Production of antibodies with peptide-CpG-DNA-liposome complex without carriers.

BACKGROUND: The screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers. RESULTS: We present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O)) induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O) enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O) without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells. CONCLUSIONS: Our overall results show that Lipoplex(O) is a potent adjuvant and that complexes of peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

Applicability of vital staining and tissue clearing to vascular anatomy and melanocytes' evaluation of temporal bone in six laboratory species.

The purpose of the present study was to define the applicability of tissue clearing to the field of otology. We combined tissue clearing with vital staining perfusion via a pumping system to examine the vascular anatomy of temporal bones in laboratory animals. We used six different types of species including Korean wild mouse, mouse, Mongolian gerbil, hamsters and Guinea pigs. A mixture of Alcian blue reagent and 4% paraformaldehyde was circulated throughout the entire circulatory system of the animal via a perfusion pump system. Transparency images were obtained from the temporal bones according to the protocol of the SunHyun 3D Imaging Kit. In examining the inner surface of the tympanic membrane, flaccid part (pars flaccida) was positioned along the entire marginal area in Guinea pig. In the Guinea pig, unlike the other species, the cortical bone of the mastoid (bullae) was easily removed using cold instruments, allowing a direct approach to the enclosed structures. The distribution and pattern of cochlea melanocytes were compared among the species. "Mobius strip"-like accumulated melanocytes in vestibules were shown in both the Korean wild mouse and mouse. The collateral blood supply to the cochlea in six different species was checked in various pattern. Combining dye infusion with tissue-clearing techniques, we documented the middle ear and transparent inner ear structures in six different species. The information and associated images will help other researchers to develop hypotheses and design experimental investigations.

Fine tuning of Rac1 and RhoA alters cuspal shapes by remolding the cellular geometry.

The anatomic and functional combinations of cusps and lophs (ridges) define the tooth shape of rodent molars, which distinguishes species. The species-specific cusp patterns result from the spatiotemporal induction of enamel knots (EKs), which require precisely controlled cellular behavior to control the epithelial invagination. Despite the well-defined roles of EK in cusp patterning, the determinants of the ultimate cuspal shapes and involvement of epithelial cellular geometry are unknown. Using two typical tooth patterns, the lophodont in gerbils and the bunodont in mice, we showed that the cuspal shape is determined by the dental epithelium at the cap stage, whereas the cellular geometry in the inner dental epithelium (IDE) is correlated with the cuspal shape. Intriguingly, fine tuning Rac1 and RhoA interconvert cuspal shapes between two species by remolding the cellular geometry. Either inhibition of Rac1 or ectopic expression of RhoA could region-distinctively change the columnar shape of IDE cells in gerbils to drive invagination to produce cusps. Conversely, RhoA reduction in mice inhibited invagination and developed lophs. Furthermore, we found that Rac1 and RhoA modulate the choices of cuspal shape by coordinating adhesion junctions, actin distribution, and fibronectin localization to drive IDE invagination.