Objective To investigate the protective effect of butyl flufenamate ointment against ultraviolet (UV)-induced skin damage,skin aging,and cutaneous squamous cell carcinoma (CSCC) in SKH-1 hairless mice.Methods A total of 128 mice were randomly and equally divided into four groups:UV group receiving UV irradiation only,butyl flufenamate ointment group and matrix cream group receiving UV irradiation after 30-minute pretreatment with topical butyl flufenamate ointment and matrix cream respectively,and blank control group receiving neither pretreatment nor irradiation.In the sunburn experiment (n =24),mice were exposed to single session of UV irradiation (1.5 minimal erythema doses (MEDs)),and 24 hours later,erythema and swelling response was observed,and skin tissue was obtained from the irradiated area on the back of mice followed by the determination of COX-2 expression using the streptavidin biotin peroxidase complex (SABC) method.To establish a photoaging (n =24) and CSCC (n =80) model,mice were exposed to four sessions of UV irradiation every week for 12 and 28 successive weeks respectively,with the irradiation dose starting at 0.9 MED and increasing gradually.After 12-week irradiation,skin tissue was resected from the back of photoaged mice and subjected to Masson staining for the evaluation of collagen changes as well as immunohistochemical analysis for the quantification of Bax,Bcl-2 and Caspase 3 expression.The initiation and progression of CSCC were observed in mice on a once-a-week basis from 12 to 28 weeks.SPSS 21.0 software was used for statistical analysis.One way analysis of variance was carried out for multiple-group comparisons of numerical data,Ridit analysis for the comparison of immunohistochemical staining intensity.Kaplan-Meier method and log-rank test were utilized for the comparison of tumor-free survival time.Results Both the degree of erythema and swelling response and expression level of COX-2 were significantly lower in the butyl flufenamate ointment group than in the other two UV-irradiated groups (all P ＜ 0.05).After 12-week irradiation,the butyl flufenamate ointment group showed milder degree of skin aging,together with higher density of collagen in dermis,weaker expression of Bcl-2 but stronger expression of Bax and Caspase 3,by comparison with the other two UV-irradiated groups (all P ＜ 0.05).During the 28 weeks of irradiation,the median tumor-free survival time was statistically longer in the butyl flufenamate ointment group than in the matrix cream group and UV group((25.0 ± 0.4) months vs.(24.0 ± 0.3) months and (23.0 ± 0.4) months,P ＜ 0.05 and 0.01 respectively).Conclusion Butyl flufenamate ointment has a certain photoprotective effect.

Objective To study cervical HPV infection in female patients with vulvar condyloma acuminatum(CA)from Shanghai area.MethodsExfoliated cells were obtained from cervices and vulva lesions of CA of 194 patients.respectively.HPV genotyping was carried out in cell samples using capture-hybridization method and gene chip techniques.Results HPV was detected in vulva lesions of all the 194 patients.Among them,74.2%(144/194)were positive for low risk(LR)-HPV,and 25.8%(50/194)for high risk(HR)-HPV.A single HPV genotype(6 or 11)was detected in 136(94.4%)patients with LR-HPV.and mixed genotypes of LR-HPV and HR-HPV in 46(86%)patients with HR.HPV.Of the 194 patients.85.6%(166/194)were complicated by cervical HPV infection.including 119(61.4%)cases of LR-HPV and 46(23.7%)cases of HR-HPV,In the case of HPV genotype.the consistence between CA lesions and cervix was 95.8%(159/166).The prevalence of LR-HPV declined sequentially from subtype 11 to 6 and 53,and that of HR-HPV from subtype 16 to 18 followed by 52,31,45 and 58.Conclusions There is a high rate of infection with HR-HPV in female patients with CA.and nearly one quarter of these patients are complicated by cervical HR-HPV infection.

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence found within several glycoprotein ligands and also in snake-venom disintegrins. Adinbitor, a protein with 73 amino acid residues including 12 cysteins and an RGD motif, was cloned from Gloydius blomhoffi brevicaudus in my laboratory. As a new member of disintegrin family, adinbitor can inhibit both human platelet aggregation induced by ADP and angiogenensis in vivo and in vitro, the typical characters of disintegrin family. To separate the effect of inhibiting platelet aggregation from that of inhibiting angiogenensis, the motif KGD was introduced into adinbitor cDNA to replace RGD by site-directed and PCR-based mutagenesis. The recombinant protein (recombinant adinbitor (KGD)) was expressed in E. coli BL21 and purified through the His· Bind affinity chromatography. Recombinant adinbitor (KGD) could inhibit ADP-induced platelet aggregation with IC50 value of 85 nmol/L. Considerably, it was a more effective inhibitor on platelet aggregation than recombinant adinbitor (RGD), which has an IC50 of 150 nmol/L. Interestingly, recombinant adinbitor (KGD) has no potency in inhibiting angiogenesis in vivo compared with recombinant adinbitor (RGD). These findings showed that KGD containing adinbitor was more suitable for inhibiting ADP-induced human platelet aggregation as a potential and specific inhibitor of human platelet aggregation, which might have promising therapeutic potential as an antithrombotic agent.

