Effects of lactoferrin on the production of interferon-λ by the human intestinal epithelial cell line HT-29.

We examined the in-vitro effects of bovine lactoferrin (LF) on the production of interferon-λ (IFN-λ), an antiviral cytokine important for the defense of enterocytes, using the human intestinal epithelial cell line HT-29. HT-29 cell cultures were treated with LF for 1 h, and the cultures were stimulated with polyinosinic-polycytidylic acid (poly I:C). LF increased the concentration of IFN-λ in the culture supernatant after stimulation in a dose-dependent manner. A similar increase in the concentration of IFN-λ was observed in the supernatant of cells washed between treatment with LF and stimulation with poly I:C. At 6 and 24 h after stimulation with poly I:C (early and late phases, respectively) treated cultures contained significantly higher concentrations of IFN-λ1 in the culture supernatant, and significantly higher IFN-λ1 and IFN-λ2 mRNA levels, than controls. These results suggest that LF activates the innate cellular immunity of the enterocytes to double-stranded RNA and increases the production of IFN-λ.

Structure of iron saturated C-lobe of bovine lactoferrin at pH 6.8 indicates a weakening of iron coordination.

The bilobal lactoferrin is an approximately 76 kDa glycoprotein. It sequesters two Fe(3+) ions together with two CO(3)(2-) ions. The C-terminal half (residues, Tyr342-Arg689, C-lobe) of bovine lactoferrin (BLF) (residues Ala1-Arg689) was prepared by limited proteolysis using trypsin. Both C-lobe and intact BLF were saturated to 100%. Both of them retained up to nearly 85% of iron at pH 6.5. At pH 5.0, C-lobe retained 75% of iron whereas intact protein could retain only slightly more than 60%. At pH 4.0 both contained 25% iron and at pH 2.0 they were left with iron concentration of only 10%. The structure of iron saturated C-lobe was determined at 2.79 Å resolution and refined to R(cryst) and R(free) factors of 0.205 and 0.273, respectively. The structure contains two crystallographically independent molecules, A and B. They were found to have identical structures with an r.m.s. shift of 0.5 Å for their C(α) atoms. A high solvent content of 66% was observed in the crystals. The average value of an overall B-factor was 68.0 Å(2). The distance of 2.9 Å observed for the coordination bond between Fe(3+) ion and N(e2) of His595 appeared to be considerably longer than the normally observed values of 1.9-2.2 Å. This indicated that the coordination bond involving His595 may be absent. Other coordination distances were observed in the range of 2.1-2.3 Å. Based on the present structure of iron saturated C-lobe, it may be stated that His595 is the first residue to dissociate from ferric ion when the pH is lowered.

Intracellular retention and subsequent release of bovine milk lactoferrin taken up by human enterocyte-like cell lines, Caco-2, C2BBe1 and HT-29.

Lactoferrin (LF) is an iron-binding glycoprotein contained in milk and other exocrine fluids, and is believed to have multiple biological functions. We investigated the intracellular dynamics of LF taken up by three lines of human enterocytes and the subsequent release of internalized LF by using two-site ELISA and confocal microscopy. LF taken up by Caco-2 cells was kept partially intact within the cells and subsequently released to the medium as degraded fragments of 30-50 kDa. The retention and subsequent release of LF by Caco-2 cells were much more abundant than those of ovalbumin, ovomucoid and lysozyme. Such results characteristic of LF were also similarly observed in C2BBe1 and HT29 cells more markedly. LF was detected as punctate signals and partially colocalized with the lactoferrin receptor, intelectin-1, in the respective cytoplasm and nuclei of Caco-2 and C2BBe1 cells. In contrast, LF within the HT-29 cells was detected as much smaller punctate signals scattered in the cytoplasm.

Antibacterial activity of the lactoperoxidase system combined with edible Laminaria hot-water extract as a source of halide ions.

Hot-water extracts prepared from nine out of 12 samples of dried edible Laminaria reduced the viable numbers of Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Esherichia coli below the detection limit after incubation for 5 min when combined with lactoperoxidase, glucose oxidase, and glucose. Some extracts showed higher bactericidal activity and a higher OI(-) concentration in the assay mixture after ultrafiltration.

Effects of a composition containing lactoferrin and lactoperoxidase on oral malodor and salivary bacteria: a randomized, double-blind, crossover, placebo-controlled clinical trial.

