BACKGROUNDTo study to immunogenicity of recombinant HEV ORF2 protein expressed in pichia pastoris.

METHODSBALB/c mice were immunized with the recombinant HEV ORF2 protein. The ability of antiserum to bind HEV was tested using affinity-capture reverse transcription polymerase chain reaction (RT-PCR). Moreover, the recombinant protein was used to immunize BALB/c mice by different routes with different adjuvants. Serum conversion rate of anti-HEV antibody and the ELISA titer were detected.

RESULTSThe antiserum could capture native HEV for RT-PCR. As to the immunization effect, the immune response by intramuscular route was better than that of the intraperitoneal route. The protein with alum and CpG adjuvant could elicits more significant immune responses than using the alum adjuvant alone. The best way was to immunize with the protein with alum and CpG adjuvant by intramuscular route with a boosted injection on the 4th week after the first immunization. The ED50 was 0.023 microgram. This is the first report that the antibody elicited by recombinant HEV ORF2 protein expressed in pichia pastoris recognizes native HEV. High immunogenicity of this kind of ORF2 was also demonstrated by inducing strong immune response in mice with good ED50 result.

CONCLUSIONSThe high immunogenicity of this kind of HEV ORF2 may make a foundation for the development of new type of hepatitis E vaccine.

Animals ; Antibody Formation ; Female ; Hepatitis E virus ; immunology ; Mice ; Mice, Inbred BALB C ; Pichia ; genetics ; Recombinant Proteins ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Proteins ; immunology

Upper urinary tract calculi with infection is a quite difficult acute urologic emergency.And what is more, upper urinary tract obstruction after radical cystectomy following urinary diversion may be fatal in the vulnerable patients with this kind of situation.Postoperative anatomy and other factors affect the upper urinary tract calculi, and urinary tract infection greatly increased the risk.But it is particularly difficult to handle with patients with poor general condition and septic shock treatment, so how to optimize the selection program is worth further studying.A 65-year-old man was admitted to hospital for combining with renal and ureteral calculi after radical cystectomy (Bricker) in March 2017.The patient underwent transurethral resection of bladder tumor in the outer court because of the discovery of bladder tumor one year before, The postoperative pathology was high-grade papillary urothelial carcinoma and he underwent regular irrigation of the bladder.Because of tumor recurrence, he came to Peking University Third Hospital for further treatment in August 2016, and the examination of urinary tract CT indicated bladder can-cer;for bilateral multiple renal pelvis and renal calices calculi, he was given laparoscopic radical cystecto-my.After four months, due to sudden chill fever, he was hospitalized.After definite diagnosis, anti-inflammatory treatment combined with left nephrostomy was given.The two-stage lithotripsy was performed.After expansion of the original left renal fistula to 24 F(1 F=0.33 mm), 24 F sheath was inducted into the kidney.We introduced a flexible ureteroscopy into the channel down to the ureteral obstruction, where we found a 1 cm oval black brown stone obstructed.A guide wire was inducted through the stones down to ileal conduit in the right lower quadrant.Through the ureteroscopy, we made ureteral calculi broken into powder, and replaced the residual stones into the renal pelvis.Then we used the nephroscopy for lithotropsy in the pelvis, and finally we introduced a flexible cystoscopy for the residual stone.The operation was successful, the operation time was 181 min, and intraoperative bleeding was 10 mL.After operation, no residual stones were found in kidney, ureter, bladder (KUB) plain films.No operation complications were related to the urinary tract.Therefore, multiple endoscopy with combination therapy of upper urinary tract calculi following urinary diversion is safe and effective, and the reasonable choice with the advantages of each instrument can improve the stone clearance rate and shorten the operation time.This can be used as a useful complement to traditional treatment.

