Lactoferrin Affects the Viability of Bacteria in a Biofilm and the Formation of a New Biofilm Cycle of Mannheimia haemolytica A2.

Respiratory diseases in ruminants are responsible for enormous economic losses for the dairy and meat industry. The main causative bacterial agent of pneumonia in ovine is Mannheimia haemolytica A2. Due to the impact of this disease, the effect of the antimicrobial protein, bovine lactoferrin (bLf), against virulence factors of this bacterium has been studied. However, its effect on biofilm formation has not been reported. In this work, we evaluated the effect on different stages of the biofilm. Our results reveal a decrease in biofilm formation when bacteria were pre-incubated with bLf. However, when bLf was added at the start of biofilm formation and on mature biofilm, an increase was observed, which was visualized by greater bacterial aggregation and secretion of biofilm matrix components. Additionally, through SDS-PAGE, a remarkable band of ~80 kDa was observed when bLf was added to biofilms. Therefore, the presence of bLf on the biofilm was determined through the Western blot and Microscopy techniques. Finally, by using Live/Dead staining, we observed that most of the bacteria in a biofilm with bLf were not viable. In addition, bLf affects the formation of a new biofilm cycle. In conclusion, bLf binds to the biofilm of M. haemolytica A2 and affects the viability of bacteria and the formation a new biofilm cycle.

Enhancing Osteoblastic Cell Cultures with Gelatin Methacryloyl, Bovine Lactoferrin, and Bioactive Mesoporous Glass Scaffolds Loaded with Distinct Parsley Extracts.

The increasing interest in innovative solutions for addressing bone defects has driven research into the use of Bioactive Mesoporous Glasses (MBGs). These materials, distinguished by their well-ordered mesoporous structure, possess the capability to accommodate plant extracts with well-established osteogenic properties, including bovine lactoferrin (bLF), as part of their 3D scaffold composition. This harmonizes seamlessly with the ongoing advancements in the field of biomedicine. In this study, we fabricated 3D scaffolds utilizing MBGs loaded with extracts from parsley leaves (PL) and embryogenic cultures (EC), rich in bioactive compounds such as apigenin and kaempferol, which hold potential benefits for bone metabolism. Gelatin Methacryloyl (GelMa) served as the polymer, and bLF was included in the formulation. Cytocompatibility, Runx2 gene expression, ALP enzyme activity, and biomineralization were assessed in preosteoblastic MC3T3-E1 cell cultures. MBGs effectively integrated PL and EC extracts with loadings between 22.6 ± 0.1 and 43.6 ± 0.3 µM for PL and 26.3 ± 0.3 and 46.8 ± 0.4 µM for EC, ensuring cell viability through a release percentage between 28.3% and 59.9%. The incorporation of bLF in the 3D scaffold formulation showed significant differences compared to the control in all assays, even at concentrations below 0.2 µM. Combinations, especially PL + bLF at 0.19 µM, demonstrated additive potential, with superior biomineralization compared to EC. In summary, this study highlights the effectiveness of MBGs in incorporating PL and EC extracts, along with bLF, into 3D scaffolds. The results underscore cytocompatibility, osteogenic activity, and biomineralization, offering exciting potential for future in vivo applications.

Antitumor activity of bovine lactoferrin and its derived peptides against HepG2 liver cancer cells and Jurkat leukemia cells.

