key: cord-0989687-mzak7czg authors: García-Montoya, Isui Abril; Cendón, Tania Siqueiros; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín title: Lactoferrin a multiple bioactive protein: An overview() date: 2011-06-25 journal: Biochim Biophys Acta Gen Subj DOI: 10.1016/j.bbagen.2011.06.018 sha: fd4b55153c383c93c61a4da80abd0c3f6dd26c6a doc_id: 989687 cord_uid: mzak7czg BACKGROUND: Lactoferrin (Lf) is an 80 kDa iron-binding glycoprotein of the transferrin family. It is abundant in milk and in most biological fluids and is a cell-secreted molecule that bridges innate and adaptive immune function in mammals. Its protective effects range from anticancer, anti-inflammatory and immune modulator activities to antimicrobial activities against a large number of microorganisms. This wide range of activities is made possible by mechanisms of action involving not only the capacity of Lf to bind iron but also interactions of Lf with molecular and cellular components of both hosts and pathogens. SCOPE OF REVIEW: This review summarizes the activities of Lf, its regulation and potential applications. MAJOR CONCLUSIONS: The extensive uses of Lf in the treatment of various infectious diseases in animals and humans has been the driving force in Lf research however, a lot of work is required to obtain a better understanding of its activity. GENERAL SIGNIFICANCE: The large potential applications of Lf have led scientists to develop this nutraceutical protein for use in feed, food and pharmaceutical applications. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders. Lactoferrin (Lf), first reported almost 50 years ago [1] [2] [3] , is a nonhemic iron-binding protein. It is a member of the transferrin family, along with serum transferrin, ovotransferrin, and melanotransferrin [4] , all of which function in iron transport, and inhibitors of carbonic anhydrase, Saxyphillin, and Pacifastin are also members of this superfamily. Lf is produced by mucosal epithelial cells in various mammalian species including humans, cows, goats, horses, dogs, and rodents, and it is also produced by fish [5] . This multifunctional glycoprotein is found in mucosal secretions including tears, saliva, vaginal fluids, semen [6] , nasal and bronchial secretions, bile, gastrointestinal fluids, urine [7] , milk and colostrum [8] . The most abundant antimicrobial proteins include lysozyme, collectin [9, 10] and lactoferrin. Lf possesses a greater iron-binding affinity, and it is the only transferrin with the ability to retain this metal over a wide range of pH values, including resistance to proteolysis. The most striking physicochemical feature of Lf is its very high affinity for iron. In both Lf and related transferrins (Tfs), two Fe + 3 ions are bound very tightly (K~1022 M) but reversibly to LF, with two synergistically bound CO 3 2− ions [11, 12] . Because of its wide distribution in various tissues, Lf is a highly multifunctional protein. Indeed, it is involved in many physiological functions, including regulation of iron absorption and immune responses; it also exhibits antioxidant activity and has both anticarcinogenic and anti-inflammatory properties. However, its antimicrobial properties are its most widely studied function [8, [12] [13] [14] [15] [16] . The antimicrobial activity of Lf is driven mostly by two mechanisms. The first involves iron sequestration in sites of infection, which deprives microorganisms of this nutrient and causes a bacteriostatic effect. The second mechanism is the direct interaction of the Lf molecule with the infectious microorganism. In some cases, positively charged amino acids in Lf can interact with anionic molecules on certain bacterial, viral, fungal and parasite surfaces, causing cell lysis. The physiological capabilities of Lf in host defense combined with current pharmaceutical and nutritional needs have led to the classification of Lf as a nutraceutical protein, and for several decades, investigators have looked for the most convenient way to produce it. Three basic approaches are currently being used. First, native Lf can be commercially purified from the milk and colostrum of several mammals. Second, recombinant Lf (rLf) can be generated from bacterial, fungal and viral expression systems. Third, transgenic plants and animals have been generated with express rLF. The nucleotide sequence of human milk Lf (hLf) was first determined by Ray et al. in 1990 [18] and compared with the amino acid sequence of human lactotransferrin determined previously [17] . 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 Several functions have been attributed to Lf. It is considered to be a key component of the innate host defense system because it can respond to a variety of physiological and environmental changes [27] . The structural features of Lf provide additional functionalities beyond the Fe + 3 homeostasis function common to all transferrins. Specifically, Lf exhibits strong antimicrobial activity against a broad spectrum of bacteria (Gram+ and Gram−), fungi, yeasts, viruses [14] and parasites [29] , although it seems to promote the growth of beneficial bacteria like Lactobacillus and Bifidobacteria [28] . It also exhibits anti-inflammatory and anticarcinogenic activities [27] and has several enzymatic functions [30] . Lf plays a key role in maintaining cellular iron levels in the body. One of the first antimicrobial properties discovered for Lf was its role in sequestering iron from bacterial pathogens. This was believed to be the sole antimicrobial action of lactoferrin because apo-lactoferrin possessed antibacterial activity [31, 32] . It was later demonstrated that lactoferrin can also kill microorganisms through an iron-independent mechanism [33] in which lactoferrin directly interacts with the bacterial cell surface [31, 34, 35] . 