Objective To develop a BP26 recombinant BCG (rBCG-BP26) vaccine,and to observe the effects of rBCG-BP26 on CD4+,CD8+ T cells in immunized mice.Methods The recombinant shuttle vector pMV261-Ag85B-BP26 was constructed by using traditional molecular biological technology.The recombinant strains were obtained by kanamycin resistance screening and PCR identification after electroporation.Western blotting was used to detect the expression of recombinant BP26 vaccine in immunized mice.Safety experiment was carried out in three different groups:the target experiment(rBCG-BP26) group,the positive control(BCG) group and the negative control(PBS) group,15 BALB/c mice in each group.Intradermal inoculations of 100 μl rBCG-BP26 [containing 106 colony forming units(CFU)],BCG,and PBS were carried out,respectively.Signs of mice in each group were observed.After immunization for 10,20,30,and 40 days,body weight was weighed,and tail blood was collected to observe the change of peripheral blood CD4+ and CD8+ T cells by flow cytometry.Results The rBCG-BP26 was successfully constructed.The expression of BP26 protein was detected in the liquid medium and the bacteria cells.The results of safety test analysis showed that there were no significant differences in signs and body weights(F=2.468,0.331,1.520,0.739,all P＞ 0.05),between PBS group[ (19.24 ± 0.54),(21.37 ± 0.66),(22.83 ± 0.62),(25.06 ± 0.37)g],BCG group[ (19.90 ± 0.02),(21.53 ± 1.57),(21.95 ± 0.55),(24.70 ± 0.39)g]and rBCG-BP26 group[ (19.16 ± 0.55 ),(20.89 ± 0.20),(22.15 ± 0.76),(24.60 ± 0.64)g].The results of flow cytometry showed that the percentages of CD4+ T cell level were lower in BCG group(26.70％,33.07％) and rBCG-BP26 group( 13.40％,26.70％) than that of the PBS group(33.85％,29.33％) and the values of CD4+/CD8+ T cells increased in rBCG-BP26 group (0.69％,1.27％,1.57％,1.70％ ) 10,20 and 30 days after immunization.Conclusions Recombinant BCG-BP26 vaccine strain can express brucella BP26 protein efficiently.Furthermore,its virulence is mild,and it can activate CD4+,CD8+ T cells in the body.It can be used as one of candidate vaccine strain against brucellosis.

Objective To construct the bp26 deletion mutant of Brucella vaccine strain M5-90 (M5-90Δbp26),to compare its immunogenicity with parental strain(M5-90),and to develope a new candidate vaccine strain of Brucella melitensis with reduced virulence that can be used to distinguish vaccinated livestock from infected animals.Methods Mutant vaccine strain of Brucella melitensis was constructed by conventional molecular biology techniques then the genetic stability of mutant M5-90Δbp26 was tested and its conventional bacteriological nature was identified; 1.0 × 109 CFU/2 ml doses of M5-90Δbp26 strain and the parental strain were used to vaccinate 3 sheep; sera were analyzed for reactivity against BP26 by Western blotting and for agglutination activity; to analyze the virulence of mutant and parental strain,mice were injected with 1.0 × 106,6.0 × 106 and 2.0 × 107 CFU/0.2 ml doses of M5-90 and M5-90Δbp26,respectively,and clinical symptoms were monitored and the death of mice was recorded.Results The M5-90Δbp26 was successfully generated and reversion was not observed in 15 generations.The size of PCR products was 629 bp while the parental strain was 1279 bp.The sequence analysis showed a 650 bp missing in M5-90Δbp26.The conventional bacteria identification tests confirmed that the mutant was depth variant strain,including mono-specific antiserum M type transformed into R,and the BK2 phage based splitting assay converted from the positive to the negative.Western blotting showed the purified BP26 protein was recognized by the serum against the parental strain while not by the serum against M5-90Δbp26 strain.Agglutination test showed the level of the serum antibody induced by M5-90Δbp26 strain(1:50) was significantly lower than that of serum induced by parental strain(＞ 1:800).Virulence test showed that M5-90Δbp26 strain was less virulent than parental strain.Conclusions M5-90Δbp26 has been successfully constructed.M5-90Δbp26 of Brucella melitensis has the characteristic of reduced virulence and has a potential as brucellosis candidate vaccine strain permitting serological discrimination between diseased and vaccinated livestock.

