The cytokine macrophage migration inhibitory factor (MIF) exhibits pro- and anti-inflammatory activities and regulates cell proliferation and survival. We investigated the effects of MIF on apoptosis. As MIF exhibits oxidoreductase activity and participates in regulating oxidative cell stress, we studied whether MIF could affect oxidative stress-induced apoptosis. We demonstrated that MIF exhibits antiapoptotic activity in various settings. MIF suppressed camptothecin-induced apoptosis in HeLa and Kym cells and HL-60 promyeloblasts. Both exogenous MIF and endogenous MIF, induced following overexpression through tetracycline (tet) gene induction, led to significant suppression of apoptosis. Apoptosis reduction by MIF was also observed in T cells. A role for MIF in redox stress-induced apoptosis was addressed by comparing the effects of rMIF with those of the oxidoreductase mutant C60SMIF. Endogenous overexpression of C60SMIF was similar to that of MIF, but C60SMIF did not suppress apoptosis. Exogenous rC60SMIF inhibited apoptosis. A role for MIF in oxidative stress-induced apoptosis was directly studied in HL-60 leukocytes and tet-regulated HeLa cells following thiol starvation or diamide treatment. MIF protected these cells from redox stress-induced apoptosis and enhanced cellular glutathione levels. As overexpressed C60SMIF did not protect tet-regulated HeLa cells from thiol starvation-induced apoptosis, it seems that the redox motif of MIF is important for this function. Finally, overexpression of MIF inhibited phosphorylation of endogenous c-Jun induced by thiol starvation, indicating that MIF-based suppression of apoptosis is mediated through modulation of c-Jun N-terminal kinase activity. Our findings show that MIF has potent antiapoptotic activities and suggest that MIF is a modulator of pro-oxidative stress-induced apoptosis.

The laminin-type globular (LG) domains of laminin ␣ chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, ␣-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin ␣5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin ␣5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified ␣ chains, such that all or part of ␣5LG was replaced with analogous segments of human laminin ␣1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing ␣5 domains LN through LG3 fused to human laminin ␣1LG4 -5. However, Lu/B-CAM did not bind other recombinant laminins containing ␣5LG3 unless ␣5LG1-2 was also present. A recombinant ␣5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the ␣5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin ␣5. Our results also suggest that the binding site for Lu/B-CAM on laminin ␣5 may overlap with that of integrins ␣3␤1 and ␣6␤1.

Macrophage migration inhibitory factor (MIF) is a cy-

The thermostability of GARDASIL (Merck & Co., Inc, Whitehouse Station, NJ, USA), a developmental vaccine against human papillomavirus (HPV), was evaluated using an enzyme immunoassay, referred to as the in vitro relative potency (IVRP) assay and differential scanning calorimetry (DSC). Gardasil samples were stored at temperatures ranging from 4 to 42 degrees C and tested for IVRP at various time points. Extrapolation of the IVRP results indicates GARDASIL is extremely stable. The half-life of the vaccine is estimated to be 130 months or longer at temperatures up to 25 degrees C. At 37 degrees C, the half-life is predicted to be 18 months and at 42 degrees C, the half-life is predicted to be approximately three months. Differential scanning calorimetry (DSC) analysis was used to evaluate the process of protein denaturation during a rapid temperature increase (as opposed to long-term storage at a specific temperature). Differences were seen among the DSC profiles of the four HPV types tested. This indicates that small differences in the amino acid structure can have a significant effect on the intermolecular contacts that stabilize the L1 proteins and the VLP assembly. For the Gardasil samples evaluated here, DSC results demonstrated the relative overall structural stability of the VLPs, but were not predictive of the excellent long-term stability observed with the IVRP assay.