A new three-layer-funnel-shaped esophagogastric anastomosis for surgical treatment of esophageal carcinoma

AIM: To reduce the incidence of postoperative anastomoticleak, stenosis, gastroesophageal reflux (GER) for patientswith esophageal carcinoma, and to evaluate the conventionalmethod of esophagectomy and esophagogastroplastymodified by a new three-layer-funnel-shaped (TLF)esophagogastric anastomotic suturing technique.METHODS: From January 1997 to October 1999, patientswith clinical stage Ⅰ and Ⅱ (Ⅱa and Ⅱb) esophagealcarcinoma, which met the enrollment criteria, were surgicallytreated by the new method (Group A) and by conventionaloperation (Group B). All the patients were followed at leastfor 6 months. Postoperative outcomes and complicationswere recorded and compared with the conventional methodin the same hospitals and with that reported previously byMcLarty etalin 1997 (Group C).RESULTS: 58 cases with stage Ⅰ and Ⅱ (Ⅱa and Ⅱb)esophageal carcinoma, including 38 males and 20 femalesaged from 34 to 78 (mean age: 57), were surgically treatedby the TLF anastomosis and 64 by conventional method inour hospitals from January 1997 to October 1999. The qualityof swallowing was improved significantly (Wilcoxon W＝2 142,P＝0.0 001) 2 to 3 months after the new operation in GroupA. Only one patient had a blind anastomatic fistula diagnosedby barium swallow test 2 months but healed up 3 weekslater. Postoperative complications occurred in 25 (43 %)patients, anastomotic stenosis in 8 (14 %), and GER in 13(22 %). The incidences of postoperative anastomotic leak,stenosis and GER were significantly decreased by the TLFanastomosis method compared with that of conventionalmethods (x2＝6.566, P ＝0.038; x2＝10.214, P＝ 0.006;x2＝21.265, P＝0.000).CONCLUSION: The new three-layer-funnel-shapedesophagogastric anastomosis (TLFEGA) hasmore advantagesto reduce postoperative complications of anastomotic leak,stricture and GER.

Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pyloriconservative region of adhesin antigen and its immunogenicity

AIM: To construct a non-resistant and attenuated Salmonella typhimurium ( S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori(Hpylon) and evaluate its immunogenicity.METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya1 delta Crp1 delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supematant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments.RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed.Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supematant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, bhe entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0×l0^10 cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response.CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro,which providing a new live oral vaccine candidate for protection and care of H pylori infection.

Expression of Helicobacter pylori Hsp60 protein and its immunogenicity

AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity.METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was transformed into BL21 (DE3) E. coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments.RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2 % of the total bacterial protein,and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself.CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

Cloning and expression and immunogenicity of Helicobacter pylori BabA_2 gene

AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori(H pylori) and to study the immunogenicity of BabA.METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore,BabA immunogenicity was studied by animal test.RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank.The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA.CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of Hpyloriinfection.

Recombinant Helicobacter pylori catalase

AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E. coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers & Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 ℃ and the activity of H. pylori catalase was high in the BL21 (DE3) E. coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

Acute diarrhea during army field exercise in southern China

AIM: During emergency period, infectious diseases can be a major threat to military forces. During field training in southern China, diarrhea is the main cause of nonbattle injury. To evaluate the causes of and risk factors for diarrhea in emergency period, we collected clinical and epidemiological data from the People's Liberation Army (PLA) during fieldtraining in southern China. METHODS: From September 25 to October 2 1997, 2636 military personnel were investigated. Fecal sample cultures for lapactic pathogens were obtained from 103 military personnel with diarrhea. In addition, a questionnaire was administered to 103 cases and 206 controls to evaluate the association between illness and potential risk factors. At the same time,another questionnaire of 1:4 case-case control was administered to 22 severe cases (each severe case paired 4 mild cases). RESULTS: The training troop's diarrhea incidence rate was significantly higher than that of garrison. The diarrhea incidence rate of officers was significantly lower than that of soldiers. A lapactic pathogen was identified in 63.1% (65/103) of the troops with diarrhea. Enterotoxigenic Escherichia coli (35.0%) and plesiomona shigelloides (16.5%) were the most common bacterial pathogens. All bacterial isolates were sensitive to norfloxacin and ceftazidine. However, almost all of them were resistant to sulfamethoxazole, trimethoprim-sulfamethoxazole,oxytetracycline, doxycycline, furazolidone, ampicillin and cloromycetin to a different degree. Risk factors associated with diarrhea includediidrinking raw water, eating outside,contacting diarrhea patients, lacking sanitation, depression,lacking sleep, which were established by multiple-factor logistic regression analysis. In addition, the unit incidence rate was associated with the density of flies and the average daily boiled water available by regression and discriminate analysis. CONCLUSION: A series of risk factors are associated with the incidence rate of diarrhea. Our results may provide a useful basis for prevention and cure of diarrhea in emergency period of PLA.

Expression of Helicobacter pyloriAlpA protein and its immunogenicity

AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.

Synthesis and properties of water-soluble oligo-(p-phenylkene vinylene)

A water-soluble oligo(p-phenylkene vinylene) [4,4′-(1,4-phenylene dithenylene)-bis-(N-methyl pyridinium iodide)] was synthesized by Witting reaction. The oligomer was characterized by NMR, elemental analysis, UV-visible spectrum, surface photovoltage spectrum and fluorescence spectrum.