BioMetals (v.27, #5)

Lactoferrin, all roads lead to Rome by Piera Valenti; Hans J. Vogel (803-806).

Lactoferrin prevents LPS-induced decrease of the iron exporter ferroportin in human monocytes/macrophages by Antimo Cutone; Alessandra Frioni; Francesca Berlutti; Piera Valenti; Giovanni Musci; Maria Carmela Bonaccorsi di Patti (807-813).
Iron balance is tightly linked to inflammation and it has been demonstrated that many proteins involved in cellular iron management are up- or down-regulated by inflammatory stimuli, ultimately leading to iron retention in the reticuloendothelial system. Ferroportin is a key player in maintenance of correct iron homeostasis, because it is the only known mammalian cellular iron exporter. In this work we show that incubation of THP-1 monocytes/macrophages with lactoferrin prevents the LPS-induced decrease of ferroportin by reducing secretion of IL-6.
Keywords: Ferroportin; Lactoferrin; Iron; Inflammation; Interleukin-6

Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes by Alexey V. Sokolov; Elena T. Zakahrova; Valeria A. Kostevich; Valeria R. Samygina; Vadim B. Vasilyev (815-828).
Copper-containing plasma protein ceruloplasmin (Cp) forms a complex with lactoferrin (Lf), an iron-binding protein, and with the heme-containing myeloperoxidase (Mpo). In case of inflammation, Lf and Mpo are secreted from neutrophil granules. Among the plasma proteins, Cp seems to be the preferential partner of Lf and Mpo. After an intraperitoneal injection of Lf to rodents, the “Cp–Lf” complex has been shown to appear in their bloodstream. Cp prevents the interaction of Lf with protoplasts of Micrococcus luteus. Upon immunoprecipitation of Cp, the blood plasma becomes depleted of Lf and in a dose-dependent manner loses the capacity to inhibit the peroxidase activity of Mpo, but not the Mpo-catalyzed oxidation of thiocyanate in the (pseudo)halogenating cycle. Antimicrobial effect against E. coli displayed by a synergistic system that includes Lf and Mpo–H2O2–chloride, but not thiocyanate, as the substrate for Mpo is abrogated when Cp is added. Hence, Cp can be regarded as an anti-inflammatory factor that restrains the halogenating cycle and redirects the synergistic system Mpo–H2O2–chloride/thiocyanate to production of hypothiocyanate, which is relatively harmless for the human organism. Structure and functions of the “2Cp–2Lf–Mpo” complex and binary complexes Cp–Lf and 2Cp–Mpo in inflammation are discussed.
Keywords: Ceruloplasmin; Lactoferrin; Myeloperoxidase; Protein–protein interactions; Synergism of antimicrobial proteins; Inflammation; Thiocyanate; Halogenative stress

Erratum to: Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes by Alexey V. Sokolov; Elena T. Zakharova; Valeria A. Kostevich; Valeria R. Samygina; Vadim B. Vasilyev (829-829).

Lactoferrin (Lf) is an iron-binding glycoprotein present in high concentration in human milk. It is a pleiotropic protein and involved in diverse bioactivities, such as stimulation of cell proliferation and immunomodulatory activities. Lf is partly resistant to proteolysis in the gastrointestinal tract. Thus, Lf may play important roles in intestinal development. Due to differences in amino acid sequences and isolation methods, Lfs from human and bovine milk as well as commercially available bovine Lf (CbLf) may differ functionally or exert their functions via various mechanisms. To provide a potential basis for further applications of CbLf, we compared effects of Lfs on intestinal transcriptomic profiling using an intestinal epithelial cell model, human intestinal epithelial crypt-like cells (HIEC). All Lfs significantly stimulated proliferation of HIEC and no significant differences were found among these three proteins. Microarray assays were used to investigate transcriptomic profiling of intestinal epithelial cells in response to Lfs. Selected genes were verified by RT-PCR with a high validation rate. Genes significantly regulated by hLf, bLf, and CbLf were 150, 395 and 453, respectively. Fifty-four genes were significantly regulated by both hLf and CbLf, whereas 129 genes were significantly modulated by bLf and CbLf. Although only a limited number of genes were regulated by all Lfs, the three Lfs positively influenced cellular development and immune functions based on pathway analysis using IPA (Ingenuity). Lfs stimulate cellular and intestinal development and immune functions via various signaling pathways, such as Wnt/β-catenin signaling, interferon signaling and IL-8 signaling.
Keywords: Human lactoferrin; Bovine lactoferrin; Cell proliferation; Immune function

Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases by Alessandra Frioni; Maria Pia Conte; Antimo Cutone; Catia Longhi; Giovanni Musci; Maria Carmela Bonaccorsi di Patti; Tiziana Natalizi; Massimiliano Marazzato; Maria Stefania Lepanto; Patrizia Puddu; Rosalba Paesano; Piera Valenti; Francesca Berlutti (843-856).
Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn’s disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account.
Keywords: Lactoferrin; Inflammation; Cystic fibrosis; Chron’s disease; Ferroportin; Infection

Effects of lactoferrin on intestinal epithelial cell growth and differentiation: an in vivo and in vitro study by Anne Blais; Cuibai Fan; Thierry Voisin; Najat Aattouri; Michel Dubarry; François Blachier; Daniel Tomé (857-874).
This study was designed to analyse the effects of human (h) and bovine lactoferrin (bLF) on the growth and differentiation of intestinal cells using the mice model supplemented with Lactoferrin (LF) and the enterocyte-like model of Caco-2 cells which spontaneously differentiate after confluency. In mice, bLF supplementation increased jejunal villus height and the expression of several intestinal brush border membrane enzymes activities. Addition of bLF or hLF to undifferentiated Caco-2 cells was able to increase cell proliferation with confluency being reached more rapidly. Moreover, when Caco-2 cells were grown in the presence of LF for 3 weeks, brush-border membrane-associated enzyme activities i.e. sucrase, alkaline phosphatase and neutral aminopeptidase, as well as the l-glutamate transporter expression were all increased indicating an increased Caco-2 cell differentiation. Accordingly, cDNA Atlas array and Western blot analysis of cell cycle proteins shown a decreased expression of Cdck2 and an increased TAF1 expression; these proteins being implicated in the regulation of numerous genes related to cellular proliferation and differentiation. These modifications were associated with an inhibition of Caco-2 cell spontaneous apoptosis. Altogether, our results indicate that LF increase in vivo and in vitro enterocyte differentiation. In addition, LF was found to increase in vitro enterocyte proliferation resulting in higher cell density in cell flasks, an effect that was likely partly due to a reduction of the cellular apoptosis. The different stimulation patterns observed for the different parameters associated with cell differentiation in relationship with specific gene regulation is discussed.
Keywords: Lactoferrin; Intestinal epithelial cell growth; Enterocyte differentiation; Apoptosis

Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating bax expression by Stéphan Hardivillé; Adelma Escobar-Ramirez; Soccoro Pina-Canceco; Elisabeth Elass; Annick Pierce (875-889).
Delta-lactoferrin (∆Lf) is a transcription factor belonging to the lactoferrin family, the expression of which inhibits cell proliferation and leads to Skp1 and DcpS gene transactivation. In this study, we showed that ∆Lf expression also induces cell death via apoptosis in HEK 293 and MCF7 cells using a cell viability assay and DNA fragmentation. Western blot analyses showed that apoptosis was caspase-9, 7 and 8 dependent. Proteolytic cleavage of the endonuclease PARP was significantly increased. The levels of expression of Bcl family members were detected by immunochemistry and showed that the Bcl-xl/Bax and Bcl-2/Bax protein ratios were decreased. We determined that the pro-apoptotic effects of ∆Lf are mainly mediated by the activation of the mitochondria-dependent death-signaling pathway. Apoptosis induction by ∆Lf is concomitant with increased cellular levels of Bax protein. Analysis of the Bax promoter region detected a ∆Lf response element located at −155 bp from the transcription start site. Both luciferase reporter gene and chromatin immunoprecipitation assays confirmed that ∆Lf interacts in vitro and in vivo specifically with this sequence. Its deletion, realized using directed mutagenesis, totally abolished ∆Lf transcriptional activity, identifying it as a ∆Lf-responsive element. These results indicate that the Bax gene is a novel ∆Lf target. Moreover we also showed that the O-GlcNAc/P interplay, which controls ∆Lf transcriptional activity, modulates Bax transactivation.
Keywords: Delta-lactoferrin; Lactoferrin; Transcription factor; Apoptosis; Tumor-suppressor; O-GlcNAcylation

Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae by Sarahí Luna-Castro; Francisco Aguilar-Romero; Luisa Samaniego-Barrón; Delfino Godínez-Vargas; Mireya de la Garza (891-903).
Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24–42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60–70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App.
Keywords: Actinobacillus pleuropneumoniae ; Adhesion; Biofilm; Bovine lactoferrin; Virulence

