Objective To investigate the relationship between the efficacy of autologous cultured melanocyte transplantation and serum levels of interleukin-17 (IL-17) and FoxP3 in patients with vitiligo.Methods Forty patients with stable vitiligo vulgaris were included in this study,and received autologous cultured melanocyte transplantation.Six months after the transplantation,treatment efficacy was evaluated,and patients were classified into the successfully treated group (n =25) and unsuccessfully treated group (n =15).Peripheral blood was collected from all the patients before the transplantation,and enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum levels of IL-17 and FoxP3.Statistical analysis was done using two independent samples t-test with the SPSS software (version 17.0).Results The successfully treated patients showed lower serum levels of IL-17 ((15.29 ± 7.86) vs.(43.88 ± 13.02) ng/L,P ＜ 0.05),but higher serum levels of FoxP3 ((6.08 ± 2.03) vs.(3.37 ± 1.81) ng/L,P ＜ 0.05) than the unsuccessfully treated patients.Conclusion The increased serum IL-17 and decreased serum FoxP3 may contribute to the failure of autologous cultured melanocyte transplantation in patients with vitiligo.

Objective: Identification of the attachment site of phage PaP3 within the genome of Pseudo-monas aeruginosa PAS. Methods:The full genome of lysogenic bacteria was cleaved by Pst Ⅰ and produce a large fragment of more than 45 000 bp, which was subsequently digested by EcoR Ⅰ. Then the fragment containing DNA sequence of phage and bacteria was cloned into pFastBacTMHT A vector, and the result of sequencing indicated the right hybrid site attR. AttL was isolated by PCR on the base of integration mechanism. And then attP and attB were indentified according to the nucleotide sequences of attR and attB. Results:A sequence of 21 bp(5'-GGTCGTAGGTTCGAATCCTAC-3') was defined to be the core site of integration, which was located at t-RNAPro gene in the genome of phage PaP3 and t-RNALys gene in the genome of Pseudomonas aeruginosa PA3. The attP and attB flanked with a set of inverted repeat and direct repeat. Conclusion:The integrated site of PaP3 within the genome of PA3 was identified and characteriged, which could be of value in investigating the mechanism of integration and gene flow between different species in the natural world.

Objective To investigate quercetin's protective effect against oxidative stress in and impact on the biological activity of mouse B10BR melanocytes. Methods B10BR cells were cultured and treated with different concentrations of quercetin followed by additional culture. Then, cell viability was measured by using MTT assay, hydrogen peroxide-induced cell apoptosis by flow cytometry, and cell morphological changes by microscopy. The tyrosinase activity in and melanin synthesis by B10BR cells were measured by dopa oxidation assay and sodium hydroxide (NaOH)-lysis method, respectively. Results After treatment with quercetin of 33.33 μmol/L for 24 hours, the survival rate of B10BR cells reached (94.22 ± 3.36)%, tyrosinase activity (107.15 ± 10.96)%, and melanin content (111.85 ± 9.49)%. A significant difference was observed in tyrosinase activity and melanin content between hydrogen peroxide-induced and 33.33 μmol/L quercetin-treated B10BR cells and those only induced by hydrogen peroxide (both P < 0.01). Flow cytometry revealed that quercetin inhibited hydrogen peroxide-induced apoptosis in melanocytes. Conclusion The protective effect of quercetin against hydrogen peroxide-induced apoptosis in melanocytes may provide a new idea for the treatment of vitiligo.

