DEFINITION
Lactoferrin is a 80kDa iron-binding glycoprotein found in milk and various external secretions such as saliva, tears, airway secretion and the granules of neutrophils (neutrophils after degranulation were observed to be the main source of lactoferrin in blood plasma), and it has been recently reevaluated because of its multifunctionality : it works not only as an iron transporter and storage, but also as an antioxidant, anticarcinogenic , immunomodulator and antiviral molecule, implying an important role in innate immunity .
THE GENE
Lactoferrin gene, whose official HGNC name is LTF (Lactotransferrin), is located in humans on the third chromosome (locus 3q21-q23 ), while in bovines it was discovered to be located on the chromosome 22 .
Three different isoforms of lactoferrin have been isolated. Lactoferrin-alpha is the iron binding form, but has no ribonuclease activity. On the other hand lactoferrin-β and lactoferrin-γ demonstrate ribonuclease activity but they are not able to bind iron.
CHARACTERISTICS OF SEQUENCE
The nucleotide sequence of human milk Lf (hLf) was first determined by Ray et al. in 1990 and compared with the amino acid sequence of human lactotransferrin determined previously.
Lf genes are highly conserved among species, with an almost identical organization and an mRNA of about 1900–2600 bp. A homology search in sequence databases revealed nucleotide sequences for the Lfs of 13 species: 3 primates, 7 even-toed ungulates, 1 pig, 1 cat, and 1 mouse. Pairwise sequence identities ranged from a minimum of ~ 78% to nearly 100%. The main outliers in this group were Lfs from primates, in which human and chimpanzee are quite similar (95–98%) when compared to orangutan (79%). On the other hand, horse and camel; pig, cow, yak, water buffalo, bull, and goat Lfs share 81 and 78% of sequence identity respectively when compared to human Lf as outgroup. The sequence relationships given above show that the Lfs form a highly conserved sequence family, and also sequence identity between Lfs and other Tfs is relatively high at 60–65%. A characteristic feature of Lfs is their highly basic character, with a pI typically greater than 9; this property is typically predictable from their sequence. Structurally, the feature that most readily distinguishes Lfs from Tfs is the peptide linker between the two lobes, thought to have evolved from an ancient duplication event, which also contains several proline residues.
CHEMICAL STRUCTURE AND IMAGES
Lactoferrin is an 80kDa glycosylated protein of ca. 700 aminoacids (whose percentage is shown in the image below) with high homologies among species.
It is a simple polypeptide chain folded into two symmetrical lobes (N and C lobes), highly homologous to one another. These two lobes are connected by a hinge region containing parts of an alpha-helix between amino acids 333 and 343, which proves additional flexibility to the molecule.
The polypeptide chain includes amino acids 1-332 for the N lobe and 334-703 for the C lobe and is made up of alpha-elix and beta-pleated sheet structures that create two domains for each lobe. Each lobe can bind or the Fe2+ or the Fe3+ ions in synergy with the carbonate ion (one carbonate ion is always bound by lactoferrin concurrently with each ferric ion) and contains a glycosylation site. Because of its ability to reversibly bind Fe ions, LF can exist both in the free (apolactoferrin) and the associated form (hololactoferrin), and it brings to a different three-dimensional conformation: free LF has an open conformation, while bound LF is a closed molecule with greater resistance to proteolysis.
Amino acids directly involved at the iron-binding site in each lobe are Asp, Tyr and His: its affinity for iron is 300 times higher than that of transferrin, and the affinity increases in weakly acidic mediums. This facilitates the transfer of iron from TF to LF during inflammation, when the pH of tissues decreases due to accumulation of lactic and other acids. An arginine chain is instead responsible for binding the carbonate ion (Image below).
Lactoferrin molecules contain (according to the species and protein) varying numbers of sites for potential glycosylation, mostly on the surface of the molecule. The degree of glycosylation varies and determines the rate of resistance to proteases or to very low pH.
Lac toferr in has demonstrated remarkable resistance to proteolytic degradation by trypsin and trypsin-like enzymes. The level of resistance is proportional to the degree of iron saturation.
SOURCES IN THE ORGANISM
Lactoferrin expression can first be detected embryonic development in two and four-cell embryos during, then throughout the blastocyst stage up to implantation. Interestingly, at the 16-cell stage, coinciding with the first major differentiation step in the embryo, lactoferrin messenger RNA (mRNA) is synthesized by the inner cells, whereas the protein is selectively taken up by the outside cells. It cannot be detected from the time of implantation until gestation and later, it can be found in neutrophils and epithelial cells forming reproductive and digestive systems.
The image shows an in situ hybridization analysis of lactoferrin expression in the developing oocyte and preimplantation embryo. Embryos were hybridized with a specific 35S-labeled antisense mouse lactoferrin probe. A–C, G–I, and N, Bright-field illumination; D–F, J–M, and O, dark-field illumination; A and D, developing oocyte; B and E, 2- to 4-cell embryo; C and F, 8- to 16-cell embryo; G and J, 16-cell embryo; H and K, 32-cell embryo; I and L, hatched blastocyst; M, oviduct, dark-field illumination; N and O, light-field (N) and dark-field (O) illumination of oviduct using sense control; scale bars, 20 μM (A–L) and 50μ M (M–O)
The predominant cell types involved in lactoferrin synthesis are of the myeloid series and secretory epithelia (Baynes and Bezwoda, 1994). In adults, higher levels of lactoferrin are present in milk and colostrum; it is also found in most mucosal secretions such as uterine fluid, vaginal secretion, seminal fluid, saliva, bile, pancreatic juice, small intestine secretions, nasal secretion and tears.
