Lactoferrin

Lactoferrin: a natural immunomodulator

Tania Siqueiros-Cendon1, Blanca Flor Iglesias-Figueroa2, Isui Abril GarcíaMontoya2, Jose Salazar-Martinez3 and Quintin Rascón-Cruz2*.

1 Laboratorio de Inmunologia, Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito 1, Nuevo Campus Universitario, CP 31125, Chihuahua, Mexico. [email protected] 2 Laboratorio de Biotecnología I, Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito 1, Nuevo Campus Universitario, CP 31125, Chihuahua, Mexico. 3 Proteo/Muuu-Technologies de México, Zaragoza 63, Zona Centro, Fco. I. Madero, Coahuila, Mexico.

* Correspondig Author. Laboratorio de Biotecnología I, Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito 1, Nuevo Campus Universitario, CP 31125, Chihuahua, Mexico. [email protected]

Abstract Lactoferrin (Lf), an iron-binding glycoprotein of the transferrin family, is expressed in most biological fluids, with particularly high levels found in mammalian milk. Its multiple activities rely not only on its capacity to bind iron but also on its capacity to interact with the molecular and cellular components of both host and pathogens. Lf can bind and sequester lipopolysaccharides (LPS), thus preventing proinflammatory pathway activation, sepsis and tissue damage. Lf is considered to be a cell-secreted mediator that bridges the innate and adaptive immune response. Much has been learned in recent years about the mechanisms by which Lf exerts its activities. However, there has been little progress in using these insights to improve pediatric care. This review looks at the recent advances in understanding the mechanisms underlying the multifunctional roles of Lf and considers future perspectives on its potential prophylactic and therapeutic applications.

1. Introduction The first literature references to Lf refer to it as a “red protein from milk”, and, from the time it was first purified [1, 2], it has been recognized as an iron-binding protein. Lf is an 80 kDa glycosylated protein of ca.700 amino acids with high homology among species, considered as a multifunctional glycoprotein widely distributed in colostrum and milk [3] as well as in other secretions, such as tears and, saliva [4]. It is released from neutrophils (PMNs) in the blood and inflamed tissues. Lf has a direct antimicrobial role, as it limits the proliferation and adhesion of microbes (e.g. bacteria, viruses and parasites) and/or kills them [5]. This effect of Lf is the result of its ability to sequester iron in biological fluids and destabilize the membranes of microorganisms [6]. The metals that it binds are the Fe2+ or Fe3+ ions, also can exist free of Fe3+ (apo-Lf). It is a simple polypeptide chain folded into two symmetrical lobes (N and C lobes), which are highly. These two lobes are connected by a hinge region containing parts of an -helix between in human LF (hLF) (Figure 1). In addition to the antimicrobial properties of Lf, its ability to modulate the overall immune response and to protect against viral infections and septic shock has already been well described [6, 5]. In this respect, it is noteworthy that Lf concentrations

are

locally

elevated

in

inflammatory

disorders,

including

neurodegenerative diseases [7], inflammatory disease, arthritis [8], and allergic inflammation [9]. Although the cellular and molecular mechanisms accounting for the immunomodulatory effects of Lf are far from being fully elucidated, both in vitro and in vivo studies suggest the existence of multiple mechanisms that include

modulation of cytokine/chemokine production, regulation of the production of reactive oxygen species, and immune cell recruitment [10]. Lf also acts as a first-line of defense by significantly impacting the development of adaptive immune responses. Iron sequestration by Lf reduces oxidative stress, thus altering the extension and specific production of cytokines [11]. Lactoferrin has a strong modulatory effect on the adaptive immune system by accelerating the maturation of T-cell precursors into competent helper cells and by the differentiation of immature B-cells into antigen-presenting cells [reviewed by 12]. Lf can also modulate both innate and adaptive immunities by acting as a Toll-like receptor (TLR4) or by interacting with lymphocytes and antigen-presenting cells (APCs) [13]. Thus, Lf may also protect infants from infections due to its large set of ability’s against direct interaction with microorganisms, but also through its immune modulatory effect.

