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Contaminants in food have severely threatened human health, and appropriate antioxidants derived from food could reduce impairment risk. Lactoferrin from milk could control iron concentration in the blood to ameliorate oxidative stress, which is also required for sperm maturation, but the underlying mechanisms remain unclear. The present study used mice with spermatogenetic dysfunction caused by bisphenol A (BPA) and cadmium (Cd) to evaluate the ameliorative effects of lactoferrin and milk (bioactive substances). BPA (50 mg/kg) and Cd (1.6 mg/kg) caused severe damage to testis, including globally decreased germ cell counts, poor sperm quality, disordered apoptosis, oxidative stress, and autophagy; however bioactive substances comprehensively ameliorated spermatogenetic dysfunction via mitigating the increased levels of BAX/BCL2, LC3II/LC3I, and P62. AMPK was involved in autophagic regulation, while ERK1/2 inhibition attenuated the protective effects of lactoferrin, including restimulating apoptosis, oxidative stress, and arrested autophagic flux. Notably, P62 was consistently stimulated with different ERK1/2 inhibitors, which was ubiquitin-dependent. The study provides evidence for the alleviative effects of lactoferrin and milk in mice with spermatogenetic dysfunction through ERK1/2 mediated the ubiquitin-dependent degradation of P62. The involved signals and molecules could be identified as novel therapeutic targets for male infertility, which contributes to expanding LF's interests in research and application.

The development of novel wound dressings with highly effective antibacterial and accelerating wound healing properties has become the focus of current research. In this study, a novel and injectable lactoferrin (LF)/lithium magnesium silicate hydrogel (LMSH) was first synthesized through a simple electrostatic interaction method. The physical and biological properties are systematically characterized. The results show that the synthesized LF/LMSH has good antibacterial properties and biocompatibility. More importantly, it can effectively promote wound healing in the rat full-thickness skin wound model after 14 days post-operation, and the healing rate can reach 99.1 %, which is much higher than that of other groups. Meanwhile, histochemical and immunofluorescent staining confirm that the prepared injectable LF/LMSH has good pro-collagen deposition, pro-angiogenic and anti-inflammatory properties. The healed wounds present a consistently thickened epidermis with more follicular and glandular structures, indicating the great potential of the prepared material for wound management.

This study identifies promising potential of a novel and safer nanocombination of bovine milk lactoperoxidase (LPO) and lactoferrin (LF) to target breast cancer in vitro and in adult female albino rat model. Favorable selective anticancer effects of the prepared nanocombination were observed, in a dose-dependent manner, against both MCF-7 and MDA cell lines, sparing normal HFB-4 cells. The administration of LPO + LFNPs markedly improved the induced-breast cancer disorders, prolonged survival and reduced the values of serum TNF-α, IL1β, CD4

Background: The ESKAPE group of pathogens which comprise of multidrug resistant bacteria, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are the cause of deadly nosocomial infections all over the world. While these pathogens have developed robust strategies to resist most antibiotics, their ability to form biofilms is one of their most combative properties. Hence there is an urgent need to discover new antibacterial agents which could prevent or destroy the biofilms made by these bacteria. Though it has been established that lactoferrin (LF), a potent iron binding antibacterial, antifungal, and antiviral protein displays anti-biofilm properties, its mechanisms of action, in addition to its iron chelation property, still remains unclear. Objective: The binding and inhibition studies of LF with the enzyme Nucleoside diphosphate Kinase (NDK) and its elastase cleaved truncated 12 kDa fragment (12-NDK). Methods: The characterization studies of NDK and 12-NDK using florescence spectroscopy, dynamic light scattering, size exclusion chromatography and ADP-glo Kinase Assay. Inhibition studies of LF-NDK using ADP-glo kinase assay, Surface Plasmon Resonance and Biofilm inhibition studies. Results: NDK and 12-NDK were cloned, expressed and purified from Acinetobacter baumannii and Pseudomonas aeruginosa. The characterization studies revealed NDK and 12-NDK from both species are stable and functional. The inhibition studies of LF-NDK revealed stable binding and inhibition of kinase activity by LF. Conclusion: The binding and inhibition studies have shown that while LF binds with both the NDK and their truncated forms, it tends to have a higher binding affinity with the truncated 12 kDa fragments, resulting in their decreased kinase activity. This study essentially gives a new direction to the field of inhibition of biofilm formation, as it proves that LF has a novel mechanism of action in other than iron sequestration.

We present a mass spectral library-based method for analyzing site-specific N-linked protein glycosylation. Its operation and utility are illustrated by applying it to both newly measured and available proteomics data of human milk glycoproteins. It generates two varieties of mass spectral libraries. One contains glycopeptide abundance distribution spectra (GADS). The other contains tandem mass spectra of the underlying glycopeptides. Both originate from identified glycopeptides in proteolytic digests of human milk and purified glycoproteins, which include tenascin, lactoferrin, and several antibodies. Analysis was also applied to digests of a NIST human milk standard reference material (SRM), leading to a GADS library of N-glycopeptides, enabling the direct comparison of glycopeptide distributions for individual proteins. Tandem spectra underlying each glycopeptide GADS peak are combined to create a second type of library that contains spectra of the underlying glycopeptide spectra. These were acquired by higher-energy (stepped) collision dissociation fragmentation followed by ion-trap fragmentation. Spectra are annotated using MS_Piano, recently reported annotation software. This data, with extensions of a widely used spectral library search and display software, provides accessible mass spectral libraries.