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. Table 1 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 pneumoniae 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 [39, 45] . 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 [60, 65] . 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) [66] . The positively charged N-terminus of Lf prevents the interaction between LPS and bacterial cations (Ca + 2 and Mg + 2 ) [67, 68] and interferes with aggregative proliferation in E. coli [52] . 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 [66] . It has also been demonstrated that the N-terminal lobe of Lf possesses a serine protease-like activity [39] . 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 [69] . 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 [48, 63] . 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 [70] [71] [72] . 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 [73, 74] . Lf possesses antiviral activity against a broad range of RNA and DNA viruses that infect humans and animals [6] . 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) [75, 76] ; inhibition of intracellular virus trafficking in the hepatitis B virus (HBV) [77] [78] [79] and human cytomegalovirus (HCMV) [80, 81] ; or direct binding of lactoferrin to the viral particle in the hepatitis C virus (HCV) [82, 83] , feline herpes virus (FHV-1) [16] , and hepatitis G virus (HGV) [84] . 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 [39, 85, 86] . Lf also binds to three of the many co-receptors of HIV [87] and the DC-SIGN receptor [88] . The interaction of Lf with surface nucleolin was shown to block the attachment and entry of HIV particles into HeLa P4 cells [87] . Fig. 2 illustrates structural in situ models of the C-and N-lobes. Interestingly, two large fragments of bLf, the C-lobe (aa 345-689) and the N-lobe (aa 1-280), have been shown to inhibit HSV infection [89] . The N-lobe the peptides 222-230 (ADRDQYELL) and 264-269 (EDLIWK) were also able to inhibit HSV-1 infection when chemically synthesized, albeit only when they were assayed together [89] . Additionally, smaller antibacterial peptides such as bLficin interfere with HSV-1 cell-to-cell movement [77] . Human respiratory syncytial virus (RSV) is inhibited by Lf at concentrations ten times lower than those found in human milk. Lf also acts against non-enveloped viruses such as adenoviruses and enteroviruses [90] . 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 [6] . The antiviral effect of Lf has also been observed in viruses that infect animals, such as the Friend virus complex, which causes erythroleukemia in rodents [92] , the feline calcivirus [93] , and the feline immunodeficiency virus [16] . It appears that Lf exhibits its antiviral activity at an early phase of the infection process [16, 75, 78, 83, 84, 91, [94] [95] [96] [97] . Several viral pathogens have been shown to use the host cell surface as herpes simplex virus, as an attachment receptor during the infection process [98, 99] . Results, also 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 [100] . Candida can colonize mucosal surfaces in individuals, and it is considered to be analogous to a commensal organism that can also become an opportunistic pathogen. Kirkpatrick et al. conducted the first studies with Candida spp. in 1971 and attributed the antifungal effect of Lf to its ability to sequester Fe + 3 [101, 102] . Both hLf and bLf, as well as Interferes with polysaccharide cell content [64] the bLf-derived peptide lactoferricin, have well-documented in vitro activity toward human pathogenic fungi, especially Candida albicans and several other Candida species. Lf also has antifungal activity [103, 104] , and it was observed that Lf could kill both C. albicans and C. krusei by altering the permeability of the cell surface, as it does in bacteria [105, 106] . Bovine Lf has been shown to be highly fungicidal for C. tropicalis and C. krusei and somewhat fungicidal for C. albicans and C. guilliermondii, while C. glabrata is almost resistant to Lf [107] . Lf exhibited activity against Cryptococcus neoformans and C. albicans via cytoplasmic and mitochondrial membrane permeabilization [108] . Although Lf's antifungal mechanism of action is through cell surface interaction rather than iron deprivation [109] , several reports demonstrated its ability to cause cell wall damage [107, 110, 111] . In addition to direct interaction with the pathogen, Fe + 3 sequestration is another important mechanism for fungicidal activity. In 2007, Zarember et al. showed that Fe + 3 sequestration by neutrophil apo-Lf is important for host defense against Aspergillus fumigatus [112] . Additionally, the in vitro antifungal activity of two peptides (hLF(1-11 aa), bLf N1-domain)) derived from human lactoferrin (hLF) were compared, and dose-dependent antifungal activity was observed [113, 114] . Lf shows an interesting antifungal effect on body tineas caused by Trichophyton mentagrophytes, against which it acts indirectly, facilitating clinical improvement of skin lesions after the peak of the symptoms. Treatment of guinea pigs with bLf reduces fungal infection on the skin of the back and limbs in tinea corpus and tinea pedis, respectively [115] . It has also been demonstrated that Lf can mediate its antifungal activity through the stimulation of host cell immune mechanisms both in vitro and in vivo [102] . A clear understanding of the antimicrobial activities of Lf has been difficult