2. Immunomodulators Immunomodulators are biological or synthetic substances capable of altering the immune response by augmenting or reducing the immune system components, including both the innate and adaptive arms of the immune response [14]. The human body has the ability to produce some peptides and small proteins with antimicrobial and immunomodulatory activities [15]. At first, it was thought that the function of these peptides was only to kill microorganisms; however it is now known that, this peptide also coordinate multiple immune system functions as a

primitive immunity mechanism [16]. These peptides are believed to contribute to the prevention or recovery of changes in the immune system by suppressing or enhancing the immune response [14]. Some immunomodulators, such as antibodies, react to unique antigen and may be specific to certain pathogens may be

nonspecific

(e.g. cytokines,

antimicrobial peptides, drugs,

and

even

microorganisms used to generate a positive host response) A clear example of this is the use of probiotics to enhance the normal microbiome of the small intestine [17].

With

this

approach,

immunomodulators

may

be

classified

as:

immunoadjuvants, immunosuppressants, or immunostimulatos. Immunoadjuvants; are substances that are required to elicit a maximal T-cell response to the microbial antigens. They are mainly used in the development of vaccines to improve the response of the organism to the vaccine [18, 19]. Immunosuppressants; they inhibit the normal functioning of the immune system; therefore, they are used in conjunction with drugs to treat autoimmune diseases and transplantation immunity [20]. Immunostimolators; are agents that act through response-mediated innate and adaptive immunity. In healthy individuals, immunostimulatorsare thought to function as a prophylactic agent and to enhance the

immune

response

to

infection.

In

immunocompromised

immunostimulators may act as natural therapy [21].

2.1 Immunomodulators in infant formulas.

individuals,

Colostrum, the first milk produced by the mammary glands of mammals after giving birth, contains elements with immunomodulatory properties; these elements have attracted the attention of some pharmaceutical and nutritional industries as a dietary supplement [22]. Lf is, present in bovine colostrum at concentration of 500 mg/100 g and is a GRAS product currently used in several countries as an infant ingredient. To date, no adverse effects regarding its use have been reported [23]. The first occurrence of an infant formula containing Lf was in Japan in 1986; this product called "BF-L dry milk", was marketed by the Morigana Milk Industry. Currently, lactoferrin can be found in various products, such as yogurt, pet food, cream, milk and other beverages [24].

2.2. Lactoferrin competence in early life response Colostrum and lactoferrin The concentration of Lf in colostrum is 5.3 ±1.9 mg/mL, and, after the first month of lactation is approximately 1 mg/mL [25]. Colostrum is the pre-milk fluid in the mammary gland of female mammals just before giving birth. After the first instance of nursing, the fluid transforms to milk over a period of 2-3 days. In all non-human mammals, colostrum is crucial to the survival of the newborn because high concentration of immune factors that are transferred to the newborn via the colostrum. In humans, some immunofactors are transferred through the placenta. Human colostrum is very important; however, if a human newborn does not receive colostrum, death is not imminent, as it is in all other mammals. In humans, as well

as other mammals, a newborn’s very first meal of colostrum significantly impacts its health and well-being, with effect lasting the rest of its life. Because a newborn´s immune system is not fully developed, it is highly susceptible to pathogens, antigens and allergens [26]. The colostrum found in breast milk contains all of the immune factors that are essential to activate, regulate and balance the immune system. Consequently, colostrum plays an important role in supporting a newborn´s immune system while it develops. Lf is the first line of defense for any entry point in the body. It is also found in small quantities in most body fluids such as saliva, tears, nasal secretions, and intestinal fluids (e.g. bile), as well as in neutrophils (the secondary granules of white blood cells). Lf is synthesized by both the mucosal lining (e.g. in the mouth or intestinal tract) and neutrophils, and it is released in response to inflammatory stimuli. The low physiologic serum levels of Lf increase significantly upon host infection. Lf receptor have been detected and isolated on activated T- and B-cells, monocytes, intestinal brush border cells, platelets and neoplastic cells [30, 31]. Mucosal immunity Lactoferrin, amylase and lysozyme are detectable and most prominent during the early fetal stages of development. Only low levels of the secretory components have been observed before week 29 of gestation, but these levels increase rapidly to adult levels one week after being born. Although the skin and gastrointestinal tracts are relatively immunologically mature prior to birth, the respiratory tract is not [26].

