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PubMed2002

[Recent advances of basic research and clinical application of lactoferrin as an antiviral reagent against chronic hepatitis C].

Hepatitis C virus(HCV), discovered in 1989, is the major causative agent of chronic viral hepatitis. Most patients progress to liver cirrhosis and hepatocellular carcinoma. In the therapy of hepatitis C, only interferon has been used effectively as an anti-HCV reagent in Japan, but its effectiveness is limited to about 30% of cases. Using human hepatocyte cell line which could support efficient HCV replication, we previously found that lactoferrin inhibited HCV infection, and demonstrated that this inhibiting activity was due to the interaction of lactoferrin with HCV. Further analysis found that the carboxyl region of lactoferrin, which partially shows amino acid sequence homology to human CD81, specifically bound to the HCV E2 envelope protein, and identified a 33 amino acids as a critical binding domain of lactoferrin. On the other hand, it has been shown that bovine lactoferrin was effective in some patients with chronic hepatitis received an 8-week course of lactoferrin treatment. Further clinical trials showed that lactoferrin is a promising candidate for adjuvant therapy with interferon in patients with chronic hepatitis C.

Antigens, CD↗

PubMed1992

Studies of the N-terminal half of human lactoferrin produced from the cloned cDNA demonstrate that interlobe interactions modulate iron release.

The factors influencing iron binding and release by lactoferrin have been addressed by comparison of the native full length molecule (Lf) with the N-terminal half of human lactoferrin (LfN) produced from the cloned cDNA expressed in baby hamster kidney (BHK) cells. The coding sequences for LfN were inserted into the expression vector pNUT between the metallothionein promoter and the human growth hormone transcription termination sequences. Transformed BHK cells were grown in roller bottles where concentrations of LfN as high as 35 mg/liter were obtained. The pure protein, produced by the transformed BHK cells, was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein blotting and immunodetection, N-terminal sequence analysis, UV-visible spectroscopy, electron spin resonance spectroscopy, and measurements of metal binding and release. By these criteria LfN was found to be correctly processed, glycosylated, and able to bind iron reversibly. Both UV-visible and electron spin resonance spectra of the half molecule were very similar to those of native lactoferrin and the full length lactoferrin produced in BHK cells, but there were marked differences in the pH at which iron release occurred. Iron release from LfN occurs in the pH range 6.0-4.0, compared with 4.0-2.5 for native lactoferrin and 6.2-4.0 for transferrin. These results suggest that the more facile release of iron from LfN compared with native lactoferrin results from the absence of stabilizing contacts between the N- and C-terminal halves and that the characteristic difference in pH stability between lactoferrins and transferrins is due primarily to differences in these interactions.

Amino Acid Sequence↗

PubMed2003

Anti-lactoferrin antibodies in patients with connective tissue diseases.

AIM: To study the prevalence of antineutrophil cytoplasmic antibodies against lactoferrin in patients with connective tissue diseases and to characterize the antibody response to lactoferrin in these patients. METHODS: Sera from 60 patients with systemic lupus erythematosus, 98 with rheumatoid arthritis, 11 with systemic sclerosis, and 6 with mixed connective tissue disease were studied. The presence of anti-lactoferrin antibodies was detected by enzyme-linked immunosorbent assay. The IgG subclass reactivity of antibodies to lactoferrin was analyzed by biotin-extravidin amplified enzyme-linked immunosorbent assay and monoclonal antibodies to human IgG subclasses. RESULTS: Anti-lactoferrin antibodies were found in 15 patients (5 patients with rheumatoid arthritis and 10 with systemic lupus erythematosus). IgG1 was the predominant subclass for antibodies to lactoferrin. CONCLUSION: Patients with connective tissue diseases are known to develop multiple auto-antibodies; anti-lactoferrin antibodies mainly of IgG1 isotype can also be found in patients with rheumatoid arthritis and more often in patients with systemic lupus erythematosus.

Adolescent↗

PubMed1992

Lactoferrin gene promoter: structural integrity and nonexpression in HL60 cells.

