RESUMO
Escherichia coli OmpT, located in the outer membrane, has been characterized as a plasminogen activator, with the ability to hydrolyze protamine and block its entry. In this investigation, a complex of low molecular weight cationic peptides purified from human urine by a combination of membrane ultrafiltration and weak cation exchange chromatography was characterized. The impact of OmpT on E. coli resistance to urinary cationic peptides was investigated by testing ompT knockout strains. The ompT mutants were more susceptible to urinary cationic peptides than ompT(+) strains, and this difference was abolished by complementation of the mutants with pUC19 carrying the ompT gene. The urinary protease inhibitor ulinastatin greatly decreased the resistance of the ompT(+) strains. Overall, the data indicate that OmpT may help E. coli persist longer in the urinary tract by enabling it to resist the antimicrobial activity of urinary cationic peptides.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Farmacorresistência Bacteriana , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Urina/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/urina , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Infecções por Escherichia coli/urina , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Humanos , Masculino , Peptídeo Hidrolases/genética , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/urinaRESUMO
Prolonged thrombocytopenia is a puzzling problem following umbilical cord blood (CB) transplantation. It might be the result of inadequate megakaryocyte progenitors and the arrest in the megakaryocyte maturation. It is an important method to solve the problem by transfusing ex-vivo expanded CB megakaryocyte progenitor cells into the patients to shorten the duration of platelet recovery. However, the most optimal condition of expansion has not been established so far. In the study, cord blood mononuclear cells (MNC) were cultured in serum-free medium with TPO, IL-3, SCF and IL-6. The numbers of MNC, CFU-MK and CD41(+) cells were measured at 0, 6, 10 and 14 days, in order to find the best cytokine combination and optimal harvest time point for clinical use. The results showed that the megakaryocyte progenitors most efficiently expanded with the cytokine combination including TPO, IL-3, SCF and IL-6. The time point of maximal CFU-MK growth is day 10. At 10 days, the numbers of CFU-MK and CD41(+) cells expanded by 6.8- and 8.8-fold respectively. In conclusion, in vitro, the cytokine cocktail including TPO, IL-3, SCF and IL-6 was the most optimal cytokine combination which stimulates the ex vivo expansion of megakaryocyte progenitors in CB MNC and serum-free medium. The maximal CFU-MK colonies were harvested at 10 days, that may be an optimal harvest time for clinical transfusion.
