The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6.

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

Corynebacterium species nasal carriage in pigs and their farmers in Bavaria, Germany: implications for public health.

Reports on cases of human diphtheria caused by toxigenic Corynebacterium ulcerans that were linked to occupational swine contact as well as isolation of C ulcerans from wild boars have suggested that pigs might serve as reservoir for human infections. Therefore, a prevalence study on Corynebacterium species nasal carriage in pigs and their farmers was performed between August 1 and December 31, 2009, in 41 swine farms from Bavaria, Germany. All 411 asymptomatic pigs and 29 of 30 healthy farmers were colonised with Corynebacterium strains of up to 11 different species. No potentially toxigenic Corynebacterium strain was isolated either from the pigs or from their farmers, respectively. The patterns of the species composition in the pigs and the farmers were very similar, suggesting a potential transmission of strains between animals and humans.

Two cases of cutaneous diphtheria associated with occupational pig contact in Germany.

In 2010, two independent cases of cutaneous diphtheria caused by toxigenic C. ulcerans were identified in Germany. Both patients had intense occupational contact with pigs. Diagnostic work-up comprising biochemical differentiation, rpoB sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis, real-time tox PCR and Elek test as well as public health measures including an intensified source tracing involving 83 asymptomatic pigs of an associated pig farm are presented.

Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin.

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.

Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry as a tool for rapid diagnosis of potentially toxigenic Corynebacterium species in the laboratory management of diphtheria-associated bacteria.

The rapid identification of the potentially toxigenic Corynebacterium species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis is essential for diagnosis and treatment of diphtheria and diphtheria-like diseases. We used matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDIT-OF MS) in comparison with classical microbiological and molecular methods on 116 Corynebacterium strains. All 90 potentially toxigenic Corynebacterium strains collected by the German National Consiliary Laboratory on Diphtheria in a period of more than ten years were correctly identified by MALDI-TOF MS. We propose an algorithm for fast and reliable diagnosis of diphtheria incorporating MALDI-TOF MS, real-time tox PCR and Elek testing.