to achieve because the mechanisms of action of Lf and the ecological niches of microbes often differ from one organism to another. The molecular mechanisms of Lf antiparasitic activity are even more complex. Antiparasitic activities of Lf usually involve interference with iron acquisition. This activity has also been shown using peptides derived from the full molecule [116, 117] . There is also evidence supporting the occurrence of a similar mechanism during amoebiasis, which is one of the leading causes of diarrhea in children under 5 years of age and is caused by Entamoeba histolytica [118] . Apo-Lf is the milk protein with the greatest amoebicidal effect against E. hystolitica in vitro because it can bind to lipids on the trophozoite's membrane, causing membrane disruption and damage to the parasite [119, 120] . Lf appears to act as a specific iron donor and could be expected to enhance infection by other parasites such as Tritrichomonas foetus [121] . It was reported that bLF bound to components of T. brucei, and that bLF hydrolysate disrupted the sites responsible for binding to parasite proteins, causing Fe + 3 deprivation [122] . Other in vitro studies show that serum transferrin as well as human and bovine Lf can bind the intracellular parasite Toxoplasma gondii, which causes toxoplasmosis and affects both humans and animals. However, Lf cannot prevent the parasite from entering the host. The mechanism of action in this case is inhibition of the intracellular growth of T. gondii in the host cells [123] . In animal models, a lactoferricin reduced infectivity of T. gondii and Eimeria stiedai sporozoites [124] . The effect of Lf on the hemoparasites Babesis caballi and Babesia equi depends on whether or not Lf is bound to Fe + 3 [125] . B. caballi was found to be significantly suppressed by apo-Lf but was not inhibited by the other types of Lf, whereas none of the Lf types had an inhibitory effect against B. equi [126] . Lf also demonstrates additive or synergistic activity with clinically used antiparasitic compounds [116, 118, 119] . Lf displays immunological properties that influence both innate and acquired immunities [127] . Its relationship with the immune system is evident from the fact that people with congenital or acquired Lf deficiency have recurring infections [128] . Oral administration of bLf seems to influence mucosal and systemic immune responses in mice [129] . Lf can modulate both specific and non-specific expression of antimicrobial proteins, pattern recognition receptors and lymphocyte movement related proteins [130] . 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 [131] [132] [133] [134] . 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 [135] , 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 [86, 136] and activate different signaling pathways [7] . 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 [6] . Leukocytes exposed to Lf modulate their cytokine production; proinflammatory cytokines, TNF-α, IL-6, and IL-1β can be modulated by Lf to increase [137] [138] [139] or decrease [131, [139] [140] [141] . Production of these factors is dependent upon the type of signal recognized by the immune [24] , and viewed using Chimera software (http://www.cgl.ucsf.edu/chimera/). system. At the cellular level, Lf increases the number of natural killer (NK) and adaptative (T strain CD4+ and CD8+) cells [142] , boosts the recruitment of polymorphonuclear cells (PMNs) in the blood [143] , induces phagocytosis [144] , and can modulate the myelopoietic process [145] . It is well documented that IL-12 plays an important role in driving development of helper T-cell type 1 immunity [146, 147] . Therefore, the role of Lf in the regulation of proinflamatory cytokines and IL-12 clearly demonstrates communication between innate and adaptative immune responses. 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 [148] . Since then, human clinical studies are showing that ingestion of LF can have a beneficial effect against progression of cancer [150] . bLf possesses antimetastatic effects and inhibits the growth of transplanted tumors [149, 151] . 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 [7, 152] . 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 [153, 154] . Interestingly, bLf and hLf exert opposite effects on angiogenesis. Whereas orally administrated bLf inhibits angiogenesis in rats [155, 156] and tumorinduced angiogenesis in mice [154] , hLf exerts a specific pro-angiogenic effect [157] . 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 [158] . Increasing evidence suggests that inhibition of the Akt signaling pathway might be a promising strategy for cancer treatment [159] . 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 [160] . Additionally, Lf induced growth arrest and nuclear accumulation of Smad-2 in HeLa cells [161] . The ability of bovine Lfcin to induce apoptosis in THP-1 human monocytic leukemic cells has also been demonstrated [162] . 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. Lf has the ability to function as an enzyme in some catalytic reactions. A remarkable similarity of certain motifs between Lf and ribonuclease A has been observed [163] . Lf has DNA binding properties [164] and can act in transcriptional activation of specific DNA sequences [165] or as a mediator of signal transduction [166] . Lf has the highest levels of amylase and ATPase activities of all the milk proteins [167] ; however, these are not its only enzymatic activities. Indeed, Lf exhibits a wide variety of activities, which can be attributed to variations in the nature of its protein characteristics; Lf has multiple isoforms, degrees of glycosylation, variations in tertiary structure (holo-or apo-Lf) and degrees of oligomerization [168, 169] . The discovery of Lf's enzymatic properties has helped to elucidate its many physiological functions. Lactoferrin was first isolated by Groves in 1960 and was recognized as a "red protein from milk" [2] . 