Deoxynivalenol (DON) is among the most prevalent contaminants in cereal crops and has been demonstrated to impair male spermatogenesis and induce oxidative stress, testicular apoptosis, and disruption of the blood–testis barrier (BTB). Lactoferrin (LF) is an iron-binding glycoprotein with multifunctions including anti-inflammation and antioxidation. Thus, this study aimed to investigate the effects of LF on the spermatogenesis and integrity of the BTB in DON-exposed mice. Thirty-two male mice were allotted to four groups for a 35-day feeding period: vehicle (basal diet), DON (12 mg/kg), LF (10 mg/d, p.o.), and DON + LF. The results showed that DON induced vacuolization of the spermatogenic epithelium, broke the adhesion junction between Sertoli cells and spermatids established by N-cadherin and induced testicular oxidative stress. LF administration restored sperm production, attenuated the DON-induced oxidative stress and reduced the breakages in adhesion junction. DON exposure enhanced the protein expression of occludin. Transcriptional profiling of the testis observed a disturbance in the expression profiles of cell adhesion and inflammatory response genes, and LF administration reversed these gene expressions. Furthermore, down-regulated signaling pathways, including the apical junction, TNFα signaling via NF-κB, and TGF-β in the DON group were observed. These were restored by LF. Enrichment analysis between DON + LF group and vehicle also confirmed the absence of these pathways. These findings indicated that LF eliminated the DON-induced detriment to spermatogenesis and cell connections between Sertoli cells and spermatids via improving antioxidant capacity and modifying the inflammatory response and cell adhesion genes.

Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are produced at high concentrations during inflammation and oxidative/halogenative stress by myeloperoxidase, an enzyme of azurophilic neutrophilic granules. Here we compared the effects of recombinant human lactoferrin (rhLTF) and its halogenated derivatives (rhLTF-Cl and rhLTF-Br) on functional responses of neutrophils. Our results demonstrated that after halogenative modification, rhLTF lost its ability to induce mobilization of intracellular calcium, actin cytoskeleton reorganization, and morphological changes in human neutrophils. Moreover, both forms of the halogenated rhLTF prevented binding of N-acetylglucosamine-specific plant lectin Triticum vulgaris agglutinin (WGA) to neutrophils and, in contrast to native rhLTF, inhibited respiratory burst of neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and by two plant lectins (WGA and PHA-L). However, we observed no differences between the effects of rhLTF, rhLTF-Cl, and rhLTF-Br on respiratory burst of neutrophils induced by phorbol 12-myristate 13-acetate (PMA), digitonin, and number of plant lectins with different glycan-binding specificity. Furthermore, all rhLTF forms interfered with PMA- and ionomycin-induced formation of neutrophil extracellular traps. Thus, halogenative modification of LTF is one of the mechanisms involved in modulating a variety of signaling pathways in neutrophils to control their pro-inflammatory activity.

The formation of complexes may be used for the development of delivery systems in foods field. The aim of this study was to explore the interaction mechanism between Lentinus edodes mycelia polysaccharide (LMP) and bovine lactoferrin (BLF), and the activity of LMP-BLF complex to inhibit oxidative stress in islet β cells. The interaction mechanisms of LMP with BLF were investigated with multi-spectroscopic techniques. The multi-spectroscopic analysis result showed that LMP bound with BLF by van der Waals force and hydrogen bond. The quenching mechanism of BLF with LMP was static quenching. Cell viability, reactive oxygen species (ROS) level, apoptosis and the related signaling pathways were detected with thiazolyl blue tetrazolium bromide (MTT) assay, 2,7-Dichlorofluorescin diacetate (DCFH-DA) staining, Hoechst 33258 staining and Western blot methods respectively. The complex alleviated apoptosis induced by hydrogen peroxide (H2O2), and inhibited oxidative stress via MAPK pathways in MIN6 cells. In addition, the complex was able to promote glucose uptake in HepG2 cells. These results will broaden our understanding of LMP-BLF complexes and the applications of polysaccharide-protein complexes in the foods field.

Lactoferrin (LF) has attracted great attention due to its various bioactivities, which depend on the degree of saturation with different cations. This study focused on the synergistic effect of LF and Zn2+ on human gingival fibroblasts (hGFs), considering antioxidant activities, cell proliferation, and collagen gene expression levels in these cells to improve the wound healing. The hGFs were cultured in an experimental medium, containing 1000 μg/mL of LF and various concentrations of ZnCl2. The cells were subjected to oxidative damage by exposure to 600 μM H2O2 for 30 min before incubation in the experimental medium. The cell proliferation rate and the relative gene expression levels of genes associated with apoptosis, antioxidant enzymes, and collagen were compared. H2O2 decomposition by LF was also measured using a colorimetric assay. LF enhanced hGF proliferation and the expression of collagen. Furthermore, LF directly scavenged H2O2 and prevented lipid peroxidation by enhancing the expression of glutathione peroxidase 4 gene expression, resulting in the prevention of apoptosis and recovery of the cells from H2O2-induced oxidative damage. The addition of ZnCl2 enhanced these results. The results indicated that LF with Zn-ion could play an important role in modulating the functions related to wound healing.