Lactoferrin is a member of the transferrin family of iron-binding proteins. It is found in several glandular epithelial tissues and human neutrophils, where it is localized to secondary granules. To examine the mechanisms controlling lactoferrin gene expression in neutrophils and defects in its expression in acute leukemia, we have cloned a lactoferrin cDNA from a chronic myelogenous leukemia library, and used it to obtain genomic clones representing the chromosomal lactoferrin gene. Using polymerase chain reaction, primer extension, and S1 analysis, we have identified the 5' end of the lactoferrin mRNA. We have defined a putative promoter region for the gene, and characterized its first two exons. In addition, we have examined the structure of these regions in DNA from HL60 cells. HL60 is a leukemic cell line that undergoes phenotypic neutrophil maturation on exposure to dimethyl sulfoxide (DMSO). However, the cells cannot be induced to express any secondary granule protein genes. We have shown that the 5' end of the lactoferrin gene, including the putative promoter region, is entirely normal in HL60. By Northern analysis, nuclear run-on studies, and primer extension assays we have shown that the gene is not transcribed in DMSO-induced HL60 cells. This supports the hypothesis that the defect in HL60 is an abnormality in the production or activity of a transacting regulator of lactoferrin gene expression.

Amino Acid Sequence↗

PubMed1990

Release of iron from phagocytosed Escherichia coli and uptake by neutrophil lactoferrin.

Escherichia coli were labeled with 59Fe and then either treated with myeloperoxidase, H2O2, and chloride or opsonized and mixed with human neutrophils. The myeloperoxidase system at pH 7.4 caused release of most of the bacterial 59Fe. A similar result has been obtained by Rosen and Klebanoff (J Biol Chem 257:13731, 1982) but at pH 5. Iron release at pH 7.4 did not require the presence of a chelator, and the majority passed through a 10,000 relative molecular mass cut-off ultrafiltration membrane. When iron-poor lactoferrin was present during incubation with myeloperoxidase, 88% of the released 59Fe was precipitated with anti-lactoferrin antiserum, indicating that it was lactoferrin-bound. When the bacteria were mixed with neutrophils in a 10:1 ratio, approximately 50% were phagocytosed. About 40% of the 59Fe was released from the ingested bacteria over a 40-minute period. Initially, most remained associated with the neutrophil phagosomes, but with time, there was gradual transfer of some of the iron to the medium. Using anti-lactoferrin antiserum, 50% to 60% of phagosomal iron and 64% to 71% of iron in the medium was shown to be bound to lactoferrin. Thus, iron is released from phagocytosed E coli. Most becomes bound to lactoferrin, and some of this is released into the surroundings of the neutrophils. This suggests that neutrophil lactoferrin may function to trap iron from ingested microorganisms, enabling its removal from sites of inflammation. This may prevent iron from catalyzing undesirable oxidative reactions, as well as making it unavailable for growth of microorganisms that survive the killing process.

Escherichia coli↗

PubMed1987

Lactoferrin derived from neutrophils inhibits the mixed lymphocyte reaction.

The effects of the products of phagocytosing neutrophils on lymphocyte transcriptional activity have been investigated using allogeneic mixed lymphocyte cultures (MLC). When MLCs are grown crowded in round-bottomed wells, uptake of tritiated uridine is inhibited by the presence of neutrophil products. Similarly, inhibitory activity is shown by purified lactoferrin. The inhibitory factor released by neutrophils is positively identified as lactoferrin, since its activity is lost in the presence of specific antibody. In addition, inhibitory activity is lost when lactoferrin is removed by a monoclonal antibody (MoAb) column and is recovered with lactoferrin from the column. The active form of lactoferrin carries iron and the apoprotein is inactive. MLCs crowded in round-bottomed wells take up considerably more uridine than when spread in flat-bottomed wells, and it is this enhanced transcriptional activity that is inhibited by neutrophil-derived lactoferrin. Enhanced uridine uptake by crowded cultures is due to factors that can be transferred in the supernatants to promote uridine uptake by spread cultures. Neutrophil lactoferrin inhibits both production and effect of the transferable factor(s).

Dose-Response Relationship, Drug↗

PubMed1987

Tear lactoferrin levels in patients with external inflammatory ocular disease.