Objective To determine the difference of macrophage RAW264.7 apoptosis induced by Brucella melitensis virulent strain 16M and attenuated strain M5-90 and elucidate the regulatory role of caspases 3,8 and 9.Methods The best multiplicity of infection (MOI) was determined through kinetic analysis of Brucella melitensis strain 16M and M5-90 induced mouse macrophages apoptosis(bacterium ∶ cell =100 ∶ 1,50 ∶ 1,10 ∶1).The infection model was established using the best MOI =50 ∶ 1.The numbers of in vivo bacteria by colony formation units were calculated after macrophages were infected for different times,including 2,4,8,12,24 and 48 h,and the infected cells were collected.The ratios of apoptosis were detected and the regulation of caspases 3,8 and 9 in apoptosis pathway was elucidated by flow cytometry.Results The numbers of 16M in vivo bacteria were 105.4,104.8,105.8,106.5,108.0 and 109.0,respectively and of M5-90 were 106.1,106.2,106.4,106.3,106.1 and 105.0,respectively.The number of in vivo bacteria of 16M was significantly increased than that of M5-90 after infected for 24 h to 48 h.The ratios of apoptosis induced by 16M after infected for 2,4,8,12,24 and 48 h was (2.67 ± 0.09)％,(13.13 ± 0.30)％,(6.56 ± 0.42)％,(6.49 ± 0.28)％,(16.07 ± 0.86)％ and (24.23 ± 1.67)％,respectively,and by M5-90 was (3.62 ± 0.02)％,(32.01 ± 2.59)％,(17.58 ± 0.44)％,(16.09 ± 0.10)％,(62.53 ± 2.70)％ and (85.53 ± 0.15)％,respectively,and by control group was [(1.90 ± 0.20)％,(1.92 ±0.16)％,(1.99 ± 0.03)％,(2.48 ± 0.11)％,(3.56 ± 0.07)％,(5.26 ± 0.33)％].The differences were statistically between groups in same time.The Brucella melitensis vaccine strain M5-90 was more powerful than virulent strain 16M in respect of inducing macrophage apoptosis after infected for 24 to 48 h.Twenty-four hours after infection,the expression of caspases 3,8 and 9 was (1.47 ± 0.05)％,(1.52 ± 0.02)％ and (2.47 ± 0.12)％,respectively,in control group and the expression was (9.70 ± 0.46)％,(6.08 ± 0.56)％ and (35.08 ± 1.64)％,respectively,after infected for 24 h induced by M5-90.The expression of caspases 3,8 and 9 was significantly higher than that control group (P ＜ 0.01).Twenty-four hours after given caspases 3,8 and 9 inhibitor,apoptosis rate in control group was (66.72 ± 1.28)％,in M5-90 group was (22.58 ± 0.55)％,(53.15 ± 1.85)％ and (29.18 ± 0.23)％,respectively,and compared with control group,apoptosis rate of caspases 3,8 and 9 was significantly lower(P ＜ 0.01).Conclusions Apoptosis of macrophage can be induced by Brucella melitensis virulent vaccine strain 16M and attenuated strain M5-90.M5-90 is stronger than that of strain 16M.Caspases 3,8 and 9 can regulate macrophage apoptosis after M5-90 infection.

OBJECTIVETo assess the association between glutathione-S-transferase gene polymorphisms GSTT1, GSTM1 and GSTP1 and onset of azoospermia.

METHODSMulti-PCR was used to detect GSTM1 and GSTT1 gene deletions. Polymorphisms of GSTP1 were determined with restriction fragment length polymorphism (RFLP) method in 236 azoospermia patients and 142 healthy fertile male controls.

RESULTSThe frequency of M1 (-/-) and P1 (Ile/Val or Val/Val) genotype was 24.65% in the control group, which was significantly higher than that of the patient group (15.68%, P=0.031). Frequency of M1 (-/-), T1 (+/+) and P1 (Ile/Val or Val/Val) genotype was 12.68% in the control group, which was significantly higher than that of the patient group (5.51%, P=0.014).

CONCLUSIONThe M1(-/-) and P1(Ile/Val or Val/Val) genotype and the M1(-/-), T1(+/+) and P1 (Ile/Val or Val/Val) genotype are associated with reduced risk of azoospermia in ethnic Chinese Han population.

Adult ; Asian Continental Ancestry Group ; Azoospermia ; genetics ; Case-Control Studies ; China ; Genotype ; Glutathione S-Transferase pi ; genetics ; Glutathione Transferase ; genetics ; Humans ; Male ; Phenotype ; Polymorphism, Genetic

INTRODUCTIONUndergraduate evidence-based practice (EBP) is usually taught through standalone courses and workshops away from clinical practice. This study compared the effects of 2 clinically integrated educational strategies on final year medical students.

MATERIALS AND METHODSFinal year medical students rotating to the general medicine service for a 2-week internship were randomly assigned to participate in a weekly EBP-structured case conference focusing on students' primary care patients (Group A, n = 47), or to receive a weekly didactic lecture about EBP (Group B, n = 47). The teaching effects of these 2 interventions were evaluated by a validated instrument for assessment of EBP related knowledge (EBP-K), attitude (EBP-A), personal application (EBP-P), and anticipated future use (EBP-F) on the first and last days of rotation.