Development of the fetal immune system The first stage of human fetal hematopoiesis occurs in the mesoderm of the yolk sac and in the extraembryonic mesenchymal tissue. Pluripotent erythroid and granulomacrophage progenitors can be detected in human embryonic structures 3–4 weeks into gestation. These primitive cells can then be detected in circulation after 4 weeks of gestation, as they migrate to the liver, which becomes the major site of hematopoiesis at 5 gestational weeks. At approximately 5 -10 weeks, the liver undergoes a dramatic increase in size as the number of nucleated cells rises. These early progenitors proliferate but undergo very little differentiation, although a discrete granulocyte/macrophage population emerges at this time. The thymus and spleen are seeded from the liver and stem cells are detectable in the bone marrow after 8-9 weeks of gestation. Hepatic hematopoiesis declines in the third trimester and ceases soon after birth [26, 27, 28]. Lactoferrin expression can be detected at 2- and 4- cell stages of embryonic development and through-out the blastocyst stage (prior to implantation). After implantation, Lf expression cannot be detected again until approximately halfway through gestation, when it can be found in the neutrophils and epithelial cells of the developing reproductive and digestive systems [29]. The Lf plasma levels are higher during pregnancy than in either male or female adults, and these levels shows a progressive increase leading up to week 29, at which point these levels remains high [32]. There are several factors that may explain this increase, such as pregnancy-associated leukocytosis, the selective increase of Lf in neutrophil granules or endometrial tissues or contribution from the decidua and mammary

glands [33]. Lf acts as a growth factor activator; compared to epidermal growth factor, the effects of Lf alone on small intestine epithelial cells are more potent and can stimulate the proliferation of endometrium stroma cells [29].

3. Development of immune competence stimulated by microorganisms The principal stimuli that induce the postnatal maturation of the mammalian immune system are signals from the microbial environment, particularly the commensal microflora of the gastrointestinal tract. Infections, particularly in the gastrointestinal and respiratory tracts, may also contribute to immune system development [34]. Lf was first identified as an important defense component of colostrum and mature milk, thereby promoting the hypothesis that its function involves the protection of the neonatal gut barriers. In concurrence with this finding, Lf receptors (LfR) were first discovered in the small intestine during a study of iron delivery to the duodenal mucosa. A recent study of LfR in the mouse small intestine suggests that LfR may act as the main iron source during the early stages of life. Subsequent binding affinity studies identified Lf on the surface of B-cell, Tcells, and monocytes, as well as on platelets, and intestinal cells [12, 35]. Lf can facilitate the adaptation of an infant´s intestine because Lf hydrolysis is minimal at the prevailing postprandrial pH in infants; Lf may have a greater biological potential in infants than in adults. Lf bi-directionally stimulates the proliferation and differentiation of the small intestinal epithelial cells, which is concentration-

dependent and affects the mass, length, and epithelial digestive enzyme expression of small intestines [36]. The constant interaction between the intestinal epithelium and the gut microbiota is a challenge for the preterm gut. Mature intestines have many physical barriers designed to limit bacterial access to the gut lumen and to prevent attachment and translocation across the intestinal epithelium. In contrast, external factors render the preterm intestine more susceptible to microbial interaction and translocation Preterm infants face many challenges in transitioning from the in utero to extrauterine environment. Failure of the preterm gut to successfully mature to accommodate bacteria and food leads to significant morbidity such as neonatal necrotizing enterocolitis [37].

4. Role of lactoferrin in the immune system 4.1 Interaction of lactoferrin with Antigen Presentation Cells Among Antigen Presentation Cells (APCs), macrophages (Mf), dendritic cells (DCs) and B-cells are of critical importance for the maintenance of tissue homeostasis and innate response; this response occurs via the major histocompatibility complex II (MHC II), as well as by linking the innate and adaptive immune responses. Mf are highly phagocytic cells that play a central role in the control of infections, either by the direct intracellular killing of microorganisms or by the secretion of cytokines to inhibit the replication of microorganisms, Mf are also involved in type II inflammation and the tissue repair processes [38, 39, 40]. DCs

are a heterogeneous population of cells highly specialized for antigen recognition; they play a key role in the immune system because they control the induction of both immunity and tolerance. B-cells utilize specific surface receptors to capture foreign antigens and present the associated epitopes to the T-cells [41, 42].

