembrane potential in A431 cells.In detail,the apoptosis rate was 8.86％ ± 0.22％,11.72％ ± 0.29％ and 31.24％ ± 0.78％ in A431 cells treated with TiO2nanoparticles of 100,200,400 mg/L followed by UV irradiation,respectively,compared to 2.69％ ± 0.28％ in the blank control group (n =3,F =256.61,P ＜ 0.05).Decreased mitochondrial transmembrane potential (expressed as total fluorescence intensity) was observed in A431 cells treated with TiO2 nanoparticles of 100,200,400 mg/L followed by UV irradiation compared with blank control group (758.48 ± 15.42,676.60 ± 14.35,557.71 ± 13.12vs.2943.65 ± 70.26,F =208.57,P ＜ 0.05,n =3),and SNK test also revealed statistical differences between these groups.Conclusions TiO2 nanoparticles combined with UV can inhibit the growth of but induce the apoptosis in A431 cells,which may be associated with the reduction in mitochondrial transmembrane potential in A431 cells,while TiO2 nanoparticles alone show no inhibitory effect on the growth of A431 cells.

OBJECTIVETo isolate the side population (SP) and non-side population (NSP) cells from A431 cells and compare their difference in tumorigenicity in mice and the expression profiles of epithelial-mesenchymal transition (EMT) markers.

METHODSA431 cells stained with Hoechst 33342 were sorted with flow cytometry. The isolated SP cells and NSP cells were inoculated into NOD/SCID mice and the tumorigenicity of the cells was observed. EMT markers E-cadherin, β-catenin, vimentin, AXL, and Erbb3 in the tumor tissues were detected by immunohisto-staining.

RESULTSThe tumors generated by SP cells were larger than those by NSP cells in NOD/SCID mice. Compared with the tumors generated by NSP cells, the cells in the periphery of tumors generated by SP cells showed up-regulated expressions of AXL, vimentin and β-catenin and down-regulated ERBB3 and E-cadherin.

CONCLUSIONThe SP cells in A431 cells have a strong tumorigenicity and show more EMT phenotypes in tissues.

Animals ; Biomarkers, Tumor ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplastic Stem Cells ; metabolism ; Side-Population Cells ; metabolism ; Skin Neoplasms ; metabolism ; pathology

Melkersson-Rosenthal syndrome (MRS) is an uncommon granulomatous disease characterized by the triad of relapsing facial paralysis, orofacial swelling, and fissured tongue. Genital swelling in MRS is rarely reported. We presented the first case of complete MRS with genital swelling in a child. Biopsy examinations of both the child's lower lip and penis showed noncaseating granuloma and intralymphatic granuloma infiltration. No symptoms or signs of other systemic disease (Crohn's disease or sarcoidosis) were observed after 2 years of follow-up. Genetic screening for CARD15/NOD2 in this patient showed negative, which further confirmed the diagnosis of MRS. Eleven other cases of suspected complete or incomplete MRS with genitalia involved were reviewed. Our case emphasizes the specific clinical feature of MRS with genitalia involved, which was genetically different from Crohn's disease and could be an independent entity. Lymphatic obstruction is responsible for localized edema in MRS.
Biopsy ; Child* ; Crohn Disease ; Diagnosis ; Edema ; Facial Paralysis ; Follow-Up Studies ; Genetic Testing ; Genitalia* ; Granuloma ; Humans ; Lip ; Lymphatic Vessels ; Male* ; Melkersson-Rosenthal Syndrome* ; Penis ; Tongue, Fissured

Melkersson-Rosenthal syndrome (MRS) is an uncommon granulomatous disease characterized by the triad of relapsing facial paralysis, orofacial swelling, and fissured tongue. Genital swelling in MRS is rarely reported. We presented the first case of complete MRS with genital swelling in a child. Biopsy examinations of both the child's lower lip and penis showed noncaseating granuloma and intralymphatic granuloma infiltration. No symptoms or signs of other systemic disease (Crohn's disease or sarcoidosis) were observed after 2 years of follow-up. Genetic screening for CARD15/NOD2 in this patient showed negative, which further confirmed the diagnosis of MRS. Eleven other cases of suspected complete or incomplete MRS with genitalia involved were reviewed. Our case emphasizes the specific clinical feature of MRS with genitalia involved, which was genetically different from Crohn's disease and could be an independent entity. Lymphatic obstruction is responsible for localized edema in MRS.
Biopsy ; Child* ; Crohn Disease ; Diagnosis ; Edema ; Facial Paralysis ; Follow-Up Studies ; Genetic Testing ; Genitalia* ; Granuloma ; Humans ; Lip ; Lymphatic Vessels ; Male* ; Melkersson-Rosenthal Syndrome* ; Penis ; Tongue, Fissured