Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics-based technology.

We describe AMIDA (autoantibody-mediated identification of antigens), a novel target identification technology based on the immunoprecipitation of disease-specific antigens by autologous serum antibodies followed by two-dimensional electrophoretic separation, and their identification via mass spectrometry. Twenty-seven potential carcinoma antigens were identified including proteins of hitherto unknown function. Validation of one of the identified antigens, cytokeratin 8, revealed its de novo expression in hyperplastic tissue, gradual overexpression with increasing malignancy, and ectopic localization on the cell surface. Furthermore, a strong prevalence of CK8-specific antibodies occurred in the serum of cancer patients already at early disease stages. In situ hybridization for one marker of unknown function, KIAA1273/TOB3, demonstrated its strong overexpression in head and neck carcinomas, thus making it a likely tumor antigen candidate. Eventually, AMIDA could foster significant improvements for the diagnosis and therapy of human diseases eliciting a humoral immune response, and allows for the rapid identification of new target molecules.

Role of glutathione in gastric mucosal cytoprotection.

Exogenous thiol compounds have been reported to protect the stomach from ethanol-induced necrotic lesions. The gastric mucosa contains high levels of an endogenous thiol, glutathion (GSH). Because of the known role of glutathione in protecting against hepatic injury, its role in gastric mucosal cytoprotection was of interest. By use of an animal model for acute gastric injury from ethanol, a close parallel relation between depletion of endogenous mucosal GSH and induction of mucosal protection was demonstrated. Surprisingly, mucosal protection varied inversely with the level of mucosal GSH obtained after treatment with specific GSH-depleting agents (diethyl maleate and cyclohexene-1-one). Depletion of gastric mucosal GSH was associated with an increase in the mucosal content of prostaglandins 6-keto F1 alpha and F2 alpha but not E2. The protective effect induced by GSH-depleting agents was partially reversed by indomethacin in some but not all studies. Although GSH depletors increased gastric juice volume, protection with these agents persisted after the volume and mucosal GSH had returned to control levels and also was not reversed by increasing the dose of ethanol threefold to overcome a possible dilutional effect. We conclude that, contrary to apparent predictions, depletion of endogenous gastric GSH protects the stomach from acute ethanol-induced injury. Although the mechanism of this protection is unknown, a mediation by endogenous release of prostaglandins seems to play a minor role since diethyl maleate was protective even in indomethacin-treated animals.

Rapid oxidation in vitro of endogenous and exogenous glutathione in bile of rats.

Biliary excretion of glutathione has recently been described but poorly characterized. Controversy has existed concerning the relative contribution of oxidized and reduced glutathione to total glutathione efflux from the liver into bile. We found that bile, unlike liver cytosol or buffer, had the unique ability to oxidize GSH rapidly (t 1/2 = 5 min) to the disulfide form by a nonenzymatic, O2, and pH-dependent chemical reaction inhibited by only certain chelating agents. Significant oxidation of GSH occurred during the collection of bile samples resulting in significant time-dependent alterations in the ratio of biliary GSH to GSSG. Thus the preponderance of GSSG in bile in the normal state reported by others represents a postexcretory in vitro artifact. Inhibition of oxidation by acidification of bile during its collection established the true contribution of GSH and GSSG to total biliary efflux.