Objective To develop a method to purify and identify anti-BP180 NC16A antibodies from the sera of patients with bullous pemphigoid (BP) or herpes gestationis.Methods The GST/NC16A fusion protein was expressed in a prokaryotic expression vector pGEX-2TBP180NC16A,and then crosslinked to glutathione sepharose beads.Anti-BP180 NC16A antibodies were isolated from the sera of 3 patients with BP and 2 patients with herpes gestationis by affinity chromatography,and analyzed by immunofluorescence,Western blot and enzyme linked immunosorbent assay (ELISA).Results The GST/NC16A fusion protein with a relative molecular mass of 37 000 was successfully expressed by the prokaryotic vector pGEX-2TBP180NC16A.Purified anti-BP180 NC16A antibodies were obtained from the sera of patients by the affinity chromatography,and ELISA revealed that the concentration of anti-BP180 NC16A was 2.4 mg/ml.The purified antibody could bind to the basement membrane zone of human skin,suggesting a strong biological activity of the antibodies.Western blot showed a single band corresponding to the expected molecular mass for anti-BP180 NC16A antibodies,indicating a high purity of the isolated antibodies.Conclusion The anti-BP180 NC16A antibodies purified by microbead-based affinity chromatography from the sera of patients with BP or herpes gestationis are highly active and specific.

ObjectiveTo develop an assay to quantitatively detect anti-BP180NC16A IgG subclasses and to assess the significance of anti-BP180NC16A lgG in bullous pemphigoid (BP).MethodsThe Glutathione S-transferase(GST)-BPI80NC16A fusion protein was expressed in E.coli system and purified by affinity chromatography.An improved enzyme-linked immunoabsorbent assay (ELISA) was developed and used to detect anti-BP180NCI6A IgG subclasses in serum samples from 10 patients with BP,5 patients with pemphigoid gestationis (PG),1 patient with linear IgA bullous dermatosis (LIBD) and 2 patients with pemphigus.Results The optimal condition for the ELISA was determined by cross assay as follows:the concentration of GSTBP180NC16A fusion protein for coating,500 μg/L; the condition for coating,4 ℃ for 12 hours; the dilution ratio of sera and secondary antibody,1∶ 100 and 1 ∶ 2000 respectively; the condition for incubation,37 ℃ for 1 hour;,the condition for the enzyme-substrate reaction,37 ℃ for 20 minutes.Of the 10 patients with BP,all were positive for anti-BP180NC16A IgG1,9 for IgG2,5 for IgG3,and 9 for IgG4.Anti-BP180NC16A IgG was undetected in any of the serum samples from 2 patients with pemphigus vulgaris or 1 patient with adult LIBD.All the 5 sera from patients with PG; were positive for all the anti-BP180NC16A IgG subclasses,which were predominated by IgGl and IgG3.ConclusionThe developed ELISA is a highly specific and reproducible semiquantitative method for the detection of anti-BP180NCI6A IgG subclasses in patients with BP and PG.

Objective: To establish a content determination method for ferric iron and ferrous iron in ferrous succinate tablets.Methods: Using a Dionex RFICTM protection column (4 mm×50 mm)and a Dionex RFICM Ion PacRCS5A analytical column (4 mm×250 mm), the eluent solution consisted of 7 mmol·L-1 dipicolinic acid, 66 mmol·L-1 potassium hydroxide, 5.6 mmol·L-1 potassium phosphate monobasic and 74 mmol·L-1 formic acid.The flow rate was 1.5 ml·min-1, the column temperature was 30.0℃ and the injection volume was 1.3 μl.Results: Fe3+ showed good linearity within the range of 0.5-15 μg·ml-1(r=1.000 0), Fe2+ showed good linearity within the range of 25-200 μg·ml-1(r=1.000 0), and the average recovery was 103.6%(RSD=2.7%, n=9)and 98.3%(RSD=1.9%, n=9), respectively.Conclusion: The method is simple, reliable and accurate, and can be used for the determination of Fe3+and Fe2+ in ferrous succinate tablets.

Objective Seborrheic keratosis are very common dermatosis occurred in the fourth to fifth decade. Recently, some encouraging results in the treatment of melain-related lesions, such as Ota`s nevus, tatoos, cafe spots, have been obtained using the Q-switched Alexandrite laser, but no reports were found in treating seborrheic keratosis. Our purpose was to assess the clinical outcomes induced by the Q-switched Alexandrite laser at 752nm. Methods Total 119 cases of seborrheic keratoses , aged from 31-76years, were treated with the Q-switched Alexandrite laser (model HT-10, Cynosure Laser Corp, USA. wavelength 752nm, pulse width 45-75ns). The energy was adjusted to 7.1-8.5J/cm 2 according to the charateristics of the lesions. The interval of two treatment was 1.5-3 months. No special care was needed. Results Sixty-seven of the 119 patients were evaluated after treatment (men 15 cases, women 52 cases, aged from 31～71 years, and course from 1 to 21 years). All the patients were treated by 1-3 times. The number of cases treated by 1, 2, and 3 times was 50 (74.6 %), 14 (20.9 %) and 3 (4.5 %), respectively. The degree of clearing was: complete clearing (17 cases, 25.4 %), significant improvement (25 cases, 37.3 %), moderate improvement (17 cases,25.4 %) and no change or mild improvement (8 cases,11.9%). The total rate of improment was 62.7%, while the improvement rate after 1 treatment was 58%. The efficacy was correlated with the area of the lesions. No side effects, such as hyperpigmentation and scars, were found. Conclusions This clinical data demonstrate the Q-switched Alexandrite laser is a safe and effective method for treatment of seborrheic keratosis and deserves to be used widely.

