The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and antimicrobial peptides (AMPs) suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs and that lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. These results suggest that there are differences in the resistance mechanisms to colistin and ceragenins/AMPs.
Use of chlorhexidine in clinical settings has led to concerns that repeated exposure of bacteria to sub-lethal doses of chlorhexidine might result in chlorhexidine resistance and cross resistance with other cationic antimicrobials including colistin, endogenous antimicrobial peptides (AMPs) and their mimics, ceragenins. We have previously shown that colistin-resistant Gram-negative bacteria remain susceptible to AMPs and ceragenins. Here, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins by serial exposure of standard strains of Gram-negative bacteria to chlorhexidine to generate resistant populations of organisms. Furthermore, we performed a proteomics study on the chlorhexidine-resistant strains and compared them to the wild-type strains to find the pathways by which bacteria develop resistance to chlorhexidine. Serial exposure of Gram-negative bacteria to chlorhexidine resulted in four- to eight-fold increases in minimum inhibitory concentrations (MICs). Chlorhexidine-resistant organisms showed decreased susceptibility to colistin (8- to 32-fold increases in MICs) despite not being exposed to colistin. In contrast, chlorhexidine-resistant organisms had the same MICs as the original strains when tested with representative AMPs (LL-37 and magainin I) and ceragenins (CSA-44 and CSA-131). These results imply that there may be a connection between the emergence of highly colistin-resistant Gram-negative pathogens and the prevalence of chlorhexidine usage. Yet, use of chlorhexidine may not impact innate immune defenses (e.g., AMPs) and their mimics (e.g., ceragenins). Here, we also show that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa . Additionally, resistance to chlorhexidine resulted in elevated expression levels of proteins associated with chaperones, efflux pumps, flagella and cell metabolism. This study provides a comprehensive overview of the evolutionary proteomic changes in P. aeruginosa following exposure to chlorhexidine and colistin. These results have important clinical implications considering the continuous application of chlorhexidine in hospitals that could influence the emergence of colistin-resistant strains.
The in vitro antimicrobial activity of two new aryl-fluoroquinolone antibiotics, A-56619 and A-56620, was compared with those of norfloxacin and several other antibiotics against 448 bacterial isolates. A-56620 was the most active agent tested. The usual 90% MIC of A-56620 was .2 ,ug/ml, except for enterococci, gentamicinresistant Serratia marcescens, and gentamicin-resistant Pseudomonas aeruginosa, for which the 90% MIC was 4 Fg/ml. A-56619 and porfloxacin were generally severalfold less active than A-56620. Cross resistance was observed between the quinolope antibiotics and other unrelated antibiotic classes. Antimicrob. Agents Chemother., abstr. no. 77, 1984). In this study, the antimicrobial spectrum of potency of A-56619 and A-56620 were studied further and compared those of with norfloxacin and several broad-spectrum cephalosporins, penicillins, and other relevant antimicrobial agents.Antimicrobial compounds of known potency were provided as follows: A-56619 and A-56620 (Abbott Laborato- The comparative in vitro antimicrobial activity for A-56619, A-56620, and the other agents evaluated is presented in Table 1. A-56619 and A-56620 had similar activity against grampositive cocci. Both antimicrobial agents were more active than norfloxacin against methicillin-resistant Staphylococcus aureus, while A-56620 was more active against the enterococci and methicillin-susceptible Staphylococcus aureus. A-56619 and'A-56620 were generally more active than the other agents tested against the gram-positive cocci. Only ampicillin was more active than A-56619 and A-56620 against the enterococci and vancomycin was of about equal activity against methicillin-resistant Staphylococcus aureus.For the gentamicin-susceptible members of the family Enterobacteriaceae, cefotaxime was generally the most active beta-lactam agent, with an MIC for 90% of strains tested (MICgo) of .0.25 ,ug/ml for all species tested, except Providencia spp., Citrobacter freundii, Enterobacter sp., and Citrobacter diversus. A-56620 was the most active quinolone carboxylic acid tested, with MIC90s ranging from 0.25 to 1.0 ,ug/ml for most of these same bacteria. However,A-56620 did demonstrate exceptional potency against Escherichia coli, Citrobacter diversus, Salmonella enteritis, and Shigella sonnei. Norfloxacin was slightly more active than A-56620 against Serratia marcescens and Proteus mirabilis. A-56619 was generally two-to fourfold less active than A-56620 against these Enterobacteriaceae and was less active than norfloxacin against Aeromonas hydrophilia, Citrobacter diversus, Citrobacter freundii, Morganella morganii, Proteus mirabilis, and Serratia marcescens.The quinolone carboxylic acids showed substantially diminished activity against gentamicin-resistant strains of Escherichia coli, Serratia
Recent clinical trials using chimeric antigen receptors (CAR) T cells have demonstrated tremendous success in eradicating hematologic malignancies. Notwithstanding the excitement generated by CAR T cell therapy, its clinical efficacy has not been effectively translated to the context of solid tumors; the physical barriers of solid malignancies and the immunosuppressive conditions at the tumor site hinder the efficacy of CAR T cells. Macrophages have the ability to infiltrate almost every tissue and frequently are recruited into tumors. Therefore, macrophages are an attractive vehicle for CAR therapy and could help solve current challenges that CAR T cells face in the treatment of solid tumors. MOTO-CAR cells are monocyte-derived human macrophages that are genetically modified by a lentiviral or adenoviral approach to express a synthetic tumor-targeting receptor and to secrete cytokines, ligands or chemokine receptors. MOTO-CAR receptors are composed of a single-chain variable fragment (ScFv) that binds to a specific tumor target, a hinge to link it to a transmembrane domain, and an engineered Toll/Interleukin-1 receptor (TIR) signaling domain. When the ScFv binds to the tumor cell via its tumor target, an activation signal is transmitted. Myd88 dependent and independent signaling cascades are elicited, activating the macrophage and polarizing it towards a proinflammatory phenotype to eliminate cancer cells in a selective way. We previously reported the expression of Thymidine Kinase 1 (TK1) on the cell membrane of the non-small cell lung carcinoma NCI-H460 and A549 cell lines. The in vitro function of TK1 MOTO-CAR cells was evaluated against these cancer cell lines, using GFP-based phagocytosis and killing assays. Additionally, cell migration and interaction was recorded using time-lapse video with a confocal microscope. Upon co-culturing, with its target TK1 specific MOTO-CARs showed a nearly 4-fold increase in killing activity when compared with the controls (p<0.01). MOTO-CAR cells were produced through a lentiviral approach with around 30% of the cells expressing MOTO-CARs and with an adenoviral approach using the Ad5f35 vector with an efficiency of 70-80% of cells being transduced. Furthermore, after transduction MOTO-CAR cells showed a consistent M1 phenotype expressing high levels of CD14, CD80, CD206 and low levels of CD163. Time-lapse videos showed migration and clustering of MOTO-CAR cells around H460 GFP + cells. Moreover, cell death was observed upon contact of MOTO-CAR cells with target cells as well as phagocytic activity. In vivo testing using an orthotopic NOD scid gamma mice model is in progress. Our preclinical data show evidence that human macrophages are a suitable vehicle for CAR therapy and have the potential to successfully extrapolate the clinical efficacy of CAR therapy to the context of solid tumors. Citation Format: Edwin J. Velazquez, John E. Lattin, Taylor D. Brindley, Zachary Z. Reinstein, Roger Chu, Lu Liu, Evita G. Weagel, Michelle H. Townsend, Kiara V. Whitley, Eliza L. Lawrence, Brandon T. Garcia, Scott Weber, Richard A. Robison, Kim L. O'Neill. Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2563.
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