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. [170] 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 [171] . 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. 3a) . An in situ model of hLfcin (Fig. 3a) and a solvated version (Fig. 3b) show structural differences at their N-and C-termini (Fig. 3a, 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 [172] . Additionally, the bLfcin peptide contains an alpha-helix (Fig. 3a-c) . This region retains high homology among other species of mammals and corresponds to amino acids 12-48 [17] . It was 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 [171] . Proteolysis of iron-free Lf could release Lf-derived active peptides in biological fluid [172] . Lf has been identified in several tissues in both humans and animals. The Lf gene has been found at the chromosome level in a set of different species; in humans it was found on chromosome 3, while in mice it was found on chromosome 9 [173] , and its size ranged from 23 to 35 kb. Lf genes have extensive homology among species, with an identical organization in cows, pigs and mice (17 exons, 15 encoding Lf) [174, 175] . The amount of Lf synthesized in the mammary gland is controlled by prolactin [176] . Its mRNA levels vary by tissue, suggesting tissue-or cell-specific regulation [177, 178] . The Lf full coding regions of 60 different species were analyzed, and it was found that the length of the gene varies from species to species (from 2055 to 2190 residues) due to deletions, insertions and mutations in the stop codon [179] . Lf is expressed both constitutively and inducibly. It is constitutively expressed on mucosal surfaces, while in some tissues it is induced by external agents. The Lf promoter contains an estrogen responsive element [REF] , and is consequently positively regulated by estrogen. The chicken ovalbumin upstream promoter transcription factor (COUP-TF) binding element overlaps the ERE of the lactoferrin gene [180] . COUP-TF binds to this element and competitively inhibits binding of the estrogen receptor to the lactoferrin ERE, thereby inhibiting transcription of the lactoferrin gene. Another negative regulator of LF gene transcription is repressor of estrogen receptor activity (REA). The absence of REA increases the expression of estrogen-induced Lf by up to 100-fold [181] . Lf can also be induced by compounds other than estrogens, such as retinoic acid, which stimulates gene expression in embryonic cells [182] . Lf expression is upregulated by estrogen with a magnitude of response that is cell-type-specific, and it is also upregulated by retinoic acids. Transcription factors such as Ets, PU.1, C/EBP, CDP, and KLF5 also modulate Lf gene expression, mainly in myeloid cells [183] . Lf expression is also modulated by oxidative stress, in response to infection, or during the early steps of embryogenesis [184] . Lf can be isolated from cow's milk by various purification methods, or it can be expressed by recombinant methods [51, 71, 185] . 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 [186] and the results showed lower infection levels. Several studies showed that infants fed with infant formulas had less intestinal iron absorption than breastfed infants [187, 188] . 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 [186, 189] . 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 [190] . Lf also exhibits synergistic activity with antifungal agents, thus reducing the minimum inhibitory concentrations of these agents against C. albicans and C. glabrata [101, 191] . As with antibacterials, Lf exhibits synergy with antiviral drugs in the treatment of hepatitis C, cytomegalovirus and HIV [6, 79, 192] . Lf delays the hypersensitivity response and limits the pathology caused by M. tuberculosis by increasing IL-12 and IL10 expression [193] . A clinical trial was conducted which demonstrated that ingestion of bLF could reduce the risk of colon cancer in humans [157] . Lf also offers applications in food preservation and safety because it can decrease bacterial counts in pork meat [194, 195] , retard lipid oxidation [196] 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 [197] . Increased levels of Lf serve as a clinical marker of inflammatory Severe Acute Respiratory Syndrome or septicemia [198] . The development of commercial production strategies for the production of recombinant Lf as a safe, effective drug and nutraceutical protein is one of the major goals in both research and industry. Purification of Lf depends on the intrinsic properties and features of the molecule, such as its net positive charge, its ability to bind Fe + 3 and its glycosylation state [6, 199, 200] . However, the need for larger amounts of Lf has led to the development of strategies to obtain a recombinant form of the protein (rLf). In global biotechnology, there are now three major competing approaches for the production of rLf: production in transgenic animal milk, production in microscopic fungi, and production in plants. To date, several rLf expression systems have been developed ( Table 2) that utilize both prokaryotic and eukaryotic organisms. The first expression systems utilized Bay Hamster Kidney Cells to express human lactoferrin [221] . The transformation of the filamentous fungi Aspergillus awamory allowed for the expression of both hLf