An aptamer affinity column for purification and enrichment of lactoferrin in milk.

As an active glycoprotein with high nutritional value, lactoferrin is widely used in food and medical treatment. Therefore, it is very important to establish an accurate and efficient detection method for lactoferrin. At present, the detection of lactoferrin in milk faces many challenges, such as low separation degree and poor parallelism. To address this issue, we developed an aptamer affinity column (AAC) for purification and enrichment of lactoferrin in milk. The column was prepared by covalent conjugation of an amino-modified aptamer with NHS-activated Sepharose. The washing buffer type (0.01 mol/L phosphate buffer) and volume (10 mL) and the sodium chlorideconcentration (1 mol/L) in the elution buffer were optimized for the AAC method. The performance of the AAC was then evaluated in terms of the column capacity, specificity, stability, and reusability. The column capacity was 500 ± 13.7 µg and the column could be reused up to ten times with a large loss in performance. The AAC method combined with high-performance liquid chromatography gave excellent linearity over a wide range, good sensitivity with a limit of detection of 3 µg/mL, and acceptable recoveries for different concentrations of lactoferrin spiked in real raw milk samples from cattle. Finally, the AAC was successfully applied to analyze lactoferrin in milk. This method could be applied to routine analysis of samples for lactoferrin in testing laboratories and dairy factories.

Bovine Lactoferrin Quantification in Dairy Products by a Simple Immunoaffinity Magnetic Purification Method Coupled with High-Performance Liquid Chromatography with Fluorescence Detection.

This study described a simple, specific, and sensitive method using immunoaffinity magnetic purification coupled with high-performance liquid chromatography-fluorescence (HPLC-FL) detection for determination of bovine lactoferrin (bLf) in dairy products. BLf was selectively extracted from dairy products using immunoaffinity beads and then detected by HPLC-FL with its intrinsic fluorescence. During the analysis, standard solutions of bLf were pretreated with Tween 20, an anti-adsorptive agent, for blocking the nonspecific binding of bLf to polypropylene tubes. The calibration curve was linear over the range of 0.8-30 µg mL-1. The validated method was successfully applied to measure bLf at the intact level in dairy products.

Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells.

Breast cancer is the most frequently diagnosed cancer among women worldwide. Here, recombinant human lactoferrin (rhLf) expressed in Pichia pastoris was tested for its potential cytotoxic activity on a panel of six human breast cancer cell lines. The rhLf cytotoxic effect was determined via a live-cell HTS imaging assay. Also, confocal microscopy and flow cytometry protocols were employed to investigate the rhLf mode of action. The rhLf revealed an effective CC50 of 91.4 and 109.46 µg/ml on non-metastatic and metastatic MDA-MB-231 cells, with favorable selective cytotoxicity index values, 11.68 and 13.99, respectively. Moreover, rhLf displayed satisfactory SCI values on four additional cell lines, MDA-MB-468, HCC70, MCF-7 and T-47D (1.55-3.34). Also, rhLf provoked plasma membrane blebbing, chromatin condensation and cell shrinkage in MDA-MB-231 cells, being all three apoptosis-related morphological changes. Also, rhLf was able to shrink the microfilaments, forming a punctuated cytoplasmic pattern in both the MDA-MB-231 and Hs-27 cells, as visualized in confocal photomicrographs. Moreover, performing flow cytometric analysis, rhLf provoked significant phosphatidylserine externalization, cell cycle arrest in the S phase and apoptosis-induced DNA fragmentation in MDA-MB-231 cells. Hence, rhLf possesses selective cytotoxicity on breast cancer cells. Also, rhLf caused apoptosis-associated morphologic changes, disruption of F-actin cytoskeleton organization, phosphatidylserine externalization, DNA fragmentation, and arrest of the cell cycle progression on triple-negative breast cancer MDA-MB-231 cells. Overall results suggest that rhLf is using the apoptosis pathway as its mechanism to inflict cell death. Findings warranty further evaluation of rhLf as a potential anti-breast cancer drug option.

[Investigation of the iron-binding capacity of the bovine lactoferrin].