The production of lactoferrin by human kidneys was also described, together with the expression and secretion throughout the collecting tubules, while in the distal part of the tubules it may be reabsorbed. These results show that the kidney produces lactoferrin in a highly ordered manner and that only a minor fraction of the urine. Therefore, lactoferrin is thought to have important functions in both the immune defense of the urinary tract and in general iron metabolism.
Immunohistochemical localization of lactoferrin (LF) in sections of a normal human kidney. Sections of the kidney were incubated with a polyclonal rabbit anti-LF antibody (B, D, and F) or with a normal rabbit serum as control (A, C, and E). (B and D) These show regions from the medulla where strong staining of distal collecting tubules is observed. In contrast, no staining was detected in tissue sections originating from the cortical region of the kidney (data not shown). All blood vessels were negative (F)
Neutrophils are an important source of lactoferrin in adults; indeed, most plasma lactoferrin originates from neutrophils. lactoferrin is predominantly stored in specific (secondary) granules. However, it can also be found in tertiary granules, but in significantly lower concentrations.
Lactoferrin is present in blood, plasma or serum in relatively low concentration: its serum levels change during pregnancy and varies also with the menstrual cycle. The concentration of lactoferrin in the blood increases during infection, inflammation, excessive intake of iron, or tumor growth.
REGULATION OF SYNTHESIS
The regulation of lactoferrin synthesis depends on the type of cells producing this protein. The amount of lactoferrin synthesized in the mammary gland is controlled by prolactin, whereas its production in reproductive tissues is determined by estrogens. Its synthesis in endometrium is influenced not only by estrogens but also epidermal growth factor. Exocrine glands produce and secrete lactoferrin in a continuous manner. In neutrophils, lactoferrin is synthetized during their differentiation from promyelocytes to myelocytes and is afterwards stored in specific granules while mature neutrophils case to produce lactoferrin.
Lactoferrin levels might vary with gender and age although the results from different studies are inconsistent.
Plasma levels change from the very beginning of pregnancy: there is a progressive rise in its concentration up to the 29th week, after which it settles at a constant level that is higher than the average. Serum lactoferrin concentration have been detected as being higher in the proliferative phase of menstrual cycle than in the secretory phase.
LACTOFERRIN RECEPTORS
The biological properties of lactoferrin are mediated by specific receptors on the surface of target cells . These receptors are typical for each cell type and can be found, for example, on mucosal epithelial cells, hepatocytes, monocytes, macrophages, polymorphonuclear leucocytes, lymphocytes, trombocytes, fibroblasts, and on some bacteria such as Staphylococcus aureus or Pseudomonas hydrophila.
Some cells have also “main receptors”, which enable them to bind not only lactoferrin, but also transferrin or lactoferrins of other species.
METABOLISM
There are two ways in which lactoferrin can be eliminated from the organism: either through receptor-mediated endocytosis of phagocytic cells (macrophages, monocytes, and other cells belonging to the reticuloendothelial system) with subsequent iron transfer to ferritin or through direct uptake by the liver. Endocytosis performed by Kupffer cells, liver endothelial cells, and hepatocytes contributes to lactoferrin removal. Kidneys seem to be involved in the removal of lactoferrin from the circulation since lactoferrin and its fragments, mainly of maternal origin, have been found in the urine of breast-fed infants.
BIOLOGICAL FUNCTIONS
Lactoferrin and iron metabolism
Although the influence of LF on iron distribution in an organism is implied by its resemblance to transferrin, it has thus far not been unequivocally proven that lactoferrin plays an important role in iron transport. This may be due to the fact that lactoferrin plasma concentrations are very low under normal conditions . On the other hand, the lactoferrin level increases when inflammation occurs. In such an environment iron exchange from transferrin is easier due to the lower pH suggesting that lactoferrin may contribute to local iron accumulation at sites of inflammation .
The relationship between biliary lactoferrin concentration and the iron status of the body has been described in rabbits: a significant increase of lactoferrin in bile was registered in anemic rabbits after acute blood loss, an observation which may be explained by the mobilization of iron stored in liver. In contrast, rabbits to who iron was administered, even in low doses, showed inhibition of lactoferrin secretion in the bile. Thus, lactoferrin might have a control function in situations when increased amounts of iron are released from its depots .
Lactoferrin from human milk seems to affect intestinal iron absorption in infants , but it depends on the organisms need for iron. Specific receptors (SI-LfR), present on enterocytes, mediate binding of lactoferrin: after lactoferrin is bound to the enterocyte, 90% of it is degraded and Fe3+ ions are released. The remaining intact 10% is transported through the cell membrane. A lack of intracellular iron may evoke increased expression of specific receptors on the surface of enterocytes and thereby elevated absorption of lactoferrin-bound iron.
Even though lactoferrin does not play the most important role in iron metabolism, its capability of binding Fe3+ ions has a significant influence on many of its other biological properties.
