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Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.

Flow cytometry represents an alternative method to agglutination assays for the accurate quantification of mixed field populations of erythrocytes observed after bone marrow transplantation. Murine monoclonal antibodies directed against the blood group ABH antigens were selected and processed in order to prepare ready-to-use fluorescent reagents. Anti-A (NaM8 7-1F6; IgG3), anti-B (NaM9-2E11: IgG3) and anti-H (NaM19-7Ell; IgM) were purified, labelled with fluorescein isothiocyanate, and used in a direct flow cytometry assay. Anti-A1 (NaM1-1C9; IgG3) was no longer active after FITC-labelling and then was used in an indirect assay. The agglutination was prevented by formaldehyde pretreatment of erythrocytes.Using artificially-made double populations of erythrocytes, measured values with mixtures of 1-100% of cells were very closely related to expected values, showing both the sensitivity and the accuracy of the method. From careful investigation of a series of bone-marrow transplanted patients, we conclude that engraftments could be demonstrated earlier by flow cytornetry than by agglutination, because minor populations (1-10%) of cells could be determined accurately only with labelled reagents. In addition, the disappearance of the donor cells on a longterm follow-up of patients enabled an earlier detection of graft failure in one case. The proposed method provides appreciable help to follow engraftment in patients and may have more general applications for the study of other haemopoietic chimaeras.

These antibodies represent precise tools to investigate GPA and related molecules in different cells and tissues.

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