Lactoferrin, an iron complexing protein in normal tears, is an important component of the nonspecific host defense system of the external eye. We measured tear lactoferrin levels in patients with contact lens-induced giant papillary conjunctivitis (GPC) by an enzyme-linked immunosorbent assay (ELISA). Patients with active GPC (N = 26) had significantly reduced tear levels of lactoferrin (0.876 +/- 0.42 mg/ml) compared with normal individuals (N = 12; 1.73 +/- 0.46 mg/ml, P less than 0.0003) and the control contact lens wearers' group (N = 11; 1.57 +/- 0.92 mg/ml, P less than 0.003). Patients with vernal conjunctivitis (N = 10), an ocular disease with similar histopathology, had slightly reduced concentrations of tear lactoferrin (1.22 +/- 0.59 mg/ml). Patients with inactive GPC (N = 7) had normal tear levels of lactoferrin (1.33 +/- 0.49 mg/ml). The lactoferrin to total protein ratio in the tears was significantly reduced in patients with GPC compared to normal subjects, control contact lens wearers, and patients with inactive GPC. The decreased tear levels of lactoferrin in patients with GPC may contribute to increased coating of lenses with bacteria and their products and enhanced ocular inflammation which may play a role in the pathogenesis of GPC.

Adenoviridae Infections↗

PubMed1986

The role of lactoferrin released by phagocytosing neutrophils in the regulation of colony-stimulating activity production by human mononuclear cells.

There remains a controversy about the alleged inhibitory effect of lactoferrin on production of colony-stimulating activity (C.S.A.) by mononuclear cells. We confirm the inhibitory action of both lactoferrin purified from human breast milk and that released from phagocytosing neutrophils. To show the inhibitory effect, it is necessary to plot the dose-response curve of medium conditioned by mononuclear cells with and without lactoferrin. Crowding cells to promote contact is essential for the fraction of C.S.A. production inhibitable by lactoferrin. Saturation of purified lactoferrin by addition of iron salts in vitro may introduce an artifact, as this lactoferrin retained its inhibitory activity at much greater dilutions than lactoferrin released from phagocytosing neutrophils.

Colony-Stimulating Factors↗

PubMed1985

[Isolation and physico-chemical properties of lactoferrin from swine neutrophils].

Lactoferrin, a non-heme iron-binding protein was isolated from pig neutrophils. The purification procedure included initial extraction of the protein in the presence of cetyltrimethylammonium bromide followed by chromatography on carboxymethyl-cellulose and Sephadex G-100. The thus obtained protein was found to be homogeneous on polyacrylamide gel (PAAG) electrophoresis at acidic values of pH. PAAG electrophoresis in the presence of sodium dodecyl sulfate revealed a single component with a molecular weight of 75 000-80 000. The resulting protein is capable of binding two atoms of iron molecule. The absorbance spectra for the pig neutrophil lactoferrin are identical to those for cow milk lactoferrin in the visible region and have a maximum at 465 nm. The amino acid composition of pig lactoferrin was determined. Isoelectric focusing of the protein obtained in a PAAG stabilized pH gradient revealed a component with pI of about 6.8. A single precipitin line was observed with rabbit antipig lactoferrin when examined by immunodiffusion. No immunological cross-reactions were observed between pig lactoferrin and bovine lactoferrin.

Amino Acids↗

PubMed1984

Uptake of lactoferrin by the liver. I. Role of the reticuloendothelial system as indicated by blockade experiments.

When 125I-labeled human lactoferrin was injected intravenously into mice it was rapidly taken up by the liver, where 75% of the dose was recovered after 15 minutes. The inhibition curve with unlabeled lactoferrin showed that the fates of labeled and unlabeled protein were similar and that the uptake capacity was saturable by milligram quantities of protein per gram of liver, suggesting the existence of numerous specific binding sites. The presence of these sites on the reticuloendothelial system was indicated by the blocking effect of dextran sulfate and latex particles. Fucoidin (fucan sulfate), which is used in the study of receptors specific for fucose, was found to inhibit the uptake of goat red blood cells as well as lactoferrin. Therefore, the inhibition exerted by fucoidin on the uptake of lactoferrin could be mediated by blockade of the reticuloendothelial system and not necessarily by competition for fucose receptors. These data indicate that the report by others that lactoferrin was taken up by a fucosyl receptor on hepatocytes is incorrect. The competition curve obtained when mouse lactoferrin was injected with its human homologue indicated that both proteins reacted with the same binding sites. However, a significant part of mouse lactoferrin was found to be taken up by the kidneys. Hepatic and renal uptakes were both reduced by prior injection of dextran sulfate.

