Objective To establish attenuated S. typhimurium expressing Helicobacter pylori (H.pylori) urease subunit B (UreB) and determine whether it could be used as oral vaccine against H.pylori Methods H.pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector PTc 01 after sequencing, then transformed into attenuated S.typhimurium SL3261 to acquire SL3261/PTc 01 UreB. The expression of H.pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA. In vitro stability of PTc 01 UreB plasmid in S. typhimurium SL3261 was confirmed by growing in Luria Bertani medium to 60 generations. Results The UreB gene fragment amplified by PCR was consistent with the sequence of the H.pylori UreB by sequence analysis. Enzyme digestion revealed that the correct PTc 01 UreB was obtained. Western blot showed that 61kD protein was expressed in SL3261/PTc 01 UreB which could be recognized by anti H.pylori UreB antiserum. Anti UreB IgA antibodies in mouse intestinal fluid and IgG antibodies in serum were determined by ELISA. After 60 generations of continuous culture, the recombinant plasmid PTc 01 UreB was stable in SL3261 and had no obvious toxicity. Conclusion The attenuated Salmonella typhimurium expressing H.pylori UreB may be used as oral vaccine against H.pylori infection.

In order to construct a recombinant attenuated Salmonella typhimurium expressing Helicobacter pylori (H. pylori) urease subunit B(UreB), UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01-UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Tewelve weeks after oral immunization of mice with this vaccine, anti-UreB IgA antibodies in mouse intestinal fluid and IgG antibodies in serum were determined by ELISA. IFN-? and IL-10 contents in the supernatant of spleen cell culture were also assessed by ELISA. The results showed that 61kD protein was expressed in SL3261/pTC01-UreB that could be recognized by anti-H. pylori UreB antiserum by Western-blot. The multiple oral immunizations with SL3261/pTC01-UreB could induce significantly H. pylori-specific mucosal IgA response as well as serum IgG responses. Moreover, there was significant increase of IFN-? and IL-10 contents in the supernatant of spleen cell culture in SL3261/pTC01-UreB group. These results suggested that the attenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection. Its effect against H. pylori infection also needs to be further evaluated in animal models.

Objective To investigate the effect of pantoprazole (proton pump inhibitor, PPI) and gefarnate (gastric mucosa protectant) on the prevention of upper gastrointestinal bleeding in patients undergoing post-percutaneous coronary intervention (PCI) dual anti-platelet therapy.Methods This research included 1263 patients taking enteric aspirin and clopidogrel after PCI.The cases were divided into 4 groups: routine treatment group (n=332), PPI group (n=318), gastric mucosa protectant group (n=299), and PPI+gastric mucosa protectant group (n=314).A follow-up for 6 months was observed including gastrointestinal symptoms, upper gastrointestinal bleeding, major adverse cardiac events (MACE), and adverse reactions.Results There were 52 cases of upper gastrointestinal bleeding within 6 months, including 21 cases from routine treatment group, 9 from PPI group, 15 from gastric mucosa protectant group, and 7 from PPI+gastric mucosa protectant group.The incidence of upper gastrointestinal bleeding among the 4 groups within 6 months was statistically different (X2=8.883, P=0.031).The routine treatment group had significant higher rate than the PPI group and the gastric mucosa protectant group (P<0.05), while among other groups there was no significant difference (P>0.05).The upper gastrointestinal bleeding occurred within 3 postoperative months in 34 out of 52 cases (65.4%).There was no statistical significance among the four groups in regard to bleeding occurrence time (X2=4.212,P=0.648).Conclusions Patients undergoing post-PCI dual anti-platelet treatment can reduce the incidence of gastrointestinal bleeding by taking pantoprazole or combined with gefarnate.Intervention against upper gastrointestinal bleeding should start on the first day after PCI and last for a minimum of 3-6 months.