In the present work, lactoferrin (Lf) based nanoparticle incorporated self-supporting gel encapsulating a flavonoid, quercetin (Q), was developed. The complex formation between Lf and Q was assessed using molecular docking and dynamics simulation that lactoferrin and quercetin showed strong interaction and binding supporting hydrophobic interaction. The microscopic, spectroscopic, and x-ray techniques were used to characterize the gel extensively. In vitro drug release was studied to understand the release pattern of quercetin from the protein gel. The viscosity of the gel and its rheological characteristics were determined using a Brookfield viscometer. Ex vivo skin permeation studies using vertical diffusion cells were carried out to understand its skin permeation properties. The gel showed strong anti-oxidant activity using the DPPH scavenging assay. The enhanced effect of the Lf-Q complex on antioxidant enzyme activity (superoxide dismutase, catalase, and malondialdehyde), was supported by molecular dynamics, surface hydrophobicity, and in vitro studies. To investigate the effect of the gel on angiogenesis, the chorioallantoic membrane assay was performed and its compatibility with erythrocytes was also assessed. Suitability for topical administration was assessed using skin irritation studies performed on Sprague Dawley rats. The overall results suggest that the developed NiPG is suitable for cutaneous localization of quercetin with enhanced antioxidant activity.

Understanding nonionic surfactant-protein interactions is fundamental from both technological and scientific points of view. However, there is a complete absence of kinetic data for such interactions. We employed surface plasmon resonance (SPR) to determine the kinetic and thermodynamic parameters of bovine lactoferrin-Brij58 interactions at various temperatures under physiological conditions (pH 7.4). The adsorption process was accelerated with increasing temperature, while the desorption rate decreased, resulting in a more thermodynamically stable complex. The kinetic energetic parameters obtained for the formation of the activated complex, [bLF-Brij58]‡, indicated that the potential energy barrier for [bLF-Brij58]‡ formation arises primarily from the reduction in system entropy. [bLF-Brij58]○ formation was entropically driven, indicating that hydrophobic interactions play a fundamental role in bLF interactions with Brij58.

For many years, natural products have played a crucial role in drug discovery and drug design as a source of active agents or as inspiration. Lactoferrin (Lf), a glycoprotein found in milk and mammalian secretions, has been extensively studied in recent years, and numerous antimicrobial, anti-inflammatory, and anticancer properties of Lf have been demonstrated in the literature. The use of lactoferrin as a co-agent or supplement to enhance the beneficial effect of drugs, or to reduce their side effects, arouses the interest of many researchers, especially since Lf is a well-studied, biocompatible, cheap, and easily accessible protein. In this mini-review, we focus on the elucidation of the role of Lf in antimicrobial or anticancer therapies, pointing to the possible mechanism underlying the determined synergism between Lf and commonly used drugs.

Mucormycosis is a deadly infection which is caused by fungi of the order Mucorales including species belonging to the genus Rhizopus, Mucor, Mycocladus, Rhizomucor, Cunninghamella, and Apophysomyces. Despite antifungal therapy and surgical procedures, the mortality rate of this disease is about 90-100% which is exceptionally high. The hypersensitivity of patients with raised available serum iron indicates that the Mucorales are able to use host iron as a critical factor of virulence. This is because iron happens to be a crucial element playing its role in the growth of cells and development. In this review, we have described Lactoferrin (Lf) as a potential iron-chelator. Lf is a naturally occurring glycoprotein which is expressed in most of the biological fluids. Moreover, Lf possesses exclusive anti-inflammatory effects along with several anti-fungal effects that could prove to be helpful to the pathological physiology of inexorable mucormycosis cases. This literature summarises the biological insights into the Lf being considered as a potential fungistatic agent and an immune regulator. The review also proposes that unique potential of Lf as an iron-chelator can be exploited as the adjunct treatment for mucormycosis infection.

Chloroquine (CQ) is a famous medicine for treatment of diseases including malaria and pneumonia caused by COVID-19, but gastrointestinal disorder caused by its oral administration is a great concern. Milk is usually recommended to be taken with CQ to reduce such effect. However, the mechanism underlying this phenomenon remains unknown. Here, we found that β-lactoglobulin (β-LG), α-lactalbumin (α-LA), bovine serum albumin (BSA), and lactoferrin (LF) in whey proteins were able to interact with CQ to form complexes as suggested by fluorescence resonance energy transfer (FRET) and molecular docking. Indeed, the crystal structure revealed that β-LG is bound to CQ through hydrophobic interactions and hydrogen bonding with a ratio of 1:1. Consequently, the formation of these protein-CQ complexes not only reduced the cytotoxicity of chloroquine to the stomach and gut cells but also facilitated its uptake by cells. This work gave an example to understand the relationship between food and drug.

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.