In this work, studies were carried out to obtain and determine the iron-binding ability of lactoferrin isolated from milk of Holstein-Friesian (black-and-white) breed of cows, which is the main stock of the Russian cattle herd (CH). Aim of the study was to obtain lactoferrin and determine its iron-binding capacity for substantiating the raw material resources of its industrial production as an easily digestible source of ferrous iron for the production of dietary supplements and/or specialized foods. MATERIAL AND METHODS: To optimize the production of lactoferrin in the conditions of dairy enterprises, we used a method of lactoferrin isolation from cow's milk in its own modification, which consisted in the degreasing of whole milk by centrifugation and double cation-exchange chromatography with successive application of the following sorbents: CM-cellulose (CM-52) and Macro-Prep High Q Support. RESULTS AND DISCUSSION: The developed modification of the method of chromatographic production of lactoferrin has shown its effectiveness and availability for production at domestic dairy enterprises. The purity of lactoferrin is about 95%, and the content is about 74 µg/cm3. Iron-binding capacity was determined in apo- and holoforms of lactoferrin. The ability of saturation of apolactoferrin with iron has been shown. CONCLUSION: The new obtained factual material allows us to express the prerequisites for further research to justify the possibility of using the iron-saturated form of hololactoferrin of cow milk of the Holstein-Frisian breed as a domestic raw material for dietary supplements and specialized foods.

Lactoferrin, chitosan and Melaleuca alternifolia-natural products that show promise in candidiasis treatment

Abstract The evolution of microorganisms resistant to many medicines has become a major challenge for the scientific community around the world. Motivated by the gravity of such a situation, the World Health Organization released a report in 2014 with the aim of providing updated information on this critical scenario. Among the most worrying microorganisms, species from the genus Candida have exhibited a high rate of resistance to antifungal drugs. Therefore, the objective of this review is to show that the use of natural products (extracts or isolated biomolecules), along with conventional antifungal therapy, can be a very promising strategy to overcome microbial multiresistance. Some promising alternatives are essential oils of Melaleuca alternifolia (mainly composed of terpinen-4-ol, a type of monoterpene), lactoferrin (a peptide isolated from milk) and chitosan (a copolymer from chitin). Such products have great potential to increase antifungal therapy efficacy, mitigate side effects and provide a wide range of action in antifungal therapy.

Bovine lactoferrin free of lipopolysaccharide can induce a proinflammatory response of macrophages.

BACKGROUND: Lactoferrin (LF) is an 80 kDa glycoprotein which is known for its effects against bacteria, viruses and other pathogens. It also has a high potential in nutrition therapy and welfare of people and a variety of animals, including piglets. The ability to bind lipopolysaccharide (LPS) is one of the described anti-inflammatory mechanisms of LF. Previous studies suggested that cells can be stimulated even by LPS-free LF. Therefore, the aim of our study was to bring additional information about this possibility. Porcine monocyte derived macrophages (MDMF) and human embryonic kidney (HEK) cells were stimulated with unpurified LF in complex with LPS and with purified LF without bound LPS. RESULTS: Both cell types were stimulated with unpurified as well as purified LF. On the other hand, neither HEK0 cells not expressing any TLR nor HEK4a cells transfected with TLR4 produced any pro-inflammatory cytokine transcripts after stimulation with purified LF. This suggests that purified LF without LPS stimulates cells via another receptor than TLR4. An alternative, TLR4-independent, pathway was further confirmed by analyses of the NF-kappa-B-inducing kinase (NIK) activation. Western blot analyses showed NIK which activates different NFκB subunits compared to LF-LPS signaling via TLR4. Though, this confirmed an alternative pathway which is used by the purified LF free of LPS. This stimulation of MDMF led to low, but significant amounts of pro-inflammatory cytokines, which can be considered as a positive stimulation of the immune system. CONCLUSION: Our results suggest that LF's ability is not only to bind LPS, but LF itself may be a stimulant of pro-inflammatory pathways.

Expression, purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe.

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343-682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.

Comparison of Proliferative Effect of Human Lactoferrin and Its Proteolytic Peptide on Normal and Transformed Epithelial Cells.

Human lactoferrin (hLF) is an iron-binding glycoprotein with a variety of functions. hLF undergoes proteolytic cleavage to smaller peptides in the stomach following ingestion. In the present study, we evaluated the effects of hLF and its proteolytic product, human lactoferrin peptide (hLFP), on the proliferation of two epithelial cells, HEK293 normal cells and KATO III gastric carcinoma cells, using an MTT assay and expression of proliferative nuclear cell antigen (PCNA), a notable proliferation marker. When the two epithelial cells were stimulated with hLF and hLFP in the presence of fetal bovine serum (FBS), hLFP stimulated proliferation of both cell types at lower concentrations than hLF by two orders of magnitude. The cancer cells exhibited proliferative responses to both hLF and hLFP at lower concentrations by 2∼3 orders of magnitude than the normal cells. Either hLF or hLFP alone did not support appreciable proliferation of these cell lines in the absence or low concentrations of FBS. Bovine serum albumin or its proteolytic product failed to promote cellular proliferation even in the presence of 10 % FBS, indicating the specificity of the proliferative activity of hLF and hLFP. These data highlight feasibility of hLF and its peptide for adjuvants for tissue culture medium.

Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction.

BACKGROUND: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells). METHODS: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections. RESULTS: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein. CONCLUSION: The present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.

Lactoferrin and ovotransferrin contribute toward antioxidative effects of Edible Bird's Nest against hydrogen peroxide-induced oxidative stress in human SH-SY5Y cells.

There are reports of improved redox outcomes due to consumption of Edible Bird's Nest (EBN). Many of the functional effects of EBN can be linked to its high amounts of antioxidants. Interestingly, dietary components with high antioxidants have shown promise in the prevention of aging and its related diseases like Alzheimer's disease. In this study, the antioxidative potentials of EBN and its constituents, lactoferrin (LF) and ovotransferrin (OVF), were determined and protective effects against hydrogen peroxide (H2O2)- induced toxicity on SH-SY5Y cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange and propidium iodide (AO/PI) staining with microscopy were examined. Results showed that EBN and its constituents attenuated H2O2-induced cytotoxicity, and decreased radical oxygen species (ROS) through increased scavenging activity. Furthermore, LF, OVF, and EBN produced transcriptional changes in antioxidant related genes that tended towards neuroprotection as compared to H2O2-treated group. Overall, the results suggest that LF and OVF may produce synergistic or all-or-none antioxidative effects in EBN.

Bovine lactoferrin enhances proliferation of human peripheral blood lymphocytes and induces cytokine production in whole blood cultures.

BACKGROUND: Lactoferrin belongs to the immunoregulatory milk proteins involved in iron metabolism as well in providing innate immunity to newborns. The protein has been the subject of numerous clinical studies. OBJECTIVES: The aim of this investigation was to evaluate the effects of bovine lactoferrins (bLF), differing in source and iron content, on spontaneous proliferation of human peripheral blood mononuclear cells (PBMC) and cytokine production by human whole blood cultures. MATERIAL AND METHODS: The following bLF preparations were used: partially iron saturated or devoid of iron bLF from milk and bLF from colostrum. The study was conducted on 12 healthy volunteers (men, 20-24 years old). The effects of bLFs on the proliferation of PBMC in four-day cultures was studied at 50-0.6 µg/mL concentration range and the rate of proliferation was determined using the MTT colorimetric method. TNF α and IL-6 levels, induced by the bLFs in 24 h whole blood cultures, were measured by ELISA. RESULTS: The lactoferrins stimulated autologous proliferation of human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner, with a comparable efficacy. This stimulation occurred both in the constant presence of bLFs in the cultures and also upon preincubation of PBMC with bLFs with subsequent exhaustive wash of cells. Only bLF from colostrum induced production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in cultures of whole blood cells. This phenomenon took place predominantly at concentration of 50 µg/mL. CONCLUSIONS: The results showed potent stimulation of the proliferative response of PBMC by bovine lactoferrin, associated with the induction of proinflammatory cytokines only in the case of colostral bLF. This observation may be of importance when high doses of bLF are used in therapy and by designing diet supplementation with this protein.

Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET.

α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF-OA was prepared and its activity and structural changes were compared with α-LA-OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF-OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF-OA was obtained by heat-treatment at pH8.0 with LD50 of 4.88, 4.95 and 4.62µM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA-OA. Similar to HAMLET, LF-OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF-OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and ß-turn and an increase of ß-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein-OA complexes.

Separation of iron-free and iron-saturated forms of transferrin and lactoferrin via capillary electrophoresis performed in fused-silica and neutral capillaries.

A capillary electrophoresis-based method for the cost-effective and high efficient separation of iron-free and iron-saturated forms of two members of transferrin family: transferrin and lactoferrin has been developed. The proposed qualitative method relying on the SDS application allowed us to separate iron-free and iron-saturated forms of these proteins, as well as human serum albumin, used as an internal standard. Owing to the distinct migration times under established conditions, the combination of transferrin and lactoferrin assays within a single analytical procedure was feasible. The performance of the method using a fused-silica capillary has been compared with the results obtained using the same method but performed with the use of a neutral capillary of the same dimensions. Neutral capillary has been used as an alternative, since the comparable resolution has been achieved with a concomitant reduction of the electroosmotic flow. Despite of this fact, the migration of analytes occurred with similar velocity but in opposite order, due to the reverse polarity application. A quantitative method employing fused-silica capillary for iron saturation study has been also developed, to evaluate the iron saturation in commercial preparations of lactoferrin.

Antifungal effect and pore-forming action of lactoferricin B like peptide derived from centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74nm and 1.4nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death.