We report a clinical trial of the effects of test tablets containing bovine lactoferrin and lactoperoxidase on oral malodor and salivary bacteria. Fifteen subjects with volatile sulfur compounds (VSCs) in mouth air above the olfactory threshold (H(2)S >1.5 or CH(3)SH >0.5 ng/10 ml) as detected by gas chromatography were enrolled in the trial. Either a test or a placebo tablet was ingested twice at 1-h intervals in two crossover phases. Mouth air was monitored for VSC levels at the baseline before ingestion of a tablet, 10 min after the first ingestion, 1 h (just before the second ingestion), and 2 h after the first ingestion. Whole saliva was analyzed at the baseline and at 2 h for bacterial numbers. At 10 min, the level of CH(3)SH was significantly lower in the test group (median [interquartile range] = 0.28 [0.00-0.68] ng/10 ml) compared to that in the placebo group (0.73 [0.47-1.00] ng/10 ml; P = 0.011). The median concentration of CH(3)SH in the test group was below the olfactory threshold after 10 min until 2 h, whereas the level in the placebo group was above the threshold during the experimental period. No difference in the numbers of salivary bacteria was detected by culturing or quantitative PCR, but terminal restriction fragment length polymorphism detected one fragment with a significantly lower copy number at 2 h in the test group (mean ± standard error, 4.89 ± 0.11 log(10) copies/10 µl) compared to that in the placebo group (5.38 ± 0.15 log(10) copies/10 µl; P = 0.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.

Inhibition of lactoperoxidase by its own catalytic product: crystal structure of the hypothiocyanate-inhibited bovine lactoperoxidase at 2.3-A resolution.

To the best of our knowledge, this is the first report on the structure of product-inhibited mammalian peroxidase. Lactoperoxidase is a heme containing an enzyme that catalyzes the inactivation of a wide range of microorganisms. In the presence of hydrogen peroxide, it preferentially converts thiocyanate ion into a toxic hypothiocyanate ion. Samples of bovine lactoperoxidase containing thiocyanate (SCN(-)) and hypothiocyanate (OSCN(-)) ions were purified and crystallized. The structure was determined at 2.3-A resolution and refined to R(cryst) and R(free) factors of 0.184 and 0.221, respectively. The determination of structure revealed the presence of an OSCN(-) ion at the distal heme cavity. The presence of OSCN(-) ions in crystal samples was also confirmed by chemical and spectroscopic analysis. The OSCN(-) ion interacts with the heme iron, Gln-105 N(epsilon1), His-109 N(epsilon2), and a water molecule W96. The sulfur atom of the OSCN(-) ion forms a hypervalent bond with a nitrogen atom of the pyrrole ring D of the heme moiety at an S-N distance of 2.8 A. The heme group is covalently bound to the protein through two ester linkages involving carboxylic groups of Glu-258 and Asp-108 and the modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is significantly distorted from planarity, whereas pyrrole rings A, B, C, and D are essentially planar. The iron atom is displaced by approximately 0.2 A from the plane of the heme group toward the proximal site. The substrate channel resembles a long tunnel whose inner walls contain predominantly aromatic residues such as Phe-113, Phe-239, Phe-254, Phe-380, Phe-381, Phe-422, and Pro-424. A phosphorylated Ser-198 was evident at the surface, in the proximity of the calcium-binding channel.

Effects of orally administered bovine lactoperoxidase on dextran sulfate sodium-induced colitis in mice.

The effect of lactoperoxidase (LPO) on dextran sulfate sodium-induced colitis was examined in mice. After 9 d of colitis induction, weight loss, colon shortening, and the histological score were significantly suppressed in mice orally administered LPO (62.5 mg/body/d) as compared to a group administered bovine serum albumin. These results suggest that LPO exhibits anti-inflammatory effects in the gastrointestinal tract.

Effects of orally administered lactoferrin and lactoperoxidase on symptoms of the common cold.

