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PubMed1993

The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei.

Five oral isolates each of Candida albicans and Candida krusei were studied for their sensitivity to the fungicidal effect of human lactoferrin. Significant inter- and intraspecies variations were observed and with most isolates the sensitivity of C. krusei to lactoferrin was greater than that of C. albicans. Fungicidal activity of lactoferrin was dose-dependent and observable only with the iron-free form of the molecule (apo-lactoferrin). Iron-saturated lactoferrin was ineffective against all isolates. Supernatant protein assays and scanning electron microscopy indicated cell surface alterations--leakage of proteins and formation of surface blebs--only in those Candida isolates that were sensitive to apo-lactoferrin. As lactoferrin is a common, non-immune, mucosal defence protein, its varying mode of action against C. albicans and C. krusei may be related to their different oral carriage rates.

Analysis of Variance↗

PubMed1985

Enzyme linked immunosorbent assay for lactoferrin. Plasma and tissue measurements.

Highly purified lactoferrin was obtained from human breast milk by sequential use of affinity chromatography and isoelectric focusing. IgG antibody to purified lactoferrin was used to develop a sensitive and reproducible enzyme linked immunosorbent assay. Characteristics of the assay included linearity over a wide range of lactoferrin concentration (3.125-200 micrograms/l) and sensitivity (lower range less than 1 microgram/l). The assay can be adapted for use on tissue cytosol as well as plasma. Healthy subjects showed plasma lactoferrin levels ranging from 187.5-450.1 micrograms/l. Pulmonary tuberculosis and acute pneumonia are associated with a 2-3-fold increase in plasma lactoferrin content while neutropenic subjects have markedly depressed lactoferrin concentrations. The assay will be useful for further delineation of lactoferrin and neutrophil function and turnover.

Antibodies↗

PubMed1989

Plasma and serum lactoferrin levels in cystic fibrosis. Relationship with the presence of cystic fibrosis protein.

Plasma lactoferrin concentrations were measured in blood of cystic fibrosis patients, heterozygotes and controls using a specific and sensitive enzyme immunoassay. 67 plasmas were studied (26 controls, 23 heterozygotes, 18 cystic fibrosis patients) and the results showed a statistically significant increase (p less than 0.05) of the level of plasma lactoferrin in cystic fibrosis patients (265 +/- 224 micrograms/l) compared to controls (168 +/- 100 micrograms/l) and heterozygotes (150 +/- 72 micrograms/l). Since it is well established that plasma lactoferrin level could be influenced by the number of neutrophils, a second set of experiments was performed on 20 cystic fibrosis patients on whom leukocyte counts were also made. When the 15 plasmas with normal neutrophils (in the range 2 to 6 giga/l) were considered, the mean lactoferrin level was 318 +/- 116 micrograms/l, still far above the normal values. For serum, a similar significant increase of lactoferrin concentration was observed in 33 cystic fibrosis patients (610 +/- 551 micrograms/l) compared to the values observed for 25 controls (237 +/- 155 micrograms/l) and 37 heterozygotes (272 +/- 231 micrograms/l). Cystic fibrosis protein (CFP) was identified in the same sera by isoelectric focusing and the intensity of the band was closely related to the increase of lactoferrin concentration in cystic fibrosis patients. In contrast, no difference in serum lactoferrin concentrations was observed between heterozygotes with or without CFP, indicating that the increased CFP concentration cannot be due only to altered granulocyte function.

Blood Proteins↗

PubMed1991

Structure, function and flexibility of human lactoferrin.

X-ray structure analyses of four different forms of human lactoferrin (diferric, dicupric, an oxalate-substituted dicupric, and apo-lactoferrin), and of bovine diferric lactoferrin, have revealed various ways in which the protein structure adapts to different structural and functional states. Comparison of diferric and dicupric lactoferrins has shown that different metals can, through slight variations in the metal position, have different stereochemistries and anion coordination without any significant change in the protein structure. Substitution of oxalate for carbonate, as seen in the structure of a hybrid dicupric complex with oxalate in one site and carbonate in the other, shows that larger anions can be accommodated by small side-chain movements in the binding site. The multidomain nature of lactoferrin also allows rigid body movements. Comparison of human and bovine lactoferrins, and of these with rabbit serum transferrin, shows that the relative orientations of the two lobes in each molecule can vary; these variations may contribute to differences in their binding properties. The structure of apo-lactoferrin demonstrates the importance of large-scale domain movements for metal binding and release and suggests that in solution an equilibrium exists between open and closed forms, with the open form being the active binding species. These structural forms are shown to be similar to those seen for bacterial periplasmic binding proteins, and lead to a common model for the various steps in the binding process.