Adverse pregnancy outcomes affect 54 million people globally per year, with at least 50% of these attributed to infection during gestation. These include inflammation of the membranes surrounding the growing fetus (chorioamnionitis), preterm prelabor rupture of membranes (PPROM), preterm birth (PTB), early-onset disease (EOD) and late-onset disease (LOD), neonatal and maternal sepsis, and maternal or fetal demise. Although universal screening and implementation of intrapartum antibiotic prophylaxis (IAP) has improved EOD outcomes, these interventions have not reduced the incidences of LOD or complications occurring early on during pregnancy such as PPROM and PTB. Thus, novel therapies are needed to prevent adverse pregnancy outcomes and to ameliorate disease risk in vulnerable populations. Lactoferrin has recently been explored as a potential therapeutic as it demonstrates strong antimicrobial and anti-biofilm activity. Lactoferrin is a glycoprotein capable of iron chelation found in a variety of human tissues and is produced in high concentrations in human breast milk. In recent studies, lactoferrin has shown promise inhibiting growth and biofilm formation of streptococcal species, including Group B Streptococcus (GBS), a prominent perinatal pathogen. Understanding the interactions between lactoferrin and GBS could elucidate a novel treatment strategy for adverse pregnancy outcomes caused by GBS infection.

Biofilms are complex bacterial structures composed of bacterial cells embedded in extracellular polymeric substances (EPS) consisting of polysaccharides, proteins and lipids. As a result, biofilms are difficult to eradicate using both mechanical methods, i.e., scraping, and chemical methods such as disinfectants or antibiotics. Bacteriophages are shown to be able to act as anti-biofilm agents, with the ability to penetrate through the matrix and reach the bacterial cells. However, they also seem to have their limitations. After several hours of treatment with phages, the biofilm tends to grow back and phage-resistant bacteria emerge. Therefore, it is now recommended to use a mixture of phages and other antibacterial agents in order to increase treatment efficiency. In our work we have paired staphylococcal phages with lactoferrin, a protein with proven anti-biofilm proprieties. By analyzing the biofilm biomass and metabolic activity, we have observed that the addition of lactoferrin to phage lysate accelerated the anti-biofilm effect of phages and also prevented biofilm re-growth. Therefore, this combination might have a potential use in biofilm eradication procedures in medical settings.

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes ( IFNB, IFNL1, and IFNL2) and IFN-stimulated genes ( ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.

The impact of covalent attachment of (-)-epigallocatechin gallate (EGCG) to lactoferrin (LF) on the structure, morphology, functionality, and allergenicity of the protein was studied. These polyphenol-protein conjugates were formed using various enzymatic (laccase- and tyrosinase-catalyzed oxidation) and nonenzymatic (free radical grafting and alkali treatment) methods. The preparation conditions for forming the enzymatic conjugates were optimized by exploring the influence of order-of-addition effects: protein, polyphenols, and enzymes. The total phenol content of the LF-EGCG conjugates was quantified using the Folin-Ciocalteu method. The nature of the conjugates formed was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy analyses. These studies showed that enzymatic cross-linking was a highly effective means of forming LF-EGCG conjugates. Analysis of these conjugates using various spectroscopic methods showed that conjugation to EGCG changed the molecular structure of LF. Atomic force microscopy showed that the four covalent cross-linking methods affected the size and morphology of these LF-EGCG conjugates formed. The antioxidant activity and emulsifying stability of LF were significantly improved by conjugation to EGCG. Finally, an enzyme-linked immunosorbent assay (ELISA) and a western blot assay indicated that conjugation of EGCG reduced the binding capacity of LF to immunoglobulin E (IgE) and immunoglobulin G (IgG), which is consistent with a decrease in allergenicity. Overall, this study suggests that LF-EGCG conjugates formed using enzymatic or nonenzymatic methods have potential applications as functional ingredients in foods.

As classical MRPs, the toxic effects of furosine, pyralline, and 5‐hydroxymethylfurfural (5‐HMF) in liver tissue are evaluated and the related mechanism is investigated here, and the protective effects of lactoferrin on liver injury caused by Maillard reaction products (MRPs) with furan ring are proved in vitro and in vivo. First, we detect the concentrations of furosine, pyralline, and 5‐HMF in several foods using ultrahigh‐performance liquid chromatography (UHPLC). Then, the effects of the three MRPs on liver cells (HL‐7702) viability, as well as liver tissue, are performed and evaluated. Furthermore, the regulations of three MRPs on necroptosis‐related pathway in liver cells are investigated. Additionally, the effects of lactoferrin in alleviating liver injury, as well as regulating necroptosis pathway, were evaluated. Results elucidate that lactoferrin protects liver injury caused by MRPs with furan ring structure through activating RIPK1/RIPK3/p‐MLKL necroptosis pathway and downstream inflammatory reaction., Lactoferrin protects liver injury caused by MRPs with furan ring structure through activating RIPK1/RIPK3/p‐MLKL necroptosis pathway and downstream inflammatory reaction.

