BioMetals (v.31, #3)

Quo vadis lactoferrin? by Hans J. Vogel; Antimo Cutone; Maria S. Lepanto; Luigi Rosa; Piera Valenti (297-300).

Physico-chemical properties influence the functions and efficacy of commercial bovine lactoferrins by Luigi Rosa; Antimo Cutone; Maria Stefania Lepanto; Mellani Jinnett Scotti; Maria Pia Conte; Rosalba Paesano; Piera Valenti (301-312).
Human and bovine lactoferrin (hLf and bLf) are multifunctional iron-binding glycoprotein constitutively synthesized and secreted by glandular epithelial cells and by neutrophils following induction. HLf and bLf possess very high similarity of sequence. Therefore, most of the in vitro and in vivo studies are carried out with commercial bLf (cbLf), available in large quantities and recognized by Food and Drug Administration (FDA, USA) as a safe substance. Physico-chemical heterogeneity of different cbLf preparations influences their effectiveness. CbLf iron-saturation affects thermal stability and resistance to proteolysis. Moreover, other metal ions such as Al(III), Cu(II), Mg(II), Mn(II), Zn(II) are chelated by cbLf, even if at lower affinity than Fe(III). Ca(II) is also sequestered by the carboxylate groups of sialic acid present on glycan chains of cbLf thus provoking the release of LPS, contributing to bactericidal activity. Similarly to more than 50% of eukaryotic proteins, cbLf possesses five N-glycosylation sites, also contributing to the resistance to proteolysis and, putatively, to the protection of intestinal mucosa from pathogens. CbLfs possess several functions as anti-microbial, anti-biofilm, anti-adhesive, anti-invasive and anti-inflammatory activities. They are also relevant modulators of iron and inflammatory homeostasis. However, the efficacy of cbLfs in exerting several functions can be erratic mainly depending from integrity, degree of iron and other metal ions saturation, N-glycosylation sites and chains, desialylated forms, Ca(II) sequestration, presence of contaminants and finally the ability to enter inside nucleus.
Keywords: Lactoferrin; Commercial bovine lactoferrins; Metal-binding ability; Lipopolysaccharide release; Iron deficiency anaemia; Anaemia of inflammation

Quality control of commercial bovine lactoferrin by Hiroyuki Wakabayashi; Koji Yamauchi; Fumiaki Abe (313-319).
Herein we review commercial bovine lactoferrin quality issues by describing an example of industrial production, the current status of global quality standardization, and quality-activity concerns for further discussion. Morinaga Milk Industry has been industrially producing bovine lactoferrin in Milei GmbH, Germany, since 1989. We delineate its production and quality as an example of safe and high-quality manufacturing. Currently, global standardization in the quality of bovine lactoferrin is progressing through Novel Food and GRAS in the EU and USA, respectively. Novel Food was applied or notified to seven lactoferrin manufacturers and GRAS was notified to three manufacturers, two of which are for infant use and one is for adult use, by the end of 2017. The specifications of these regulations are relatively high, including more than 95% lactoferrin purity in protein, which means that such companies can supply relatively high-grade lactoferrin. There appear to be several concerns regarding lactoferrin quality affecting activities, including contamination of lipopolysaccharide (LPS) and angiogenin, purity, and degradation of lactoferrin sample. Although LPS is immunologically toxic when invading the body, it is distributed normally in foods and the gut. However, an industrial lactoferrin sample may contain LPS at a maximum LPS/lactoferrin molecule ratio = 1/1724, which means 99.9% of the lactoferrin molecule is LPS-free. It is difficult to speculate that LPS contained in a lactoferrin sample affects its activities. Finally in order to achieve good and reproducible results, we make proposals to researchers a use of high-grade lactoferrin, careful storage, and indication the manufacturers’ names and specifications in the paper.
Keywords: Lactoferrin; Quality; Specification; Novel Food; GRAS; LPS