Antibacterial activity
The antibacterial activity of Lf has been documented in the past, both in vitro and in vivo for Gram-positive and Gram-negative bacteria and some acid-alcohol resistant bacteria. Table1 shows the bacteria against which Lf has shown an inhibitory effect and the mechanism used by Lf to exert its effect. Particular attention should be given to bacteria listed in Table 1 because some of these are known to be resistant to antimicrobials, such as the strains of Staphylococcus aureus , Listeria monocytogenes, methicillin-resistant Klebsiella pneumonia and Mycobacterium tuberculosis , among others. Lf has also been proven effective against strains of Haemophillus influenzae and Streptococcus mutans that were inhibited by an iron-independent interaction with the cell surface.
The sequestration of iron away from bacterial pathogens inhibits bacterial growth, limits the use of this nutrient by bacteria at the infection site and downregulates the expression of their virulence factors. Lf's bactericidal function has been attributed to its direct interaction with bacterial surfaces. In 1988, it was shown that Lf damages the external membrane of Gram-negative bacteria through an interaction with lipopolysaccharide (LPS). The positively charged N-terminus of Lf prevents the interaction between LPS and bacterial cations (Ca+ 2 and Mg+ 2) and interferes with aggregative proliferation in E. coli . The interaction between Lf and LPS or other surface proteins also potentiates the action of natural antibacterials such as lysozyme, which is secreted from the mucosa at elevated concentrations along with Lf.
It has also been demonstrated that the N-terminal lobe of Lf possesses a serine protease-like activity. In H. influenzae , Lf is able to cleave proteins in arginine-rich regions, and the protease active site is situated in the N-terminal lobe, thus attenuating virulence and preventing colonization.
In vitro and in vivo studies have shown that Lf has the ability to prevent the attachment of certain bacteria to the host cell. The attachment-inhibiting mechanisms are unknown, but it has been suggested that Lf's oligomannoside glycans bind bacterial adhesins, preventing their interaction with host cell receptors.
Biofilm formation, which was proposed as a colonial organization adhesion method for Pseudomonas aeruginosa, is a well-studied phenomenon in patients suffering from cystic fibrosis. Through biofilm formation, bacteria become highly resistant to host cell defense mechanisms and antibiotic treatment. It is well known that some bacterial strains require high levels of iron to form biofilms. Thus, Lf's function as an iron chelator has been hypothesized to effectively inhibit biofilm formation through iron sequestration.
Antiviral activity
Lf possesses antiviral activity against a broad range of RNA and DNA viruses that infect humans and animals . Initial work suggested that only enveloped viruses were affected by Lf, and that this activity was due to several factors, including inhibition of virus–host interaction in the herpes simplex virus (HSV); inhibition of intracellular virus trafficking in the hepatitis B virus (HBV) and human cytomegalovirus (HCMV); or direct binding of lactoferrin to the viral particle in the hepatitis C virus (HCV), feline herpes virus (FHV-1), and hepatitis G virus (HGV).
The human immunodeficiency virus (HIV) remains a major medical challenge because current treatments of the syndrome that it causes are not completely effective. In vitro studies show that, among human plasma and milk proteins, Lf exerts a strong activity against HIV. This effect is due to inhibition of viral replication in the host cell. Lf also binds to three of the many co-receptors of HIV and the DC-SIGN receptor.
Lf also acts against non-enveloped viruses such as adenoviruses and enteroviruses. Several mechanisms of action have been proposed for the antiviral activity of Lf. One of the most widely accepted hypotheses is that Lf binds to and blocks glycosaminoglycan viral receptors , especially heparan sulfate (HS). The binding of Lf and HS prevents the first contact between virus and host cell, thus preventing infection.
Lactoferrin antiviral activity will be more deeply explained later.
Immunomodulatory and anti-inflammatory activity
Lf displays immunological properties that influence both innate and acquired immunities. Its relationship with the immune system is evident from the fact that people with congenital or acquired Lf deficiency have recurring infections. Oral administration of bLf seems to influence mucosal and systemic immune responses in mice. Lf can modulate both specific and non-specific expression of antimicrobial proteins, pattern recognition receptors and lymphocyte movement related proteins. The role that Lf plays in regulating innate immune responses confirms its importance as a first line host defense mechanism against invading pathogens, modulating both acute and chronic inflammation. Most intriguing is the ability of Lf to induce mediators from innate immune cells that subsequently impact adaptive immune cell function. Lf's positive charge allows it to bind to negatively charged molecules on the surface of various cells of the immune system, and it has been suggested that this association can trigger signaling pathways that lead to cellular responses such as activation, differentiation and proliferation. Lf can be transported into the nucleus, where it can bind DNA and activate different signaling pathways.
In addition to inducing systemic immunity, Lf can promote skin immunity and inhibit allergic responses. It activates the immune system against skin allergens, causing dose-dependent inhibition of Langerhans cell migration and the accumulation of dendritic cells in lymph nodes.
Leukocytes exposed to Lf modulate their cytokine production; proinflammatory cytokines, TNF-α, IL-6, and IL-1β can be modulated by Lf to increase or decrease. Production of these factors is dependent upon the type of signal recognized by the immune system. At the cellular level, Lf increases the number of natural killer (NK) and adaptative (T strain CD4+ and CD8+) cells, boosts the recruitment of polymorphonuclear cells (PMNs) in the blood, induces phagocytosis, and can modulate the myelopoietic process.