and murine Lf (mLf) [215, 216] . As shown in Table 2 , expression systems were developed in yeasts, bacteria, insects and plants, which have produced human recombinant Lf (rhLf) [205, 207, 212, [217] [218] [219] [230] [231] [232] [233] [234] [235] [236] [237] [238] [239] [240] [241] [242] , caprine Lf (cLF) [208] , bovine Lf (bLf) [201, 203, 206, 214] , equine Lf (eLf) [211] , porcine Lf (pLF) [209, 220] , yak Lf [210] , and Kumin Lf [204] , as well as Lf peptides, including Lfc [202, 203, 206, 214] , which reached expression levels of 0.1 mg/L in a plant model [231] and 1200 mg/L in P. pastoris [212] . Use of viral vectors has allowed for the expression of Lf by insect infection, either in cell culture or directly in the organism, where the expression of both hLf and pLf has been successful. This practice has resulted in the production of transgenic Spodoptera frugiperda and Bombyx mori, which express 205 μg of pLf per infected pupa [220] and up to 65 mg/L in larvae [218] . Lf has also been expressed in higher eukaryotic organisms, including both plants and animals. Using microinjection and direct infection with viral vectors in the mammary gland, transgenic animals have been created that produce milk containing recombinant Lf. These animals include goats [223, 224] , mice [225, 226] , rabbits [227] and cows [228, 229] . Levels of up to 2 g/L have been achieved in transgenic goat milk. Molecular farming, which involves the utilization of plants as bioreactors, is well depicted as a tool for the production of valuable therapeutic and industrial proteins. This process is advantageous because of the lack of contamination from human or animal pathogens and endotoxins in the final product. Furthermore, higher plants are able to synthesize proteins with the proper folding, glycosylation and functional activity, and plant cells can direct the protein of interest to environments that reduce its degradation. hLf was expressed in plant expression models (Table 2) and bLf was only expressed in tobacco plants [235] . However, none of these nuclear transformation methods are able to promote the accumulation of a large amount of rLf. Therefore, new promoters or regulatory elements as well as other transformation methods should be tested to increase accumulation and stability of recombinant proteins. The expression of Lf in plant models could significantly improve crop quality by increasing its resistance to some diseases; additionally, it provides a source for high quality Lf protein. Lf is a multifunctional protein that takes part in a large number of important physiological processes. The beneficial effect of Lf in the treatment of various infectious diseases caused by bacteria, fungi, protozoa, and viruses in animals and humans is described above; nevertheless, much research and many experiments still need to be performed to obtain a better understanding of its activity. The usefulness of Lf has led scientists to develop this health-enhancing nutraceutical protein for use in food and pharmaceutical applications. [245] (h = human, b = bovine, c = caprine, p = porcine, e = equine, Lfc = lactoferricin, NR = not reported). 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structure of camel apolactoferrin at 2.6 Å resolution and structural basis of its dual role Antiinflammatory activities of lactoferrin Neonatal small bowel epithelia: enhancing anti-bacterial defense with lactoferrin and Lactobacillus GG Bovine lactoferrin: benefits and mechanism of action against infections Association of lactoferrin with lysozyme in granules of human polymorphonuclear leukocytes Killing of Actinobacillus actinomycetemcomitans by human lactoferrin Antibacterial activity of lactoferrin and a pepsin derived lactoferrin peptide fragment Lactoferrin: an important host defense against microbial and viral attack Bactericidal effect of lactoferrin on Legionella pneumophila: effect of the physiological state of the organism Lactoferrin: a multifunctional protein with antimicrobial properties Effect of food components on growth of Basillius stearothermophilus Residues in colostrum following antibiotic dry cow therapy Inhibition of bacteria by lactoferrin and other iron-chelating agents Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae Identification of the bactericidal domain of lactoferrin Potential antimicrobial effects of human lactoferrin against oral infection with Listeria monocytogenes in mice Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans Bactericidal activity of human lactoferrin: influence of physical conditions and metabolic state of the target microorganism Enhanced antimicrobial activity of lactoferrin by binding to the bacterial surface Ultrastructural effects of antimicrobial peptides from bovine lactoferrin on the membranes of Candida albicans and Escherichia coli Antibacterial activity of lactoferrin and lactoferricin against oral Streptococci Lactoferrin impairs type III secretory system function in enteropathogenic Escherichia coli Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages Human milk fractions inhibit the adherence of diffusely adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) to HeLa cells Recombinant human lactoferrin is effective in the treatment of Helicobacter felis-infected mice Inhibition of Helicobacter pylori infection by bovine milk glycoconjugates in a BAlb/cA mouse model Iron acquisition by Helicobacter pylori: importance of human lactoferrin Metal complexes of lactoferrin and their effect on the intracellular multiplication of Legionella pneumophila Iron availability influences aggregation, biofilm, adhesion and invasion of Pseudomonas aeruginosa