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes food-borne disease in humans ranging from watery diarrhea to bloody diarrhea and severe hemorrhagic colitis, renal failure and hemolytic uremic syndrome. Cattle, the most important source of E. coli O157:H7 transmission to humans, harbor the bacteria in their gastrointestinal tract without showing clinical symptoms. Prevention of E. coli O157:H7 infections in ruminants could diminish the public health risk. However, there is no specific treatment available nor a vaccine or a therapeutic agent which completely prevents E. coli O157:H7 infections in cattle. This paper provides an overview of latest research data on eradicating enterohemorrhagic E. coli O157:H7 in ruminants by use of bovine lactoferrin administration. The article provides insights into the anti-microbial and immunomodulatory activities of bovine lactoferrin against E. coli O157:H7 infections in cattle.
Keywords: Cattle; Escherichia coli O157:H7; Bovine lactoferrin; Rectal administration; Shiga toxin; Interleukin 6

A review of the design and modification of lactoferricins and their derivatives by Ya Hao; Na Yang; Da Teng; Xiumin Wang; Ruoyu Mao; Jianhua Wang (331-341).
Lactoferricin (Lfcin), a multifunction short peptide with a length of 25 residues, is derived from the whey protein lactoferrin by acidic pepsin hydrolysis. It has potent nutritional enhancement, antimicrobial, anticancer, antiviral, antiparasitic, and anti-inflammatory activities. This review describes the research advantages of the above biological functions, with attention to the molecular design and modification of Lfcin. In this examination of design and modification studies, research on the identification of Lfcin active derivatives and crucial amino acid residues is also reviewed. Many strategies for Lfcin optimization have been studied in recent decades, but we mainly introduce chemical modification, cyclization, chimera and polymerization of this peptide. Modifications such as incorporation of d-amino acids, acetylation and/or amidation could effectively improve the activity and stability of these compounds. Due to their wide array of bio-functions and applications, Lfcins have great potential to be developed as biological agents with multiple functions involved with nutritional enhancement, as well as disease preventive and therapeutic effects.
Keywords: Lactoferricin; Modification; Nutritional enhancement; Preventive and therapeutic activity; Biological agent

Antirotaviral potential of lactoferrin from different origin: effect of thermal and high pressure treatments by José Antonio Parrón; Daniel Ripollés; Sergio José Ramos; María Dolores Pérez; Zeynep Semen; Pedro Rubio; Miguel Calvo; Lourdes Sánchez (343-355).
Rotaviral gastroenteritis causes a high rate of infant mortality and severe healthcare implications worldwide. Several studies have pointed out that human milk and dairy fractions, such as whey and buttermilk, possess antirotaviral activity. This activity has been mainly associated with glycoproteins, among them lactoferrin (LF). Thermal treatments are necessary to provide microbiological safety and extend the shelf life of milk products, though they may diminish their biological value. High hydrostatic pressure (HHP) treatment is a non-thermal method that causes lower degradation of food components than other treatments. Thus, the main objective of this study was to prove the antirotaviral activity of LFs from different origin and to evaluate the effect of several thermal and HHP treatments on that activity. LF exerted a high antirotaviral activity, regardless of its origin. Native LFs from bovine, ovine, swine and camel milk, and the human recombinant forms, at 1 mg/mL, showed neutralizing values in the range 87.5–98.6%, while human LF neutralized 58.2%. Iron saturation of bovine LF did not modify its antirotaviral activity. Results revealed interspecies differences in LFs heat susceptibility. Thus, pasteurization at 63 °C for 30 min led to a decrease of 60.1, 44.5, 87.1, 3.8 and 8% of neutralizing activity for human, bovine, swine, ovine and camel LFs, respectively. Pasteurization at 75 °C for 20 s was less harmful to the activity of LFs, with losses ranging from 0 to 13.8%. HHP treatment at 600 MPa for 15 min did not cause any significant decrease in the neutralizing activity of LFs.
Keywords: Lactoferrin; Antirotaviral activity; Pasteurization; High hydrostatic pressure