Objective To evaluate the antitumor effect of dacarbazine (DTIC) on B16-F1 melanoma after CDK2 gene silencing.Methods Cultured B16-F1 melanoma cells were divided into 4 groups:control group receiving no treatment,CDK2-shRNA group infected with a recombinant lentivirus pUL-CDK2-shRNA,DTIC group cultured in 96-well plates followed 12 hours later by the treatment with 250 μmol/L DTIC,CDK2-shRNA + DTIC group infected with pUL-CDK2-shRNA followed 12 hours later by the treatment with 250 μmol/L DTIC.MTT assay was performed to evaluate the growth inhibition of B16-F1 melanoma cells,and coefficient of drug interaction (CDI) was calculated.AnnexinV-FITC/PI double staining was conducted to detect cell apoptosis.C57BL/6 mice were subcutaneously injected with B16-F1 cells at exponential growth phase into the right groin to establish melanoma-bearing mouse models.Twenty mouse models were randomly and equally divided into 4 groups:control mouse group injected with phosphate-buffered solution (PBS) into tumors,CDK2-shRNA mouse group injected with pUL-CDK2-shRNA into tumors,DTIC mouse group injected with DTIC into the abdominal cavity,and CDK2-shRNA + DTIC mouse group treated with pUL-CDK2-shRNA and DTIC.The animal experiment lasted 18 days,and the tumor growth curve was drawn.After 18-day treatment,all the mice were sacrificed,and tumors were isolated and weighed.The tumor growth inhibition rate was calculated,and the tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL).Results After 72-hour culture,compared with the control group,the CDK2-shRNA group,DTIC group,and CDK2-shRNA + DTIC group showed significantly decreased relative cell survival rates (40.6％ ± 2.8％,45.2％ ± 3.7％,28.7％ ± 2.1％,respectively;F =458.04,P ＜ 0.05),but significantly increased cell apoptosis rates (25.1％ ± 3.3％,15.6％ ± 2.2％,45.6％ ± 3.5％,respectively;F =115.46,P ＜ 0.05).Additionally,CDK2-shRNA + DTIC group showed significantly lower relative cell survival rates (P ＜ 0.01),but higher cell apoptosis rates (P ＜ 0.01) compared with the DTIC group.The CDI value was less than 0.7.On the sixth day after the in vivo treatment,the tumor volumes in the control mouse group,CDK2-shRNA mouse group,DTIC mouse group,and CDK2-shRNA + DTIC mouse group were (185.44 ± 68.97) mm3,(83.91 ± 14.33) mm3,(123.70 ± 20.85) mm3,and (34.54 ± 10.72) mm3 respectively.From then on,the CDK2-shRNA mouse group,DTIC mouse group,and CDK2-shRNA + DTIC mouse group showed significantly decreased tumor growth rates compared with the control mouse group (F =11.819,P ＜ 0.05),and the tumor growth rate was significantly lower in the CDK2-shRNA + DTIC mouse group than in the DTIC mouse group (P =0.04).The calculated tumor growth inhibition rates in the CDK2-shRNA mouse group,DTIC mouse group and CDK2-shRNA + DTIC mouse group were 52.2％,41.2％ and 86.4％ respectively.Compared with the control mouse group,the CDK2-shRNA mouse group,DTIC mouse group,and CDK2-shRNA + DTIC mouse group showed significantly increased tumor cell apoptosis indice (32.93％ ± 3.72％,21.62％ ± 3.54％,63.29％ ± 4.74％ respectively;F =222.25,P ＜ 0.05).Moreover,the tumor cell apoptosis index was significantly higher in the CDK2-shRNA + DTIC mouse group than in the DTIC mouse group (P ＜ 0.01).Conclusion CDK2 gene silencing can enhance the inhibitory effect of DTIC on the growth of melanoma,and show a synergistic effect with DTIC,likely by increasing the apoptosis of tumor cells.

Aim To observe the effects of cytokine signaling inhibition protein-3(SOCS3) on the liver fibrosis progression and reverse.Methods C57BL/6 mouse model was established by subcutaneous injection of carbon tetrachloride(CCl4).After a successful model of fibrosis, one-month normal diet was given to induce the reverse fibrosis model, while normal mice of the same gender and weight were as control group.Mice were sacrificed at 1, 2, 3, 4, 5, 6, 7, 8 weeks, respectively, then the liver tissue was harvested for the observation of its injury by hematoxylin and eosin(HE) staining.Then Masson staining was applied for the detection of changes in collagen, and the immunohistochemistry(IHC) for the observation of type Ⅰ Collagen(Colla-1), alpha smooth muscle actin(α-SMA), transforming growth factor beta 1(TGF-β1) and SOCS3 protein expression.In vitro formation of fibrosis was induced by TGF-β1 stimulating HSC-T6 cell lines, which was then reversed by MDI medium, with co-incubation of HSC-T6 cells with plasmid in the process of the reverse.Western blot was employed to detect SOCS3, Colla-1, α-SMA, TGF-β1 expression.Results The expression of SOCS3 and TGF-β1 increased in mouse model of fibrosis with the worsening fibrosis process and decreased in the reverse process.Over-expression SOCS3 in the reverse process reduced the development of liver fibrosis;meanwhile, the expression of TGF-β1 was also reduced accordingly.Conclusion SOCS3 may influence the development of the liver fibrosis and its reverse via regulating the expression of TGF-β1.