Animals↗

PubMed1982

Modulation of classical C3 convertase of complement by tear lactoferrin.

Lactoferrin isolated fom normal human tears was shown to inhibit the complement-mediated lysis of antibody-coated red cells. The anti-complementary effect of lactoferrin on serum complement could be reversed by adding Fe3+ but not by Mg++ or Ca++. Lactoferrin did not inhibit the formation of EAC14 but markedly blocked the assembly of the EAC142 enzyme. Once the C3 convertase was formed lactoferrin did not affect the function of the enzyme and only had a minor effect on the intrinsic decay of C2 from the convertase. Inhibition of the C3 convertase formation was not seen by preincubating EAC14 intermediates with lactoferrin, but only occurred when lactoferrin and C2 were incubated together with EAC14 cells. Our findings suggest that lactoferrin may play an anti-inflammatory role by modulating activation of the complement system.

Calcium Chloride↗

PubMed1994

Faecal flora in the newborn. Effect of lactoferrin and related nutrients.

Bifidobacteria, lactobacilli and staphylococci are the predominant organisms in the faeces of breast fed babies whereas in formula fed babies coliforms, enterococci and bacteroides predominate. In vitro studies suggest that the mechanisms responsible are probably related to the acid base properties of the formula and 'immunological' proteins such as lactoferrin and sIgA. In human babies however the addition of bovine lactoferrin to an infant formula has little effect on the faecal flora and does not move it in the direction of the breast fed baby. There are various possible explanations of this lack of effect, e.g., inactivation of the lactoferrin when it is added to a formula, and immunological responses in the intestine to a foreign protein. We consider the most likely explanation is that other factors necessary for the optimum activity of lactoferrin were not present or in inappropriate concentration, e.g. sIgA, lysozyme, citrate, bicarbonate. If human lactoferrin is added to an infant formula it may be these other factors will require attention if the lactoferrin is to have a significant effect. An iconoclastic interpretation which cannot be completely excluded is that the hypothesis of lactoferrin bacteriostatic activity is based on in vitro studies and is not a reflection of what happens in babies. The faecal flora of a breast fed baby is very different from that of a baby receiving either cow's milk or a modern infant formula (Figure 1). Among breast fed babies bifidobacteria lactobacilli and staphylococci are the predominant organisms, whereas in formula fed babies the predominant organisms are enterococci, coliforms, and bacteroides (1).

Animals↗

PubMed1995

Neutralization of heparin activity by neutrophil lactoferrin.

Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin-induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.

Amino Acid Sequence↗

PubMed1994

Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin.

Lactoferrin is an iron-binding protein which is synthesized by mucosal epithelium and neutrophils and released by these cells in response to inflammatory stimuli. It promotes neutrophil aggregation and manifests iron-dependent and -independent antimicrobial properties in vitro. Since lactoferrin binds to glycosaminoglycans (GAGs) and sulfated polysaccharides can inhibit its clearance in vivo and in vitro, we sought to examine its interaction with the GAGs chondroitin sulfate and heparin. Amino-terminal sequencing of proteolytic fragments of human lactoferrin that were fractionated by GAG chromatography suggested that the amino-terminal 6 kDa of the secreted protein mediates its interaction with GAGs. Synthetic peptides were used to show that the first 33 residues of human lactoferrin can bind well to solid-phase or solution-phase GAGs. The first 33 residues bound fluoresceinamine-labeled heparin with an IC50 (611 nM) which approximated that of the intact protein (124 nM). In contrast, when the first six residues (GRRRRS) were removed from this peptide, it then bound poorly to heparin (IC50 = 49 microM). Our results suggest that the GRRRRS sequence at the amino terminus of human lactoferrin acts synergistically with an RKVR sequence at positions 28-31 to form the predominate functional GAG-binding site of human lactoferrin. Molecular modeling of the crystalline structure of lactoferrin supports a synergistic activity between these two sites since it shows that they juxtapose each other on the surface of the folded protein. Solid docking calculations indicate that they can form a cationic cradle as a binding site for chondroitin sulfate.