Bovine lactoferrin (bLf) is a natural glycoprotein, and it shows broad-spectrum antimicrobial activity. However, reports on the influences of bLf on probiotic bacteria have been mixed. We examined the effects of apo-bLf (between 0.25 and 128 mg/mL) on both aerobic and anaerobic cultures of probiotics. We found that bLf had similar effects on the growth of probiotics under aerobic or anaerobic conditions, and that it actively and significantly (at concentrations of >0.25 mg/mL) retarded the growth rate of Bifidobacterium bifidum (ATCC 29521), B. longum (ATCC 15707), B. lactis (BCRC 17394), B. infantis (ATCC 15697), Lactobacillus reuteri (ATCC 23272), L. rhamnosus (ATCC 53103), and L. coryniformis (ATCC 25602) in a dose-dependent manner. Otherwise, minimal inhibitory concentrations (MICs) were 128 or >128 mg/mL against B. bifidum, B. longum, B. lactis, L. reuteri, and L. rhamnosus (ATCC 53103). With regard to MICs, bLf showed at least four-fold lower inhibitory effect on probiotics than on pathogens. Intriguingly, bLf (>0.25 mg/mL) significantly enhanced the growth of Rhamnosus (ATCC 7469) and L. acidophilus (BCRC 14065) by approximately 40–200 %, during their late periods of growth. Supernatants produced from aerobic but not anaerobic cultures of L. acidophilus reduced the growth of Escherichia coli by about 20 %. Thus, bLf displayed a dose-dependent inhibitory effect on the growth of most probiotic strains under either aerobic or anaerobic conditions. An antibacterial supernatant prepared from the aerobic cultures may have significant practical use.
Keywords: Lactoferrin; Probiotic; Lactic acid bacterium; Bifidobacterium

Lactoferrin and bifidobacteria by Hirotsugu Oda; Hiroyuki Wakabayashi; Koji Yamauchi; Fumiaki Abe (915-922).
We herein summarized the effects of lactoferrin (LF) on bifidobacteria. Many in vitro studies previously reported the growth-promoting (bifidogenic) effects of LF on bifidobacteria. The involvement of bound iron, sugar chains, and LF peptides has been proposed in this bifidogenic mechanism. Peptides in the LF pepsin hydrolysate (LFH) showed stronger bifidogenic activity than natural LF; therefore, we speculated that peptides may be the bifidogenic active principle of LF. LF or its peptides may be recognized by LF-binding proteins on the surface of bifidobacterial cells, and the cationic nature or disulfide bonds of LF or its peptides may play a crucial role in its recognition by these proteins. Of the bifidobacterial species so far identified, human LF and peptides in human LFH were more likely to show bifidogenic activity especially to Bifidobacterium bifidum, and bovine LF (bLF) and peptides in bovine LFH (bLFH) to B. breve and B. infantis. In animal studies, the administration of LF to mice or piglets increased bifidobacteria levels in the intestine. In human trials, the administration of LF-containing formula to infants increased bifidobacteria levels in the feces; however, human milk achieved better results than LF-containing formula. In the case of breast-fed infants, LF may show bifidogenic activity synergistically with other milk components such as human milk oligosaccharides. As bLFH showed stronger bifidogenic activity than natural bLF, especially to B. breve and B. infantis in vitro, and these species are known to be infant-specific species, bLFH may be a beneficial ingredient in formula.
Keywords: Lactoferrin; Bifidobacteria; Peptide; Infant; Formula

The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B by Ari Morgenthau; Sarathy K. Partha; Paul Adamiak; Anthony B. Schryvers (923-933).
A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein’s C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.
Keywords: Cationic antimicrobial peptides; Lactoferrin receptor; Lipoprotein; Bactericidal; Outer membrane protein; Neisseria