OBJECTIVES: Lactoferrin (LF) and lactoperoxidase (LPO) are present in human saliva. LF has been demonstrated to show antibacterial and antiviral activities. In saliva, LPO catalyzes the hydrogen peroxide-dependent oxidation of thiocyanate to hypothiocyanite that exhibits antimicrobial and antiviral properties. A randomized, open-label, parallel-group clinical trial was conducted to examine the effectiveness of sucking tablets containing LF and LPO (LF+LPO) in alleviating symptoms of the common cold and/or influenza infection. METHODS: A total of 407 subjects were randomized into two groups, treatment and non-treatment groups, and each group was further classified into subgroups habitually wearing a face mask, washing their hands, or gargling. The common cold, influenza, and gastrointestinal symptoms were used to evaluate the effectiveness, and the incidence and duration of symptoms were statistically analyzed. RESULTS: The incidence and duration of common cold, gastrointestinal symptoms, and influenza infection were not statistically different between treatment and non-treatment groups. LF+LPO tablets were moderately effective in reducing the incidence and duration of common cold symptoms in the subgroup that did not gargle and especially to shorten significantly the duration of fever higher than 38°C in the subgroup that did not wear a face mask. CONCLUSION: The results suggested that the effect of ingestion of the tablet is not obvious in alleviating common cold symptoms but may be helpful when the subjects do not follow precautionary measures such as gargling and the use of a protective face mask.

Effects of orally administered bovine lactoferrin and lactoperoxidase on influenza virus infection in mice.

Milk contains a wide variety of host protective factors against infectious microbes. Among these protective factors, lactoferrin (LF) and lactoperoxidase (LPO) have been reported to exhibit antiviral activities as well as immuno-modulatory effects. In the present study, the effects of orally administered LF and LPO were assessed in a mouse influenza virus infection model. BALB/c mice were intranasally infected with 6.6x10(2) p.f.u. of influenza virus A/PR/8/34(H1N1). Bovine LF or LPO was administered once daily at a dose of 62.5 mg per mouse by gavage, starting 1 day before infection. Mice given LF or LPO showed a significantly lower lung consolidation score on day 6 after infection compared with the control mice that were given water instead. Concurrently, the number of infiltrated leukocytes recovered from bronchoalveolar lavage fluid (BALF) on day 6 was significantly lower in mice given LF or LPO. However, the virus yield in the BALF was not affected by these treatments. The serum level of IL-6, a pro-inflammatory cytokine, positively correlated with the lung consolidation score in each group and was significantly lower on day 6 in the mice given LPO. These results suggest the potential of oral administration of LF or LPO to attenuate pneumonia in influenza-virus-infected mice through the suppression of infiltration of inflammatory cells in the lung.

Inactivating effects of the lactoperoxidase system on bacterial lyases involved in oral malodour production.

The main components of oral malodour have been identified as volatile sulfur compounds (VSCs), including hydrogen sulfide (H(2)S) and methyl mercaptan (CH(3)SH). The lactoperoxidase (LPO) system (consisting of LPO, glucose oxidase, glucose and thiocyanate) was previously shown to exhibit antimicrobial activities against some oral bacteria in vitro and suppressive effects on VSCs in mouth air in a clinical trial. Here, we examined the in vitro effects of the LPO system on the activities of the bacterial lyases involved in the production of VSCs by oral anaerobes. The exposure of crude bacterial extracts of Fusobacterium nucleatum and Porphyromonas gingivalis or purified methionine γ-lyase to the LPO system resulted in the inactivation of their lyase activities through l-cysteine and l-methionine, which was linked to the production of H(2)S and CH(3)SH, respectively. The exposure of living F. nucleatum and P. gingivalis cells to the LPO system resulted in the suppression of cell numbers and lyase activities. The inactivation of the crude bacterial extracts of F. nucleatum and purified methionine γ-lyase by the LPO system was partly recovered by the addition of DTT. Therefore, the LPO system may inactivate bacterial lyases including methionine γ-lyase by reacting with the free cysteine residues of lyases. These results suggested that the LPO system suppresses the production of VSCs not only through its antimicrobial effects, but also by its inactivating effects on the bacterial lyases of F. nucleatum and P. gingivalis.

Preparation and antimicrobial action of three tryptic digested functional molecules of bovine lactoferrin.

Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species.

A lactoferrin-receptor, intelectin 1, affects uptake, sub-cellular localization and release of immunochemically detectable lactoferrin by intestinal epithelial Caco-2 cells.

