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Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications.

Goldenseal (Hydrastis canadenisis) is a native American medicinal plant used as an immune stimulant. Astragalus (Astragalus membranaceus) is a widely used herbal product in China, other Asian countries, and the United States as an immune stimulant to be taken on first clinical signs of infection. In this study, the innate effects of goldenseal and Astragalus on pro-inflammatory cytokines produced by cultured macrophages were examined using two different commercial preparations of goldenseal and Astragalus. Both goldenseal and Astragalus were found to exhibit little to no direct effect on stimulation of mouse macrophages (J774A.1 cells), with only Astragalus able to affect production of tumor necrosis factor (TNF)-alpha when used in high concentrations. However, both goldenseal and Astragalus were able to modify responses from lipopolysaccharide-stimulated macrophages, with identified immunomodulatory effects to reduce production of TNF-alpha, interleukin (IL)-6, IL-10, and IL-12 in a dose-dependent manner. The results obtained indicate that both goldenseal and Astragalus exhibit abilities to modulate macrophage responses during stimulation. Therefore, it is hypothesized that their historical use as therapeutic agents may be due to reduction in the pro-inflammatory response that indirectly leads to limiting of clinical symptoms during infection. Both products differ in their immune stimulatory patterns, offering insight into differential use and therapeutic potential of these products to regulate macrophage immune responses and activation events.