Effectiveness of human, camel, bovine and sheep lactoferrin on the hepatitis C virus cellular infectivity: comparison study.

PURPOSE: The prevalence of HCV infection has increased during recent years and the incidence reach 3% of the world's population, and in some countries like Egypt, may around 20%. The developments of effective and preventive agents are critical to control the current public health burden imposed by HCV infection. Lactoferrin in general and camel lactoferrin specifically has been shown to have a compatitive anti-viral activity against hepatitis C virus (HCV). The purpose of this study was to examine and compare the anti-infectivity of native human, camel, bovine and sheep lactoferrin on continuous of HCV infection in HepG2 cells. MATERIAL AND METHODS: Used Lfs were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified Lfs were evaluated in two ways; 1. the pre-infected cells were treated with the Lfs to inhibit intracellular replication at different concentrations and time intervals, 2. Lfs were directly incubated with the virus molecules then used to cells infection. The antiviral activity of the Lfs were determined using three techniques; 1. RT-nested PCR, 2. Real-time PCR and 3. Flowcytometric. RESULTS: Human, camel, bovine and sheep lactoferrin could prevent the HCV entry into HepG2 cells by direct interaction with the virus instead of causing significant changes in the target cells. They were also able to inhibit virus amplification in HCV infected HepG2 cells. The highest anti-infectivity was demonstrated by the camel lactoferrin. CONCLUSION: cLf has inhibitory effect on HCV (genotype 4a) higher than human, bovine and sheep lactoferrin.

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct.

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3 kb hLF minigene to the regulatory elements of the ß-casein gene. The transgenic goat produced more than 30 mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future.

Quantification of protein deposits on silicone hydrogel materials using stable-isotopic labeling and multiple reaction monitoring.

This study was designed to use multiple reaction monitoring (MRM) for accurate quantification of contact lens protein deposits. Worn lenses used with a multipurpose disinfecting solution were collected after wear. Individual contact lenses were extracted and then digested with trypsin. MRM in conjunction with stable-isotope-labeled peptide standards was used for protein quantification. The results show that lysozyme was the major protein detected from both lens types. The amount of protein extracted from contact lenses was affected by the lens material. Except for keratin-1 (0.83 ± 0.61 vs 0.77 ± 0.20, p = 0.81) or proline rich protein-4 (0.11 ± 0.04 vs 0.15 ± 0.12, p = 0.97), the amounts of lysozyme, lactoferrin, or lipocalin-1 extracted from balafilcon A lenses (12.9 ± 9.01, 0.84 ± 0.50 or 2.06 ± 1.6, respectively) were significantly higher than that extracted from senofilcon A lenses (0.88 ± 0.13, 0.50 ± 0.10 or 0.27 ± 0.23, respectively) (p < 0.05). The amount of protein extracted from contact lenses was dependent on both the individual wearer and the contact lens material. This may have implications for the development of clinical responses during lens wear for different people and with different types of contact lenses. The use of MRM-MS is a powerful analytical tool for the quantification of specific proteins from single contact lenses after wear.

Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria. MATERIAL AND METHODS: Saliva from 10 healthy volunteers was tested individually for its anti-A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti-A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI-TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans-BEC binding assay. RESULTS: Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans. CONCLUSION: The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway.

Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.

Lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with Burkholderia cenocepacia iron-modulated biofilm.

In cystic fibrosis (CF) high iron concentration in airway secretion plays a pivotal role in bacterial multiplication and biofilm formation as well as in inflammatory response. Burkholderia cenocepacia, an opportunistic facultative pathogen responsible for chronic lung infections and cepacia syndrome, recurrently infects CF patients. Lactoferrin (Lf), an iron binding multifunctional glycoprotein synthesized by exocrine glands and neutrophils, has been found at higher concentration in the airway secretions of infected CF patients than in healthy subjects. Here the influence of milk derivative bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, on invasiveness of B. cenocepacia iron-modulated biofilm, as well as on inflammatory response by infected CF bronchial (IB3-1) cells, is reported. bLf did not significantly affect invasion efficacy by biofilmforming B. cenocepacia clinical strains. Conversely, the addition of bLf to cell monolayers during infection significantly decreased the pro-inflammatory Interleukin (IL)-1beta and increased the anti-inflammatory IL-11 expression compared to that observed in cells infected in the absence of bLf. The bLf ability to modulate genes expressed following B. cenocepacia infection seems related to its localization to the nucleus of infected IB3-1 cells. These results provide evidence for a role of bLf in the protection of infected CF cells from inflammation-related damage, thus extending the therapeutic potential of this multifunctional natural protein.