This study aimed to investigate the preventive effects of lactoferrin (Lf) on chronic alcoholic liver injury (ALI) in female mice. Female C57BL/6J mice were randomly divided into four groups: control group (CON), ethanol administration group (EtOH), low-dose Lf treatment group (LLf), and high-dose Lf group (HLf). In the last three groups, chronic ALI was induced by administering 20% ethanol ad libitum for 12 weeks. Mice in the CON and EtOH groups were fed with AIN-93G diet. Meanwhile, 0.4% and 4% casein in the AIN-93G diet were replaced by Lf as the diets of LLf and HLf groups, respectively. HLf significantly reduced hepatic triglyceride content and improved pathological morphology. HLf could inhibit cytochrome P450 2E1 overexpression and promote alcohol dehydrogenase-1 expression. HLf activated protein kinase B and AMP-activated protein kinase (AMPK), as well as upregulating nuclear-factor-erythroid-2-related factor-2 expression to elevate hepatic antioxidative enzyme activities. AMPK activation also benefited hepatic lipid metabolism. Meanwhile, HLf had no obvious beneficial effects on gut microbiota. In summary, Lf could alleviate chronic ALI in female mice, which was associated with redox balance and lipid metabolism regulation.

The milk-derived bovine lactoferrin (bLf) is an iron-binding glycoprotein with remarkable selective anticancer activity towards highly metastatic cancer cells displaying the proton pump V-ATPase at the plasma membrane. As studies aiming to dissect the bLf mechanisms of action are critical to improve its efficacy and boost its targeted clinical use, herein we sought to further uncover the molecular basis of bLf anticancer activity. We showed that bLf co-localizes with V-ATPase and cholesterol-rich lipid rafts at the plasma membrane of highly metastatic cancer cells. Our data also revealed that bLf perturbs cellular trafficking, induces intracellular accumulation of cholesterol and lipid rafts disruption, downregulates PI3K, and AKT or p-AKT and inhibits glycolysis of cancer cells harbouring V-ATPase at the plasma membrane lipid rafts. Altogether, our results can lay the foundation for future bLf-based targeted anticancer strategies as they unravel a novel cascade of molecular events that explains and further reinforces bLf selectivity for cancer cells displaying plasmalemmal V-ATPase.

A 56-day feeding trial was conducted to investigate the effects of dietary lactoferrin (LF) supplementation on growth performance and intestinal health of juvenile orange-spotted groupers (Epinephelus coioides) fed high soybean meal (SBM) diets. The control diet (FM) and high soybean meal diet (SBM60) were prepared to contain 48% protein and 11% fat. Three inclusion levels of 2, 6, and 10 g/kg LF were added into the SBM diets to prepare three experimental diets (recorded as LF2, LF6, and LF10, respectively). The results showed that supplementation of LF in SBM diets increased growth rate in a dose dependent manner. However, feed utilization, hepatosomatic index, whole-body proximate composition and the abundance and diversity of intestinal microbiota did not vary across dietary treatments (P > 0.05). After dietary intervention with LF, the contents of intestinal malondialdehyde, endotoxin, and D-lactic acid, as well as plasma low density lipoprotein cholesterol, high density lipoprotein cholesterol and total cholesterol were lower, and the intestinal activities of glutathione peroxidase, lipase, trypsin, and protease were higher in LF2-LF10 groups than that in SBM60 group (P < 0.05). Supplementation of LF in SBM diets increased the muscle layer thickness of middle and distal intestine and the mucosal fold length of middle intestine vs SBM60 diet. Furthermore, supplementation of LF in SBM diets resulted in an up-regulation of the mRNA levels for IL-10 and TGF-β1 genes and a down-regulation of the mRNA levels of IL-1β, IL-12, IL-8, and TNF-α genes vs SBM60 diet (P < 0.05). The above results showed that dietary LF intervention could improve the growth and alleviate soybean meal induced enteritis in juvenile orange-spotted groupers. The dietary appropriate level of LF was at 5.8 g/kg, through the regression analysis of percent weight gain against dietary LF inclusion levels.

Alzheimer's Disease (AD) represents the most frequent type of dementia in elderly people. There are two major forms of the disease: sporadic (SAD) - whose causes are not completely understood - and familial (FAD) - with clear autosomal dominant inheritance. The two main hallmarks of AD are extracellular deposits of amyloid-beta (Ab) peptide and intracellular deposits of the hyperphosphorylated form of the tau protein (P-tau). An ever-growing body of research supports the infectious hypothesis of sporadic forms of AD. Indeed, it has been documented that some pathogens, such as herpesviruses and certain bacterial species, are commonly present in AD patients, prompting recent clinical research to focus on the characterization of Antimicrobial Peptides (AMPs) in this pathology. Literature also demonstrates that Ab can be considered itself as an AMP thus representing a type of innate immune defense peptide that protect the host against a variety of pathogens. Beyond Ab, other proteins with antimicrobial activity, such as lactoferrin, defensins, cystatins, thymosin β4, LL37, histatin 1 and statherin have been shown to be involved in AD. Here we have summarized and discussed these findings and explored the diagnostic and therapeutic potential of AMPs in AD.

Pancreatic cancer is a devastating gastrointestinal tumor with limited Chemotherapeutic options. Treatment is restricted by its poor vascularity and dense surrounding stroma. Quinacrine is a repositioned drug with an anticancer activity but suffers a limited ability to reach tumor cells. This could be enhanced using nanotechnology by the preparation of quinacrine-loaded Undaria pinnatifida fucoidan nanoparticles. The system exploited fucoidan as both a delivery system of natural origin and active targeting ligand. Lactoferrin was added as a second active targeting ligand. Single and dual-targeted particles prepared through nanoprecipitation and ionic interaction respectively were appraised. Both particles showed a size lower than 200 nm, entrapment efficiency of 80% and a pH-dependent release of the drug in the acidic environment of the tumor. The anticancer activity of quinacrine was enhanced by 5.7 folds in dual targeted particles compared to drug solution with a higher ability to inhibit migration and invasion of cancer. In vivo, these particles showed a 68% reduction in tumor volume compared to only 20% for drug solution. In addition, they showed a higher animals' survival rate with no hepatotoxicity. Hence, these particles could be an effective option for the eradication of pancreatic cancer cells.

