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 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 detect 10 kinds of human milk oligosaccharides (HMOS) and compare their amounts during lactational stage.Methods Breast milk samples in different stages of lactation as colostrum (day 0-7 postpartum),transitional milk (day 8-15 postpartum),and mature milk (day 16-180 postpartum) were collected and 10 HMOS in those samples were detected and quantified by ultra high performance liquid chromatography-fluorescence detection after fluorescence labeling by using standard curves.Correlations between HMOS and lactation day were conducted by Person correlation analysis method,while the differences among three stages were calculated by ANOVA test.Results Ten HMOS were successfully separated and quantified under chosen chromatographic parameters.2'FL,3'SL,6'SL,LNT,LNnT and LNFP-I were negatively correlated and 3'FL was positively correlated with lactation days.They were different in three lactational stages (P ＜ 0.05),while P1,LNFP-V and LNnFP-V showed no correlation and difference (P ＞ 0.05).Conclusion The amount of HMOS changed during lactational stages.Seven HMOS were correlated with lactation days and different in three lactational stages (P ＜ 0.05).

Objective To find out the distribution of TCM constitution on the patients with stroke in Beijing. Method The TCM Constitution Questionnaire was used to evaluate 489 patients with stroke in three hospitals in Beijing. Results The qi-deficiency constitution (140 patients, 28.63%), yin-deficiency constitution (95 patients, 19.43%), blood-stasis constitution(89 patients, 18.20%) and phlegm-heat constitution (73 patients, 14.93%) were the most common constitution, while qi-depression constitution (22 patients, 4.50%) and yang-deficiency constitution (20 patients, 4.09%) were the less common. The top three constitution in middle aged wereqi-deficiency (33 patients, 27.27%), phlegm-heat (23 patients, 19.01%) and blood stasis (22 patients, 18.18%). The top three constitution in the aged groupwere qi-deficiency (107 patients, 29.08%), yin-deficiency (76 patients, 20.65%) and blood-stasis (67 patients, 18.21%). Conclusions Stroke is closely related with imbalance of TCM constitution. The prevention and treatment of stroke patients with different ages should vary according to the constitutions.

Objective To undertake an updated genetic spectrum analysis in patients with hereditary spinocerebellar ataxia (SCA) in mainland China. Methods SCA 1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy (DRPLA) nucleotide repeat mutations were detected in 430 families with autosomal dominant SCA (ADCA) and 237 patients with sporadic ataxias by PCR and DNA sequencing. Subsequently, point and Indel (Insertion/deletion) mutation analyses of SCA5, SCA11, SCA13, SCA14, SCA15/16/29, SCA27, SCA31 and SCA35 were detected in 91 families with ADCA and 196 patients with sporadic ataxias excluded from SCA1, 2, 3, 6, 7, 8, 10, 12, 17 and DRPLA genotypes via PCR and Denaturing High Performance Liquid Chromatography (PCR-DHPLC), Multiplex ligation-dependent probe amplification and DNA direct sequencing analysis. Results Among the 430 ADCA families, there were 25 SCA1 (5.81%), 27 SCA2 (6.28%), 267 SCA3/MJD (62.09%), 8 SCA6 (1.86%), 8 SCA7 (1.86%), 1 SCA12 (0.23%), 1 SCA17 (0.23%) and 2 SCA35 (0.47%), and the remaining 91 families (21.16%) were genetically unidentified. Among the 237 sporadic SCA patients, there were 6 SCA1 (2.53%), 9 SCA2 (3.80%), 23 SCA3/MJD (9.70%) and 3 SCA6 (1.27%), and the remaining 196 (82.7%) were genetically unidentified. No pathogenic point mutation causing SCA5, SCA11, SCA13, SCA14, SCA27 or SCA31 subtypes was found. Conclusion SCA3/MJD is substantially the most common subtype in patients with ADCA and sporadic forms in mainland China, followed by SCA2, SCA1, SCA6 and SCA7. While SCA12, SCA17 and SCA35 are seldom found, SCA5, SCA8, SCA10, SCA11, SCA13, SCA27, SCA31 and DRPLA are very rare. The high proportion of genetically unidentified cases further verify that SCAs are of highly genetic heterogeneity, suggesting that other disease-causing genes might be involved in the negative ADCA pedigrees, and other etiological factors may involve in those sporadic cases other than genetics.

OBJECTIVETo evaluate the response rate and adverse reactions of xeloda, an analogue of 5-fluorouracil, in the treatment of relapsed and metastatic breast cancer.

