Elevation of circulating phospholipase A2 (PLA2) activity is associated with sepsis and septic shock. Elevated levels of PLA2 activity also are seen in association with chronic inflammatory disorders such as rheumatoid arthritis. The relationship between these phospholipases is unclear. We have developed a highly specific enzyme-linked immunosorbent assay (ELISA) capable of measuring human synovial PLA2 in plasma, using monoclonal antibodies raised to recombinant synovial PLA2. This ELISA has been used to quantitate circulating PLA2 levels in patients clinically diagnosed with sepsis. These elevated levels positively correlated with the elevation seen in plasma PLA2 enzyme activity. The antibodies also have been used to purify immunoreactive PLA2 from plasma of patients with sepsis, thus enabling characterization of the purified protein by amino-terminal sequence analysis. We conclude from this study that the increase in PLA2 activity seen in association with sepsis and septic shock results from a dramatic elevation in levels of a circulating PLA2 enzyme. This inflammatory PLA2 is indistinguishable, both immunologically and chemically, from that associated with rheumatoid arthritis. Therapeutic agents directed towards inhibition of this inflammatory PLA2 enzyme may have utility in the treatment of both chronic and acute inflammatory disease.
The self-oncoprotein ErbB-2 is overexpressed in a number of malignancies. The presence of endogenous anti-ErbB-2 Ab and T cell immune responses to this protein in cancer patients has made ErbB-2 an attractive target for active immunization. However, the finding that murine anti-ErbB-2 Abs can have stimulatory, inhibitory, or no effects on cancer cell growth suggests that an inappropriately induced immune response may have an adverse effect. To ensure the induction of a beneficial Ab response, it is important to identify the epitopes recognized by these Abs. In this study we have used phage-displayed ErbB-2 gene fragment libraries and synthetic peptides to epitope-map a panel of anti-ErbB-2 mAbs. The epitopes of three mAbs, N12, N28, and L87, were successfully located to C531-A586, T216-C235, and C220-C235 of ErbB-2, respectively. It was found that while N12 inhibited tumor cell proliferation, N28 stimulated the proliferation of a subset of breast cancer cell lines overexpressing ErbB-2. The peptide region recognized by N12, (C531-A586; EP531), was used as an immunogen to selectively induce an inhibitory immune response in mice. Mice immunized with the GST fusion peptide (GST-EP531) recognized the peptide region EP531 as well as native ErbB-2. More importantly, Igs purified from mouse sera were able to inhibit up to 85% of tumor cell proliferation. In conclusion, our study provides direct evidence of the function-epitope relationship of anti-ErbB-2 Abs and also emphasizes the value of inducing a potent tumor inhibitory polyclonal Ab response by rationally selecting regions of ErbB-2 used for immunization.
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