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Today it is generally accepted that B cells require cognate interactions with CD4+T cells to develop high-affinity antibodies against proteins. CD4+T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+T-cell epitopes presented by HLA-DRB1*1501 to CD4+T cells during immune responses against FVIII. CD4+T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

5-Azacytidine (5-aza-CR) is a DNA-hypomethylating antineoplastic agent used because of its inhibitory activity on DNA methyltransferases. Today, it is approved as an epigenetically active drug therapy for treatment of myelodysplastic disorders, with a contraindication as to pre-existing liver diseases. Because the mechanism of its hepatotoxicity is still unknown, we investigated the pharmacodynamic properties of 5-aza-CR with regard to death receptor/ligand-induced apoptosis and the mode of execution of cell death. In a time- and concentration-dependent manner, primary murine, human hepatocytes and HepG2 cells exposed to 5-aza-CR became highly sensitive toward cell death induced by CD95L, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, or TNF. Cell death was characterized as apoptotic by membrane blebbing, chromatin condensation, and exposure of phosphatidylserine on the outer membrane. Neither 5-aza-2'-deoxycytidine nor the common DNA methyltransferase inhibitors S-(5'-adenosyl)-L-homocysteine or RG 108 showed any significant effects under these conditions. Despite the complete protection of HepG2 by high concentrations of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk), effector caspase-3/7 activity was completely abolished at approximately a 20-fold lower concentration of z-VAD-fmk. Under these conditions, the serine protease inhibitors N,alpha-tosyl-L-phenylalanine chloromethyl ketone, N,p-tosyl-L-lysine chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively, conferred protection against death receptor ligands. We conclude that this caspase-independent apoptosis is executed by a yet-unidentified serine protease.

Two new prenylated stilbenes, artochamins F (1) and G (2), and their four novel derivatives, artochamins H–K (3–6), were isolated from the stems of Artocarpus chama. Their structures were elucidated mainly by NMR spectroscopy and mass spectrometry. The structure of 3 was confirmed by X-ray crystallographic analysis. The origin of 3–6 could be assumed biogenetically from 2. The cytotoxicity of these compounds against HepG2 cells was evaluated. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Abstract 2188