Amino Acid Sequence↗

PubMed1998

Interaction of human milk lactoferrin with ATP.

Human lactoferrin exhibits many unique properties. It is known as one of the most important factors that provide nonspecific defense of cells against bacteria, viruses, and carcinogenesis, as well as an important component of a specific system responsible for the passive immunity of newborns. As a compound with extremely broad spectrum of functions many of which were not elucidated so far, lactoferrin is intensely studied. In this study we obtained electrophoretically and immunologically homogenous preparations of lactoferrin from human milk. Using various methods, we were the first to show that the fraction of lactoferrin, which displays an increased affinity for Sepharose Blue, forms complexes with ATP with a stoichiometry of 1 mole ATP per mole protein. It is shown that the ATP-binding site is located in the C-terminal domain of the lactoferrin molecule. The binding of ATP results in the dissociation of tetrameric forms of the protein and a change in the mode of interaction of lactoferrin with polysaccharides and other proteins. The data may be used in analysis of the possible reasons for multifunctional properties of lactoferrin and possible ways of regulation of its functions.

Adenosine Triphosphate↗

PubMed1997

Direct evidence for the secretion of lactoferrin and its binding to sperm in the porcine epididymis.

Lactoferrin has been for the first time purified from the porcine cauda epididymal fluid as a 70 kDa protein. Both Western and Northern blot analyses show that lactoferrin is synthesized in the regions from the distal caput to the cauda epididymis and secreted into the luminal fluid. Lactoferrin is first secreted as a 75 kDa glycoprotein and its carbohydrate moieties are gradually digested to form 70 kDa protein in the cauda epididymis. Lactoferrin has already bound to the surface of the epididymal sperm because the anti-lactoferrin antiserum induces the mature sperm tail-to-tail agglutination. These results strongly suggest new physiological functions of lactoferrin on the sperm maturation in the epididymis.

Animals↗

PubMed1986

In vivo study of lactoferrin and murine rebound myelopoiesis.

Lactoferrin has been reported to inhibit the production of colony-stimulating factor (CSF); thus we sought to study its possible effects on myelopoiesis in vivo. The characteristics of rebound myelopoiesis in C57BL mice injected with a sublethal dose of cyclophosphamide (CY) were used to test lactoferrin for any granulopoietic activity. An experimental group received daily injections of 50 micrograms of human lactoferrin beginning 24 hr after CY injections. By measuring the total nucleated cellularity of the femoral marrow, the peripheral blood count, and the incorporation of tritiated thymidine by the marrow in six replicate experiments, no statistically significant difference was noted between the lactoferrin injected groups and the control groups. Neither the route of lactoferrin administration (i.v. or i.p.) nor the sex of the animal altered the myelopoietic recovery. Lactoferrin had no stimulatory or inhibitory effect on murine rebound myelopoiesis in vivo contrary to the reported in vitro results.

Animals↗

PubMed2004

Liposomal lactoferrin induced significant increase of the interferon-alpha (IFN-alpha) producibility in healthy volunteers.

Interferon-alpha (IFN-alpha) producibility has been widely accepted as one of the important markers to evaluate the immune status. In this study, preliminary clinical tests were carried out to confirm the immunomodulatory activity of liposomal lactoferrin including IFN-alpha producibility and NK activity. In a primary open trial, the liposomal lactoferrin was administered to five healthy males for one week and various immunological indices were evaluated. Furthermore, ten healthy males were administered 319 mg per day of liposomal or non-liposomal lactoferrin for four weeks, and immune status was monitored at 0, 1 and 4 weeks after the intake as well as three weeks after stopping it. In this double-blinded comparative study, the IFN-alpha producibility was significantly increased only in the liposomal lactoferrin group during administration and decreased 3 weeks after stopping it, while the IFN-alpha producibility was unchanged in the non-liposomal lactoferrin group. Although the biological mechanism of IFN-alpha producibility enforced by liposomal lactoferrin has not been wholly understood, it is suggested to be a novel active constituent having preventive and therapeutic effects on inflammatory diseases, cancer and infectious diseases such as chronic hepatitis C.

Adult↗