The antimicrobial properties of copper surfaces against a range of important nosocomial pathogens

Hospital acquired infections (HAI) are a major problem worldwide and controlling the spread of these infections within a hospital is a constant challenge. Recent studies have highlighted the antimicrobial properties of copper and its alloys against a range of different bacteria. The objective of this study was to evaluate the antimicrobial properties of copper compared to stainless steel against a range of clinically important pathogens. These pathogens consisted of five isolates of each of the following organisms; meticillin resistantStaphylococcus aureus (MRSA),Pseudomonas aeruginosa, Escherichia coli, vancomycin-resistant Enterococci (VRE) and Panton-Valentine Leukocidin positive community acquired-MSSA (PVL positive CA-MSSA). MRSA,P. aeruginosa, E. coli, and CA-MSSA isolates were not detectable after a median time of 60 minutes. No detectable levels for all VRE isolates were determined after a median time of 40 minutes. However, for all isolates tested the stainless steel had no effect on the survival of the bacteria and levels remained similar to the time zero count. The results of this study demonstrate that copper has a strong antimicrobial effect against a range of clinically important pathogens compared to stainless steel and potentially could be employed to aid the control HAI.     

Nucleotide sequence of DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3

DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3 was sequenced following amplification by the polymerase chain reaction. A comparison with the deduced amino acid (aa) sequence of the C. trachomatis serovar L2 showed that the L3 had three extra aa and 55 aa substitutions.     

Projecting human pharmacokinetics of therapeutic antibodies from nonclinical data: What have we learned?

The pharmacokinetics (PK) of therapeutic antibodies is determined by target and non-target mediated mechanisms. These antibody-specific factors need to be considered during prediction of human PK based upon preclinical information. Principles of allometric scaling established for small molecules using data from multiple animal species cannot be directly applied to antibodies. Here, different methods for projecting human clearance (CL) from animal PK data for 13 therapeutic monoclonal antibodies (mAbs) exhibiting linear PK over the tested dose ranges were examined: simple allometric scaling (CL versus body weight), allometric scaling with correction factors, allometric scaling based on rule of exponent and scaling from only cynomolgus monkey PK data. A better correlation was obtained between the observed human CL and the estimated human CL based on cynomolgus monkey PK data and an allometric scaling exponent of 0.85 for CL than other scaling approaches. Human concentration-time profiles were also reasonably predicted from the cynomolgus monkey data using species-invariant time method with a fixed exponent of 0.85 for CL and 1.0 for volume of distribution. In conclusion, we expanded our previous work and others and further confirmed that PK from cynomolgus monkey alone can be successfully scaled to project human PK profiles within linear range using simplify allometry and Dedrick plots with fixed exponent.Key words: monoclonal antibody, pharmacokinetics, clearance, allometric scaling, species-invariant time method     

Production of stable bispecific IgG1 by controlled Fab-arm exchange

The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques.     

High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has generated interesting data worthy of further investigation.     

Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats

Antibody-drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ～45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.     

Effects of chelating agent and environmental stresses on microbial biofilms: relevance to clinical microbiology

Aims: To determine the effect of pH, temperature, desiccation, ethylenediaminetetraacetic acid (EDTA) and desferrioxamine B (DFO) on Panton‐Valentine leukocidin‐positive community acquired methicillin‐susceptible Staphylococcus aureus (PVL +ve CA‐MSSA) biofilm formation. Methods and Results: Biofilms from PVL +ve CA‐MSSA (clinical isolate) were subjected to pH, temperature, desiccation, EDTA and DFO. PVL +ve CA‐MSSA were more resistant to pH and heat than their planktonic equivalents. Desiccation studies demonstrated that PVL +ve CA‐MSSA biofilms were more refractory to the treatment than planktonic cells. Significant inhibition of PVL +ve CA‐MSSA biofilm formation was observed in the presence of 1 mmol l^?1 EDTA. Low concentrations (2·5 μmol l^?1) of DFO enhanced the growth of PVL +ve CA‐MSSA biofilms. At higher concentrations (1 mmol l^?1), DFO did inhibit the growth but not as much as EDTA. A combination of EDTA and DFO inhibited PVL +ve CA‐MSSA biofilm formation at lower concentrations than either alone. Conclusions: This study demonstrates that PVL +ve CA‐MSSA biofilms are resistant to environmental stress but their growth can inhibited effectively by a mixture of EDTA and DFO. Significance and Impact of the Study: The inhibition of biofilm formation by PVL +ve CA‐MSSA using chelating agents has not been previously reported and provides a practical approach to achieve the disruption of these potentially important biofilms formed by an emerging pathogen.