The immunomodulatory nature of lactoferrin (LF) derives from its ability to bridge innate and adaptive immunity in obtaining physiological equilibrium. LF is an attractive molecule for treatment of diseases that compromise immune homeostasis. Oral delivery is a preferable method for LF administration; however, its bioavailability is affected by protein degradation and absorption. The aim of this study was to evaluate the systemic effects of orally and intravenously (IV) administered recombinant human LF (rhLF) on blood cell transcriptome profiling. Rats were administered a single dose of rhLF by gavage or IV. The transcriptome profiles from the control and the rhLF-treated rats after 3, 6, and 24 h were analyzed using a Clariom D microarray. The results showed differentially expressed genes in response to IV as well as oral administered rhLF including coding and noncoding RNAs. Moreover, a comparison of the differentially expressed genes between oral and IV administration of LF, after 6 h, revealed that the majority (72.8%) of the genes altered in response to oral administration of rhLF were the same as for the IV treatment. The pathway profiles showed similarities in up-regulation of specific genes involved in oxidative stress and inflammatory responses for both routes of treatments. These findings provide evidence of the systemic signal transduction effects of orally administered rhLF.

Lactoferrin as a nutritional enteral supplement has emerged as a novel preventative therapy against serious infections in preterm infants, although neonatal studies have demonstrated variable results, in part due to the lack of pharmacokinetic data and differences in the products tested. We conducted a prospective, dose escalation (100, 200, and 300 mg·kg–1·day–1) safety study of bovine lactoferrin (Glanbia Nutritionals, USA) dissolved in sterile water (100 mg·mL–1) for 30 days in preterm infants with birth weight –1·day–1, respectively)] over a 10-month period. No AEs related to the study solution occurred, and lactoferrin was tolerated by each group. During lactoferrin supplementation, one bloodstream infection occurred in each group, but there were no incidences of urinary tract infections and no cases of necrotizing enterocolitis. Postnatal cytomegalovirus acquisition was detected in the group treated with 200 mg·kg–1·day–1 (n = 2). There were no adverse effects on hepatic, renal, or hematologic function. All of the patients survived to discharge. Bovine lactoferrin at doses up to 300 mg·kg–1·day–1 is safe in preterm infants. Future studies examining higher doses of lactoferrin, length of treatment, and potency of different products will aid in determining the optimal approach for the use of lactoferrin to prevent infections in preterm infants.

We previously conducted two randomized controlled trials with bovine lactoferrin (bLF) for the prevention of late-onset sepsis (LOS) in infants with a birth weight –1·day–1], and used the same control (maltodextrin). We fitted multivariate Cox regression models to estimate the effect of bLF on the risk of development of the composite outcome, adjusting for covariates. We included 335 neonates with a mean birth weight of 1162 ± 244 g; 27.5% were

Lactoferrin supplementation may help prevent infections in preterm infants, but the efficacy has varied with different doses and products. We assessed the absorption and excretion of bovine lactoferrin (bLF) in 31 infants receiving 100, 200, or 300 mg·kg–1·day–1 of enteral bLF for 30 days. bLF and human lactoferrin (hLF) in infant saliva, blood, urine, and stool, as well as expressed (EBM) or donor breast milk (DBM) that were collected (i) before the treatment was initiated, (ii) at study day 22, and (iii) one week after treatment cessation, were measured using ELISA. During treatment, bLF was absorbed from the gastrointestinal tract and detected in plasma, saliva, and urine, as well as excreted in stool. Levels of bLF in the saliva and stool began to decline within 12 h after dosing, and bLF was undetectable in all samples one week after treatment. The concentrations of hLF exceeded those of bLF across sample types and time-points. Infants receiving EBM demonstrated higher levels of hLF in the saliva and stool than those receiving DBM. Neither bLF nor hLF levels varied by patient characteristics, bLF dosage, or infection status. This is the first study demonstrating bLF absorption into the bloodstream and distribution to saliva and urine in preterm infants. Future studies should further explore LF pharmacokinetics because higher and more frequent dosing may improve the clinical benefit of LF supplementation.

Milk contains bioactive molecules with important functions as defensive proteins; among them are the whey protein lactoferrin and proteins of the milk fat globule membrane (MFGM) present in buttermilk. The aim of this study has been to investigate the effects of lactoferrin, whey, and buttermilk as modulators of intestinal innate immunity and oxidative stress on intestinal epithelial cells, to evaluate its potential use for the development of functional foods. The mRNA expression levels of innate immune system Toll-like receptors (TLR2, TLR4, and TLR9), lipid peroxidation (malondialdehyde + 4-hydroxyalkenals) and protein expression levels of carbonyl were analyzed in enterocyte-like Caco-2/TC7 cells treated for 24 h with different concentrations of lactoferrin, whey, or buttermilk. None of the substances analyzed caused oxidative damage; however, whey significantly decreased the levels of lipid peroxidation. Furthermore, both lactoferrin and whey reduced the oxidative stress induced by lipopolysaccharide. With respect to TLR receptors, lactoferrin, whey, and buttermilk specifically altered the expression of TLR2, TLR4, and TLR9 receptors, with a strong decrease in the expression levels of TLR4. These results suggest that lactoferrin, whey, and buttermilk are potentially interesting ingredients for functional foods because they seem to modulate oxidative stress and the inflammatory response induced by the activation of TLRs.

