20 research outputs found

    Rise and Fall of an Anti-MUC1 Specific Antibody

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    So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology

    Dialysis-associated peritonitis in children

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    Peritonitis remains a frequent complication of peritoneal dialysis in children and is the most common reason for technique failure. The microbiology is characterized by a predominance of Gram-positive organisms, with fungi responsible for less than 5% of episodes. Data collected by the International Pediatric Peritonitis Registry have revealed a worldwide variation in the bacterial etiology of peritonitis, as well as in the rate of culture-negative peritonitis. Risk factors for infection include young age, the absence of prophylactic antibiotics at catheter placement, spiking of dialysis bags, and the presence of a catheter exit-site or tunnel infection. Clinical symptoms at presentation are somewhat organism specific and can be objectively assessed with a Disease Severity Score. Whereas recommendations for empiric antibiotic therapy in children have been published by the International Society of Peritoneal Dialysis, epidemiologic data and antibiotic susceptibility data suggest that it may be desirable to take the patient- and center-specific history of microorganisms and their sensitivity patterns into account when prescribing initial therapy. The vast majority of patients are treated successfully and continue peritoneal dialysis, with the poorest outcome noted in patients with peritonitis secondary to Gram-negative organisms or fungi and in those with a relapsing infection

    Muc2 Protects against Lethal Infectious Colitis by Disassociating Pathogenic and Commensal Bacteria from the Colonic Mucosa

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    Despite recent advances in our understanding of the pathogenesis of attaching and effacing (A/E) Escherichia coli infections, the mechanisms by which the host defends against these microbes are unclear. The goal of this study was to determine the role of goblet cell-derived Muc2, the major intestinal secretory mucin and primary component of the mucus layer, in host protection against A/E pathogens. To assess the role of Muc2 during A/E bacterial infections, we inoculated Muc2 deficient (Muc2−/−) mice with Citrobacter rodentium, a murine A/E pathogen related to diarrheagenic A/E E. coli. Unlike wildtype (WT) mice, infected Muc2−/− mice exhibited rapid weight loss and suffered up to 90% mortality. Stool plating demonstrated 10–100 fold greater C. rodentium burdens in Muc2−/− vs. WT mice, most of which were found to be loosely adherent to the colonic mucosa. Histology of Muc2−/− mice revealed ulceration in the colon amid focal bacterial microcolonies. Metabolic labeling of secreted mucins in the large intestine demonstrated that mucin secretion was markedly increased in WT mice during infection compared to uninfected controls, suggesting that the host uses increased mucin release to flush pathogens from the mucosal surface. Muc2 also impacted host-commensal interactions during infection, as FISH analysis revealed C. rodentium microcolonies contained numerous commensal microbes, which was not observed in WT mice. Orally administered FITC-Dextran and FISH staining showed significantly worsened intestinal barrier disruption in Muc2−/− vs. WT mice, with overt pathogen and commensal translocation into the Muc2−/− colonic mucosa. Interestingly, commensal depletion enhanced C. rodentium colonization of Muc2−/− mice, although colonic pathology was not significantly altered. In conclusion, Muc2 production is critical for host protection during A/E bacterial infections, by limiting overall pathogen and commensal numbers associated with the colonic mucosal surface. Such actions limit tissue damage and translocation of pathogenic and commensal bacteria across the epithelium

    Ligand-Binding Mass Spectrometry to Study Biotransformation of Fusion Protein Drugs and Guide Immunoassay Development: Strategic Approach and Application to Peptibodies Targeting the Thrombopoietin Receptor

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    The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc (“peptibodies”) using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins

    Aquaponics: The Basics

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    Aquaponics is a technology that is part of the broader integrated agri-aquaculture systems discipline which seeks to combine animal and plant culture technologies to confer advantages and conserve nutrients and other biological and economic resources. It emerged in the USA in the early 1970s and has recently seen a resurgence, especially in Europe. Whilst aquaponics broadly combines recirculating fish culture with hydroponic plant production, the application of the term aquaponic is broad and many technologies claim use of the name. Combining fish culture with aquatic-based, terrestrial plant culture via aquaponics may be better defined via its nutrient resource sharing credentials. Aquaponics applies several principles including, but not limited to, efficient water use, efficient nutrient use, lowered or negated environmental impact and the application of biological and ecological approaches to agricultural fish and plant production. Water sources are important so that the nutrients required for fish and plant production are available and balanced, and system water chemistry is paramount to optimised fish and plant production. Systems may be configured in several ways, including those that are fully recirculating and those that are decoupled. Aquaponics importantly seeks to apply methods that provide technical, biological, chemical, environmental and economic advantages

    Urbanization Forecasts, Effects on Land Use, Biodiversity, and Ecosystem Services

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    Several studies in recent years have forecasted global urban expansion and examined its potential impacts on biodiversity and ecosystem services. The amount of urban land near protected areas (PAs) is expected to increase, on average, by more than three times between 2000 and 2030 (from 450,000 km2 circa 2000) around the world. During the same time period, the urban land in biodiversity hotspots, areas with high concentrations of endemic species, will increase by about four times on average. China will likely become the nation with the most urban land within 50 km of its PAs by 2030. The largest proportional change, however, will likely be in Mid-Latitudinal Africa; its urban land near PAs will increase 20 ± 5 times by 2030. The largest urban expansion in biodiversity hotspots, an increase of over 100,000 km2, is forecasted to occur in South America. The forecasts of the amount and location of urban land expansion are subject to many uncertainties in their underlying drivers including urban population and economic growth. Nevertheless, the direct impacts of urban expansion on biodiversity and ecosystem services will likely be significant. The forecasts point to the need to reconcile urban development and biodiversity conservation strategies. Urbanization will also have impacts on food and food security. While the direct loss of cropland to urban expansion is of concern to the extent that high-yielding croplands are lost, the indirect impacts of urbanization due to dietary changes to more meat-based food products can also be substantial. Presently, regional and global studies that forecast impacts of future urban expansion on biodiversity and ecosystem services are in their infancy and more analyses are needed especially focusing on interactive effects of factors that drive urbanization. We conclude by highlighting the knowledge gaps on implications of future urbanization and suggest research directions that would help fill these gaps
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