Objective To compare the clinical results of the ureteral reimplantation with the traditional open approach and laparcocopic pneumovesical approach. Methods A retrospective review of 108 patients who underwent ureteral reimplantation from December 2004 to October 2010 was conducted.The patients were divided into open and pneumovesical groups according to the surgical approach.Perioperative results were compared between the two groups in terms of operative time,postoperative intravenous antibiotics duration,catheterization duration,postoperative stay,average total cost and surgical cost,respectively.And the pneumovesical group was divided into two stages by time,compared the operative time of the two stages and between the later stage and the open group. Results The postoperative hospital stay of pneumovesical group was shorter than the open group (6.8 ± 1.9 d and 8.9 ± 2.9 d,P =0.002 ).For catheterization duration,the pneumovesical group was shorter than the open group as well (5.2 ± 1.2 d and 6.2 ±2.2 d,P=0.057).For the postoperative intravenous antibiotics duration,the pneumovesical group was 5.0 ± 1.3 h,the open group was 5.4 ±1.6 h (P =0.159).For the total cost,the pneumovesical group was 16 067.9 ±4 295.8 RMB,the open group was 15 617.7 ± 5 486.5 R MB (P =0.168).For the surgical cost,the pneumovesical group was 9369.4 ± 1366.6 RMB,the open group was 7397.9 ± 1797.3 RMB ( P =0.083 ).Operative duration of the pneumovesical group and open group were 3.2 ± 1.1 h and 2.3 ± 1.1 h ( P =0.003).For pneumovesical group,the mean operative durations of the two stages were 3.6 h and 2.8 h (P =0.286).And the later stage of pneumovesical group was a little longer than the open group,but no significant difference ( P =0.234 ).No major complication was found in the 2 groups during the operative time and the postoperative hospital stay.Twenty-four patients (38 ureters) of the pneumovesical group were followed up with micturating cystourethrography ( MCU),ureterovesical reflux recurred in 3 patients.Two patients changed from grade Ⅲ to grade Ⅰ and 1 patient changed from grade Ⅴ to grade Ⅲ after the surgery.Five patients (9 ureters) of the open group were followed up,1 patient found bladder diverticulum; 1 patient found ureteral stricture 6 months after the surgery and got improved after secondary ureteral relimplantation surgery. Conclusions The pneumovesical approach is shorter than the open group in postoperative hospital stay and catheterization reserved duration.The pneumovesical approach is a safe and effective option for ureteral reimplantation.

Objective To characterize the immunogenicity in gene immunization of the conserved regions of hepatitis C virus(HCV) based on different delivery strategies. Methods We first constructed a novel DNA vaccine encoding a fusion gene(from partial NS3 and Core) of HCV. Then we compared different protocols based on naked DNA injection twice or DNA injection with gene electrotransfer(GET) in BALB/c mice. The immune response was measured by antibody ELISA and by IFN-gamma ELISPOT. Results Our data showed that a protocol based on intradermally injection of DNA with optimal GET induced the strongest humoral and cellular immunity, and DNA with GET induced a substantially higher anti-NS3/Core T cell re-spoase than naked DNA injection. Conclusion Our data suggest that DNA vaccines encoding NS3/Core fu-sion protein of HCV immunized by the present strategy could merit further study in the context of future prophylactic and therapeutic HCV T cell based vaccines.

OBJECTIVETo analyze the retention rate and its influencing factors of HIV/AIDS patients by Chinese medicine (CM) maintenance treatment in the first 5 provinces in China.

METHODSKaplan-Meier method was used to analyze the retention rate of treatment in patients. Cox hazard regression model was used to assess factors that might influence the treatment time of Chinese medication.

RESULTSTotally 2,353 patients took part in this four-year study. Of them, 1,156 (49. 1%) were male, 2,344 (99. 6%) were Han nationality, 2,260 (96%) were married, 2,219 (94.3%) had junior middle-schooling or below, the average age was 41.52 +/- 8.98 years, 1,758 (74.7%) received paid blood donation, 478 (20.3%) received blood transfusion, 737 (31.3%) were absent of symptoms, 963 (40.9%) received combined treatment of CM and Western medicine. The median time within the four years was 44.84 months. The average retention rate for 1, 2, 3, 4 years of CM treatment were 86.6%, 78.4%, 72.2%, 65.6%, respectively. The results of Cox model indicated that the drop-out risk could be reduced in combined treatment of CM and Western medicine patients (HR=0. 805, P<0.01) and AIDS patients (HR=0. 769, P<0.01). The drop-out risk could be increased by the infection route of paid blood donation (HR =1. 373, P<0.01).

CONCLUSIONSThe four-year retention rate of the 2 353 patients by CM treatment in the first 5 provinces in China was 65. 6%. Route of infection, whether or not in combination of Western medicine, and staging showed influence on CM maintenance treatment time.

Acquired Immunodeficiency Syndrome ; therapy ; Adolescent ; Adult ; China ; Drugs, Chinese Herbal ; therapeutic use ; Factor Analysis, Statistical ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Patient Acceptance of Health Care ; statistics & numerical data ; Phytotherapy ; Proportional Hazards Models ; Young Adult

BACKGROUNDTo determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P. pastoris).

METHODSBy using the rHEV ORF2 protein from E.coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P. pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E. The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested.

RESULTSThe minimum concentration of coated antigen with which HEV IgG could be detected was 12.5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1:5 120. No cross-reaction was found with sera from patients with any other types of hepatitis. The 37 degree C acceleration test showed that the rORF2 was highly stable within 12 months at 4 degrees C. The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P. pastoris, especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E.Coli.

CONCLUSIONThere may be more extensive conformational epitopes in the rHEV ORF2 from P. pastoris. The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E.

Gene Expression ; Hepatitis Antibodies ; blood ; Hepatitis Antigens ; genetics ; immunology ; Hepatitis E ; immunology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

AIMTo prepare lung targeting azithromycin cationic liposomes and to observe its tissue distribution in mice.