Liver cancer and leukemia are the fourth and first causes, respectively, of cancer death in children and adults worldwide. Moreover, cancer treatments, although beneficial, remain expensive, invasive, toxic, and affect the patient's quality of life. Therefore, new anticancer agents are needed to improve existing agents. Because bovine lactoferrin (bLF) and its derived peptides have antitumor properties, we investigated the anticancer effect of bLF and LF peptides (LFcin17-30, LFampin265-284 and LFchimera) on liver cancer HepG2 cells and leukemia Jurkat cells. HepG2 and Jurkat cells were incubated with bLF and LF peptides. Cell proliferation was quantified by an MTT assay, and cell morphology and damage were visualized by light microscopy or by phalloidin-TRITC/DAPI staining. The discrimination between apoptosis/necrosis was performed by staining with Annexin V-Alexa Fluor 488 and propidium iodide, and the expression of genes related to apoptosis was analyzed in Jurkat cells. Finally, the synergistic interaction of bLF and LF peptides with cisplatin or etoposide was assessed by an MTT assay and the combination index. The present study demonstrated that bLF and LF peptides inhibited the viability of HepG2 and Jurkat cells, inducing damage to the cell monolayer of HepG2 cells and morphological changes in both cell lines. bLF, LFcin17-30, and LFampin265-284 triggered apoptosis in both cell lines, whereas LFchimera induced necrosis. These results suggested that bLF and LF peptides activate apoptosis by increasing the expression of genes of the intrinsic pathway. Additionally, bLF and LF peptides synergistically interacted with cisplatin and etoposide. In conclusion, bLF and LF peptides display anticancer activity against liver cancer and leukemia cells, representing an alternative or improvement in cancer treatment.

Analysis of the intestinal IgA response in mice exposed to chronic stress and treated with bovine lactoferrin.

Immunoglobulin (Ig) A, an antibody with a pivotal role in gut homeostasis, can be modulated by stress and bovine lactoferrin (bLf). The aim of the present study was to analyze the impact of chronic stress on the IgA response in the small intestine during bLf treatment. Male BALB/c mice (n=6 mice/group) underwent 1 h of chronic stress by immobilization for 7 consecutive days or were left unstressed, and were untreated or treated with bLf (50, 500 or 5,000 µg). Plasma corticosterone expression levels were determined by ELISA. The distal small intestine was dissected to analyze: i) total IgA, secretory IgA and IgG, as well as and specific IgA and IgG antibody levels in the intestinal liquid by ELISA; ii) α­chain and polymeric immunoglobulin receptor (pIgR) protein expression in epithelial cell extracts analyzed by western blotting; iii) the mRNA expression levels of α­/J­chains, pIgR, IL­2, IL­4, IL­5 and IL­6 in whole mucosal samples by reverse transcription­quantitative PCR. Data were analyzed by one­way ANOVA, and the differences were analyzed by the Holm­Sidák post hoc test and were considered significant if P<0.05. Results from the present study revealed the upregulatory effects of chronic stress on the total antibody levels, protein (α­chain; 78­kDa pIgR) and mRNA (α­ and J­chains; pIgR; IL­6) expression levels were restricted by bLf under stress. The effects of chronic stress on the downregulation of IL­2 and IL­4 mRNA expression were not changed by bLf under stress. The corticosterone response in unstressed mice treated with 5,000 µg bLf and the specific­IgG levels in the unstressed and stressed groups treated with bLf at all doses were increased. The findings suggested an effect of bLf in maintaining homeostasis under stress.

Bovine lactoferrin as a Modulator of Neuroendocrine Components of Stress.

Stress is a condition that maintains the homeostasis of the organism through the activation of different neuroendocrine pathways and secretion of a wide array of chemical mediators, including corticotropin-releasing hormone (CRH), neurotransmitters and glucocorticoids hormones. These molecules fulfill important physiological functions, but under stressful conditions, they can induce or aggravate a pathological state depending on type, severity and duration of stress. For this reason, the search for compounds that modulate the activity of the neuroendocrine pathways is crucial for the control of diseases associated with stressful situations. Bovine lactoferrin (bLf) is an iron-binding multifunctional glycoprotein that exhibits modulatory properties on the neuroendocrine system. Bovine lactoferrin affects the production and secretion of neuroendocrine components of the hypothalamus-pituitary-adrenal (HPA) axis. Neuroendocrine mechanisms of bLf entail either the down- or up-modulation of adrenal corticosteroids via HPA pathway activation, nitric oxide (NO) generation and opioid nervous system pathway activation. This manuscript is focused on reviewing the current contributions of bLf modulatory actions on the response of hormones, neurotransmitters involved in stress and behavior. Sustained use of drugs for stress-associated dysfunctions loses efficacy and requires the dose increase by tolerance and drug dependence. Therefore, bLf may be included as a therapeutic and/or adjunctive agent of drugbased therapies for the treatment of stress-associated emotional-disturbances.