METHODSTwenty-two breast cancer patients who had recurrent and metastatic measurable foci were treated from Dec. 1999 to Feb. 2000. Xeloda was given, as a single drug, at a dose of or 2,510 mg/m2/d, bid, for two weeks followed by one week rest as one cycle, at least for one cycle in each patient.

RESULTSAmong these 22 patients, there was no complete response. Rates of partial response 8(36.4%), stable disease 10(45.5%), progressive disease 4(18.2%), and clinical benefit response (CR + PR + SD) 18(81.8%). The response rate in patients who had failed in previous chemotherapy of taxanes and/or anthracycline was 30.0%-33.3%. The common adverse reactions were hand-foot syndrome, skin pigmentation, nausea, vomiting, anorexia and fatigue. Mild-moderate anemia and leukopenia were observed in 36.4% of patients. Stomatitis, dizziness, diarrhea and chest distress were present in some. One patient developed degree IV myelosuppression. Total bilirubin and alanine transaminase (ALAT) mild elevation occurred in a few patients.

CONCLUSIONXeloda is an effective drug in the treatment of patients with relapsed and metastatic breast cancer, especially for those who have failed in chemotherapy with taxanes and/or anthracycline. Xeloda is well tolerated but has mild adverse reactions.

Adult ; Aged ; Antimetabolites, Antineoplastic ; adverse effects ; therapeutic use ; Breast Neoplasms ; drug therapy ; pathology ; Capecitabine ; Deoxycytidine ; adverse effects ; analogs & derivatives ; therapeutic use ; Female ; Fluorouracil ; analogs & derivatives ; Humans ; Middle Aged ; Neoplasm Metastasis ; Recurrence

Objective:To investigate the efficacy and prognostic survival of pembrolizumab combined with apatinib and chemotherapy in the treatment of human epidermal growth factor receptor-2 (HER2)-negative advanced gastric cancer.Methods:Patients with HER2-negative advanced gastric cancer were selected from December 2019 to December 2022 as the study subjects. Forty-five patients who received chemotherapy therapy (fluorouracil+cisplatin) were randomly collected and included in control group, and 52 patients who were treated with pembrolizumab combined with apatinib were randomly selected and enrolled as observation group. The difference in short-term efficacy was compared. The levels of serum tumor markers and immune function (CD3 +, CD4 +, CD8 +, CD4 +/CD8 +) were recorded. The long-term efficacy and adverse reactions of patients were compared. Measurement data conforming to the normal distribution were expressed as xˉ± s, and the mean comparison between groups was performed by independent sample t test. Chi-square test was used to compare the rate or composition ratio among enumeration data. P<0.05 was considered statistically significant. Results:At 6 months after treatment, the disease control rate in observation group was significantly higher than that in control group (78.85% (41/52) vs 57.78% (26/45)) ( χ2=5.01, P=0.025), but there was no statistical significance in objective response rate between groups (36.54% (19/52) vs 24.45% (11/45)) ( χ2=1.65, P=0.199). The levels of pepsinogen I, tissue polypeptide specific antigen, carcinoembryonic antigen, carbohydrate antigen 199 and CD8 + in both groups were reduced after treatment, and the levels were lower in observation group than those in control group ( t=6.06, 6.78, 4.68, 11.21, 3.45, all P<0.001). The levels of CD3 +, CD4 + and CD4 +/CD8 + were enhanced significantly in the two groups, and the observation group had higher levels after treatment ( t values were 2.10, 3.74, and 5.19; P values were 0.028, <0.001, and <0.001). After 1 year of follow-up, the survival rate in observation group with 59.62% (31/52) was significantly higher than 37.78% (17/45) in control group ( χ2=4.60, P=0.032). The progression-free survival time ((10.22±1.62) months vs (8.13±1.57) months, t=6.43, P<0.001) and overall survival time ((11.62±1.84) months vs (9.73±1.71) months, t=5.21, P<0.001) in observation group were significantly longer compared to control group. There were no statistical differences in the incidence rates of bone marrow suppression ( χ2=1.92, P=0.165), hand-foot syndrome ( χ2=3.47, P=0.062), gastrointestinal reaction ( χ2=0.32, P=0.574), hypertension ( χ2=0.94, P=0.333) and proteinuria ( χ2=2.39, P=0.122) between the two groups. Conclusion:Compared with chemotherapy, pembrolizumab combined with apatinib shows good short-term efficacy and long-term efficacy in patients with HER2-negative advanced gastric cancer.