RESUMO
The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslational modifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band on Coomassie-stained gels; activity assays were normal and showed <0.002 IU of activated factor IX (FIXa) per IU of FIX. The molecule has >97% γ-carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX.
RESUMO
Pseudomonas aeruginosa establishes chronic infections by forming biofilms; however studies of the virulence have focused on the planktonic form. Few in vitro co-culture models exist to study biofilm infections. We present a novel in vitro co-culture method examining the interactions between mature P. aeruginosa biofilms and human lung epithelial cells.
Assuntos
Biofilmes/crescimento & desenvolvimento , Técnicas de Cocultura/métodos , Plâncton/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/microbiologia , Apoptose , Aderência Bacteriana , Humanos , Interleucina-8 , Modelos BiológicosRESUMO
Phenotypic tolerances to antibiotics of mature and young Pseudomonas aeruginosa PAO1 biofilms and released planktonic bacteria were compared for four antibiotics. Resistance levels were similar for gentamicin and ciprofloxacin but differed for ceftazidime and meropenem. ß-Lactamase mapping showed that, after 5 h of ceftazidime exposure, mature biofilms produced more ß-lactamase than young biofilms, facilitating the growth of released planktonic bacteria. This shows the importance of early treatment and choice of antibiotics for P. aeruginosa biofilm infections.