Immunization with heat shock proteins (hsp) isolated from cancer cells has been shown to induce a protective antitumor response. The mechanism of hsp-dependent cellular immunity has been attributed to a variety of immunological activities mediated by hsp. Hsp have been shown to bind antigenic peptides, trim the bound peptides by intrinsic enzymatic activity, improve endocytosis of the chaperoned peptides by APCs, and enhance the ability of APCs to stimulate peptide-specific T cells. We have investigated the potential capacity of hsp70 and gp96 to function as a mediator for Ag-specific CTL stimulation in an in vitro model for human melanoma. Repetitive stimulation of PBLs by autologous DCs loaded with melanoma-derived hsp did not increase the frequency of T cells directed against immunodominant peptides of melanoma-associated Ags Melan-A and tyrosinase. In contrast, repeated T cell stimulation with peptide-pulsed DCs enhanced the number of peptide-specific T cells, allowing HLA/peptide multimer-guided T cell cloning. We succeeded in demonstrating that the established HLA-A2-restricted CTL clones recognized HLA-A2+ APCs exogenously loaded with the respective melanoma peptide as well as melanoma cells processing and presenting these peptides in the context of HLA-A2. We were not able to show that these melanoma-reactive CTL clones were stimulated by autologous dendritic cells pulsed with melanoma-derived hsp. These results are discussed with respect to various models for proving the role of hsp in T cell stimulation and to recent findings that part of the immunological antitumor activities reported for hsp are independent of the chaperoned peptides.
Beneath glycation, oxidation reactions may take place at cereal proteins during production of malt. The extent of oxidative chemical changes at malt proteins has not yet been studied. In the present short communication, malt protein was characterized by the determination of free thiol groups and degree of methionine oxidation as well as the sites that are reactive to covalent modification by 2,4-dinitrophenylhydrazine (DNPH, “protein carbonylation”). Protein carbonylation in pale malts was around 1.5 nmol/mg protein and increased with increasing malt colour. Investigations on the protein pellet isolated for determination of carbonylation revealed that solubility and colour may disturb the quantification of carbonyl sites in roasted malts. Free thiols decreased with increasing malt colour already in pale malts (EBC < 10). The formation of methionine sulfoxide (MetSO) was intensified with increasing malt colour. An amount of 7–20% of methionine was converted to MetSO in pale and dark malt, whereas nearly 60% of methionine was oxidized to MetSO in roasted malts. The formation of methionine sulfone was negligible. This study shows that malt proteins suffer from oxidation during kilning, and future studies will have to show whether this supports the pro- or antioxidant activity of malt.
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