Objective To investigate the effect of nuclear translocation of E2p45 related factor 2 (Nrf2)on the biological activity of melanocytes.Methods Plasmid vectors containing wild-type nrf2 gene (pcDNA-nrf2) and nls-deleted nrf2 gene (pcDNA-nrf2△nls) were constructed.B10BR normal murine melanocytes were classified into three groups,i.e.,untransfected group,wild-type nrf2 group transfected with pcDNA-nrf2,and mutated nrf2 group transfected with pcDNA-nrf2△nls.Each of the above groups were further divided into three subgroups:control subgroup receiving no treatment,hydrogen peroxide (H2O2) subgroup treated with H2O2 of 200 μmol/L for 24 hours,and combined subgroup pretreated with tert-butyl hydroquinone (TBHQ) followed by treatment with H2O2 of 200 μmol/L for 24 hours.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,dopa oxidation assay to determine tyrosinase activity,Transwell assay to estimate cell migration ability,Western blot to quantify the expressions of Nrf2 and his tag fusion protein.Results TBHQ significantly enhanced the nuclear expression of Nrf2 in B10BR cells transfected with pcDNA-nrf2 or pcDNA-nrf2△nls (both P ＜ 0.01).No significant difference was observed in tyrosinase activity between untreated wild-type nrf2 group,mutated nrf2 group,and untransfected group (P ＞ 0.05).There was a statistical decrease in tyrosinase activity in the two H2O2-treated transfected groups compared with the untreated transfected groups (both P ＜ 0.05),and the decrease was reversed by TBHQ pretreatment in the wildtype nrf2 group (P ＜ 0.05),but not in the mutated nrf2 group (P ＞ 0.05).Further more,the proliferative activity of B10BR cells experienced no obvious changes in the wild-type nrf2 group (P ＞ 0.05),but was significantly reduced in the untransfected group (P ＜ 0.05) and mutated nrf2 group (P ＜ 0.01) after the H2O2 treatment compared with the corresponding untreated groups.TBHQ could protect the pcDNA-nrf2-transfected B10BR cells,but not pcDNA-nrf2△nls-transfected B10BR cells,from H2O2-induced oxidative damage.Transwell assay showed no significant difference in migration ability among these nine groups (P ＞ 0.05).Conclusions Abnormal nuclear translocation of Nrf2 could affect antioxidant activity of,proliferative activity of and tyrosinase activity in melanocytes.TBHQ may enhance the tyrosinase activity in,proliferative activity and antioxidant activity of melanocytes via activating the nuclear expression of wild type Nrf2.

Objective To evaluate the effect of calcipotriol on the proliferation of and cytokine secretion by melanocytes and perilesional CD8+ cytotoxic T lymphocytes (CTLs) from patients with vitiligo.Methods Melanocytes isolated from abdominal skin and CD8+ CTLs from perilesional skin of patients with vitiligo were subjected to successive culture in vitro.After several passages,the melanocytes and CD8+ CTLs were cultured alone or in combination with or without the presence of various concentrations of calcipotriol for 24 to 48 hours.MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium,inner salt) method was used to evaluate the proliferative activity of cells,enzyme-linked immunosorbent assay to determine the levels of interleukin (IL)-6,tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the culture supematant of cells,flow cytometry to detect cell apoptosis.Some co-cultured melanocytes and CTLs were treated with calcipotriol of 10-8 mol/L and anti-IL-6 antibody of various concentrations (0,1,2,2.5,5,10 mg/L) for two days followed by enumeration of cells.The concentrations of 108 and 10-9 mol/L (calcipotriol) were chosen for relevant tests.Results There was a marked apoptosis in MCs after coculture with CD8+ CTLs.The 24-hour treatment with calcipotriol of 104 and 10-9 mol/L had no obvious effect on the proliferation of melanocytes cultured alone (both P ＞ 0.05),but accelerated the proliferation of melanocytes cocultured with CTLs (both P ＜0.05) as well as that of CD8+ CTLs cultured alone or in combination with melanocytes (all P ＜0.05).A statistical decrease was observed in IL-6,TNF-α and IFN-γlevels in the supernatant of cocultured melanocytes and CTLs compared with those in the supernatant of melanocytes and CTLs cultured alone,and calcipotriol of 10-9 mol/L intensified the decrease in supernatant IL-6 level (t =2.89,P ＜0.05),but no statistical changes were noted for the level of TNF-α or IFN-γin the supernatant of the coculture system after treatment with calcipotriol of 104 or 104 mol/L compared with that before treatment (both P ＞ 0.05).In the coculture system pretreated with calcipotriol of 10-8 mol/L,the number of CD8+ CTLs significantly decreased (t =3.15,P ＜0.05),whereas that of melanocytes significantly increased (t =3.53,P ＜0.05) after the treatment with anti-IL-6 antibody of 5 mg/L.Conclusions Perilesional CD8+ CTLs have a specific cytotoxic effect on melanocytes,and calcipotriol may inhibit the cytotoxic effect of CD8+ CTLs by suppressing the secretion of IL-6,which may partly explain the therapeutic mechanism of calcipotriol for vitiligo.