RESULTSAll scores improved significantly after the 2-week EBM-teaching for both groups. When compared to Group B, students in Group A had significantly higher post-intervention scores of EBP-K (21.2 ± 3.5 vs 19.0 ± 4.6; ie. 57.8 ± 72.9% vs 29.1 ± 39.1%; P <0.01) and EBP-P (18.7 ± 4.3 vs 15.3 ± 3.9; ie. 28.5 ± 25.5 % vs 14.1 ± 18.7 %; P <0.001). In contrast, the scores of EBP-A and EBP-F were similar between the 2 groups.

CONCLUSIONStructured case conference, when compared to the didactic lectures, significantly improved EBP-K and EBP-P for final year medical students.

Adult ; Education, Medical, Undergraduate ; Evidence-Based Medicine ; education ; Female ; Humans ; Male ; Surveys and Questionnaires ; Taiwan ; Teaching ; methods ; Young Adult

Background/Aims: Hypopharyngeal multichannel intraluminal impedance-pH (HMII-pH) technology incorporating 2 trans-upper esophageal sphincter impedance channels has been developed to detect pharyngeal reflux. We used the HMII-pH technique to validate the candidate pharyngeal acid reflux (PAR) episodes based on the dual-pH tracings and determined the interobserver reproducibility. Methods: We conducted a cross-sectional study in tertiary centers in Taiwan. Ninety patients with suspected laryngopharyngeal reflux and 28 healthy volunteers underwent HMII-pH test when off acid suppressants. Candidate PAR episodes were characterized by pharyngeal pH drops of at least 2 units and reaching a nadir pH of 5 within 30 seconds during esophageal acidification. Two experts manually independently identified candidate PAR episodes based on the dual-pH tracings. By reviewing the HMII-pH tracings, HMII-pH-proven PAR episodes were subsequently confirmed. The consensus reviews of HMII-pH-proven PAR episodes were considered to be the reference standard diagnosis. The interobserver reproducibility was assessed. Results: A total of 105 candidate PAR episodes were identified. Among them 84 (80.0%; 95% CI, 71.0-87.0%) were HMII-pH-proven PAR episodes (82 in 16 patients and 2 in 1 healthy subject). Patients tended to have more HMII-pH-proven PAR episodes than healthy controls (median and percentile values [25th, 75th, and 95th percentiles]: 0 [0, 0, 3] vs 0 [0, 0, 0], P = 0.067). The concordance rate in diagnosing HMII-pH-proven PAR episodes between 2 independent observers was 92.2%. Conclusion Our preliminary data showed that 80.0% (71.0-87.0%) of the proposed candidate PAR episodes were HMII-pH-proven PAR episodes, among which the interobserver reproducibility was good.

Brucellosis is a zoonotic disease that causes animal and human diseases. Vaccination is a major measure for prevention of brucellosis, but it is currently not possible to distinguish vaccinated animals from those that have been naturally infected. Therefore, in this study, we constructed the Brucella (B.) abortus 2380 wbkA mutant (2308DeltawbkA) and evaluated its virulence. The survival of 2308DeltawbkA was attenuated in murine macrophage (RAW 264.7) and BALB/c mice, and it induced high protective immunity in mice. The wbkA mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon. Antibodies to 2308DeltawbkA could be detected in sera from mice, implying the potential for use of this protein as a diagnostic antigen. The WbkA antigen would allow serological differentiation between infected and vaccinated animals. These results suggest that 2308DeltawbkA is a potential attenuated vaccine against 16M. This vaccine will be further evaluated in sheep.
Animals ; Antibodies ; Brucella abortus* ; Brucella* ; Brucellosis ; Humans ; Immunization* ; Immunoglobulin G ; Interferons ; Macrophages ; Mice* ; Sheep ; Staphylococcal Protein A ; Vaccination ; Virulence ; Zoonoses