Lactoferrin (Lf) is a major protein in human milk. Multiple biological functions of Lf are postulated to be mediated by a Lf receptor (LfR). The Lf receptor (LfR) plays an important role in absorption of Lf and Lf-bound iron by intestinal epithelial cells. Here, we cloned and characterized the promoter from a ~ 3.1 kb 5′-flanking region of the human LfR gene. Neither a TATA box nor a CCAAT box is found at the typical positions. The transcription start site was identified as 298 bp upstream of the translation start codon (+ 1) by 5′ RLM-RACE. A series of deletions of 5′-flanking sequences of the human LfR gene were cloned into a promoter-less pGL3 luciferase reporter and transiently transfected into an intestinal enterocyte model (Caco-2 cells). A fragment of − 299/+ 63 elicited the maximal promoter activity in transfected Caco-2 cells, suggesting that functional transcription factor binding sites appear in the region of − 299/+ 63. Bioinformatics analysis indicates that the − 299/+ 63 fragment contains two putative Sp1 binding sites. The promoter activity was significantly decreased when the Sp1 binding sites were mutated by site-directed mutagenesis. Additionally, the promoter activity was dramatically inhibited by treating cells with an Sp1 inhibitor. Binding of Sp1 to the promoter was confirmed by EMSA. Moreover, after Sp1 expression was significantly suppressed by RNA interference, LfR was significantly decreased at both RNA and protein levels. In conclusion, the LfR gene promoter contains downstream core promoter elements, and the Sp1 binding sites play critical roles in transcriptional regulation of the LfR gene.
Keywords: Lactoferrin receptor; Promoter; Caco-2 cells; Transcription activation

Role of lactoferrin and its receptors on biliary epithelium by Romina Mancinelli; Francesca Olivero; Guido Carpino; Diletta Overi; Luigi Rosa; Maria Stefania Lepanto; Antimo Cutone; Antonio Franchitto; Gianfranco Alpini; Paolo Onori; Piera Valenti; Eugenio Gaudio (369-379).
Human lactoferrin is an iron-binding glycoprotein present at high concentrations in breast milk and colostrum. It is produced by many exocrine glands and widely distributed in a variety of body fluids. This protein has antimicrobial, immunomodulatory, antioxidant, and anticancer properties. Two important hLf receptors have been identified: LDL receptor related protein (LRP1), a low specificity receptor, and intelectin-1 (ITLN1), a high specificity receptor. No data are present on the role of hLf on the biliary epithelium. Our aims have been to evaluate the expression of Lf and its receptors in human and murine cholangiocytes and its effect on proliferation. Immunohistochemistry and immunofluorescence (IF) were conducted on human healthy and primary biliary cholangitis (PBC) liver samples as well as on liver samples obtained from normal and bile duct ligated (BDL) mice to evaluate the expression of Lf, LRP1 and ITLN1. Cell proliferation in vitro studies were performed on human cholangiocyte cell lines via 3-(4,5-dimetiltiazol-2-il)-2,5-diphenyltetrazolium assay as well as IF to evaluate proliferating cell nuclear antigen (PCNA) expression. Our results show that mouse and human cholangiocytes express Lf, LRP1 and ITLN1, at higher extent in cholangiocytes from BDL and PBC samples. Furthermore, the in vitro addition of bovine Lf (bLf) has a proliferative effect on human cholangiocyte cell line. The results support a proliferative role of hLf on the biliary epithelium; this pro-proliferative effect of hLf and bLf on cholangiocytes could be particularly relevant in human cholangiopathies such as PBC, characterized by cholangiocyte death and ductopenia.
Keywords: Biliary epithelium; Cholangiocytes; Lactoferrin; Intelectin-1; Primary biliary cholangitis

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets? by Clement Chan; Vahid F. Andisi; Dixon Ng; Nick Ostan; Warren K. Yunker; Anthony B. Schryvers (381-398).
A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.
Keywords: Lactoferrin-binding protein; Transferrin binding protein; Vaccine; Antimicrobial peptides

The ferroportin-ceruloplasmin system and the mammalian iron homeostasis machine: regulatory pathways and the role of lactoferrin by Maria Carmela Bonaccorsi di Patti; Antimo Cutone; Fabio Polticelli; Luigi Rosa; Maria Stefania Lepanto; Piera Valenti; Giovanni Musci (399-414).
In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.
Keywords: Lactoferrin; Ferroportin; Ceruloplasmin; Iron; Homeostasis; Inflammation