Objective To investigate the intracellular signal transduction pathways involved in the protective effect of nicotinic acid against ultraviolet B(UVB)-induced damage in human skin keratinocytes.Methods Cultured human keratinocyte HaCaT cells were divided into several groups to be treated with nicotinic acid,UVB irradiation,LY294002(an inhibitor of Akt),U0126(an inhibitor of extracellular signal-regulated kinase(ERK)1/2),SB203580(an inhibitor of P38)alone or in combination for different durations.Then,Western blot was performed to quantify the phosphorylation levels of the protein kinase B(Akt)/MAPK pathwayassociated proteins including Akt,P38,JNK and ERK1/2,MTT assay to evaluate the activity of HaCaT cells,enzyme-linked immunosorbent assay to determine the levels of endothelin-1(ET-1)and basic fibroblast growth factor(bFGF)in the culture supernatant of HaCaT cells,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)to evaluate the apoptosis in HaCaT cells.Results As Western blot showed,phosphorylated Akt,P38,JNK and ERK1/2 were markedly activated within 60 minutes after pretreatment with nicotinic acid and irradiation with UVB(all P ＜ 0.01),and the activation was more significant for phosphorylated Akt,P38,and ERK1/2 within 2 hours(all P ＜ 0.01).Nicotinic acid effectively suppressed the UVB-induced cell death and apoptosis in HaCaT cells.The levels of supernatant ET-1 and bFGF were significantly decreased in HaCaT cells treated with the above 3 inhibitors followed by UVB irradiation than in those treated with the inhibitors alone(all P ＜ 0.05),and nicotinic acid pretreatment only reversed the decrease in supernatant bFGF in HaCaT cells treated with SB203580 followed by UVB irradiation.Conclusion The Akt signaling pathway may play a regulatory role in the protection by nicotinic acid against UVB-induced damage in HaCaT cells.

Objective To express and purify the epitope peptide of human melanin-concentrating hormone receptor 1, and to evaluate its performance in the detection of autoantibodies in vitiligo patients. Methods The target gene encoding the epitope peptide of human melanin-concentrating hormone receptor 1 was synthesized, cloned to prokaryotic expression vector pGEX-4T-2 which was then transferred to E. coli BL21. The protein expression was induced by isopropy-β-D-thiogalactoside (IPTG) and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Blocking ELISA was carried out with membrane proteins extracted from melanocytes as the blocking antigen. The antigenicity of the peptide was detected in sera from 100 patients with progressive vitiligo and 30 healthy human controls. Results The recombinant expression vector was successfully constructed, and the target protein was successfully expressed in E.coli, which was evidenced by SDS-PAGE and Western blot. With the glutathione S-transferase (GST) purification kit, the purity of the recombinant protein reached 100% when the sampling weight was less than 0.625 μg.The binding of the target protein with serum IgG antibodies from vitiligo patients could be blocked by natural membrane antigen of melanocytes. Of the 100 sera from patients with progressive vitiligo, 36 were reactive with the target protein. Conclusions The epitope peptide of human melanin-concentrating hormone receptor 1 has been successfully expressed and purified. The purified protein can bind with serum IgG antibodies from vitiligo patients, and may be applied to the detection of autoantibodies against human melanin-concentrating hormone receptor 1.

Objective To discuss the value of transcatheter artery embolization for the treatment of GI massive hemorrhage. Methods Seventy-eight GI massive bleeding cases underwent emergency angiography. Intraarterial embolization was performed in 86 arteries of the 78 patients. All patients were followed up for 1 to 10 years. Results Bleeding stopped immediately in 100% of the patients. Bleeding recurred in 16 cases from 48 hours to 37 monthes. Conclusion Arterial embolization for digestive tract bleeding is safe and effective during emergency angiography to buy a time for definite treatment for some patients.