Intelectin 1 (IntL) is known as a lectin expressed in intestinal epithelia and also as a receptor for an iron-binding protein, lactoferrin (LF). Uptake of LF with bound iron by enterocytes via receptor-mediated endocytosis has been well investigated, whereas subsequent fate of endocytized LF and LF/IntL complexes remains largely unknown. In the present study, we examined contribution of IntL to the uptake, sub-cellular localization and subsequent release of LF by intestinal Caco-2 IntL-transfectants using two-site ELISA and fluorescence confocal microscopy. LF taken up by IntL-transfectants was immunochemically detected mostly as intact protein in the cell lysates, and it was a little larger in amount than that of the mock-transfectants. In the IntL-transfectants cultured on porous membrane, LF taken up from the apical side was detected immunochemically as punctate signals in the apical-side cytoplasmic region near nucleus. The LF signals were co-localized with IntL and, in a time-dependent manner, partially with early endosome antigen 1 (EEA1), but not with alkaline phosphatase. LF taken up, retained and subsequently released by the IntL-transfectants was larger in amount than that of mock-transfectants. Moreover, uptake of LF altered sub-cellular localization of IntL and markedly enhanced the IntL signals within the cells.

Twenty-five years of research on bovine lactoferrin applications.

Lactoferrin (LF) was identified as a milk protein in 1960. Large-scale manufacturing of bovine LF (bLF) was established more than 20 years ago. Using this commercially available material, research for bLF applications has advanced from basic studies to clinical studies, and bLF has been applied to commercial food products for the last 25 years. During this period, it was found that LF is digested by gastric pepsin to generate a multi-potent peptide, lactoferricin. It was also demonstrated that oral administration of bLF augments host protection against infections via antimicrobial action and immunomodulation of the host. In addition, researchers have demonstrated that oral administration of bLF prevents cancer development. In this review, we look back on 25 years of bLF research and development.

Orally administered lactoperoxidase ameliorates dextran sulfate sodium-induced colitis in mice by up-regulating colonic interleukin-10 and maintaining peripheral regulatory T cells.

We previously demonstrated orally administered bovine lactoperoxidase (LPO) ameliorated dextran sulfate sodium-induced colitis in mice. Here, we examine the mechanism of action of LPO. Three days after colitis induction, expression of interferon-gamma mRNA in colonic tissue was significantly decreased in mice administered LPO; while mRNA expression of interleukin (IL)-10 and regulatory T cell (Treg) marker, Foxp3, were significantly increased. The proportion of CD4+CD25+ Tregs in peripheral CD4+ T cells was also significantly elevated when LPO was administered. Nine days after colitis induction, the severity of colitis symptoms, including body weight loss and colon shortening, was reduced and expression of IL-10 mRNA was increased in mice administered LPO. The proportion of CD4+CD25+ Tregs in peripheral leukocytes was also significantly elevated when LPO was administered. These results suggest LPO ameliorates colitis by up-regulating colonic anti-inflammatory cytokines and maintaining peripheral regulatory T cells.

Recombinant human intelectin binds bovine lactoferrin and its peptides.

Intelectin (IntL), a lectin that exists on the brush border membrane of the small intestine, plays a role in the innate immune response and also acts as a receptor for lactoferrin (LF), an iron-binding glycoprotein found in milk and other secretions. Similar to human LF (hLF), bovine LF (bLF) has been shown to induce proliferation and differentiation of human enterocytes and to modulate their cytokine productions. To evaluate the interaction between human IntL (hIntL) and bLF, recombinant hIntL (rhIntL) conjugated with a tag sequence was examined for its ligand-binding capacity by using microtiter plates coated with LF or other proteins. Interestingly, rhIntL showed higher binding for bLF than hLF. It also bound pepsin hydrolysate of bLF, but to a lower degree than native bLF. A very low binding of rhIntL was observed for bovine serum albumin or transferrin. These findings suggest that hIntL acts as a receptor for bLF and its digested fragments.

In vitro and in vivo effects of a composition containing lactoperoxidase on oral bacteria and breath odor.

The antimicrobial activity of a composition containing bovine lactoperoxidase (LPO), glucose oxidase, glucose and buffer salts was tested against oral bacteria in vitro. A preliminary in vivo study was conducted to test the effect on breath odor of the tablets containing this composition. Suspension of the composition in filter-sterilized saliva or phosphate-buffered saline containing sodium thiocyanate (NaSCN) at a physiological concentration showed bactericidal activity against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Although hydrogen peroxide (H(2)O(2)) was not detected in the suspension, hypothiocyanite (OSCN(-)) was detected at an average concentration of 0.161 mM. Three tablets made with the composition were continuously sucked by three subjects, and the levels of volatile sulfur compounds (VSCs) in their oral air samples were monitored over a 2 h period by compact gas chromatography. Ingestion of the tablets reduced the average levels of VSCs in the oral air, whereas non-treatment or ingestion of the control tablets without enzymes did not. These results suggest that the composition shows bactericidal activity through the formation of OSCN(-) in saliva and is effective for reducing breath odor, although further clinical studies are needed.