Animals↗

PubMed1982

Lactoferrin binding to lysozyme-treated Micrococcus luteus.

When the cell lysis of Micrococcus luteus by hen egg white or human lysozyme is performed in the presence of bovine or human lactoferrin, a temporary increase of the turbidity of the solution as followed at 450 nm is observed. Examination of the suspension under light microscopy has proven that the protoplasts produced upon lysozyme action are agglutinated by lactoferrin. The rate of agglutination depends on pH, lactoferrin, lysozyme and cells concentrations. Agglutination is maximal at pH 5.5. Around 1.4 X 10(6) binding sites for lactoferrin per cell have been determined through a Scatchard plot analysis. The binding to the cells is not mediated by the glycosidic moiety of lactoferrin but rather by a charge-to charge interaction as succinylation of about four out of the 39 lysines of lactoferrin completely abolishes its ability to agglutinate the cells. Binding does not depend on ionic iron nor on the iron content of lactoferrin itself.

Animals↗

PubMed1991

Comparison of bovine, sheep and goat milk lactoferrins in their electrophoretic behavior, conformation, immunochemical properties and lectin reactivity.

1. The biochemical properties of bovine, goat and sheep lactoferrin were compared. Molecular weights of the three lactoferrins were estimated to be 78,000 to 80,000 as determined by SDS-PAGE. By IEF, microheterogeneity was observed for all of them. 2. Partial antigenic identity was observed between bovine lactoferrin and goat or sheep lactoferrin by immunodiffusion method. 3. CD spectra at the u.v. region of the three lactoferrins suggested their similar secondary and tertiary structural profiles. 4. Reactivities with peroxidase-conjugated lectins showed that the carbohydrate compositions of the three ruminants' lactoferrin were the same but not identical with that of human lactoferrin.

Animals↗

PubMed1996

Complete sequence analysis of rat transferrin and expression of transferrin but not lactoferrin in the digestive glands.

Rat milk and digestive juices contain transferrin but not lactoferrin, which is a major iron-binding protein in these secretions of human and mouse. To compare the structure of rat transferrin to that of transferrins and lactoferrins in other species, we isolated a cDNA clone containing the entire coding region of transferrin from rat liver and determined its sequence. The amino-acid sequence of rat transferrin had 69.8% identity with that of human transferrin and 48.8% identity with that of human lactoferrin. Rat transferrin, like other transferrins, had the potential N-linked glycosylation site only in the C-terminal domain, although lactoferrins characterized so far contained the glycosylation sites in both the N- and C-terminal domains. Southern and Northern analyses showed that there was the gene specifically hybridized with the mouse lactoferrin cDNA in rat genomic DNA, but only the transferrin mRNA was detected in mammary gland, submaxillary gland and pancreas of rat. These results suggest that the rat lactoferrin gene is silent in the mammary gland, and transferrin can serve as a functional substitute for lactoferrin in rat.

Amino Acid Sequence↗

PubMed2005

Characterization of the infection-responsive bovine lactoferrin promoter.

The concentration of lactoferrin in bovine milk is dramatically increased in response to infection. The high levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. However, molecular mechanisms of how the lactoferrin gene is regulated in the mammary gland in response to infection remain unknown. In this study, we isolated and characterized the 5' flanking region of the bovine lactoferrin gene. An 8.2 kilobase (kb) fragment of the bovine lactoferrin gene, containing 4.4 kb of 5' flanking region, exon 1, intron 1, and exon 2, was isolated from a bovine genomic library on two overlapping bacterial artificial chromosome (BAC) clones. Sequence analysis of the isolated lactoferrin gene revealed that the promoter region contains a high GC content, a non-canonical TATA box, multiple stimulating protein 1 (SP1)/GC elements, and other putative binding sites for transcription factors including nuclear factor-kappaB (NF-kappaB), activator protein 1 (AP1), signal transducer and activator of transcriptions 3 and 5 (STAT3 and STAT5), and steroid hormone receptors. To demonstrate that the isolated promoter is functional, 4.4 kb of 5' flanking region was inserted upstream from the firefly luciferase gene and the chimeric construct was transiently transfected into murine mammary epithelial cells. Transfection studies showed that the basal promoter activity is quite potent, being similar in strength to that of the simian virus 40 (SV40) promoter/enhancer. In addition, a 24-h treatment with Escherichia coli lipopolysaccharide (LPS) significantly stimulated its activity up to 2.3-fold in a dose-dependent manner. Furthermore, promoter deletion analysis indicated that the sequence up to -543 was sufficient for basal activity, whereas the sequence up to -1029 was required for maximal basal activity. The basal activity of the promoter is affected by both positive regulatory regions (-2462/-1879 and -1029/-75) and a negative regulatory region (-1407/-1029). LPS-responsive regions of the promoter were localized to the region from -1029 to -543 containing one STAT3 site and two NF-kappaB sites, and the region from -4355 to -2462 containing three AP1 sites and six NF-kappaB sites. Taken together, our findings suggested that the lactoferrin promoter responds to infection via the NF-kappaB pathway.