It is well documented that IL-12 plays an important role in driving development of helper T-cell type 1 immunity. Therefore, the role of Lf in the regulation of proinflamatory cytokines and IL-12 clearly demonstrates communication between innate and adaptative immune responses.
Anticarcinogenic activity
The anti-tumor properties of Lf were discovered about a decade ago and have been confirmed by numerous laboratory studies which have shown that bovine lactoferrin (bLf) significantly reduces chemically induced tumorigenesis when administered orally to rodents. Since then, human clinical studies are showing that ingestion of LF can have a beneficial effect against progression of cancer: bLf possesses antimetastatic effects and inhibits the growth of transplanted tumors. Similar to its role in inflammation, Lf has the ability to modulate the production of cytokines in cancer. Lf can induce apoptosis and arrest tumor growth in vitro; it can also block the transition from G1 to S in the cell cycle of malignant cells. Additionally, treatment of tumors in mice with recombinant human Lf (rhLf) inhibits their growth, increases the levels of anticarcinogenic cytokines such as IL-18, and activates NK cells and CD8+ T lymphocytes. Interestingly, bLf and hLf exert opposite effects on angiogenesis. Whereas orally administrated bLf inhibits angiogenesis in rats and tumor-induced angiogenesis in mice, hLf exerts a specific pro-angiogenic effect. Recently, colorectal cancer was inhibited by bLf in animal models, and human Lf reduced the risk of colon carcinogenesis as demonstrated by a clinical trial. Increasing evidence suggests that inhibition of the Akt signaling pathway might be a promising strategy for cancer treatment. In breast cancer, Lf is able to limit the growth of tumor cells, and addition of exogenous Lf to the culture media of breast cancer cell lines (MDA-MB-231) induced cell cycle arrest at the G1/S transition. Additionally, Lf induced growth arrest and nuclear accumulation of Smad-2 in HeLa cells. The ability of bovine Lfcin to induce apoptosis in THP-1 human monocytic leukemic cells has also been demonstrated. Although the results achieved by several researchers point to a clear anti-tumor role for Lf, the mechanisms by which it exerts these effects are not fully understood. Thus, further work on this subject is required.
BIOACTIVE PEPTIDES DERIVED FROM LACTOFERRIN
Lactoferrin was first isolated by Groves in 1960 and was recognized as a “red protein from milk”. Moderate proteolysis led to a release of two Lf fragments, namely the N- and C-terminal lobes. Enzymatic treatment of bLf with pepsin produced a low molecular weight peptide with antibacterial properties against a large number of Gram-positive and Gram-negative bacteria, in addition to fungi. Bellamy et al. identified a region of amino acids at the N-terminus that retains its biological activity when separated from the full molecule; this was termed lactoferricin (Lfc-B) and was shown to exhibit greater antimicrobial activity than Lf: The activity that is exerted by this region corresponds to residues 17–41 of bLf. The region also includes two Cys residues linked by a disulfide bridge that contains many hydrophobic and positively charged residues.
The tertiary structure of Lfcin is markedly different when compared to its homologous region of intact Lf (Fig): an in situ model of hLfcin (Fig.a) and a solvated version (Fig.b) show structural differences at their N- and C-termini (Fig.a, b). A single β-sheet strand replaces the long α-helix observed in the Lf structure; such a structure may be better suited to recognize bacterial membrane topology. Additionally, the bLfcin peptide contains an alpha-helix. This region retains high homology among other species of mammals and corresponds to amino acids 12–48.
It was also found that minimal variations in the amino acid sequence change the antimicrobial activity of the peptide. For example, Lfampin 268–284 and Lfampin 265–284, chemically synthesized fragments from the N-terminal sequence of bLf, differ in only three amino acids (265Asp-Leu267-Ile268) but exhibit different strengths of antimicrobial activity. Proteolysis of iron-free Lf could release Lf-derived active peptides in biological fluid.
CLINICAL APPLICATIONS
Lf can be isolated from cow's milk by various purification methods, or it can be expressed by recombinant methods. Because of the multiple functions of Lf, it has been used for clinical trials and industrial applications. One of the first applications of Lf was in infant formula. Currently it is added to immune system-enhancing nutraceuticals, cosmetics, pet care supplements, drinks, fermented milks, chewing gums, and toothpaste. The ability of Lf to prevent nososcomial infection in infants was tested and the results showed lower infection levels. Several studies showed that infants fed with infant formulas had less intestinal iron absorption than breastfed infants. It was proposed that Lf also promotes the proliferation of lactic acid bacteria in the bowel, such as Bifidobacterium and Lactobacillus, which protect the host from harmful bacteria.
The activity of Lf and its bioactive peptides has been documented both in vitro and in vivo against a wide variety of pathogens. Clinical trials demonstrated the efficiency of Lf for use in treating infections and inflammatory diseases. For example, Lf was tested as a second treatment against H.pylori in patients with recurring infections. The patients supplemented with bLf showed a greater recovery from infection. Lf also exhibits synergistic activity with antifungal agents, thus reducing the minimum inhibitory concentrations of these agents against C.albicans and C. glabrata .