Objective To validate the repeatability of the simplified cough score and its responsiveness to effective treatment and investigate the relationship between the simplified cough score and cough symptom score.MethodsA total of 119 patients with chronic cough referred to our respiratory clinic were recruited into the study between June 2010 and February 2011. Cough severity was evaluated by the simplified cough score,cough symptom score,Leicester cough questionnaire,and cough reflex sensitivity detection,and the correlations among them were analyzed.The change ratio,effect size,and standardized response mean of the simplified cough score were calculated after a 2-week course of effective treatment.The repeatability of the simplified cough score was assessed in 99 untreated patients with stable chronic cough.ResultsThe intraclass correlation coefficient in a 3-day test-retest interval of simplified cough score was 0.90 ( 95 ％ CI =0.84 - 0.92,P =0.00 ) for daytime and 0.89 ( 95 ％ CI =0.91 - 0.96,P =0.00 ) for nighttime. There was an obvious positive linear correlation between the simplified cough score and cough symptom score ( daytime:r =0.82,P =0.00 ; nighttime:r =0.92,P =0.00 ),a significant negative linear correlation between the cough score and Leicester cough questionnaire,and a weak but significant negative correlation between the simplified cough score and cough threshold C2 or C5 to capsaicin. After a 2-week course of effective treatment,the change ratio,effect size,and standardized response mean were 46.71％,1.16,and 1.05 for daytime and 71.87％,1.09,and 1.10 for nighttime,respectively.ConclusionThe simplified cough score is a reliable and valid tool for evaluation of cough severity in clinical practice.

Objective To investigate the local immune response in condyloma acuminatum treated with aminolevulinic acid-photodynamic therapy (ALA-PDT).Methods In vitro and in vivo studies were performed.A previously established keratinocyte cell line human papilloma virus (HPV) 16E7/HaCaT which stably expresses HPV16E7 protein was used in this study.Peripheral blood mononuclear cells (PBMCs) were separated from 10 healthy volunteers.After pretreatment with ALA-PDT,HPV16E7/HaCaT cells were cocultured with the PBMCs for 3 hours in a Transwell chamber followed by the observation of chemotactic migration of PBMCs.Tissue samples were obtained from the lesions of 10 patients with condyloma acuminatum before,and at 1,2,3 and 48 hours after the first session of ALA-PDT.Immunohistochemistry was conducted to determine the number of CD4+ T cells,CD8+ T cells and CD68+ macrophages as well as CD4/CD8 T-cell ratio in the tissue samples.Results After 3-hour coculture with HPV16E7/HaCaT cells pretreated by ALA-PDT,PBMCs showed apparent chemotactic migration.Immunohistochemistry revealed a statistical increase in the number of CD4+ T cells,CD8+ T cells and CD4/CD8 T-cell ratio at 48 hours (all P ＜ 0.05),as well as in the number of CD68+ macrophages at 3 hours and 48 hours (both P ＜ 0.05) after the first session of ALA-PDT.Conclusion ALA-PDT may induce local antiviral immune response in condyloma acuminatum.

Objective To investigate the pathogenic characteristics of Vibrio cholera strains isola-ted from Hubei province in 2012 , and to identify the source of infection by analyzing their genetic correla-tions.Methods The biochemical identification , toxin gene detection and drug susceptibility test were car-ried out to analyze a total of 35 Vibrio cholera strains isolated from three epidemic areas .Comparison of ge-nomic DNA fingerprints and cluster analysis among isolates of Vibrio cholera was conducted by using pulsed-field gel electrophoresis ( PFGE ) .Results All of the 35 strains were Vibrio cholera O139 , of which 71.42%were toxic strains.The drug resistance rates of Vibrio cholera strains to tetracycline, cotrimoxazole and rifampincin were 57.14%, 88.57%and 80.00%, respectively.Analysis of genomic DNA fingerprints of the isolates showed highly similar with similarity values ranging from 80%-100%.Most of the strains iso-lated from the same epidemic area fell into the same one cluster with 100% homology in genome Only a strain isolated from turtle in Jingzhou area was belong to a different cluster .Conclusion The Vibrio cholera O139 strains were the dominant strains causing the outbreaks of cholera in Hubei province in 2012 .Most of them were toxigenic strains .A large majority of the strains had developed resistance to tetracycline , cotri-moxazole and rifampincin , but all strains showed high susceptibility to ceftriaxone and imipenem .Vibrio cholera strains isolated from the same epidemic area were mainly belonged to the same one cluster , sugges-ting the same source of infection .However, the strains varied among different epidemic area .Follow-up in-vestigations of three outbreaks of cholera in this study were all associated with food infection .Therefore , more attention should be paid to food sanitation and safety measurement .Although a non-toxigenic strain iso-lated from turtle was not associated with the epidemic of cholera , surveillance for seafood and aquatic prod-ucts would still be necessary .