Objective:To investigate the efficacy of systemic glucocorticoid treatment and its related factors in progressive vitiligo patients with vitiligo disease activity (VIDA) scores ≥ 2 points.Methods:A total of 272 progressive vitiligo patients with VIDA scores ≥ 2 points and skin lesion area < 1% of body surface area, who received no systemic glucocorticoid treatment, were collected from Department of Dermatology, the Third People′s Hospital of Hangzhou from June 2018 to June 2019. The area and type of skin lesions, VIDA scores, predisposing factors and special clinical markers (trichrome vitiligo, confetti-like depigmentation, Koebner phenomenon and inflammatory vitiligo) were analyzed. These patients were randomly divided into 3 groups by a random number table: topical glucocorticoid group (62 cases) , oral prednisone + topical glucocorticoid group (76 cases) and compound betamethasone injection + topical glucocorticoid group (134 cases) , and the latter two groups were also called as the systemic and topical glucocorticoid group. The patients in the topical glucocorticoid group were treated with halometasone cream or 0.05% clobetasol propionate cream once a day; during the oral prednisone treatment, the dose was adjusted once every 7 days, and gradually reduced from 30 mg/d to 20, 15, 10 and 5 mg/d, and the treatment lasted 35 days; during the treatment with compound betamethasone injection, intramuscular injection was performed once every 20 days at a dose of 1 ml for 2 sessions. The stable disease rate (defined as the proportion of patients experiencing no progression during the study among the analyzed patients) was calculated in these groups after 3 months of treatment, and changes in vitiligo types were evaluated after 1 year of follow-up. Statistical analysis was carried out by using Kruskal-Wallis H test, χ2 test and Fisher′s exact test. Results:After 3-month treatment, there was a significant difference in the expansion rate of skin lesion area among the 3 groups ( H = 12.468, P < 0.001) , and the expansion rate of skin lesion area was significantly lower in the oral prednisone + topical glucocorticoid group and compound betamethasone injection + topical glucocorticoid group than in the topical glucocorticoid group ( P < 0.001, = 0.005, respectively, α = 0.016 7) ; among the patients with slowly progressive vitiligo (VIDA scores = 2 or 3 points) , the stable disease rate was significantly higher in the systemic and topical glucocorticoid group than in the topical glucocorticoid group ( χ2 = 23.973, 11.877, respectively, both P < 0.001) ; the stable disease rate also significantly differed among the patients with different VIDA scores (VIDA scores = 2, 3 or 4 points) in the systemic and topical glucocorticoid group ( χ2 = 17.122, P < 0.001) . After 3-month treatment, the patients with predisposing factors or special clinical markers showed significantly decreased stable disease rate (47.3% [35/74], 41.2% [47/114], respectively) compared with those without predisposing factors or special clinical markers (70.6% [96/136], 87.5% [84/96]; χ2 = 11.098, 47.548, respectively, both P < 0.001) . After 1 year of follow-up, the proportion of patients with localized vitiligo converted into non-localized vitiligo was significantly higher in the topical glucocorticoid group (41.9%, 26/62) than in the systemic and topical glucocorticoid group (21.9%, 46/210; χ2 = 10.328, P = 0.006) , and higher in the group with predisposing factors or special clinical markers than in that without predisposing factors or special clinical markers respectively (both P < 0.01) . Conclusions:Early systemic glucocorticoid treatment should be performed in the progressive vitiligo patients with high VIDA scores, predisposing factors and special clinical markers.

Objective To investigate the consensus and controversies on delineation of radiotherapy target volume for patients with non-small cell lung cancer(NSCLC). Methods Study questionnaires were designed by radiation oncologists in Peking University School of Oncology. The forms were sent through email to radiation oncologists in 10 radiation departments in China and 2 departments in US in November,2007. The brief introduction and PET/CT digital data of one patient with NSCLC were sent to radiation oncologists in 10 departments in Beijing. On Jan. 12,2008,the case discussion was held by more than 300 radiation oncologists from Beijing,Tianjin, Hebei, Liaoning Province and Inner Mongolia Autonomous Region. Results All receivers of the questionnaire responded . The set up error was 5-7 mm . For patients with locally advanced NSCLC treated with radiotherapy concurrently with near full dose chemotherapy,ll out ot 12 responding departments defined planning target volume(PTV) of primary tumor as gross tumor volume(GTV) plus 6-8 nun plus set-up error and respiratory movements ,and only one defined PTV as GTV plus set-up error and respiratory movements. For PTV of the mediastinal lymph nodes in the same patient,9 out of 12 responding departments defined PTV as GTV plus 6-8 mm plus set-up error and respiratory movements,and 3( of China) out of 12 defined PTV as GTV plus set-up error and respiratory movements. Stereotactic body .radiotherapy with high fraction dose was used in 11 out of 12 responding departments with fraction dose varying from 6 to 20 Gy,including 6 of which defined PTV of primary tumor as GTV plus 6-8 mm plus respiratory movements and set-up error, and 5 defined PTV of early stage lung cancer as GTV plus respiratory movements and set-up error. The consensus on delineation of primary tumor of the case discussion was that the appropriate window width and window level were 1600 Houasfield Units(HU) and -600 HU for lung window,and 400 HU and 20 HU for mediastinal window. The controversies was focused on whether the CTV for metastatic lymph nodes should be restricted as GTV plus 6-8 mm or enlarged to enclose all the involved lymph node region. Conclusions PIT of primary tumor and mediastinal metastatic lymph nodes should be GTV plus 6-8 mm plus respiratory movements plus set-up error. The basic controversies of target delineation are focused on the fraction dose and PIT range for early stage NSCLC, and on the possibility of defining the PIT as GTV plus respiratory movements and set-up error when treated with concurrent radiotherapy and full dose chemotherapy for locally advanced NSCLC.