Bovine lactoferrin promotes energy expenditure via the cAMP-PKA signaling pathway in human reprogrammed brown adipocytes by Kanae Nakamura; Tsunao Kishida; Akika Ejima; Riho Tateyama; Satoru Morishita; Tomoji Ono; Michiaki Murakoshi; Keikichi Sugiyama; Hoyoku Nishino; Osam Mazda (415-424).
Lactoferrin (LF) is a multifunctional protein in mammalian milk. We previously reported that enteric-coated bovine LF reduced the visceral fat in a double-blind clinical study. We further demonstrated that bovine LF (bLF) inhibited adipogenesis and promoted lipolysis in white adipocytes, but the effect of bLF on brown adipocytes has not been clarified. In this study, we investigated the effects of bLF on energy expenditure and cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway using human reprogrammed brown adipocytes generated by gene transduction. bLF at concentrations of ≥ 100 μg/mL significantly increased uncoupling protein 1 (UCP1) mRNA levels, with the maximum value observed 4 h after bLF addition. At the same time point, bLF stimulation also significantly increased oxygen consumption. Signaling pathway analysis revealed rapid increases of intracellular cAMP and cAMP response element-binding protein (CREB) phosphorylation levels beginning 5 min after bLF addition. The mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were also significantly increased after 1 h of bLF stimulation. H-89, a specific PKA inhibitor, abrogated bLF-induced UCP1 gene expression. Moreover, receptor-associated protein (Rap), an antagonist of low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced bLF-induced UCP1 gene expression in a dose-dependent manner. These results suggest that bLF promotes UCP1 gene expression in brown adipocytes through the cAMP-PKA signaling pathway via the LRP1 receptor, leading to increased energy expenditure.
Keywords: Lactoferrin; Brown adipocytes; Energy expenditure; UCP1

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin by E. T. Zakharova; A. V. Sokolov; N. N. Pavlichenko; V. A. Kostevich; I. N. Abdurasulova; A. V. Chechushkov; I. V. Voynova; A. Yu. Elizarova; N. N. Kolmakov; M. G. Bass; I. V. Semak; A. I. Budevich; P. M. Kozhin; N. K. Zenkov; V. M. Klimenko; O. V. Kirik; D. E. Korzhevskii; E. B. Menshchikova; V. B. Vasilyev (425-443).
Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson’s disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.
Keywords: Lactoferrin; Hypoxia-inducible factors HIF-1a/HIF-2a; Erythropoietin; Nrf2; Neuroprotection; Parkinson’s disease; Experimental autoimmune encephalomyelitis; Ischaemia; Stroke

Efficacy of bovine lactoferrin in the post-surgical treatment of patients suffering from bisphosphonate-related osteonecrosis of the jaws: an open-label study by Francesco Calvani; Antimo Cutone; Maria Stefania Lepanto; Luigi Rosa; Valentino Valentini; Piera Valenti (445-455).
Osteonecrosis of the jaws is an emerging pathological condition characterized by un-exposure or exposure of the necrotic bone, independently from the etiology. This term is usually referred to medication-related osteonecrosis of the jaws due to severe adverse reaction to certain medicines, as bisphosphonates, used for the treatment of cancer and osteoporosis. The management of patients with Bisphosphonate-Related Osteonecrosis of the Jaws (BRONJ) remains challenging because surgical and medical interventions may not eradicate this pathology. The goal of treatment of patients at risk of developing BRONJ or of those who have active disease is the preservation of quality of life by controlling pain, managing infection, and preventing the development of new areas of necrosis. The treatment of osteonecrosis consists in the surgical removal of necrotic bone followed by antibiotic therapy and application of sterile greasy gauze until the wound closure. The classical medical treatment has been compared with the innovative one consisting in the application of sterile greasy gauze soaked with bovine lactoferrin (bLf) after surgery. Here, for the first time, bLf efficacy on wound repair in subjects suffering from BRONJ with the progressive destruction of bone in the mandible or maxilla has been demonstrated. The positive results consist in a significant shorter time of wound closure (1 or 2 weeks) compared to that observed with classical surgical treatment (2–3 months). These promising results are an interesting tool for the innovative treatment of this pathology and for increasing the quality of life of these patients.
Keywords: Lactoferrin; Osteonecrosis of the jaw; BRONJ; MRONJ; Wound repair