Objective To investigate the predictive values of preoperative radiological features-intratumoral arteries and tumoral morphological classification on microvascular invasion (MVI) and on prognosis in patients with hepatocellular carcinoma (HCC).Methods A total of 220 consecutive HCC patients who underwent curative hepatectomy at Nanjing Drum Tower Hospital from January 2008 to December 2014 were retrospectively analyzed.The predictive values of preoperative radiological features and clinical data on MVI were analyzed by the univariate analysis and multivariate logistic regression methods.The prognosis of HCC patients was analyzed by the Kaplan-Meier survival analysis and the Cox proportional hazards models.Results Univariate analysis and multivariate logistic regression showed intratumoral arteries and tumoral morphological classification using preoperative CT[called the radiological predictorsof microvascular invasion (RPMVI)] to be independent predictors of MVI.The AUROC for RPMVI inpredicting MVI was O.830 (95％ CI,O.769 ～ 0.891,P ＜0.05).The Cox multivariate analysis identified Child-Pugh grading,tumor size ＞ 5 cm,RPMVI,MVI and non-anatomical liver resection to be independent risk factorsof overall survival (OS),while tumor size ＞ 5 cm,RPMVI,MVI and non-anatomical liver resection to be independent risk factor sofre currence-free survival (RFS).The 1-,3-,and 5-year OS rates were 83.3％,61.7％,and 40.1％ in patients with RPMVI and 97.1％,76.5％,and 69.6％ in patients without RPMVI (P ＜0.05),respectively.The 1-,3-,and 5-year RFS rates were 61.9％,36.9％,and 28.4％ in patients with RPMVI and 81.6％,61.9％,and 52.2％ in patients without RPMVI (P ＜ 0.05),respectively.Conclusions RPMVI is a novel radiological marker that accurately predicted histological MVI in HCC patients preoperatively.Similar to MVI,RPMVI was found to be an independent risk factor for prognosisin HCC patients,and it may provide the important information for surgical treatment planning in HCC patients.

Background/Aims: Diagnosis of isolated laryngopharyngeal reflux symptoms (ILPRS), ie, without concomitant typical reflux symptoms (CTRS), remains difficult. Mean nocturnal baseline impedance (MNBI) reflects impaired mucosal integrity. We determined whether esophageal MNBI could predict pathological esophagopharyngeal reflux (pH+) in patients with ILPRS. Methods: In this cross-sectional study conducted in Taiwan, non-erosive or low-grade esophagitis patients with predominant laryngopharyngeal reflux symptoms underwent combined hypopharyngeal multichannel intraluminal impedance-pH monitoring when off acid suppressants. Participants were divided into the ILPRS (n = 94) and CTRS (n = 63) groups. Asymptomatic subjects without esophagitis (n = 25) served as healthy controls. The MNBI values at 3 cm and 5 cm above the lower esophageal sphincter (LES) and the proximal esophagus were measured. Results: Distal but not proximal esophageal median MNBI values were significantly lower in patients with pH+ than in those with pH– (ILPRS in pH+ vs pH–: 1607 Ω vs 2709 Ω and 1885 Ω vs 2563 Ω at 3 cm and 5 cm above LES, respectively; CTRS in pH+ vs pH–: 1476 vs 2307 Ω and 1500 vs 2301 Ω at 3 cm and 5 cm above LES, respectively, P < 0.05 for all). No significant differences of any MNBI exist between any pH– subgroups and healthy controls. The areas under the receiver operating characteristic curve in the ILPRS group were 0.75 and 0.80, compared to the pH– subgroup and healthy controls (P < 0.001 for both), respectively. Interobserver reproducibility was good (Spearman correlation 0.93, P < 0.0001). Conclusion Distal esophageal MNBI predicts pathological reflux in patients with ILPRS.

Background/Aims: Obstacles exist in facilitating hepatitis C virus (HCV) care cascade. To increase timely and accurate diagnosis, disease awareness and accessibility, in-hospital HCV reflex testing followed by automatic appointments and a late call-back strategy (R.N.A. model) was applied. We aimed to compare the HCV treatment rate of patients treated with this strategy compared to those without. Methods: One hundred and twenty-five anti-HCV seropositive patients who adopted the R.N.A. model in 2020 and another 1,396 controls treated in 2019 were enrolled to compare the gaps in accurate HCV RNA diagnosis to final treatment allocation. Results: The HCV RNA testing rate was significantly higher in patients who received reflex testing than in those without reflex testing (100% vs. 84.8%, P<0.001). When patients were stratified according to the referring outpatient department, a significant improvement in the HCV RNA testing rate was particularly noted in patients from non-hepatology departments (100% vs. 23.3%, P<0.001). The treatment rate in HCV RNA seropositive patients was 83% (83/100) after the adoption of the R.N.A. model, among whom 96.1% and 73.9% of patients were from the hepatology and non-hepatology departments, respectively. Compared to subjects without R.N.A. model application, a significant improvement in the treatment rate was observed for patients from non-hepatology departments (73.9% vs. 27.8%, P=0.001). The application of the R.N.A. model significantly increased the in-hospital HCV treatment uptake from 6.4% to 73.9% for patients from non-hepatology departments (P<0.001). Conclusions The care cascade increased the treatment uptake and set up a model for enhancing in-hospital HCV elimination.