Macrophages— Mf are Antigen Presenting Cells (APCs) and their role in the innate immune response involves inducing the phagocytosis of foreign particles and subsequently releasing pro-inflammatory mediators. Mf also enables cross-talk between innate and adaptive immune system to stimulate antigen-specific T-cells. Active binding studies reveled that Lf receptors are located on the surface of Mf in both bovine and human model [43, 44]. Lactoferrin also contributes to the suppression of pro-inflammatory cytokines and type I interferon (IFN /) induction [45, 46]. Lf also affects the ability of Mf present antigens for the antigen specific CD4+ T-cells in the adaptive immune system. In addition to regulating these Mf, activities of Mf. Lf can also increase phagocytic activity in Mf that are infected or have not yet been activated (47, 48]. IL-12, one of the major cytokines produced by Mf, is an important modulator of IFN. The main role of IL-12 at the site of infection it to recruit Mf [49, 50], which acts as a co-stimulator to maximize secretion of IFN from differentiated TH1 cells and memory T-cells [51, 52].

Dendritic Cells — Dendritic cells (DCs) are a group of functionally related phagocytic cells that can manipulate T-cell differentiation [53] and redirect memory T-cell functions [54, 55]. DCs play an important role in triggering T-cell responses

that lead to the secretion of Th1 cytokines [56, 57]. It has also been shown that defensin 2, another key innate immunity molecule, and acts directly on DCs to induce their functional maturation and enable them to elicit a Th1 response [58, 59]. The ability of lactoferrin to promote antigen-specific delayed-type hypersensitivity (DTH) responses and to activate bacille Calmette-Guerin (Mycobacterium strain) (BCG)-specific T-cells suggests that Lf plays a role in the initiation of T-cell activation, through the modulation of dendritic cell function [60]. Dendritic cells possess Lf receptors because both bovine and human Lf has been discovered to bind to the surface of peripheral blood-derived dendritic cells [61]. It was shown that the ability of dendritic cells to migrate upon antigen stimulation or capture is important in the promotion of antingen-specific immune responses [62]. Lf acts as an alarmin to promote the recruitment and activation of APCs and antigen-specific immune responses. It has also been reported as a novel maturation factor for human dendritic cells [10, 63]. Lactoferrin is a strong mediator of dendritic cell function. This observation, together with the above-described impact on Mf, suggests that lactoferrin exerts its effect on cells involved in the commitment of pathogens (antigens) that can direct the development of adaptive immunity (Figure 2).

4.2 Lactoferrin modulates antigen-specific adaptive immune responses

A key immunomodulatory function (e.g. APC activation, maturation, migration, and antigen presentation) that may be mediated by Lf is to bridge innate and adaptive cell functions, for both T- and B- cell responses. B lymphocytes— Considerable attention has been paid to Lf because of its potential role in the maturation and function of immune system cells [64]. Significally, Lf leads to an increase in the expression of the complement 3 receptor (C3R) and acquisition of surface IgD. Also of importance is the interaction between Lf and the Lf receptor on T- and B- lymphocytes [65]. Structural changes in the Nterminal basic region and the basic characteristics of the whole molecule contribute to its interaction with B lymphocytes [66]. Oral administration of Lf increases the secretion of IgA and IgG in murine mucosa with intestinal secretion [67, 68]. These data suggests that Lf acts on B-cells, a well-known antigen presenter, to allow subsequent interaction with T-cells that favor the elevation of the antibody response (Figure 2). T lymphocytes— The effect of lactoferrin on T-cell populations can be further delineated in terms of the cellular subset that are specifically targeted. The adaptive immune response is dominated by T-cell activity, which includes various functions. T-helper cell type 1 (TH1) and type 2 (TH2) stimulate and activate Mf, resulting in intracellular killing events that eliminate intracellular pathogens [69, 70]. Lf accelerates T-cell maturation by inducing the expression of CD4 surface markers through the activation of a transduction pathway [71]. The expression of Lf receptors has been reported on all T-cell subsets. Both bovine and human Lf is capable of binding to surface receptors on the human T-cell line (Jurkat) [65, 72].