METHODSThe azithromycin cationic liposomes were prepared by thin film method with freeze-thawing steps. HPLC method was established and validated for the determination of azithromycin in tissues of mice.

RESULTSThe particle size of the liposomes was 6.582 microm with zeta potential of +19.5 mV. The entrapment efficiency was more than 75%. The liposomes was stable in 6 months stored at 4 degrees C. The release in vitro was characterized by Higuchi equation. Azithromycin liposomes and free azithromycin solution were injected intravenously at a dose of 80 mg x kg(-1) to mice. Compared with solution, liposomes were characterized by slower clearance, increased half-life and the AUC increased by 7.4 fold in lung.

CONCLUSIONThin film method with freeze-thawing steps could increase the entrapment efficiency and increase the particle size of azithromycin liposomes. After modification of lipid membrane with stearylamine, the cationic liposomes were prepared. The azithromycin concentration and AUC increased in lung after iv administration to mice of the cationic liposomes. This offered a good information for preparing liposomes targeting on the lung.

Animals ; Anti-Bacterial Agents ; administration & dosage ; chemistry ; pharmacokinetics ; Area Under Curve ; Azithromycin ; administration & dosage ; chemistry ; pharmacokinetics ; Cations ; Drug Carriers ; Drug Compounding ; methods ; Drug Delivery Systems ; Female ; Liposomes ; Lung ; metabolism ; Male ; Mice ; Particle Size ; Tissue Distribution

BACKGROUNDTo observe the protective effect of hepatitis E virus (HEV) ORF2 recombinant protein expressed in prokaryote cell cynomolgus macaques (cynos) against challenging with wild-type HEV.

METHODSCynos were immunized with HEV ORF2 recombinant protein and then challenged with wild-type HEV, the unimmunized cynos were used as control. Blood samples were collected and tested to see if there were dynamic changes of ALT and antibody to HEV before and after challenge with wild-type HEV.

RESULTSAll the five unimmunized cynos re-presented hepatitis 3 weeks after challenging with wild-type HEV. However, all the five immunized cynos showed no hepatitis and pathological changes.

CONCLUSIONSCynos can be efficiently protected by immunization with HEV ORF2 recombinant protein against wild-type HEV. This protein can be a promising candidate for HEV vaccine.

Animals ; Female ; Hepatitis Antibodies ; blood ; Hepatitis E ; immunology ; prevention & control ; Hepatitis E virus ; immunology ; Macaca mulatta ; Male ; Recombinant Proteins ; immunology ; Viral Proteins ; immunology

OBJECTIVETo investigate the impact of dissected lymph node number on the prognosis of patients with advanced cancer of cardia and stomach fundus.

METHODSClinical data of 236 patients with advanced cancer of cardia and stomach fundus undergone D(2) radical resection were reviewed retrospectively. Five-year survival rate and post-operative complication rate were followed up and their relationships with dissected lymph node number were analyzed respectively.

RESULTSThe 5-year survival rate of the entire cohort was 37.5%. Among those patients with the same stage, the more lymph nodes (LNs) resected, the better survival outcomes achieved(Log-rank trend test P=0.0013). A cut point analysis yielded the ability to detect the significant survival differences. The best long-term survival outcomes were observed with LN counts of more than 20 for stage II(P=0.0136), more than 25 for stage III(P<0.0001), more than 30 for stage IV(P=0.0002) or more than 15 for the entire cohort (P=0.0024), with greatest comparative discrepancies. The post-operative complication rate was 15.7% and was not significantly correlated with dissected lymph node number(P=0.101).

CONCLUSIONSThe prognosis of patients with advanced cancer of cardia and stomach fundus is associated with the number of resected LNs when D(2) lymphadenectomy is carried out. Suitable increment of dissected lymph node number would not increase the post-operative complication rate.

Adult ; Aged ; Cardia ; pathology ; Female ; Gastric Fundus ; pathology ; Humans ; Lymph Node Excision ; Lymph Nodes ; surgery ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Retrospective Studies ; Stomach Neoplasms ; diagnosis ; pathology ; surgery

OBJECTIVETo establish chemical pattern recognition method for the identification and evaluation of Atractylodes macrocephala.

METHODThe chemical constituents in methanol extract of 32 samples of A. macrocephala were determined by HPLC. The fingerprints were obtained and were handled by hierarchical clustering analysis and stepwise discriminant analysis.

RESULT AND CONCLUSIONAccording to the result of classification, all samples collected were devided into three Grades--the superior, the ordinary and the fake. Chemical pattern recognition method was established. It may be of practical value for the quality control of A. macrocephala.

Atractylodes ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Cluster Analysis ; Pattern Recognition, Automated ; Plants, Medicinal ; chemistry ; Quality Control ; Rhizome ; chemistry