As with antibacterials, Lf exhibits synergy with antiviral drugs in the treatment of hepatitis C, cytomegalovirus and HIV. Lf delays the hypersensitivity response and limits the pathology caused by M. tuberculosis by increasing IL-12 and IL10 expression. A clinical trial was conducted which demonstrated that ingestion of bLF could reduce the risk of colon cancer in humans. Lf also offers applications in food preservation and safety because it can decrease bacterial counts in pork meat, retard lipid oxidation and limit the growth of microbes.
Lf can also be used as a molecular marker; detection of urinary Lf via electrochemical immunosensors aids in the diagnosis of urinary tract infections. Increased levels of Lf serve as a clinical marker of inflammatory Severe Acute Respiratory Syndrome or septicemia.
ANTIVIRAL ACTIVITY OF LACTOFERRIN
For decades it has been generally accepted that breast-feeding is beneficial for the newborn: comparative studies between bottle-fed and breast-fed children showed that those who had received their mothers' milk were less susceptible to several diseases such as diarrhea, rotavirus and RSV infections (Lopez Alarcon et al., 1997 ).
Several constituents in breast milk may have a potentially protective effect. Not only proteins of the non-specific immune system (lysozyme, lactoperoxidase, LF), but also specific immunoglobulins (IgM, IgG and secretory IgA), lipid components, cytokines or prostaglandins help in the protection of the newborn. Later studies have shown that at least part of the antiviral properties of breast milk can be attributed to a direct antiviral activity of LF . LF comprises antiviral activity against a wide range of human and animal viruses, both RNA- and DNA-viruses. An overview of these antiviral activities and the possible mechanism underlying those activities of LF will be given below.
The antiviral activity of hLf was first demonstrated in mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P) . Since 1994,
potent antiviral activity of hLf and bLf has been demonstrated against both enveloped and naked viruses.
Hepatitis C virus (HCV)
Hepatitis C virus (HCV) is a member of the flaviviridae family: it is an enveloped virus that contains a positive, single strand RNA genome and whose unique feature is the ability to cause a persistent infection. Therefore, HCV is associated with the cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.
Ikeda et al. , in 1998, observed the antiviral effect of lactoferrin on HCV replication in cultured human hepatocytes and noticed that it was lost after heat treatment, indicating that the natural conformation of this protein is needed to exert its antiviral effect.
They also later noticed that Lactoferricin (LFcin), a tryptic digest obtained from the N-terminal region of the N-lobe, strongly bactericidal and fungicidal, was totally ineffective against HCV. This further illustrates the need for the natural conformation of LF for its antiviral activity. Time of addition assays indicated that LF probably interferes with adsorption of HCV to the target cells: it is most effective if administered before or simultaneous with the viral inoculum.
LF can prevent adsorption to target cells by the fact that it binds to the envelope proteins of HCV E1 and E2 (Yi et al., 1997 ). In addition, it was shown that LF interfered with binding of HCV E2 in vivo, since anti-human LF antibodies, in the presence of LF, were able to co-precipitate secreted and intracellular forms of E2, which were transiently expressed in HepG2 cells; according to its inability in protecting from HCV, LFcin couldn't bind to these envelope proteins E1 or E2.
Liao et al. recently compared the effects of recombinant camel lactoferrin (rcLF), native camel lactoferrin (ncLF) and their N and C fragments on HCV infection. The inhibitory effects on HCV entry into Huh7.5 cells were evaluated by incubation of HCV with LF prior to infection or pre-treatment of the cells with LF prior to infection. The inhibitory effect on HCV amplification in Huh7.5 cells was determined by LF treatment of HCV-infected cells. Nested RT-PCR was performed to amplify intracellular HCV 5' non-coding RNA sequences. The results show that rcLF and ncLF and their fragments could prevent HCV entry into Huh7.5 cells by direct interaction with the virus and inhibited virus amplification. Therefore, the N and C lobes of ncLF are sufficient to elicit anti-HCV effects in Huh7.5 cells; rcLF and its N lobe displayed similar HCV inhibitory effects to their native counterparts and may constitute an efficient and cost-effective approach for potential clinical applications.
LF may thus well be among the candidates for an anti-HBV reagent that could prove effective
in treatment of patients with chronic hepatitis: Kaito et al. evaluated the effect of combined triple therapy of lactoferrin, interferon and ribavirin in patients with Cronic Hepatitis C (CHC). What they observed was that the mean HCV RNA titer significantly decreased at the end of lactoferrin monotherapy (which was well tolerated and no patient showed any adverse event):
Statistically significant differences were observed between the baseline HCV RNA titer (2653 ± 2405 × 104 copies/mL) and after 8 weeks of therapy (2408 ± 2643 × 104 copies/mL) in the Lf monotherapy group (P < 0.05). In the control group, HCV RNA titer was measured over 8 weeks in 31 out of 45 patients. The HCV RNA titers did not change significantly.
There were more responders in the Lf monotherapy group (26%, 11/42) than in the control monotherapy group (6%, 2/31) (P < 0.05). Serum levels of anti-Lf antibody were significantly elevated in the Lf non-responder group compared with the Lf responder group after 8 weeks of Lf monotherapy (P < 0.01):
As no patients became negative for serum HCV RNA after Lf monotherapy, they performed the second stage of their study using combined triple therapy of Lf, IFN and ribavirin: fifty patients were treated with these three compounds while sixty-one control patients received only IFN and ribavirin. They found a virological response rate of 55% (6/11) in the Lf responder group, only 6% (2/31) in the non-responder group and 18% (8/45) in the control group.
