Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane-penetrating loop.

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is one of two major virulence factors of Bacillus anthracis, the causative agent of anthrax disease. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the cytotoxic enzymes of the toxin are extruded. The PA pore is formed by a cooperative conformational change in which the membrane-penetrating loops of PA associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target for monoclonal antibody-based therapy. cAb29 is a highly effective neutralizing antibody against PA. Here, the cryo-EM structure of PA in complex with the Fab portion of cAb29 was determined. It was found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to the static surface-exposed loop of the D3 domain of the same subunit, thereby preventing pore formation. These results provide the structural basis for the antibody-based neutralization of PA and bring into focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

Non-invasive diagnosis of chronic beryllium disease in workers exposed to hazardous dust in Israel.

OBJECTIVES: Chronic beryllium disease (CBD) is caused by prolonged occupational exposure to beryllium and is characterised by various clinical presentations, mostly pulmonary. The inflammatory process involves non-caseous granulomas and proliferation of CD4+ cells. CBD is diagnosed by lung biopsy showing tissue granuloma formation, and by the beryllium lymphocyte proliferation test (BeLPT) for past exposure and sensitisation to beryllium. The induced sputum (IS) technique was developed for diagnosing asthma, chronic obstructive pulmonary disease and interstitial lung diseases. A CD4/CD8 ratio >2.5 in T cells from IS is a positive result for granulomatous lung diseases. We previously revealed that dental technicians are exposed to excessive levels of beryllium. The efficacy of IS (CD4/CD8 >2.5) and BeLPT in diagnosing CBD in 17 workplaces where beryllium was present was evaluated. METHODS: All consecutive patients with a clinical suspicion of CBD referred to our institution for diagnosis and management were enrolled. Results of the gold standard lung biopsy with BeLPT were compared to the non-invasive IS+BeLPT. Kappa and McNemar tests evaluated agreement levels. Correlations between demographic and clinical parameters and a confirmed diagnosis of CBD were analysed. RESULTS: The two approaches were compared in 57 of 98 subjects. There was a high level of agreement (kappa 0.920) between IS+BeLPT and biopsy+BeLPT. IS+BeLPT had a specificity of 97.3% and sensitivity of 87.5%. 21 of 87 exposed workers (24%) had CBD, of whom 12 were dental technicians (p=0.044 dental technicians versus all other occupations). CONCLUSIONS: This study demonstrated that the CD4/CD8 ratio in IS together with positive/negative BeLPT findings can be used in diagnosing CBD.

Inhibitors of protein tyrosine phosphorylation: preliminary assessment of activity by time-resolved fluorescence.

Epidermal growth factor (EGF) receptor (ErbB1)-associated tyrosine kinase inhibitors may act as potential chemotherapeutic agents. In order to assess the inhibitory activity of these compounds, we developed a simple and sensitive assay based on time-resolved fluorescence. In this technique, crude cell lysates bearing the ErbB1 receptor were captured in microtitre plates immobilized with monoclonal anti-ErbB1 antibody SG 565. Subsequently, the phosphotyrosine content of the cell lysates was quantified by a europium-labelled anti-phosphotyrosine antibody. Thus, genistein, a tyrosine kinase inhibitor, was capable of reducing by half the tyrosine phosphorylation caused by the binding of EGF to A431 cells, whereas 6-carboxymethyl genistein did not inhibit protein tyrosine phosphorylation. This assay is simple to perform, does not use radioactive substrates, and can be useful for screening EGF receptor tyrosine kinase inhibitors from natural products or synthetic compounds. Moreover, the assay has a high signal:noise ratio and is suitable for large-scale screening.