Primary infection with Mycobacterium tuberculosis (Mtb) results in the formation of a densely packed granulomatous response that essentially limits the entry and efficacy of immune effector cells. Furthermore, the physical nature of the granuloma does not readily permit the entry of therapeutic agents to sites where organisms reside. The Mtb cell wall mycolic acid, trehalose 6,6′-dimycolate (TDM), is a physiologically relevant molecule for modelling macrophage-mediated events during the establishment of the tuberculosis-induced granuloma pathogenesis. At present, there are no treatments for tuberculosis that focus on modulating the host’s immune responses. Previous studies showed that lactoferrin (LF), a natural iron-binding protein proven to modulate inflammation, can ameliorate the cohesiveness of granuloma. This led to a series of studies that further examined the effects of recombinant human LF (rHLF) on the histological progression of TDM-induced pathology. Treatment with rHLF demonstrated significant reduction in size and number of inflammatory foci following injections of TDM, together with reduced levels pulmonary pro-inflammatory cytokines TNF-α and IL-1β. LF facilitated greater penetration of fluoroquinolone to the sites of pathology. Mice treated with TDM alone demonstrated exclusion of ofloxacin to regions of inflammatory response, whereas the animals treated with rHLF demonstrated increased penetration to inflammatory foci. Finally, recent findings support the hypothesis that this mycobacterial mycolic acid can specifically recruit M1-like polarized macrophages; rHLF treatment was shown to limit the level of this M1-like phenotypic recruitment, corresponding highly with decreased inflammatory response.

In this commentary, we summarize the current evidence from randomized controlled trials on enteral lactoferrin supplementation in preterm neonates. Our recently completed systematic review includes 12 randomized controlled trials performed all over the world. Our meta-analysis suggests clinical benefit in decreasing late-onset sepsis, late-onset fungal sepsis, length of stay in the hospital and urinary tract infections. There were no adverse effects. There was no statistically significant decrease in necrotizing enterocolitis, mortality or neurodevelopmental impairment in lactoferrin supplemented preterm infants. There was significant statistical heterogeneity in the effects of lactoferrin on late-onset sepsis between larger and smaller studies, which may reflect either small study biases, differences in the effectiveness, dose or duration of supplemental lactoferrin products, or differences in underlying population risk, or any or all of these.

Myeloperoxidase (MPO) is a unique heme-containing peroxidase that can catalyze the formation of hypochlorous acid (HOCl). The strong interaction of MPO with low-density lipoproteins (LDL) promotes proatherogenic modification of LDL by HOCl. The MPO-modified LDL (Mox-LDL) accumulate in macrophages, resulting in the formation of foam cells, which is the pathognomonic symptom of atherosclerosis. A promising approach to prophylaxis and atherosclerosis therapy is searching for remedies that prevent the modification or accumulation of LDL in macrophages. Lactoferrin (LF) has several application points in obesity pathogenesis. We aimed to study LF binding to Mox-LDL and their accumulation in monocytes transformed into macrophages. Using surface plasmon resonance and ELISA techniques, we observed no LF interaction with intact LDL, whereas Mox-LDL strongly interacted with LF. The affinity of Mox-LDL to LF increased with the degree of oxidative modification of LDL. Moreover, an excess of MPO did not prevent interaction of Mox-LDL with LF. LF inhibits accumulation of cholesterol in macrophages exposed to Mox-LDL. The results obtained reinforce the notion of LF potency as a remedy against atherosclerosis.

Lactoferrin is a multifunctional protein found in the secretions of mammals. The antimicrobial activity of lactoferrin was the first to be discovered and was assumed to be solely dependent on its iron-chelating ability. However, lactoferrin has been reported to display proteolytic activity towards bacterial virulence factors and to modulate the host defence by stimulating the immune system and balancing pathogen-induced inflammation. Here, we review the current understandings of the antimicrobial effect, interaction with host cells, and innate immune modulation of lactoferrin, and put forward this moonlighting protein as a possible alternative for antibiotics.