These associated changes to the surface of molecules that regulate T-cell function suggest that Lf is capable of modulating T-cell and NK cell activity due to T-cell proliferation. Indeed, Lf can potentiate the restoration of humoral immune response of the host, suggesting a possible mechanism for cell reconstitution through proliferative pathways [73]. Lf induces TH1 polarization in diseases in which the ability to control infection or tumor relies on a strong immune response; however, Lf may also reduce TH1 cytokines to prevent excessive inflammatory responses [74].

5. Response against microorganisms The antimicrobial effects of Lf have been demonstrated in vivo, for the parenteral administration of Lf in several experimental animal models, most notably in mice [75]. Additionally, recent studies have shown that, in healthy human volunteers, bovine Lf reinforces the immune system and antioxidant status [76]. Furthermore, several in vitro studies have indicated that the structure of Lf plays an important role in antimicrobial activity (e.g. the interaction with viruses and bacterial toxins), as the glycosylation sites of the Lf protein expose its outer surface and allow it to maintain contact with microorganisms [46]. Some of the mechanisms through which Lf triggers an immune response against pathogens are summarized in Table 1.

Anti-bacterial activity. It is well known that Lf interacts directly with bacterial LPS thereby preventing the interaction between the endotoxin containing LPS binding protein (LBP) and CD14 [77, 78, 79]. In addition this Lf-LPS interaction triggers a signaling cascade that results in the release of pro-inflammatory mediators, such as cytokines and chemokines, as well as small molecules, such as lipid mediators and reactive oxygen species [40]. In addition to LPS, other pro-inflammatory microbial molecules (e.g. unmethylated CpG-containing oligonucleotides), can be neutralized by Lf. The ability to bind unmethylated CpG-containing oligonucleotides allows Lf to have anti-inflammatory effects on B-cells, supporting the possibility that Lf may interact with TLR-9 [12, 80]. This mechanism is attributed to the N-terminal domain of Lf and its ability to bind large amounts of iron [81, 77], thereby preventing the host cell from inhibiting the production of pro-inflammatory cytokines (e.g., TNF-, IL-1, IL-6 and IL-8) [6, 46]. The contribution of Lf to pro-inflammatory cytokine release not only enhances phagocytosis and cell adhesion, but it also provides protection against pathogens and their metabolites [12, 78]. However, when Lf has been administrated via orally in Staphylococcus aureus-infected models, an increase in the host response can be observed as increase in TNFlevels and a decrease in IL-5 and IL-10 levels [12]. Antiviral activity. Although it was initially suggested that Lf only exercises its antiviral activity against enveloped viruses, it is currently believed that Lf has antiviral activity against a broad spectrum of RNA and DNA viruses as well. Its main contribution to this antiviral defense is ability to bind to cell membrane glycosaminoglycans [78, 29]. In the presence of some viruses (e.g. vesicular

stomatitis virus), Lf can increase the phagocytic activity of Mf [12]. On the other hand, It been demonstrated that Lf has strong interactions with the protein gp120 of the Human Immunodeficiency Virus, wherein Lf binds to the DC-SING receptor of dendritic cells to block their interactions and subsequently inhibit the transmission of the virus [6, 82]. Antifungal activity. Candida albicans is one of the most common causes of vaginal infections [83]. The adherence of this fungus to the vaginal epithelium can be prevented by the action of Lf because of its ability to sequester iron [81], [78]. In addition, Lf has the ability to induce apoptosis in this yeast, which was first reported by Madeo et al in 1997 [84]. In 2000, Wakabayashi et al., reported that Lf inhibits the in vivo growth of Trichophyton mentagrophytes and Trichophyton rubrum, two of the principal etiological agents of dermatophytosis [85]. This growth inhibition occurs because Lf enhances the inflammatory response involved in the cellmediated immunity that is required to cure this mycosis [86]. Antiparasitic activity. The mechanisms involved in the antiparasitic activity of Lf are complex [81, 78]. Lf exhibits activity against Entamoeba histolytica, Trichomonas faetus, Trypanosoma cruzi, Trypanozoma brucei, Plasmoduim falciparum, Toxoplasma gondii and Eimeria stiedai. Is not clear whether the antimicrobial properties of Lf are related to the direct action against microbes or to the activation of the immune system, but several lines of evidence now indicate that both forms of action are involved. This hypothesis is supported by reports that Lf can modulate and direct changes in the balance of T-cell immunity. In studies involving Toxoplasma gondii infection it has been found that the oral administration of Lf