This response was significantly higher in the Lf responder group than in the control group, suggesting an important role of Lf treatment in HCV eradication. The type of response to Lf might be useful as a predictive parameter in the continuation of the combination therapy with IFN and ribavirin.
Rotavirus
Rotavirus is a member of the reoviridae-family whose genome consists of 10 different segments of double stranded RNA, packaged within a three-shelled capsid. Rotavirus infections are the most frequent cause of non-bacterial gastroenteritis in neonates and children in the world, causing approximately 1 million death cases world-wide every year.
LF displays a potent inhibition of simian rotavirus SA11 replication in vitro (Superti et al., 1997). In these studies, apo-LF was as potent in inhibiting rotavirus as the metal saturated LF isoforms, but apo-LF had a 600 times higher selectivity index, due to its lack of toxicity.
The antiviral mechanism of LF against rotavirus lies in the prevention of adsorption of the virus to the target cell, since LF is capable of binding virus particles, as determined with flow cytometry of virus binding to target cells, thus preventing both rotavirus hemagglutination and viral binding to susceptible cells. Since in contrast with many other viruses, rotavirus does not bind to glycosaminoglycans as heparan sulphates and for this reason it is thought that LF cannot compete with rotavirus for binding to its cellular receptors.
Moreover, rotavirus antigen synthesis in intestinal cultured cells was markedly inhibited when LF was added during the viral adsorption step or when it was present in the first hours of infection, suggesting that it interferes with the early phases of rotavirus
infection. Therefore, LF not only prevents infection, but also maintains an antiviral effect after the virus has entered the target cell.
Manganese- or zinc-saturated bLF had slightly decreased activity, compared with apo- or ironsaturated bLF, and the removal of sialic acid enhanced the anti-rotavirus activity (Superti et al., 2001 ):
Dose–response curves of apo-lactoferrin (□), Fe3+-lactoferrin (♢), Zn2+-lactoferrin (○), and Mn2+-lactoferrin (▵) towards SA-11 rotavirus cytopathic effect in cultured HT-29 cells.
Respiratory syncytial virus (RSV)
Infections with respiratory syncytial virus (RSV), a member of the paramyxoviridae family, are the most common cause of acute lower airway infections in infants and children. Breast milk has a protective effect against illness from RSV infections, but little is known about its components that play a role in the antiviral effect against RSV.
Even if it is thought that immunoglobulins and lipids are the most important components of breast milk, neutralising activity in it could not be related to presence of immunoglobulins (Laegreid et al. in 1986 suggested that both immunoglobulin and non-immunoglobulin components in human milk can neutralize RSV).
Sano et al. noticed that RSV-induced IL-8 secretion from Hep-2 cells was down-regulated by LF, which also decreased RSV infectivity. Interactions of LF with RSV F protein, the most important surface glycoprotein for viral penetration, were examined so as to clarify the mechanism of this effect and LF was shown to directly interact with the F(1) subunit, which involves antigenic sites of F protein.
RSV-induced IL-8 secretion from HEp-2 cells was altered by pre-incubation of RSV with LF. (A) RSV was pre-incubated in the absence or presence of LF for 2 h, and inoculated to HEp-2 cells. The cells were washed and further incubated for 5 h. IL-8 secretion into media was measured and expressed as percent of the secretion induced by RSV alone. (B) RSV was pre-incubated with or without LF (100 μg/ml) in the presence of 0.2 M mannose, and RSV-induced IL-8 secretion was compared.
LF reduced RSV entry into HEp-2 cells. (A) RSV infectivity to HEp-2 cells. HEp-2 cells were inoculated with RSV, which was pre-incubated with or without LF (100 μg/ml) for 2 h. The cells were further incubated for 5 h and RSV titers in the media were examined by plaque forming assay. (B) RSV uptake by HEp-2 cells. RSV was labeled with FITC and incubated in the absence or presence of LF (100 μg/ml) for 1 h, and added to HEp-2 cells.
The RSV F protein mediates the fusion of the virion to the cellular membrane, and it is the most important protein not only in virus propagation but also in virus-induced pro-inflammatory response. Therefore, it is considered that the interaction of LF with F protein must be the important mechanism by which LF modulates the RSV infection to HEp-2 cells. It was also found by immunoprecipitation that LF binds to RSV G protein. Although the data are not shown, we also found by immunoprecipitation assay that both SP-A and LF bound to RSV G protein. The physiological meaning of the binding to G protein remains to be clarified.
Herpesviridae: Herpes Simplex Virus (HSV)
Herpes simplex virus type 1 and 2 (HSV-1 and -2) are members of the α-herpes virus family. The genome of all herpes viruses consists of DNA and infection with HSV can be persistent or latent. Reactivation of HSV-1 and -2 causes mild disease in immunocompetent subjects. However, reactivations in immunocompromised patients such as AIDS-patients, transplant recipients and premature neonates can be quite severe and even life threatening. Several groups have firstly reported antiviral effect of bovine and human LF against both HSV-1 and -2. Both apo-LF, as well as holo-LF were capable of inhibiting both viruses (Hasegawa et al., 1994). Later, Fujihara et al. (Fujihara and Hayashi, 1995) reported antiviral activity of LF against HSV-1 in vitro, but also in vivo in a mouse cornea infection model. Topical administration of 1% LF solution significantly decreased infection, however, virus replication was not fully inhibited.
