Biomimetic cell-cell adhesion capillary electrophoresis for studying Gu-4 antagonistic interaction between cell membrane receptor and ligands.

We report a new method: biomimetic cell-cell adhesion capillary electrophoresis (BCCACE) to screen drugs targeting interactions between cell membrane receptors and ligands under an environment close to physiological conditions, in which the cell membrane receptors/ligands can maintain their natural conformations and bioactivity without being isolated and purified. Firstly, we screened twenty-one lactose derivatives by cell-immobilized capillary electrophoresis and obtained Gu-4 with the best activity (K = 3.58 ±â€¯0.22 × 104) targeting macrophage antigen-1 (Mac-1). Then, BCCACE was performed as follows: HEK 293 cells overexpressed with receptor (intercellular adhesion molecules-1, ICAM-1) were cultured and immobilized on the inner wall of capillaries as stationary phase, which simulated the endothelial cells lining on the inner surface of blood vessels. HEK 293 cells overexpressed with ligand Mac-1 as samples were used to simulate the neutrophils cells in blood vessels. And Gu-4 added into the running buffer solution as the antagonist was used to simulate the drug in blood. The results showed that Gu-4 (40 µM) could selectively inhibit cell-cell adhesion by targeting the interaction between Mac-1 and ICAM-1. Finally, the pharmaceutical efficacy assays of Gu-4 at cellular and animal levels were carried out using the concentration of 40 µM and the dose of 20 mg kg-1 respectively, which showed the anti-cancer metastasis activity of Gu-4 and the validity of the method. This method simulated a complete three-dimensional vascular model, which can easily obtain the suitable blood concentration of drugs. This system simulated the interaction between leukocytes and vascular endothelial cells in the bloodstream antagonized by drugs, and obtained the effective concentration of the antagonist. It can be used as an accuracy and efficient drug screening method and will be expected to become a new method to screen drugs targeting cell-cell adhesion.

Protective Immune Responses Elicited by Fusion Protein Containing PsaA and PspA Fragments.

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA-PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA-PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA-PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA-PspA induced IL-17A secretion. Mice immunized with PsaA-PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA-PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA-PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.