The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment. In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 ± 1.5 times during 8 days of incubation; mean ± SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 ± 0.2 times; mean ± SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture.

Objective. To clarify the consequences of the valine/leucine polymorphism at position 247 of the ␤ 2 -glycoprotein I (␤ 2 GPI) gene in patients with antiphospholipid syndrome (APS), by investigating the correlation between genotypes and the presence of anti-␤ 2 GPI antibody.

IgG anti-laminin-1 auto-Abs are associated with recurrent miscarriages and the subsequent pregnancy outcome of recurrent aborters.

␤ 2 -Glycoprotein I ( ␤ 2 -GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that ␤ 2 -GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for ␤ 2 -GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by ␤ 2 -GPI and subsequently by anti-␤ 2 -GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1liposomes was enhanced more than 10-fold in the presence of both ␤ 2 -GPI and an anti-␤ 2 -GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-␤ 2 -GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with ␤ 2 -GPI. We suggest that autoimmune atherogenesis linked to ␤ 2 -GPI interaction with oxLDL and Abs may be present in APS.

Binding of beta(2)-glycoprotein I (beta(2)-GPI)-dependent anticardiolipin antibodies (aCL) derived from antiphospholipid syndrome (APS) is significantly reduced in aCL ELISA due to loss of the phospholipid (PL) binding property of beta(2)-GPI by plasmin treatment. In the present study, the treatment generated a nicked form of beta(2)-GPI and resulted in loss of antigenicity for the autoantibodies detected in ELISA, using an beta(2)-GPI directly adsorbed polyoxygenated carboxylated plate, the assay system of which was not related to PL binding. The nicked form bound to neither Cu(2+)-oxidized low-density lipoprotein (oxLDL) nor to beta(2)-GPI-specific lipid ligands isolated from oxLDL, the result being a complete loss of subsequent binding of anti-beta(2)-GPI autoantibodies. The conformational change in the nicked domain V was predicted from its intact structure determined by an X-ray analysis (implemented in Protein Data Bank: 1C1Z), molecular modeling and epitope mapping of a monoclonal anti-beta(2)-GPI antibody, i.e. Cof-18, which recognizes the related structure. The analysis revealed that novel hydrophobic and electrostatic interactions appeared in domain V after the cleavage, thereby affecting the PL binding of beta(2)-GPI. Such a conformational change may have important implications for exposure of cryptic epitopes located in the domains such as domain IV.