and Burkholderia cenocepacia Anti-bacterial activity of bovine milk lactoferrin against some mastitis causative pathogens with special regard to mycoplasma Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with fibrosis cystic In vitro adhesion and invasion inhibition of Shigella dysentariae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins A bactericidal effect for human lactoferrin Correction of the iron overload defect in 2-microglobulin knockout mice by lactoferrin abolishes their susceptibility to tuberculosis Lactoferrin augments BCG vaccine efficacy to generate T helper response and subsequent protection against challenge with virulent Mycobacterium tuberculosis Periodontitis, periodontopathic bacteria and lactoferrin Antimicrobial effect of lactoferrin and its amidated and pepsin-digested derivatives against Salmonella enteriditids and Pseudomona fluorescence El hierro y la virulencia bacteriana Damage of the membrane of enteric Gramnegative bacteria by lactoferrin and transferrin Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli Elucidation of the antistaphylococcal action of lactoferrin and lysozyme Geme III, Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites Problem pulmonary pathogens: Pseudomonas aeruinosa A component of innate immunity prevents bacterial biofilm development The effect of recombinant human lactoferrin on growth and the antibiotic susceptibility of the cystic fibrosis pathogen Burkholderia cepacia complex when cultured planktonically or as biofilms Lactoferrin increases the susceptibility of S. epidermidis biofilms to lysozyme and vancomycin Suppression of bacterial biofilm formation by iron limitation Anti-HSV activity of lactoferrin and lactoferricin is dependent on the presence of heparan sulphate at the cell surface Inhibition with lactoferrin of in vitro infection with human herpes virus Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1 Lactoferrin inhibits hepatitis B virus infection in cultured human hepatocytes Lactoferrin inhibits HIV-1 replication in vitro and exhibits synergy when combined with zidovudine Inhibition of cytomegalovirus infection by lactoferrin in vitro and in vivo Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts Characterization of antiviral activity of lactoferrinagainst hepatitis C virus infection in human cultured cells Lactoferrin markedly inhibits hepatitis C virus infection in cultured human hepatocytes Effects of bovine lactoferrin on in vitro replication of feline herpesvirus Lactoferrin inhibits herpes simplex virus type 1 adsorption to Vero cells Antiviral effects of milk proteins: acylation results in polyanionic compounds with potent activity against human immunodeficiency virus types 1 and 2 in vitro Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells Lactoferrin prevents dendritic cellmediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN-gp120 interaction Bovine lactoferrin peptidic fragments involved in inhibition of herpes simplex virus type 1 infection Antiviral activity of lactoferrin towards naked viruses Lactoferrin inhibits enterovirus infection of human embryonal rhabdomyosarcoma cells in vitro Protective influence of lactoferrin on mice infected with the polycythemia-inducing strain of Friend virus complex Cessation of feline calicivirus shedding coincident with resolution of chronic gingivostomatitis in a cat The anti-HSV activity of lactoferrin and lactoferricin is dependent on the presence of heparan sulfate at the cell surface Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides Bovine lactoferrin inhibits adenovirus infection by interacting with viral structural polypeptides Antiviral activity of human lactoferrin: Inhibition of alphavirus interaction with heparan sulfate Brief overview on cellular virus receptors Heparan sulfate: anchor for viral intruders? Delineation of the glycosaminoglycanbinding site in the human inflammatory response protein lactoferrin Inhibition of growth of Candida albicans by iron-unsaturated lactoferrin: relation to host-defense mechanisms in chronic mucocutaneous candidiasis Modulation of in vitro fungicidal activity of human lactoferrin against Candida albicans by extra cellular cation concentration and target cell metabolic activity Bactericidal activity of human lactoferrin: sensitivity of a variety of microorganisms Killing of Candida albicans by lactoferricin B, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin Cooperative anti-Candida effects of lactoferrin or its peptides in combination with azole antifungal agents Synergistic fungistatic effects of lactoferrin in combination with antifungal drugs against clinical Candida isolates In vitro susceptibility of Candida species to lactoferrin Fungicidal activity of human lactoferrin-derived peptides based on the antimicrobial αβ region Interaction between lactoferrin and ovotransferrin and Candida cells The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei Modulation of the anti-Candida activity of apo-lactoferrin by dietary sucrose and tunicamycin in vitro Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion Human antimicrobial peptides antifungal activity against Aspergillus fumigatus Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin Lactoferrin given in food facilitates dermatophytosis cure in guinea pig models Iron chelators as therapeutic agents against Pneumocystis carinii Inhibition of growth of Pneumocystis carinii by lactoferrins alone and in combination with pyrimethamine, clarithromycin and minocycline Microbicidal action of lactoferrin and lactoferricin and their synergistic effect with metronidazole in Entamoeba histolytica Amoebicidal