Objective To investigate the relationship of polymorphisms at codons 333 and 637 of TAP-1 allele gene to recurrent condyloma acuminatum (CA) in a Chinese population. Methods Amplificatory refraction mutation system-PCR (ARMS-PCR) was performed to detect TAP1 polymorphic residues at codon 333 in 88 patients with recurrent CA and 81 age- and sampling date-matched controls and at codon 637 in 60 patients with recurrent CA and 60 age- and sampling date-matched controls. Results The frequencies of AA,GG and AG genotypes at codon 333 of TAP-1 gene were 86.36%, 0, 13.64%, respectively, in patients with recurrent CA, 79.01%, 0 and 20.99%, respectively, in the controls, and no significant difference was observed between the two groups (χ2 = 1.604, P ＞ 0.05). However, a significant difference was observed in the frequencies of AA, GG and AG genotypes at codon 637 of TAP-1 gene between the patients and controls (3.33% vs 10.0%, 95.00% vs 60.00%, 1.67% vs 30.00%, χ2 = 21.551, P ＜ 0.01 ). Conclusions The recurrence of CA may be associated with the polymorphism at codon 637, but not with that at codon 333, of TAP-1 gene.

Bullous pemphigoid (BP) is a group of autoimmune blistering skin diseases.There are special measures for the assessment of disease severity and therapeutic response in autoimmune bullous skin diseases.Common scoring indexes include autoimmune bullous skin disorder intensity score (ABSIS),bullous pemphigoid disease area index (BPDAI),and so on.ABSIS has been validated,and is widely applied in evaluation of pemphigus vulgaris and other autoimmune blistering diseases.BPDAI is still in the process of verification,and has been applied in only a few clinical trials.The evaluation of diseases by ABSIS and BPDAI is both based on a global assessment of skin and mucosal lesions,and degree of subjective pruritus.

Objective: To study the related substances in clozapine and its preparations. Methods:Comparative analysis was adopted according to the method respectively described in Chinese pharmacopoeia and United States pharmacopoeia. Results:The impurity profile of clozapine from domestic and abroad was basically the same, and that of clozapine and its preparations was also basically the same. The method in United States pharmacopoeia was better than that in Chinese pharmacopoeia in terms of in terms of separation of the impurities. Conclusion: The current legal inspection standard for the related substances in clozapine described in Chinese pharmacopoeia should be improved.

Objective To measure the expressions of IL-10, IL-23 and CD86 in lesions of epidermodysplasia verruciformis (EV), and to explore the relationship between cellular immune abnormality and EV pathogenesis. Methods Immunohistochemistry was used to measure the expressions of IL-10, IL-23 and CD86 in tissue samples from 10 patients with EV and 10 normal human controls. Results Three cytokines were observed in all the samples of EV, with the expression score ranging from 3 to 6 and expression intensity from moderate to high. However, of the control specimens, only 1 was positive for IL-10 with the expression score being 3, and expression intensity being moderate. Conclusion The pathogenesis of epidermodysplasia verruciformis may be correlated with the expression abnormality of some cytokines secreted by keratinocytes.

Objective To evaluate the prognostic accuracy of the score of toxic epidermal necrolysis (SCORTEN) scoring system in patients with toxic epidermal necrolysis (TEN) or Stevens-Johnson syndrome (SJS).Methods Clinical data were collected from 39 patients with SJS/TEN hospitalized in Peking Union Medical College Hospital during April 1992 and March 2014,and retrospectively analyzed.Among the 39 patients,13 had died,and the other 26 patients,who were matched to the dead patients in a ratio of 2:1 for age,all had a definite diagnosis and were discharged with improved conditions.The SCORTEN scoring system was used to evaluate the 39 patients with SJS/TEN and calculate expected mortality.The expected mortality and actual mortality were compared between different groups stratified by age in the 39 patients.The receiver operating characteristic (ROC) curve was drawn to assess the prognostic accuracy of the SCORTEN scoring system.Results According to the SCORTEN scoring system,15 out of the 39 patients scored 1 point,14 scored 2 points,6 scored 3 points,and 4 scored 4 points.The total number of expected deaths was 6.808,while that of actual deaths was 13.There was no significant difference between the expected mortality and actual mortality in every SCORTEN score-based group.The area under curve (AUC) was 0.832 8,indicating a good predictive ability of the SCORTEN scoring system.Conclusion The SCORTEN scoring system can predict mortality in TEN/SJS patients at early stage.

Objective To induce the mutation of HPV-16 E7 in two zinc-binding motifs near the C terminus by polymerase chain reaction (PCR) and evaluate the effect of this mutation on the antigen-specific immunity of HPV-16 E7. Methods HPV-16 E7 fragment was amplified by PCR and cloned into pGEM-3zf vector. Two site mutations at 58 and 91 animo acid sites in the open reading frame of HPV-16 E7 were induced by PCR, and then the molecular clones of HPV-16 E7 wild type (pcDNA3.1/E7) and mutant (pcDNA3.1/ME7) were successfully reconstructed. Western blot and immunofluorescence were used to detect the expression of E7 protein. Results Intracellular fluorescence signals were observed in the cells transfected with pcDNA3.1/E7 and pcDNA3.1/ME7 24 hours after transfection, but the signals in the cells transfected with pcDNA3.1/ME7 disappeared 48 hours after tansfection. Twenty-four and 48 hours after transfection with pcDNA3.1/ME7, E7 protein was not detected by Western blot. Conclusions The stability of HPV-16 E7 protein is reduced by mutations (C58G, C91G) near two zinc-binding motifs. It is suggested that the zinc-binding motifs near the C terminus of HPV-16 E7 may be important for maintaining the stability of E7 protein.