Objective To establish a human keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein. Methods HPV16E7 gene was amplified from CaSki cells using PCR and inserted into the eukaryotic expression plasmid pcDNA3.1. Then, the recombinant expression plasmid pcDNA3.1-HPV16E7 was transfected into HaCaT cells followed by G418 selection and identification by RT-PCR and Western blot. Results The recombinant eukaryotic expression plasmid pcDNA3.1-HPV16E7 was successfully identified by restriction enzyme digestion pattern and sequence analysis. Agarose gel electrophoresis of RT-PCR products detected the 297-bp fragment of HPV16E7 cDNA, and Western blot confirmed the stable expression of HPV16E7 protein. Conclusion A human keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein is successfully established.

Objective To establish a model for cutaneous squamous cell carcinoma by irradiation of SKH-1 hairless mice with solar-simulated ultraviolet (solar UV), and to explore the biological characteristics of the model. Methods A total of 91 SKH-1 hairless mice were randomly divided into seven experimental groups (n = 10) and seven control groups (n = 3). The mice in experimental groups were irradiated with minimal erythema dose of solar UV 4 times per week for various durations (4, 8, 12, 16, 20, 24, 28 weeks), while the control mice received no irradiation. The general status and skin appearance of mice were observed during the treatment process. Mice were killed immediately after the last irradiation at different time points and pathological examination was carried out to observe the histological changes of skin lesions. Results Papules measuring equal to or more than 1 mm in diameter began to develop in some mice in experimental group 10 weeks after the first irradiation; tumors began to appear in 39.3% (11/28) of the remaining mice in experimental group on week 20, and in 100% (10/10) of the remaining mice on week 28. The cumulative dose approximated to 26.99 J/cm2 for UVB and 242.91 J/cm2 for UVA after 28-week irradiation. No tumor was observed in the control mice. Pathological examination revealed characteristic changes of squamous cell carcinoma in 30% of the mice on week 12, 33.3% on week 16, 60% on week 20, 87% on week 24, and 100% on week 28. Conclusions Ultraviolet could induce the hyperplasia of skin in SKH-1 hairless mice, and even cause the development of cutaneous squamous cell carcinoma after prolonged irradiation.

Objective To study the enhancement patterns of hilar bile duct wall of ischemic-type biliary lesion (ITBL) on contrast-enhanced ultrasound (CEUS).Methods Eighteen healthy subjects,18 orthotropic liver transplantation (OLT) recipients without complications,and 36 patients,which were subdivided into 2 groups according to the final diagnosis:patients with (n =24) and without (n =12)ITBL,were enrolled in this study.The patients without ITBL had anastomotic biliary stricture (n =3),cholangitis (n =4),biliary sludge (n =1),and acute rejection (n =4),respectively.The images of baseline sonography and CEUS were retrospectively analyzed in consensus by 2 readers.The enhancement time and level of hilar bile duct wall,hepatic artery and liver parenchyma were recorded.Results Hilar bile duct wall became enhancing earlier than liver parenchyma in all of 4 groups.During arterial phase,hyper-or isoenhancing bile duct walls were present in most cases in the groups of healthy subjects,OLT recipients without complications and patients without ITBL.However,non-or hypo-enhancement of hilar bile duct wall were present in 16 (66.7％) ITBL patients,which is different from the other groups (P ＜0.05).Conclusions The main features of ITBL differing from the other groups were non-or hypo-enhancement of hilar bile duct wall in arterial phase.It may be a diagnostic index to apply in detecting ITBL with CEUS.

Objecfive To investigate the association between the exon 2,3,4 of MHC class Ⅰ chain-related gene-B(MICB)and ulcerative colitis(UC)in Chinese Han.Methods Using polymerase chain reaction single-stranded conformation polymorphism,allele frequency of MICB exon 2,3 and 4 in 105 patients with UC and 213 age and sex matched healthy controls were genotyped.All of the studied individuals were Chinese Han.Results Allele frequency of MICB 0106 was increased in patients with UC as compared with normal controls(19.0%vs 8.9%,P=0.000,Pc＜0.001,OR=2.402,95%CI:1.488-3.879).The frequency of MICB 0106 was increased significantly in patients with extensive colitis (24.4%vs 8.9%,P=0.000,Pc＜0.001,OR=3.294,95%CI:1.800-6.027),moderate and severe disease(24.1%vs 8.9%,P=0.000.Pc＜0.001,OR=3.294 95%CI:1.893-5.576)and in those with extra intestinal manifestations(20.5%vs 8.9%,P=0.002,Pc=0.012,OR=2.626,95%CI:1.418-4.861).Furthermore,MICB 0106 allele was higher in frequency in the male patients with UC (22.1%vs 8.0%,P=0.001,Pc=0.006,OR=3.276,95%CI:1.737-6.178)and the patients more than 40 years old(28.8%vs 8.3%,P=0.000,Pc＜0.001,OR=4.500,95%CI:2.381-8.504)as compared with healthy controls.Conclusion MICB 0106 allele is positively associated with UC,especially with extensive colitis,moderate and severe disease,presence of extra intestinal manifestations,male gender and age of more than 40 years in Chinese Han in Hubei province.