promotes Th2 cell responses in the intestinal mucosa, which is characterized by decreased levels of IFN- and elevated levels of IL-10 [6, 12]. 6. Lactoferrin from other species As we discussed in a previous paper [78], the different benefits of Lf have led to an interest in using molecular strategies to develop recombinant Lf from different species to increase its availability. Because of its great antimicrobial capacity, lactoferrin could be used as nutraceutical protein or as a coadunate drug. Even though colostrum contains high Lf levels, industrial companies will required to produce or purify Lf without affecting alimentary industry uses of milk. Currently, highly purified bLf and hLf can be produced [91]. In addition to human and bovine Lf, lactoferrin from other species (e.g. mouse, rat, chimpanzee, boar, sheep, goat, buffalo, camel and dog) has been sequenced and found to vary in light from 2112 to 2530 pb [92, 93]. It has been possible to produce recombinant Lf specific to human, bovine, equine, porcine, caprine, yak, and Kunming by using various expression systems (e.g., bacteria, fungi, yeast, cell lines, insects, mammals and plants). While Lf has been shown to be produced in quantities ranging from 0.756 mg/L to 10.6 g/L, human Lf remains the most expressed among all of the different expression systems [78, 94, 95, 96, 97, 98, 99, 100].

7. Future applications of lactoferrin Lactoferrrin has multiple activities, it can bind a significant number of compounds and substances, such as lipopolysaccharides, heparin, glycosaminoglycans, DNA,

and metal ions (e.g. Fe, Al, Mn, Co, Cu, Zn) [29, 101], is involved in iron homeostasis, has a wide range antimicrobial activity against bacteria, virus, fungi and parasites; and has anti-inflammatory, immunomodulatory, anticarcinogenic and enzymatic activities [102, 81]. Antibiotic-resistant microorganisms are extremely dangerous to humans, and extensive scientific research has resulted in the development of new antibiotics with different effects in an effort to solve the issue. The scientific community has targeted that Lf is a promising candidate to help break the vicious cycle of antibiotic resistances [103, 5]. It has been demonstrated that oral Lf supplementation in human newborns can prevent infection or decrease the severity of an existing infection [104, 105, 106]. A human study found that nutritional supplementation with colostrum was equally efficient in preventing episodes of the flu compared to a vaccine [22, 107]. In clinical trials, the administration of bovine lactoferrin suppressed carcinogenesis in the colon and other organs, and human studies have recently shown that Lf inhibits the growth of adenomatous polyps and can reduce the risk of colon carcinogenesis [108]. There have been reports on ability of Lf affecting metabolism by reducing triacylglycerol and cholesterol levels [109]; a study in Japan revealed that the use of oral lactoferrin can reduce visceral fat in humans [110]. In addition to its application to human health, Lf has industrial application. One a study has shown that Lf can be used to extend the shelf-life of various meat patties and other meat products, fresh ground pork with added Lf had lower total plate counts [111]. Lactoferrin can also be used as an alternative animal feed additive, to reduce or eliminate the impact of antibiotic consumption on animal husbandry and to strengthen natural immune systems of livestock. Along these lines, a study found that early-weaned piglets