5' Flanking Region↗

PubMed2004

Inhibition of herpes simplex virus infection by lactoferrin is dependent on interference with the virus binding to glycosaminoglycans.

Previous reports have indicated that lactoferrin inhibits herpes simplex virus (HSV) infection during the very early phases of the viral replicative cycle. In the present work we investigated the mechanism of the antiviral activity of lactoferrin in mutant glycosaminoglycan (GAG)-deficient cells. Bovine lactoferrin (BLf) was a strong inhibitor of HSV-1 infection in cells expressing either heparan sulfate (HS) or chondroitin sulfate (CS) or both, but was ineffective or less efficient in GAG-deficient cells or in cells treated with GAG-degrading enzymes. In contrast to wild-type HSV-1, virus mutants devoid of glycoprotein C (gC) were significantly less inhibited by lactoferrin in GAG-expressing cells, indicating that lactoferrin interfered with the binding of viral gC to cell surface HS and/or CS. Finally, we demonstrated that lactoferrin bound directly to both HS and CS isolated from surfaces of the studied cells, as well as to commercial preparations of GAG chains. The results support the hypothesis that the inhibition of HSV-1 infectivity by lactoferrin is dependent on its interaction with cell surface GAG chains of HS and CS.

Animals↗

PubMed2005

Antiviral activity of human lactoferrin: inhibition of alphavirus interaction with heparan sulfate.

Human lactoferrin is a component of the non-specific immune system with distinct antiviral properties. We used alphaviruses, adapted to interaction with heparan sulfate (HS), as a tool to investigate the mechanism of lactoferrin's antiviral activity. Lactoferrin inhibited infection of BHK-21 cells by HS-adapted, but not by non-adapted, Sindbis virus (SIN) or Semliki Forest virus (SFV). Lactoferrin also inhibited binding of radiolabeled HS-adapted viruses to BHK-21 cells or liposomes containing lipid-conjugated heparin as a receptor analog. On the other hand, low-pH-induced fusion of the viruses with liposomes, which occurs independently of virus-receptor interaction, was unaffected. Studies involving preincubation of virus or cells with lactoferrin suggested that the protein does not bind to the virus, but rather blocks HS-moieties on the cell surface. Charge-modified human serum albumin, with a net positive charge, had a similar antiviral effect against HS-adapted SIN and SFV, suggesting that the antiviral activity of lactoferrin is related to its positive charge. It is concluded that human lactoferrin inhibits viral infection by interfering with virus-receptor interaction rather than by affecting subsequent steps in the viral cell entry or replication processes.

Animals↗

PubMed1999

Unique epitopes of lactoferrin expressed in human cytotrophoblasts involved in immunologic reactions.

OBJECTIVE: Lactoferrin is an iron-binding protein that has been implicated in protection against infections and allogeneic recognition reactions and in the control of cell growth. We studied the biochemical characteristics and expression of the unique lactoferrin epitopes (LF(1)) in human placentas. STUDY DESIGN: Immunohistologic studies of normal human term placentas were done by using monoclonal antibodies to LF(1). Double-antibody experiments were done by using monoclonal antibodies to markers of inflammation (macrophages, human leukocyte antigen [HLA-DR]). LF(1) was studied immunochemically by using lactoferrin fragments produced by the reaction of lactoferrin with trypsin or N-glycanase. RESULTS: Anti-LF(1) monoclonal antibodies reacted with most interstitial cytotrophoblasts in the basal plate and with villous cytotrophoblasts of some but not all chorionic villi. Cytotrophoblasts expressing LF(1) were associated with large numbers of HLA-DR-reactive macrophages. Anti-LF(1) monoclonal antibodies reacted with 2 distinct tryptic fragments of lactoferrin, and these reactivities were not affected by treatment with N-glycanase. CONCLUSION: Placental cytotrophoblasts express unique epitopes of lactoferrin (LF(1)). Such expression is increased in the presence of activated macrophages. This expression could be an extraembryonic response to inflammation and maternal allogeneic recognition as an effort to protect trophoblastic cells. The LF(1) epitopes might represent conserved polypeptide epitopes on 2 homologous lobes of lactoferrin.