Other groups have recently confirmed the in vitro antiviral activity of LF against both HSV-1 and -2, among them Jessen et al. in 2008 explained both lactoferrin and lactoferricin ability to inhibit HSVcell-to-cell spread: plaque reduction assays following pre-attachment of HSV-1 to the cell surface had previously demonstrated that both bovine Lf and Lfcin caused a decreased number of plaques compared to the positive control (Andersen et al., 2004), but this could have been due to inhibition of intracellular growth of virus or to inhibition of cell-to-cell spread. As a consequence, they investigated the role of Lf and Lfcin in inhibiting cell-to-cell spread of the virus. The results (figure below) illustrated that bovine Lf strongly inhibited plaque formation by both HSV-1 and HSV-2, while human Lf showed a somewhat lesser reduction in plaque formation of 20–30%. Plaque size was also significantly reduced, compared to the untreated positive control, consistent with an interference with the viral cell-to-cell spread. Both bovine and human Lfcin moderately reduced the number of plaques in HSV-1-infected cells, while no effect was observed towards HSV-2. In this case, the plaque size was not influenced by the presence of the peptides.
Number of plaques observed in an infectious center assay after 48 h incubation in the presence of bovine/human Lf (bLf/hLf) and bovine/human Lfcin (LfcinB/LfcinH), after infection with (A) HSV-1 or (B) HSV-2.
Investigating the mechanism of inibition, they demonstred that Glycosaminoglycan were involved. The initial attachment of HSV to cells occurs through binding of the viral glycoprotein(s) gC or gB to HS (Heparan Sulfates) of host cells. In the absence of HS, virus can bind to chondroitin sulfate proteoglycans (CS), although with lower efficiency. When bovine Lfcin was added after the initial viral entry period, a significant plaque inhibition was observed compared to the control. To reveal if this effect could be linked to the presence of heparan- or chondroitin sulfate, these glycosaminoglycans were separately removed by enzymatic treatement after initial infection. When infected cells were treated with chondroitinase-ABC or heparinase-III after viral entry, in the absence of bovine Lfcin, no plaque reduction activity was observed, either in terms of total plaque counts or plaque size. This indicates that enzymatic removal of either of the glycosaminoglycans alone had no effect on the plaque formation. However, when the infected cells were exposed to bovine Lfcin, and then incubated with chondroitinase-ABC for 2 days to remove chondroitin sulfate from the cell surface, the antiviral activity of bovine Lfcin was abolished, resulting in no significant change in plaque formation when compared to the positive control. When the same experiment was done with heparinase-III, to remove heparan sulfate from the cell surface, the number of plaques still decreased significantly compared to the positive control (fig. below). In addition to reducing the number of plaques, bovine Lfcin also reduced the plaque size in the absence of heparan sulfate, confirming that the peptide continued to have an effect on the viral cell-to-cell spread mechanism. This clearly illustrates that Lfcin is able to inhibit plaque formation in the absence of heparan sulfate, probably by interacting with chondroitin sulfate, indicating that inhibition of cell-to-cell spread by Lfcin is dependent on a completely different glycosaminoglycan to that used in inhibition of initial infection.
After the initial binding of the virus to the host cell, BLfcin also has anti-HSV activity intracellularly, which was investigated by Marr et al. : the cellular uptake and intracellular trafficking of HSV-1 in Vero cells treated with BLf or BLfcin was followed, using immunofluorescence microscopy to visualize both intracellular HSV-1 capsids and host cell microtubules.
Despite the high viral concentration, both BLf and BLfcin were able to significantly reduce the number of HSV-1 capsids entering the cells, compared to the untreated infection control cells (Fig. below). HSV-1 cellular uptake was not completely inhibited by the presence of either BLf or BLfcin, however. In the control-infected cells, HSV-1 proteins were already found in the nucleus 1.5 h after viral infection (Fig. a). For cells treated with BLf or BLfcin, however, the HSV-1 capsids appeared to remain associated with microtubules at the periphery of the cells (Fig. b and c), indicating a possible interference with trafficking of the HSV-1 capsids along microtubules towards the nucleus.
BLf and BLfcin reduction of HSV-1 uptake and delay of the trafficking of HSV-1 towards the nucleus. Immunofluorescence microscopy of Vero cells infected with HSV-1 in the absence of BLf/BLfcin (Control; a, d, g, j), or in the presence of BLf (b, e, h, k) and BLfcin (c, f, i, l). The arrows indicate the few HSV-1 capsids that entered the cells treated with BLf/BLfcin at 1.5 and 4 h post-infection. The arrowheads in (a) point to the viral proteins already accumulating in the nucleus at 1.5 h post-infection.
Furthermore, HSV-1 capsid trafficking and viral protein accumulation within the nucleus appeared to be delayed in the presence of BLf and BLfcin; The delay should not be attributed to the reduction of viral uptake, because control cells infected at an m.o.i. of 2.5, which have numbers of internalized virus capsids similar to that shown in Fig. b and c, revealed a larger numbers of fluorescent-labeled spots in the nucleus than the BLf- or BLfcin-treated cells infected at an m.o.i. of 25.
