ABSTRACT
Objective To evaluate bacteriophage therapy for gut-derived sepsis caused by Acinetobacter baumannii in mice.Methods Lyric phages of Acinetobacter baumannii were isolated from environment by using double-layer agar method.A murine model of gut-derived sepsis was established by oral administration of ampicillin-resistant Acinetobacter baumannii and injection of ampicillin and cyclophosphamide into peritoneal cavities of mice.Bacteriophage therapy were given 1 d before infection (group 1),2 d(group 2) and 6 d(group 3) after infection.The survival of the mice was observed,mice without bacteriophage therapy were as control.Independent-sample t test was performed to compare inflammatory cytokines levels in peripheral blood and liver,number of bacteria in liver and spleen between mice with and without bacteriophage therapy.Results The minimal lethal dose of Acinetobacter baumannii for mice with gut-derived sepsis was 1 × 107 CFU/mL.The survival of the mice in group 2 (4/6 survived),which were treated with bacteriophage 2 d after inoculation of Acinetobacter baumannii,was higher than those of group 1 (2/6 survived),group 3 (3/6 survived) and the control group (phage-untreated,0/6 survived).Interleukin (IL)-1 β,tumor necrosis factor (TNF)-α and IL-6 in peripheral blood in mice with bacteriophage therapy were (105 ±6) ng/L,(105 ± 11) ng/L and (104 ± 12)ng/L,which were lower than those in control group (t =5.04,9.05 and 9.33,P ＜ 0.01) ; IL-1 β and TNF-α in liver of mice with bacteriophage therapy were (104 ± 9) ng/L and (104 ± 11) ng/L,which were lower than those in the controls (t =13.70 and 12.80,P ＜0.01),but IL-6 levels were not of statistical difference between therapy and control groups (t =1.06,P ＞ 0.05).Number of bacteria in liver and spleen in mice with bacteriophage therapy were (2.9 ± 1.3) × 103CFU/g and (8.3 ±7.6) × 102 CFU/g,which were also lower than those in control group (t =9.16 and 8.96,P ＜ 0.01).Conclusions Bacteriophage therapy can be effective against gut-derived sepsis caused by Acinetobacter baumannii.
ABSTRACT
Objective To construct a subtractive cDNA library of genes differentially expressed in human lymphoma cell line Jurkat cells treated with phosphonoformate (PFA), and to clone genes associated with its immune regulation. Methods Using suppression subtractive hybridization (SSH) technique, the mRNA was isolated from Jurkat cells treated with phosphonoformate or 0 9 percent sodium chloride, respectively, and then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. The tester cDNA, which was hybridized with driver cDNA twice and amplified by nested polymerase chain reaction (PCR) twice, was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109. The clones, which were selected randomly, were amplified by PCR, and then they were sequenced and analyzed in GenBank with Blast search. Results The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA was constructed successfully. The amplified library contained 46 positive clones, which contained 200- 1 000 bp of inserts. Fourteen clones were analyzed by sequencing and bioinformatics, which were identified as eleven known genes and three genes with unknown function. Conclusions The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA using SSH technique was constructed successfully, which brought some new clues for the study of the immune regulation mechanism of PFA