Objective : To investigate the characteristics of mortality of falls among the elderly in Haishu District, Ningbo City, Zhejiang Province, so as to provide insights into the improvement of fall control strategy. Methods: Data pertaining to surveillance of falls in Haishu District from 2017 to 2022 were collected from the Ningbo Municipal Digital Disease Prevention and Control Platform. The trends in mortality of falls in Haishu District from 2017 to 2022 were evaluated using annual percent change (APC). The distribution of demographic characteristics, locations of falls, injury sites, interval from fall occurrence to death and type of falls specified by age were descriptively analyzed. Results: A total of 1 206 deaths occurred due to falls among the elderly at ages of 60 years and older in Haishu District from 2017 to 2022, including 414 men and 612 women, with a male to female ratio of 0.67∶1. The average age was (85.20±8.42) years. The average annual crude mortality and standardized mortality of were 101.10/105 and 85.40/105 from 2017 to 2022, respectively, with no obvious changing trends (APC=5.797% and 5.337%, all P>0.05). The standardized death rate of falls in females was higher than that of males (102.60/105 vs. 69.55/105, P<0.05). The average annual mortality of falls appeared a tendency towards a rise with age among the elderly at ages of 60 years and older, with the highest mortality of 147.18/105 seen in the elderly at ages of 85 years and older. Falls predominantly occurred at home (836 cases, 81.48%) and on the same level (881 cases, 85.87%). The injury sites were mainly lower limbs (388 cases, 37.82%) and head (308 cases, 30.02%). In addition, the interval from fall occurrence to death mainly ranged from 24 hours to 6 months (757 cases, 73.79%). Conclusion The mortality of falls appeared a tendency towards a rise with age among the elderly at ages of 60 years and older in Haishu District from 2017 to 2022, which was higher in women than in men. The location of falls was predominantly at home and the type of falls predominantly at the same level. High attention to residential environments, and reinforcement of gender- and age-specific interventions are recommended.

OBJECTIVETo establish attenuated Salmonella typhimurium producing Helicobacter pylori (H. pylori) urease subunit B (UreB) and determine whether it could be used as an oral vaccine against H. pylori.

METHODSH. pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, and then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01-UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA assay. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the supernatant of spleen cells culture were also assessed by ELISA. In vitro stability of pTC01-UreB plasmid in SL3261 was confirmed by growing in Luria Broth (LB) medium to 80 generations.

RESULTSThe UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB as evidenced by sequence analysis. Enzyme digestion revealed that the correct pTC01-UreB was obtained. Western blot showed that a 61kDa protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum. After 80 generations of continuous culture, the recombinant plasmid pTC01-UreB was stable in SL3261 and had no obvious toxicity. Multiple oral immunizations with SL3261/pTC01-UreB could significantly induce H. pylori-specific mucosal IgA response as well as serum IgG response. Moreover, there were significant increases of IFN-gamma and IL-10 in the SL3261/pTC01-UreB group. Finally, no obvious side effects for mice and no change in gastric inflammation were observed.

CONCLUSIONAttenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection.

Administration, Oral ; Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; blood ; immunology ; Female ; Helicobacter Infections ; prevention & control ; Helicobacter pylori ; immunology ; Immunization ; Interferon-gamma ; biosynthesis ; Interleukin-10 ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Plasmids ; Protein Subunits ; Salmonella typhimurium ; genetics ; Urease ; immunology ; Vaccines, Attenuated ; immunology ; Vaccines, Synthetic ; immunology

Objectives To investigate the prevalence of non-alcoholic fatty liver (NAFLD) disease and its correlation with chronic metabolic diseases in two primary school students in Shanghai. Methods One thousand ifve hundred and thirty-two 7-11 year-old students from two primary schools were enrolled in Septamber-October 2011. The anthropometric indices, blood pressure, screening for pseudoacanthosis nigricans and liver ultrasonography of all subjects were recorded. Results The overall prevalence of NAFLD, obesity, abdominal obesity, pseudoacanthosis nigricans, high systolic blood pressure and high diastolic blood pressure was 6.5%, 26.7%, 16.3%, 5.1%, 1.7%and 1.9%, respectively. The prevalence of NAFLD and abdomi-nal obesity in students of central urban area was signiifcantly higher than that in suburban area (P<0.01). The binary regression analysis revealed a signiifcant association between NAFLD in students with sex, age, obesity, abdominal obesity, pseudoacan-thosis nigricans and high systolic blood pressure (P<0.05). Conclusions NAFLD has close correlation with chronic metabolic disease in children. It is time to adopt prevention, detection and treatment in NAFLD children with symptoms of chronic meta-bolic diseases.