These observations may reflect an essential mode of antiviral activity of BLf and BLfcin, since intact microtubules play a crucial role in the efficient nuclear targeting of several viruses.
Human Immunodeficiency virus (HIV)
Infection with human immunodeficiency virus (HIV), a member of the lentiviridae, causes AIDS. The genome consists of single stranded RNA that is packaged in a capsid. The capsid is surrounded with an envelope, which contains glycoproteins that are involved in the entry of the target cell. Data about LF levels in plasma or saliva of HIV-infected subjects are conflicting: an increase in LF levels but also decreases in LF levels were observed. However, the observed decreases in LF levels were eminent in tears and plasma of symptomatic AIDS patients, who are more often subject to opportunistic infections. Semba et al. in 1998 also demonstrated a linear correlation between low maternal serum LF levels and perinatal transmission of HIV to the neonate. All these clinical data demonstrate that LF is involved in the antiviral defense against HIV in vivo.
Native bLf or hLf (iron saturation 10–20%) were observed to inhibit the HIV-1-induced cytopathic effect. When negatively charged groups were added to Lf by succinylation, there was a strong antiviral effect on HIV-1 and HIV-2, whereas addition of positive charges to Lf through amination resulted in a loss of anti-HIV activity. Both native Lf and the charged-modified protein bind strongly to the V3 loop of the gp120 envelope protein, resulting in inhibition of virus-cell fusion and entry of the virus into cultured cells [108]. Both HIV-1 replication and syncytium formation were also inhibited efficiently, in a dose-dependent manner, when added prior to HIV infection
or during the viral adsorption step, thus that Lf blocks HIV binding to or entry into cultured cells. Modest inhibition of HIV infection was obtained with bLfcin, indicating that other domains within the native bLF protein may be required to inhibit HIV-1 entry.
Carthagena et al. evalueted whether human LF (hLF) and its exposed domain LF-33 represented by the peptide (LF-33-GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP), which constitutes the glycosaminoglycans recognizing site of the human LF (hLF) and is involved in LF-HIV gag binding and endotoxines neutralization, may inhibit early steps of HIV mucosal transmission.
They investigated whether hLF and LF-33 peptide were able to inhibit the attachment of free R5-tropic HIV-1JR-CSF and X4-tropic HIV-1NDK particles to HT-29 (colorectal) and HEC-1 (human endometrial) CD4-negative epithelial cells. Pre-incubation of cells with human LF at 10 µg, 30 µg and 60 µg, leads to a decrease of 38%, 56% and 76% of the attachment of HIV-1JR-CSF and of 43%, 58% and 60% of the attachment of HIV-1NDK, on HEC-1 cells (Fig. below A), respectively; and of 24%, 47% and 64% of the attachment of HIV-1JR-CSF, and of 49%, 70% and 68% of the attachment of HIV-1NDK, on HT-29 cells (Fig. B).
The LF-33 peptide exhibited a lower inhibitory effect on HIV-1 attachment to epithelial cells as compared to hLF. Indeed, the observed reductions of HIV-1JR-CSF binding on HEC-1 cells at 10 µg, 30 µg and 60 µg of LF-33 peptide were 28%, 41% and 38%, respectively, and those of HIV-1NDK bound on HEC-1 cells were 30%, 27% and 30%, respectively (Fig. C). The decreases of HIV-1JR-CSF attachment to HT-29 cells at 10 µg, 30 µg and 60 µg of LF-33 peptide were of 52%, 52% and 47%, respectively, and those of HIV-1NDK attachment on HEC-1 cells were 39%, 32% and 31% respectively (Fig. D). Irrelevant peptide control used instead LF-33 showed not effect on virus attachment (not shown).
In addition, the hLF, but not the LF-33 peptide, inhibited up to 40% the transfer in trans of HIV-1JR-CSF and HIV-1NDK, from immature dendritic cells to CD4 T lymphocytes, likely in a DC-SIGN-dependent manner: the percentages of HIV-1JR-CSF transfer by hLF were decreased by 56% and 61%, respectively (Fig. below, B); and those of HIV-1NDK transfer were 44% and 64%, respectively. No inhibitory effect was observed when the LF-33 peptide was used. Unexpectedly, the incubation with the LF-33 peptide enhanced 3 to 6 fold the HIV-1 transfer from iDC to autologous CD4 T cells, as shown in Fig. D, likely because the peptide increased the HIV-1 attachment to iDC, as demonstrated by binding inhibition experiments (Fig. C).
Altogether, these findings demonstrate that hLF can interfere with HIV-1 mucosal transmission by blocking virus attachment to epithelial cells and by inhibiting virus transfer from dendritic cells to CD4 T cells, two crucial steps of HIV dissemination from mucosae to lymphoid tissue.
CONCLUSIONS
All these datas demonstrate that Lf can be considered not only a primary defence factor against mucosal infections, but also a polyvalent regulator, which interacts with several viral and host components involved in infectious processes. Its capability to exert antiviral activity, through its binding to host cells and/or viral particles, strengthens the idea that this glycoprotein is an important brick in the mucosal wall, effective against viral attacks, and candidates it as a potential protective weapon against virus infections.