Amidohydrolases↗

PubMed1998

Effect of substratum on growth, cell morphology and lactoferrin synthesis and secretion in bovine mammary cell culture.

The role of extracellular matrix in morphology, growth and lactoferrin synthesis and secretion in bovine mammary cells from a developing gland is poorly defined. In this study, bovine mammary cells from a hormone-primed developing gland were isolated and cultured on plastic, collagen, embedded within collagen, or on EHS-matrix, with the hormones prolactin, insulin, and cortisol in the presence or absence of fetal calf serum. Mammary cells on plastic or collagen spread and formed confluent cells sheets, while those embedded within collagen or on EHS-matrix maintained their acinar-like structure. Histological and ultrastructural analysis of cells showed that cells on plastic and collagen grew in multilayers, while those embedded within collagen or on EHS-matrix lacked any lumen structure. The ultrastructure of cells on different substrata more resembled an undifferentiated phenotype. Mammary cells secreted lactoferrin in increasing concentrations throughout the culture period. The total amount secreted in culture was regulated by extracellular matrix and fetal calf serum. Cells embedded within collagen in serum-free cultures secreted the lowest amounts of lactoferrin (up to 619 ng/ml; day 14), while those on collagen and supplemented with fetal calf serum secreted up to 4920 ng/ml at day 14. Fetal calf serum induced higher lactoferrin secretion within each substratum on which the cells were cultured. No intracellular accumulation of lactoferrin was noted in cells on plastic or collagen or those embedded within collagen, whereas those on EHS-matrix accumulated more than 500 ng/ml of lactoferrin intracellularly/intracinarly. Furthermore, when cultured on a similar substratum, cells from a developing gland secreted higher lactoferrin than cells from a lactating gland.

Animals↗

PubMed2000

Systemic or local co-administration of lactoferrin with sensitizing dose of antigen enhances delayed type hypersensitivity in mice.

Lactoferrin (LF), a major defense protein synthesized and stored in granulocytes has been implicated in maintaining immune homeostasis during an insult-induced metabolic imbalance. In this study, we demonstrated that lactoferrin augments the delayed type hypersensitivity (DTH) response to specific antigens in mice. Lactoferrin (LF) was given to mice orally or intraperitoneally (i.p. ) at the time of immunization, or subcutaneously (s.c.) in a mixture with the immunizing doses of the following antigens, sheep red blood cells (SRBC), Calmette-Guerin bacillus (BCG) or ovalbumin (OVA). A DTH reaction was determined 24 h after administration of an eliciting dose of antigen as a specific increase in foot pad swelling. Lactoferrin enhanced DTH reaction to all studied antigens in a dose-dependent manner. Lactoferrin (LF) given to mice in conjunction with antigen administered in an incomplete Freund's adjuvant induced the DTH response at the level of control mice given antigen in a complete Freund's adjuvant. In addition, LF remarkably increased DTH response to a very small, otherwise non-immunogenic SRBC dose. The increase in DTH response was less pronounced for orally administered LF than for any other routes of administration, however, statistically significant augmentation was demonstrated for each antigen studied. Although the costimulatory action of LF was accompanied by the appearance of bovine lactoferrin-specific cellular responses in mice, it is very unlikely that such responses will be generated in humans, since bovine lactoferrin is a dietary antigen to which a tolerance has been acquired. Considering the involvement of LF in generation of stimulatory signals during the induction phase of an antigen specific immune responses, we suggest that LF may be useful for development of safer and more efficacious vaccination protocols.

Adjuvants, Immunologic↗

PubMed1999

Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin.

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.

