Bovine lactoferrin(Metrodora Therapeutics)

Enterovirus eibovi (EV-E) is a bovine RNA virus with debated pathogenic potential. The aim of this study was to evaluate the in vitro effects of bovine lactoferrin (bLF), an antimicrobial and immunomodulatory glycoprotein, on EV-E-infected bovine peripheral blood mononuclear cells (PBMC). The PBMC viability, phagocytic function, and lymphocyte proliferation were examined using spectrophotometric assays. Flow cytometry was used for immunophenotyping of lymphocytes, and real-time quantitative PCR for evaluation of immune-related gene expression. The safe concentration range of bLF (1-25 µg/mL) was determined on the basis of the viability and proliferation of uninfected PBMC. Bovine lactoferrin significantly influences the activity of enterovirus-infected cells, with high (10 and 25 µg/mL) and low (1, 2.5, and 5 µg/mL) protein concentrations exerting different effects. Low concentrations of bLF primarily affected unstimulated cells, increasing the expression of IFNα, IFNβ, IL2, and NFκB genes. In contrast, high concentrations of lactoferrin predominantly modulated the activity of infected cells that were additionally stimulated with a mitogen or antigen: They increased T lymphocyte proliferation; reduced phagocyte activity; increased the expression of IFNα, IL10, and NFκB; and decreased the expression of IFNγ. The increased expression of IL10 in response to high protein concentrations was also observed in unstimulated cells. The only common effect of both high and low concentrations of bLF was a decrease in the percentage of WC1+ γδ T cells following concanavalin A stimulation. Thus, bLF modulated the immune response of EV-E-infected PBMC in a concentration-dependent manner: Lower concentrations promoted early antiviral responses by inducing type I interferons and IL-2, whereas higher concentrations upregulated the anti-inflammatory cytokine IL-10, suggesting a protective effect against immune overstimulation. These findings indicate that bLF may serve as an immunomodulatory agent during enteroviral infections in cattle, with promising implications for both therapeutic and prophylactic applications.

Lactoferrin (Lf) is an iron-binding glycoprotein involved in various biological functions, including iron metabolism and immune response. Bovine lactoferrin (bLf) has gained attention due to its potential therapeutic applications. This study investigates the effects of bLf on human erythrocyte membranes, focusing on Na,K-ATPase (NKA) modulation. Whole blood and erythrocyte ghost membranes were treated with different concentrations of bLf, and multiple assays were performed to assess enzyme activity, lipid peroxidation, and lipid membrane composition. First, we demonstrate that Fe⁺² is not able to increase the activity of the NKA. Bovine lactoferrin, on the other hand, significantly increases NKA activity, particularly affecting the α3 isoform. Additionally, bLf treatment led to an increase in total phospholipids and cholesterol content, suggesting modifications in the erythrocyte membrane. Molecular docking analysis revealed a strong interaction between bLf and α3-NKA, but not α1 isoform, supporting a potential regulatory role. Furthermore, bLf reduced Fe³⁺ and Thiobarbituric Acid Reactive Substances (TBARS) levels, indicating a protective effect against oxidative stress. These findings suggest that bLf modulates erythrocyte membrane properties, and could be specific to α3-NKA activation, reinforcing the role of Fe⁺³ in modulating the NKA activity.