activity of milk, apo-lactoferrin, sIgA and lysozyme Microbicidal effect of the lactoferrin peptides lactoferricin 17-30, actoferrampin 265-284, and lactoferrin chimera on the parasite Entamoeba histolytica Tritrichomonas foetus: iron acquisition from lactoferrin and transferrin The detection of bovine lactoferrin binding protein on Trypanosoma brucei Toxoplasma gondii: inhibition of the intracellular growth by human lactoferrin Reduction of the infectivity of Toxoplasma gondii and Eimeria stiedai sporozoites by treatment with bovine lactoferricin Seroprevalence of Babesia equi in three breeding systems of equines Short report: inhibitory effect of lactoferrin on in vitro growth of Babesia caballi Interaction of lactoferrin with cells involved in immune function Lactoferrin deficiency as a consequence of a lack of specific granules in neutrophils from a patient with recurrent infections. Detection by immunoperoxidase staining for lactoferrin and cytochemical electron microscopy The mode of oral bovine lactoferrin administration influences mucosal and systemic immune responses in mice Modulation of immunerelated gene expression in small intestine of mice by oral administration of lactoferrin Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthma Lactoferrin: a modulator of immune and inflammatory responses Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation Lactoferrin molecular structure, binding properties and dynamics of lactoferrin Lactoferrin interacts with deoxyribonucleic acid: a preferential reactivity with double-stranded DNA and dissociation of DNA-anti-DNA complex Activation of macrophages by lactoferrin: secretion of TNF-alpha Lactoferrin regulates the immune responses in post-surgical patients Lactoferrin regulates the release of tumor necrosis factor alpha and interleukin in vivo Lactoferrin increases the output of neutrophil precursors and attenuates the spontaneous production of TNF-alpha and IL-6 by peripheral blood cells Bovine lactoferrin decreases histopathological changes in the liver and regulates cytokine production by splenocytes of obstructive jaundiced rats Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by T-cell-dependent augmentation of natural killer cell activity P-selectin-dependent leukocyte recruitment and intestinal mucosal injury induced by lactoferrin Phagocytosis-enhancing effect of lactoferrin on bovine peripheral blood monocytes in vitro and in vivo Interleukin-12 and the regulation of innate resistance and adaptive immunity Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy A. Varadhachary, Phase I trial of oral talactoferrin alfa in refractory solid tumors Orally administered bovine lactoferrin induces caspase-1 and interleukin-18 in the mouse intestinal mucosa: a possible explanation for inhibition of carcinogenesis and metastasis Regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin Activation of mucosal intestinal immunity in tumor-bearing mice by lactoferrin Bovine lactoferrin inhibits tumor-induced angiogenesis Orally administered bovine lactoferrin systemically inhibits VEGF-mediated angiogenesis in the rat Bovine lactoferrin inhibits tumor-induced angiogenesis Effect of orally administered bovine lactoferrin on the growth of adenomatous colorectal polyps in a randomized, placebo-controlled clinical trial Takahiro Kozu Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway Apoptosis of stomach cancer cell SGC-7901 and regulation of Akt signaling way induced by bovine lactoferrin Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells Lactoferrin induces growth arrest and nuclear accumulation of Smad-2 in HeLa cells Apoptosis in human leukemic cells induced by lactoferricin, a bovine milk protein-derived peptide: involvement of reactive oxygen species Purification and characterization of a high molecular weight ribonuclease from human milk Lactoferrin binds to neutrophilic membrane DNA Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA Signal transduction and metabolism in chondrocytes is modulated by lactoferrin Multiple enzymatic activities of human milk lactoferrin Lactoferrin contains structural motifs of ribonuclease Multiple molecular forms of human lactoferrin Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin Distinct bactericidal activities of bovine lactoferrin peptides LFampin 268-284 and Lfampin 265-284; Asp-Leu-Ile makes a difference Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties Assignment of the lactotransferrin gene to human chromosome 3 and to mouse chromosome 9 Differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse, rat, and hamster Lactoferrin gene expression and regulation: an overview Lactoferrin is a marker for prolactin response in mouse mammary explants Lactoferrin in milk from different species An iron-binding protein common to many external secretions Bioinformatics analysis of lactoferrin gene for several species COUP-TF acts as a competitive repressor for estrogen receptor-mediated activation of the mouse lactoferrine gene Genetic deletion of the repressor of estrogen receptor activity (REA) enhances the response to estrogen in target tissues in vivo Induction of lactoferrin expression in marine cells by retinoic and estrogen Factors regulating lactoferrin gene expression Multifunctional roles of lactoferrin: a critical overview Lactoferrin research, technology and applications Italian Task Force for the Study and Prevention of Neonatal Fungal Infections, Italian Society of Neonatology. Bovine lactoferrin supplementation for prevention of late-onset sepsis in very low-birth-weight neonates: a randomized trial Iron absorption from infant milk formula and the