Amino Acid Sequence↗

PubMed1997

Growth inhibitory effect of bovine lactoferrin on Toxoplasma gondii tachyzoites in murine macrophages: role of radical oxygen and inorganic nitrogen oxide in Toxoplasma growth-inhibitory activity.

To study the effector pathway of Toxoplasma growth-inhibitory activity induced by lactoferrin in murine macrophage, the role of reactive oxygen intermediates (O2-) and inorganic nitric oxide (NO) was examined. Production of O2- was diminished in cultures of macrophages supplemented with lactoferrin and the effect of lactoferrin was dose and time dependent. Production of NO was enhanced in cultures of macrophages supplemented with interferon-gamma, but not with lactoferrin. These findings suggest that this Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages is not mediated by O2- or NO molecules. A competitive inhibitor of the L-arginine dependent effector pathway, NG-monomethyl-L-arginine (NG MMA), virtually abolished the inhibitory effects induced by interferon-gamma. Similarly, the inhibitory activity induced by lactoferrin was also diminished in cultures supplemented with NG MMA. From these findings, it appears that the Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages may be mediated by an L-arginine-dependent effector pathway that does not involve NO production.

Animals↗

PubMed2002

Lactoferrin immunomodulation of DTH response in mice.

Improved nontoxic adjuvants, especially adjuvants capable of inducing cell-mediated immunity (CMI), are needed for research in immunology and for development of human and veterinary vaccines. Bovine Lactoferrin, an effector molecule shown to directly participate in host defense, was assessed at various concentrations as an adjuvant component for induction of DTH responses to sheep red blood cells (SRBC). Subcutaneous immunization with Lactoferrin enhanced delayed type hypersensitivity (DTH) in CBA mice in a dose-dependent fashion; DTH responses were most significantly increased when sensitization was accomplished using Lactoferrin at 50 microg/dose and 250 microg/dose. Furthermore, Lactoferrin admixed with suboptimal dose of SRBC enhanced DTH responses by over 17-fold. Peritoneal cells collected from mice intraperitoneally injected with a 100 microg/dose of Lactoferrin demonstrated modest, but significant, production of TNF-alpha, IL-12 and MIP-1alpha when cultured in vitro, compared to saline-injected controls. J774A.1 murine macrophages stimulated with Lactoferrin resulted in increased TNF-alpha protein production, and upregulated IL-12 and IL-15 mRNA. Levels of message for chemokines MIP-1alpha and MIP-2 were also increased in a dose-dependent way. Taken together, these results indicate that Lactoferrin as an adjuvant may stimulate macrophages to generate a local environment likely to push immune responses towards development and maintenance of CMI.

Adjuvants, Immunologic↗

PubMed1985

Effect of ACTH and oxytocin treatment on lactoferrin and citrate in cows' milk.

Six dairy cows were treated before milkings with either oxytocin (Pitocin, 20 i.u.) or ACTH (Synacthen, 150 i.u.), principally to determine their effect on the ratio of citrate: lactoferrin concentrations in the milk. With ACTH treatment, after 3 d milk yield and citrate concentration decreased significantly, lactoferrin and bovine serum albumin (BSA) concentrations increased significantly. Somatic cell counts (SCC) increased temporarily in the milk of three of the cows which previously had greater than 100 000 cells/ml. Lactoferrin yield remained fairly constant but citrate yield was significantly reduced. The citrate: lactoferrin molar ratio decreased from 1373 to 606. With oxytocin treatment, after 4 d milk yield first increased and then significantly decreased, citrate concentration decreased significantly while there were no significant changes in lactoferrin or BSA concentration or in the yield of any other milk constituents. The citrate: lactoferrin molar ratio decreased from 1621 to 1301. There were no significant changes in SCC either during treatment or 4 d after treatment but there was a significant rise at 16 d after treatment. It was concluded that in lactating cows both hormones affected citrate and lactoferrin concentrations in the direction that would improve the antibacterial properties of milk, but that this was accompanied by adverse effects on milk secretion. The extent of the change was not sufficient to be likely to produce inhibition of coliform bacteria.

Adrenocorticotropic Hormone↗

PubMed1999

Preparation and characterization of the N and C monoferric lobes of buffalo lactoferrin produced by proteolysis using proteinase K.

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (< 14.4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin-proteinase K complex and its sequence determined to be Val-Ala-Gln-Gly-Gly-Ala-Ala-Gly-Leu-Ala. Circular dichroism studies indicated a high alpha-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.

Amino Acid Sequence↗