Bovine Lactoferrin can Decrease the In Vitro Biofilm Production and Show Synergy with Antibiotics Against Listeria and Escherichia coli Isolates.

BACKGROUND: Bovine Lactoferrin (bLf) has been reported as antimicrobial, antiviral, immunomodulatory and anticancer protein. Escherichia coli and Listeria spp. are food-borne bacteria that can produce illness in human being and mammals, the emergent antimicrobial drug resistance has been reported in these pathogens. OBJECTIVE: The aim for this study was to evaluate the bLf effect on in vitro biofilm production and the synergic effect of antibiotics on E. coli and Listeria isolates. METHODS: E. coli and Listeria specimens were isolated from bovine carcasses and slaughterhouses surfaces, respectively. Biofilm formation was analyzed with or without bLf, incubated for 48 h and spectrophotometry, cell viability was analyzed by colony-forming unit (CFU) and the synergistic effect of bLf with ampicillin, oxytetracycline, and streptomycin was evaluated through the fractional concentration index (FCI). RESULTS: Our results show that a low bLf concentration (0.8 µM) can diminish the in vitro biofilm production in Listeria isolates; also improves the in vitro oxytetracycline and streptomycin activity against E. coli, and ampicillin activity against Listeria isolates. CONCLUSION: bLf can affect the biofilm production in Listeria isolates from slaughterhouses surfaces and shown synergic effect with ampicillin. Also has a synergic effect with oxytetracycline and streptomycin against E. coli isolates from bovine carcasses.

Naringenin-lactoferrin binding: Impact on naringenin bitterness and thermodynamic characterization of the complex.

Naringenin (NG) is a flavonoid with many bioactive properties, however, its bitterness limits its use in foods. It is known that complex formation with proteins can mask this undesirable sensory property. Therefore, a trained panel evaluated the effect of bovine lactoferrin (LF) on NG bitterness using time-intensity analysis. LF reduced the maximum bitterness intensity and overall bitterness perception for NG by 27% and 33%, respectively. Isothermal titration nanocalorimetry (ITC), molecular docking (DC), and molecular dynamics (MD) were used to characterize NG-LF binding. These techniques provided similar values of ΔG° for binding ( [Formula: see text]  = -33.42 kJ mol-1; [Formula: see text]  = -32.22 kJ mol-1; [Formula: see text]  = -31.84 kJ mol-1). ITC showed that the complex formation is primarily entropy driven and DC suggested that NG binds at a hydrophobic site in LF. Here are presented strategic tools for promoting NG incorporation in food and health products.

Surface plasmon resonance study of interaction between lactoferrin and naringin.

Lactoferrin (LF) is a glycoprotein that serves as a potential vehicle for small bioactive molecules in food. In an effort to improve this functionality, the kinetic and thermodynamic interaction of LF with naringin (NR) was studied by surface plasmon resonance (SPR). The results demonstrated that the association rate constant between LF and NR was 5.00 × 104 M-1 s-1, while the dissociation rate of the complex was 0.36 s-1, at 25 °C. The stable complex predominated over free molecules (ΔG25°C0=-29.35 kJ mol-1), and the binding constant was 1.39 × 105 M-1, at 25 °C. The association of LF and NR to form an intermediate complex occurred in multi-steps. Nevertheless, the intermediate complex formation from the dissociation of the stable complex occurred in a single step with the activation energy independent of temperature. This study provides an important basis to explore LF as a vehicle for bioactive molecules.

Lactoferrin and Peptide-derivatives: Antimicrobial Agents with Potential Use in Nonspecific Immunity Modulation.