Orally administered lactoperoxidase increases expression of the FK506 binding protein 5 gene in epithelial cells of the small intestine of mice: a DNA microarray study.

Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.

Bovine lactoferrin: benefits and mechanism of action against infections.

Ingestion of bovine lactoferrin (bLF) has been reported to show anti-infective, anti-cancer, and anti-inflammatory effects. In particular, it has become evident that oral bLF had a beneficial effect on infections of both digestive and nondigestive tract tissue in various animal models. Furthermore, the effects of bLF have been indicated in clinical studies on patients with Helicobacter pylori infection, chronic hepatitis C, tinea pedis, and other diseases. Immunomodulation in the intestine and systemic sites has been suggested to mediate the protective effects of oral bLF against infection. Recently, we demonstrated the beneficial effects of oral bLF in influenza virus infected mice. BLF administration reduced the lung consolidation score and the number of infiltrating leukocytes in bronchoalveolar lavage fluid. We also investigated the effect of oral bLF on the transcription of genes related to immunity in the small intestine of mice using the quantitative RT-PCR method. We found that intake of bLF increased the expression of IL-12p40, IFN-beta, and NOD2. Thus, oral bLF activates the transcription of important immune-related genes in the small intestine, and such transcriptional activation may promote systemic host immunity.

Transfer of antigen-pulsed dendritic cells induces specific T-Cell proliferation and a therapeutic effect against long-term Helicobacter pylori infection in mice.

Helicobacter pylori causes persistent infection of the stomach and results in chronic gastritis and peptic ulcers. Jaws II cells, derived from mouse bone marrow, were pulsed with live or formalin-killed or whole-cell sonicates (WCS) of H. pylori. Representative cell surface molecules were expressed at substantial levels on Jaws II cells, indicating that appropriate maturation of the cells was achieved with the three H. pylori antigens without any significant differences. H. pylori WCS-pulsed Jaws II cells secreted a significant amount of tumor necrosis factor alpha into the culture supernatant. The naïve T cells exposed to the WCS-pulsed Jaws II cells showed significant proliferation and gamma interferon (IFN-gamma) and interleukin-10 (IL-10) production in vitro. A 2-log reduction in the number of colonizing bacteria was observed in the mice treated with the WCS-pulsed Jaws II cells; however, no significant reductions were achieved in mice treated with Jaws II cells pulsed with other H. pylori antigens. Up-regulated production of IFN-gamma and IL-10 was observed in the stomachs of the mice treated with the WCS-pulsed Jaws II cells, which is consistent with the result obtained in vitro. There were no differences in gastritis scores or H. pylori-specific antibody titers among the mice treated with Jaws II cells pulsed with the three different H. pylori antigens. The results suggest that Th1 cell-mediated immunity in combination with Th2 cell-mediated immunity plays a role in reducing colonizing bacterial numbers in mice with chronic H. pylori infections.

Polymerase chain reaction--restriction fragment length polymorphism analysis of clarithromycin-resistant Helicobacter pylori infection in children using stool sample.

BACKGROUND: To analyze clarithromycin-resistant Helicobacter pylori infection in children, we developed a method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using stool samples. MATERIALS AND METHODS: Twenty-three children without significant upper abdominal symptoms were included (mean age 7.0 years). Of these, 18 and five were diagnosed as H. pylori-positive and -negative, respectively, by the H. pylori stool antigen test (HpSA). The DNA from the stool samples was purified using the QIAamp DNA Stool Minikit (QIAGEN). The PCR was performed on the purified DNA using oligonucleotide primers designed to amplify the 23S rRNA gene of H. pylori. The PCR products were reacted with restriction enzymes MboII, BceAI, and BsaI to detect mutations A2142G, A2142C, and A2143G, respectively. RESULTS: Sixteen of the 18 HpSA-positive samples were PCR-positive, and all five HpSA-negative samples were PCR-negative. Thus, the PCR had 89% sensitivity and 100% specificity, with 91% accuracy in reference to HpSA. Of the 16 PCR-positive samples, one and four were digested with MboII and BsaI, respectively, indicating 31% prevalence of CAM-resistance. CONCLUSIONS: We conclude that the PCR-RFLP using stool samples is a rapid and reliable method to noninvasively detect clarithromycin-resistant H. pylori infection in children. It may be useful before choosing regimens of H. pylori eradication.