Shun’ichi Dosako

Inhibitory Effects of Bovine Lactoferrin on the Adherence of Enterotoxigenic Escherichia coli to Host Cells

Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC). We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo. In the in vitro study, BLF was found to inhibit the adherence of ETEC. This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells. In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC.

Interaction between human lactoferrin and B lymphocytes

Our previous studies demonstrated that B lymphocytes incorporated human lactoferrin (LF) and then released it to an extracellular medium. We did competition experiments for better understanding of the interaction between LF and B lymphocytes, using a reverse hemolytic plaque forming assay. Competition between human and bovine LF reduced the number of plaque forming cells (PFC), but human transferrin (TF) showed no inhibitory effect on the PFC counts. These suggested that the receptor on B lymphocytes to human LF did not share with TF but bovine LF. The competition between a synthetic N-terminal nonapeptide of human LF (Gly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp) and human LF demonstrated reduced number of PFC dose-dependently. Mild tryptic human LF also reduced the human LFPFC counts, although removal of the C-terminal region did not affect the PFC counts. From these results, we concluded that highly cationic areas of human LF, such as N-terminal, accounted for the interaction with B lymphocytes.

Suspension Culture of Mouse Hybridoma HB8852 Cells with a Protein-Free Medium

A protein-free, ERDF-based medium was developed containing ethanolamine, sodium selenite, and inorganic iron as growth factors. Mouse hybridoma HB8852 cells were cultured in a spinner flask with the protein-free medium to evaluate the cell growth and monoclonal antibody production, which were comparable to those with a chemically defined, serum-free medium (ITES-ERDF) and a 10% FCS-ERDF medium. However, the cell viability was slightly lower. SDS-PAGE analysis of the spent, protein-free medium revealed the recovery of monoclonal antibodies in the medium with high purity. It is concluded that the medium is highly beneficial to produce and purify monoclonal antibodies in a large-scale mammalian cell culture.