optimal level of iron supplementation Exclusive breast-feeding for 9 months: risk of iron deficiency Effects of the lactoferrin (LF) on the growth performance, intestinal microflora and morphology of weanling pigs Effect of lactoferrin supplementation on the effectiveness and tolerability of a 7-day quadruple therapy after failure of a first attempt to cure Helicobacter pylori infection Activated lactoferrin's ability to inhibit Candida growth and block yeast adhesion to the vaginal epithelial monolayer Effect of lactoferrin in patients with chronic hepatitis C: combination therapy with interferon and ribavirin Lactoferrin modulation of antigen-presentingcell response to BCG infection Effect of bovine lactoferrin and casein peptide powder on microbial growth and glucose utilization by microorganisms in pork meat during storage at 4°C, Pak Activated lactoferrin-a new approach to meat safety Effects of natural phenolic compounds on the antioxidant activity of lactoferrin in liposomes and oil-in-water emulsions Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis Expression profile of immune response genes in patients with severe acute respiratory syndrome Bovine lactoferrin and lactoferricina derived from milk: production and applications Structure and iologicalactions of lactoferrin Multimerization and fusion expression of bovine lactoferrin derivative Lfcin B15-W4, 10 in Escherichia coli Expression of the cationic antimicrobial peptide lactoferricin fussed with the anionic peptide in Escherichia coli Fusion expression of bovine lactoferricin in Escherichia coli Cloning, expression and characterization of Kunming mice lactoferrin and its N-lobe Probiotic Lactobacillus casei expressing human lactoferrin elevates antibacterial activity in the gastrointestinal tract Expression of bovine lactoferrin C-lobe in Rhodococcus erythropolis and its purification and characterization Producing human lactoferrin by high density fermentation recombinant Pichia pastoris Expression and purification of goat lactoferrin from Pichia pastoris expression system Expression, characterization and purification of recombinant porcine lactoferrin in Pichia pastoris Molecular cloning and expression of yak (Bos grunniens) lactoferrin cDNA in Pichia pastoris Expression, purification and characterization of equine lactoferrin in Pichia pastoris High-level expression and production of human lactoferrin in Pichia pastoris Expression of recombinant porcine lactoferrin N lobe in Pichia methanolica and its antibacterial activity Heterologous expression of bovine lactoferricin in Pichia mehtanolica A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic Production of biologically active recombinant human lactoferrin in Aspergillus oryzae Expression of human lactoferrin cDNA in insect cells High level expression of functionally active human lactoferrin in silkworm larvae Recombinant functional human lactoferrin expressed in baculovirus system Expression of porcine lactoferrin by using recombinant baculovirus in silkworm, Bombyx mori L. and its purification and characterization Expression of cloned human lactoferrin in baby-hamster kidney cells Recombinant pseudoadenovirus nanostructure with the human lactoferrin gene: production and study of lactoferrin expression and properties in vivo Expression of active recombinant human lactoferrin in the milk of transgenic goats High-level expression of human lactoferrin in the milk of goats by using replication-defective adenoviral vectors Characterization of recombinant human lactoferrin secrete in milk of transgenic mice Expression analysis of a bovine B-casein/human lactoferrin hybrid gene in transgenic mice Adenoviral vector mediates high expression levels of human lactoferrin in the milk of rabbits Large scale production of recombinant human lactoferrin in the milk of transgenic cows Py r l , Transgenic cows that produce recombinant human lactoferrin in milk are not protected from experimental Escherichia coli intramammary infection Expression of human lactoferrin N-lobe in Nicotiana benthmiana with potato virus x-based agroinfection Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial proteins Expression of human lactoferrin cDNA confers resistance to Ralstonia sdanacearum in transgenic tobacco plants High expression of a human lactoferrin in transgenic tobacco cell cultures Transgenic plants expressing antimicrobial lactoferrin protein are resistant to a fungal pathogen Expression and localization of recombinant human lactoferrin in transgenic Javanica rice cv rojolele Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain Expression, characterization, and biologic activity of recombinant human lactoferrin in rice Expression of full-length bioactive antimicrobial human lactoferrin in potato plants Recombinant human lactoferrin: a valuable protein for pharmaceutical products and functional foods Production of human lactoferrin in transgenic cell suspension cultures of sweet potato Transgenic ginseng cell lines that produce high levels of a human lactoferrin Partial resistance to bacterial wilt in transgenic tomato plants expressing antibacterial lactoferrin gene N-Glycosylation potential of maize: the human lactoferrin used as a model Genetic modification of alfalfa (Medicago sativa L.) for quality improvement and production of novel compounds Generation of transgenic barley lines producing human lactoferrin using mutant alpha-tubulin gene as the selective marker This work was supported in part by an internal grant from Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, and Muuu-Technologies de México, and García-Montoya thanks CONACYT for the MC studies grant.