Lactoferrin (Lf) is a conserved cationic non-heme glycoprotein that is part of the innate immune defense system of mammals. Lf is present in colostrum, milk and mucosal sites, and it is also produced by polymorphonuclear neutrophils and secreted at infection sites. Lf and Lf N-terminus peptide-derivatives named lactoferricins (Lfcins) are molecules with microbiostatic and microbicidal action in a wide array of pathogens. In addition, they display regulatory properties on components of nonspecific immunity, including toll-like receptors, proand anti-inflammatory cytokines, and reactive oxygen species. Mechanisms explaining the ability of Lf and Lfcins to display both up- and down-modulatory properties on cells are not fully understood but result, in part, from their interactions with membrane receptors that elicit biochemical signal pathways, whereas other receptors enable the nuclear translocation of these molecules for the modulation of target genes. The dual role of Lf and Lfcins as antimicrobials and immunomodulators is of biotechnological and pharmaceutical interest. Native Lf and its peptide-derivatives from human and bovine sources, the recombinant versions of the human protein, and their synthetic peptides have potential application as adjunctive agents in therapies to combat infections caused by multi-resistant bacteria and those caused by fungi, protozoa and viruses, as well as in the prevention and reduction of several types of cancer and response to LPS-shock, among other effects. In this review, we summarize the immunomodulatory properties of the unique multifunctional protein Lf and its N-terminus peptides.

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity.

In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly.

Modulatory Effects of Oral Bovine Lactoferrin on the IgA Response at Inductor and Effector Sites of Distal Small Intestine from BALB/c Mice.

Bovine lactoferrin (bLf) up-modulates intestinal IgA that is essential for homeostasis and which might confer protection to the distal small intestine that is vulnerable to inflammation. This study analyzed the effects of bLf administered orally on the IgA response at inductive (Peyer's patches) and effector (lamina propria) sites of the distal small intestine in mice. Groups of five healthy male BALB/c mice were orally treated with 5 mg of bLf for 7, 14, 21, or 28 days. Then, mice were killed and the distal small intestine was dissected. Intestinal fluid samples were analyzed to determine IgA and IgM levels by enzyme-immuno assay. Peyer's patches and lamina propria were analyzed for IgA(+) or IgM(+) plasma cells, B, CD4(+) T and CD8(+) T cells as well as CD4(+) T cells positive for either pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon-γ and interleukin (IL)-12] or for IgA-producing ILs (IL-4, -5, -10 and -6) by cytofluorometry. Antibodies, antibody-secreting cells, and B and T responses in both Peyer's patches and lamina propria were higher in bLf-treated than bLf-untreated mice. The generation of IL-10 and IL-6 CD4(+) T cells in Peyer's patches or TNF-α and IL-12 CD4(+) T cells in lamina propria showed similar response patterns. On days 14 and 28, cytokine/IL CD4(+) T cell responses were increased in Peyer's patches or decreased in lamina propria. The effect of bLf on the elicitation of IgA indicates a potential application of bLf as a nutraceutical to control inflammation in the distal small intestine.

Inhibition of Mayaro virus infection by bovine lactoferrin.

Mayaro virus (MAYV) is an arbovirus linked to several sporadic outbreaks of a highly debilitating febrile illness in many regions of South America. MAYV is on the verge of urbanization from the Amazon region and no effective antiviral intervention is available against human infections. Our aim was to investigate whether bovine lactoferrin (bLf), an iron-binding glycoprotein, could hinder MAYV infection. We show that bLf promotes a strong inhibition of virus infection with no cytotoxic effects. Monitoring the effect of bLf on different stages of infection, we observed that virus entry into the cell is the heavily compromised event. Moreover, we found that binding of bLf to the cell is highly dependent on the sulfation of glycosaminoglycans, suggesting that bLf impairs virus entry by blocking these molecules. Our findings highlight the antiviral potential of bLf and reveal an effective strategy against one of the major emerging human pathogens in the neotropics.

Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligand.

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.