Comparing invasive effects of five foodborne bacterial pathogens in human embryonic intestine 407 cells and human ileocecum HCT-8 cells

Objective To refine the infectious doses of enteric bacterial pathogens in animal assays and vaccine clinical trials by studying the invasion kinetics of five bacterial pathogens with human intestinal cells. Methods Utilizing in vitro cultured cell invasion assays with gentamicin-killing step, the invasive effects were analyzed in foodborne pathogens including Salmonella, Shigella, Yersinia, Escherichia coli (E. coli) O157 and opportunistic pathogens Citrobacter in human embryonic intestine 407 cells and ileocecum HCT-8 cells at multiplicities of infection (MOIs) of 0.04–4 000.00 E. coli HS served as a noninvasive control. Results The study results showed that the bacterial invasive efficiency and the average number of internalized bacteria per host cell changed with different starting MOIs. Higher starting MOIs did not always produce more bacterial internalization. The bacterial invasion effects varied with different bacterial strains and host cell lines. E. coli O157:H7 did invade human ileocecum HCT-8 cells. Conclusions This study shows that these bacteria possess different invasive patterns at various starting MOIs and also in different cell lines. The results could help to figure out the appropriate infectious doses of the bacteria in animal assays and in vaccine clinical trials. The bacterial invasion kinetics is also valuable in evaluating the safety and efficacy of live attenuated bacterial vaccines.

Comparing invasive effects of five foodborne bacterial pathogens in human embryonic intestine 407 cells and human ileocecum HCT-8 cells

Objective: To refine the infectious doses of enteric bacterial pathogens in animal assays and vaccine clinical trials by studying the invasion kinetics of five bacterial pathogens with human intestinal cells. Methods: Utilizing in vitro cultured cell invasion assays with gentamicin-killing step, the invasive effects were analyzed in foodborne pathogens including Salmonella, Shigella, Yersinia, Escherichia coli (E. coli) O157 and opportunistic pathogens Cit-robacter in human embryonic intestine 407 cells and ileocecum HCT-8 cells at multi-plicities of infection(MOIs)of 0.04–4 000.00 E.coli HS served as a noninvasive control. Results: The study results showed that the bacterial invasive efficiency and the average number of internalized bacteria per host cell changed with different starting MOIs.Higher starting MOIs did not always produce more bacterial internalization. The bacterial in-vasion effects varied with different bacterial strains and host cell lines.E.coli O157:H7 did invade human ileocecum HCT-8 cells. Conclusions: This study shows that these bacteria possess different invasive patterns at various starting MOIs and also in different cell lines.The results could help to figure out the appropriate infectious doses of the bacteria in animal assays and in vaccine clinical trials.The bacterial invasion kinetics is also valuable in evaluating the safety and efficacy of live attenuated bacterial vaccines.

Construction of RIP140 recombinant adenovirus and its expression in neonatal rat cardiomyocytes / 中国药理学通报

Aim The limited transfection efficiency for plasmid in primary neonatal rat cardiomyocytes,which are terminal differentiated cells,and long foreign DNA (the RIP140 gene sequence are as long as 3.5 kb) cause us to choose a better system to study RIP140 gene expression in primary non-replicative cells. Methods Full-length of RIP140 was cloned into pAdTracker-CMV shuttle vector,and then recombined with virus backbone pAdEasy-1 vector in BJ5183 bac-teria.Positive recombinant plasmid was confirmed by sequence analysis and restriction enzyme determina-tion,and then transfected into AD293 cells for amplifi-cation.Titers of virus particles were determined by Tis-sue Culture Infectious Dose 50 (TCID50 )method and cell vitality was analyzed by CCK-8 kit in cardiomyo-cytes.RIP140 gene was identified by Western blot. Results Sequence analysis suggested that full-length RIP140 gene was cloned correctly into AdEasyTM sys-tem.Virus titers of Ad-RIP140 and Ad-GFP were 1011.3 and 1011.7 PFU·mL-1 ,respectively.Cell vitali-ty was not affected when the Multiplicity of Infection (MOI)was lower than 200.Green fluorescent protein (GFP)and Western blot analysis showed RIP140 gene was remarkably increased in cardiomyocytes for 12h in-fection by Ad-RIP140 (P<0.05 ).Conclusion Re-combinant adenovirus containing RIP140 gene was suc-cessfully constructed and effectively expressed in car-diomyocytes.These will be helpful for further research on the function of RIP140 in cardiomyocytes.