Epitope recognition by anti-cathepsin D autoantibodies in endometrial cancer patients.

OBJECTIVE: This study analyzed a model for the identification of specific epitopes recognized by autologous tumor-reactive humoral responses of endometrial cancer patients as potential markers for the monitoring of cancer. METHODS: The presence of circulating pro- and mature forms of cathepsin D and antibodies reactive with this enzyme were identified by Western immunoblot and quantitated by an enzyme immunoassay. Specific immunoreactivities with 34- and 52-kDa cathepsin D forms were analyzed by Western immunoblot using sera from endometrial cancer patients (n = 40) and normal volunteers (n = 15). Subsequently, reactivities with specific cathepsin D epitopes were defined by a peptide-specific ELISA. RESULTS: Circulating pro-forms of cathepsin D were detected in 31 of 40 endometrial cancer patients tested and none of the control volunteers. Circulating IgG reactive with cathepsin D could be demonstrated in 29/31 patients with circulating procathepsin D, while an anti-cathepsin D response was not detectable in normal controls. This response appeared to be directed against the pro-peptide portion of cathepsin D. Using a peptide-specific ELISA, the frequencies of antibody production against specific epitopes within the pro-peptide were defined. CONCLUSION: There is a demonstrable tumor-reactive immune response elicited in endometrial cancer patients, directed against specific antigenic epitopes, some of which are conserved among these patients. Since these proteins are recognized as non-self, due at least in part to posttranslational processing errors, defining these epitopes will be useful as a means of diagnosis, assessment of therapeutic success, and, ultimately, identification of immunotherapeutic targets.

Modulation of proliferation and chemosensitivity by procathepsin D and its peptides in ovarian cancer.

Since the presence of precursors (pro-forms) of the aspartyl endoprotease, cathepsin D, appears to be linked with tumor progression, their presence was examined in sera and tumor tissues of ovarian cancer patients. The role of cathepsin D pro-forms was further assessed in the dysregulated proliferation and chemoresistance observed in advanced ovarian cancer. Cathepsin D was isolated from sera of ovarian cancer patients (n = 20) and normal volunteers (n = 11), as well as from solubilized normal ovarian epithelium (n = 8) and ovarian epithelial tumor tissue (n = 12). The specific molecular forms of cathepsin D were analyzed in these samples by Western immunoblot. Multiple circulating molecular weight forms of cathepsin D were identified in ovarian cancer patients ranging from 24 to 60 kDa, while in normal controls, a major band was observed at 34 kDa in all samples and minor bands corresponding to 27 and 48 kDa were detected in approximately half of the controls. To assess its consequences on ovarian cancer, the 52-kDa protein was immunoprecipitated from culture medium of an exponentially growing ovarian tumor cell line and was further purified by reverse-phase high-pressure liquid chromatography. Its effect on proliferation was assayed by determining cell doubling times and their chemosensitivity was measured in a standard cytotoxicity assay using cisplatin. In addition, decapeptides corresponding to the pro-portion of cathepsin D were analyzed in parallel. Procathepsin D and one decapeptide, peptide 2, as well as IGF-II (as a known positive) increased cell proliferation, with doubling times of 28.4, 28.8, and 30.3 h, respectively, versus untreated UL-1 cells (36.4 h). Procathepsin D treatment of UL-1 tumor cells significantly increased the cisplatin LD(50) (74.9 microgram/ml) over untreated (33.9 microgram/ml) as well as IGF-II-treated (38.8 microgram/ml) cells. Peptide 2 also showed a significant increase in LD(50) (69.5 microgram/ml) compared to untreated and peptide 1-treated cells (37.1 microgram/ml). There are several unique forms of cathepsin D expressed and accumulated by ovarian tumors and these forms are detectable in the sera of those with ovarian cancer. The presence of these procathepsin D can increase the proliferation of these tumor cells, while decreasing their sensitivity to chemotherapeutic agents. While procathepsin D and IGF-II both enhance proliferation, only procathepsin D (and peptide 2) appears to modulate chemosensitivity, suggesting a separate receptor or pathway for this consequence.

Is EAG the answer to the M-current?

The resemblance between the Drosophila EAG current and the mammalian M-current is very strong, but final confirmation for a member of the extended EAG family encoding the M-current remains to be determined.

Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo.

The ability of magainin 2 to augment antibiotic therapy was examined. Susceptibility to magainin 2 was determined on Escherichia coli incubated in the presence and absence of sublethal concentrations of antibiotics both in vitro and in vivo. Experiments in buffer and normal human serum revealed that E. coli exposed to sublethal amounts of cefepime, a beta-lactam antibiotic, was significantly more susceptible to the antimicrobial activity of magainin 2. Bacteria incubated with subinhibitory concentrations of other beta-lactam type antibiotics, but not amikacin (an aminoglycoside) or ciprofloxacin (a quinolone), were also more susceptible to magainin 2 in normal human serum. Bacteria were less susceptible to magainin 2 when they were examined in heat-inactivated serum. Complement was shown to be required for magainin 2 activity in serum by using C8-deficient sera. The combination of magainin 2 and cefepime was shown to be more antimicrobial in normal human serum for a variety of bacterial strains. Magainin 2 was completely inactive as a therapeutic agent when it was administered alone (2 mg per mouse) but significantly increased the survival of mice when it was administered with a low level of cefepime.