In this minireview we discuss the role of lactoferrin (LTF) in detoxifying hematoma after intracerebral hemorrhage (ICH). Subsequent to ICH, neutrophils enter the ICH-affected brain, where they release various granule contents, including LTF. LTF is an iron-binding glycoprotein that binds Fe3+ with high affinity. Unlike other iron-binding proteins, LTF can retain Fe3+ at the low pH associated with inflamed tissue. LTF’s ability to sequester Fe3+ is of particular importance to ICH pathogenesis, because large quantities of free iron, which is pro-oxidative and pro-inflammatory, are generated in the ICH-affected brain owing to blood hemolysis. LTF delivered to ICH-affected brain, either as a therapeutic agent or through infiltrated polymorphonuclear neutrophils (cells containing high levels of LTF), could limit the pathogenesis of ICH. LTF is a protein with a high isoelectric point (8.7), a property that enables it to bind to negatively-charged apoptotic cells or proteins. Here, LTF could act as a bridging molecule that couples the apoptotic cells to LTF receptors on the cellular membranes of microglia/macrophages to facilitate the efferocytosis/erythrophagocytosis of apoptotic cells and damaged red blood cells. Thus, by virtue of sequestrating iron and facilitating efferocytosis, LTF may contribute to hematoma detoxification and hematoma/inflammation resolution after ICH.

Lactoferrin (Lf) samples from several manufacturers were evaluated in vitro. The purity and protein form of each Lf were examined by SDS–PAGE, Western blot, and proteomics analysis. Assays were conducted to evaluate uptake of Lfs and iron from Lfs by enterocytes as well as Lf bioactivities, including effects on intestinal cell proliferation and differentiation, IL-18 secretion, TGF-β1 transcription, and growth of enteropathogenic Escherichia coli (EPEC). Composition of the Lfs varies; some only contain a major Lf band (∼80 kDa), and some also contain minor forms. All Lfs and iron from the Lfs were absorbed by Caco-2 cells, with various efficiencies. The bioactivities of the Lfs varied considerably, but there was no consistent trend. All Lfs promoted intestinal cell proliferation, secretion of IL-18, and transcription of TGF-β1. Some Lfs exhibited pro-differentiation effects on Caco-2 cells. Effects of pasteurization (62.5 °C for 30 min, 72 °C for 15 s, or 121 °C for 5 min) on integrity, uptake, and bioactivities were examined using Dicofarm, Tatua, and native bovine Lfs. Results show that pasteurization did not affect protein integrity, but variously affected uptake of Lf and its effects on intestinal proliferation, differentiation, and EPEC growth. To choose a Lf source for a clinical trial, assessment of bioactivities is recommended.

Piglets, especially weaning piglets, show a lower level of immunity and higher morbidity and mortality, owing to their rapid growth, physiological immaturity, and gradual reduction of maternal antibodies, which seriously affects their growth and thus, value. It is important that piglets adapt to nutrient digestion and absorption and develop sound intestinal function and colonization with gut microbiota as soon as possible during their early life stage. Lactoferrin is a natural glycoprotein polypeptide that is part of the transferrin family. It is widely found in mucosal secretions such as saliva and tears, and most highly in milk and colostrum. As a multifunctional bioactive protein and a recommended food additive, lactoferrin is a potential alternative therapy to antibiotics and health promoting additive for piglet nutrition and development. It is expected that lactoferrin, as a natural food additive, could play an important role in maintaining pig health and development. This review examines the following known beneficial effects of lactoferrin: improves the digestion and capacity for absorption in the intestinal tract; promotes the absorption of iron and reduces the incidence of iron deficiency anemia; regulates intestinal function and helps to balance the microbial biota; and enhances the resistance to disease of the piglets via modulating and enhancing the immune system.

In this short review, we outline the major events that led to the development of iron acquisition systems in Gram-negative bacteria and mammals since the beginning of life on earth. Naturally, the interaction between these organisms led to the development of a wonderfully complex set of protein systems used for competition over a once prevalent (but no longer) biocatalytic cofactor. These events led to the appearance of the lactoferrin gene, which has since been exploited into adopting countless new functions, including the provision of highly bactericidal degradation products. In parallel to lactoferrin’s evolution, evolving bacterial receptors have countered the bactericidal properties of this innate immunity protein.

Many pathogenic bacteria can protect themselves from the effects of antibiotics and the host immune response system by forming biofilms. Biofilms are polymer-entrapped bacterial cells, which adhere to each other and are often attached to a surface. Eradication of bacterial biofilms typically requires much higher concentrations of antibiotics than are normally needed to kill cultured planktonic cells, raising serious clinical concerns. In an attempt to prevent the formation of biofilms or to break up existing biofilms of pathogenic bacteria, herein we have used the standard crystal violet assay as well as the Calgary biofilm device to test several lactoferrin- and lactoferricin-derived antimicrobial peptides for their antibiofilm activity against Pseudomonas aeruginosa PAO1. Our results revealed that the short bovine lactoferricin-derived RRWQWR-NH2 (20–25) hexapeptide has no activity against P. aeruginosa PAO1. Moreover, the longer human lactoferricin-derived peptide GRRRRSVQWCA (1–11) and the bovine lactoferrampin (268–284) peptide were also almost devoid of activity. However, several different “mix-and-match” dimeric versions of the two lactoferricin-derived peptides proved quite effective in preventing the formation of biofilms at low concentrations, and in some cases, could even eradicate an existing biofilm. Moreover, the full-length bovine lactoferricinB (17–41) peptide also displayed considerable antimicrobial activity. Some of the longer lactoferricin-derived dimeric peptides acted through a bactericidal mechanism, whereas others seemed to interfere in cell-signalling processes. Taken together, our results indicate that synthetic dimeric peptides comprising short naturally occurring human and bovine lactoferricin constructs could be further developed as antibiofilm agents.