receiving rice bran expressing porcine recombinant-Lf as a feed additive, had improve antimicrobial characteristics and IgG concentration [112, 113, 114]. It has been proposed that Lf could be used to control diseases caused by fungal pathogens in crops [95]. In cell culture applications, there is a concern regarding potential contamination when using protein and peptides from animals; therefore, the expression of recombinant Lf in rice has been studied as a cell culture component to enhance the growth of intestinal cells, hybridoma cells, human embryonic kidney cells and osteoblasts. This study concluded, concluding that Lf is effective in promoting mammalian cell growth and increasing cell productivity [115]. Human Lf is safe, and is considerate by FDA to be a GRAS product with no contraindications in either in pediatric or adult patients [103, 75]. Some pharmaceutical industries (e.g., Venture LLC, Biopharming, Ventria Bioscience, AusBioMed, Max biocare, Morinaga Milk Industry Co. LTD) are currently commercializing human and bovine Lf in different products including a nutraceutical powder, a vitamin supplement for children, infant formula, beverages and a cell growth promoter. The bio-pharmacy company Agennix has a product, talactoferrin, which is currently undergoing clinical trials for consideration as a GRAS product. Its proposed used include the treatment of diverse carcinomas, severe sepsis, and diabetic foot ulcers. Infant formulas supplemented with Lf are among the products in which industry is particularly interested. As discussed the Morinaga Milk Industry was the first to add Lf in infant formula in an attempt to approximate a mother’s milk in terms of nutritional value and balance.

8. Conclusion Lf is a versatile molecule molded by nature selection to be the first-line defense in mammals. Its ability to exert a multiple regulatory effects due to its cationic nature that allows to bind a large number of surface molecules or metal ions during the microorganism development and inducing host immune-modulatory activation, influencing both adaptive and innate immunities. Due to its plethora of abilities, it is required the development expression systems of Lf for food and pharmaceutical application. Particularly, the role of Lf as multifunctional nutraceutical protein for preventive strategies in newborn nutrition.

Acknowledgments This work was supported in part by an internal grant from Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Mexico and Proteo/MuuuTechnologies de México.

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Table I. Lactoferrin mechanism of action against bacteria

Mechanism of action Enhancing of phagocytosis

Biofilms inhibition

Positive domain union with negative charges on microorganisms Modification of the interactions of microbes, with the host cells, or with the extracellular matrix Inhibits LPSmediated activation Induction of apoptosis

Target Gram-Positive bacteria  S. mutans  S. epidermidis  S. aureus Gram-Negative bacteria:  P. aeruginosa  B. cepacia  B. cenocepacia  Porphyromonas gingivalis Virus:  Vesicular Stomatitis Virus Fungi:  Candida spp.  A. Fumigatus Parasites:  E. histolytica  B. caballi  T. cruzi Gram-Negative bacteria:  Porphyromonas gingivalis  Prevetella intermedi  P. aeruginosa  B. cepacia  B. cenocepacia  E. coli  M. bovis Gram-Positive bacteria:  S. epidermidis Gram-Negative bacteria:  E. coli Parasites:  T. gondii  E. stiedai Gram-Positive bacteria:  B. subtilis  K. pneumoniae  S. mutans Virus:  Rotavirus  Enterovirus Gram-Negative bacteria

Fungi: 

Reference 1,77, 6, 87, 88, 46, 76

77, 89,6,78

6, 90

78, 77

6, 79, 40

84 Candida albicans

Figure 1. Predicted structure of lactoferrin from EU812318 (bLF) sequence using PDB ID: 1FCK (human) as a template, showing two-lobe, four-domain polypeptide. Modeled using Protein Model Portal, and viewed using Chimera software (http://www.cgl.ucsf.edu/chimera/).

Figure 2. Schematic representation of the influence of host Lf on immune cells. a) B- and T- lymphocytes maturation; b) B- lymphocytes activation; c) B- and Tlymphocytes interaction; d) IgA and IgG secretion; e) T- lymphocytes proliferation; i) prevents interaction between LPS and CD14 as a TLR4; f) IL5 and IL10 secretion decreased; g) regulates of NF activation of monocytes; h) phagocytic activity of macrophages. B = B lymphocytes; T = T lymphocytes; Mac = macrophages; Mon = Monocytes; Neu = Neutrophils.