Bovine and human lactoferricin peptides: chimeras and new cyclic analogs by Mauricio Arias; Lindsey J. McDonald; Evan F. Haney; Kamran Nazmi; Jan G. M. Bolscher; Hans J. Vogel (935-948).
Lactoferrin (LF) is an important antimicrobial and immune regulatory protein present in neutrophils and most exocrine secretions of mammals. The antimicrobial activity of LF has been related to the presence of an antimicrobial peptide sequence, called lactoferricin (LFcin), located in the N-terminal region of the protein. The antimicrobial activity of bovine LFcin is considerably stronger than the human version. In this work, chimera peptides combining segments of bovine and human LFcin were generated in order to study their antimicrobial activity and mechanism of action. In addition, the relevance of the conserved disulfide bridge and the resulting cyclic structure of both LFcins were analyzed by using “click chemistry” and sortase A-catalyzed cyclization of the peptides. The N-terminal region of bovine LFcin (residues 17–25 of bovine LF) proved to be very important for the antimicrobial activity of the chimera peptides against E. coli, when combined with the C-terminal region of human LFcin. Similarly the cyclic bovine LFcin analogs generated by “click chemistry” and sortase A preserved the antimicrobial activity of the original peptide, showing the significance of these two techniques in the design of cyclic antimicrobial peptides. The mechanism of action of bovine LFcin and its active derived peptides was strongly correlated with membrane leakage in E. coli and up to some extent with the ability to induce vesicle aggregation. This mechanism was also preserved under conditions of high ionic strength (150 mM NaCl) illustrating the importance of these peptides in a more physiologically relevant system.
Keywords: Lactoferrin; Lactoferricin; Peptide cyclization; Click chemistry; Sortase A

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis by Sakawrat Kanthawong; Aekkalak Puknun; Jan G. M. Bolscher; Kamran Nazmi; Jan van Marle; Johannes J. de Soet; Enno C. I. Veerman; Surasakdi Wongratanacheewin; Suwimol Taweechaisupapong (949-956).
LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.
Keywords: Burkholderia pseudomallei ; Burkholderia thailandensis ; Antimicrobial peptide; LFchimera; Lactoferrin

Mechanism of action of the tri-hybrid antimicrobial peptide LHP7 from lactoferricin, HP and plectasin on Staphylococcus aureus by Di Xi; Xiumin Wang; Da Teng; Ruoyu Mao; Yong Zhang; Xiaojie Wang; Jianhua Wang (957-968).
The tri-hybrid peptide-LHP7 has the potent activity against Gram-positive and Gram-negative as well as fungi, but its mechanism of action has remained elusive. The effluences of LHP7 on the Staphylococcus aureus cell membrane and targets of intracellular action were investigated. LHP7 exhibited an inhibitory effect on the S. aureus growth, similar to those achieved by plectasin, vancomycin and gramicidin. The membrane integrity studies confirmed that LHP7 disrupted the cell membrane, indicating a membrane permeabilizing killing action. A marginal decline in the intensity fluorescence indicated no significant depolarization of the membrane potential following LHP7 treatment. Furthermore, electron microscopy showed that cell shrinkage, cell wall thickening, cellular content leakage, and cell disruption were observed in the cells treated with LHP7. A gel retardation assay showed that LHP7 bound to the genomic DNA of S. aureus or plasmid DNA at a mass ratio of 2.5–10 (peptide/DNA). Circular dichroism indicated that LHP7 inserted into the groove of DNA. The cell cycle analysis showed that after the treatment with LHP7 for 30 and 60 min, the proportion of cells in I-phase increased from 8.71 to 12.09 % and from 8.71 to 15.68 %, indicating that LHP7 induced arrest of cells in the I-phase. These results would conduce to elucidate its underlying antibacterial mechanism.
Keywords: Cationic peptide; LHP7; Antibacterial mechanism; Membrane permeability; Staphylococcus aureus

Bactericidal effect of bovine lactoferrin and synthetic peptide lactoferrin chimera in Streptococcus pneumoniae and the decrease in luxS gene expression by lactoferrin by Nidia León-Sicairos; Uriel A. Angulo-Zamudio; Jorge E. Vidal; Cynthia A. López-Torres; Jan G. M. Bolscher; Kamran Nazmi; Ruth Reyes-Cortes; Magda Reyes-López; Mireya de la Garza; Adrian Canizalez-Román (969-980).
Streptococcus pneumoniae (pneumococcus) is responsible for nearly one million child deaths annually. Pneumococcus causes infections such as pneumonia, otitis media, meningitis, and sepsis. The human immune system includes antibacterial peptides and proteins such as lactoferrin (LF), but its activity against pneumococcus is not fully understood. The aim of this work was to evaluate the bactericidal effect of bovine lactoferrin (bLF) and the synthetic LF-peptides lactoferricin (LFcin17–30), lactoferrampin (LFampin265–284), and LFchimera against S. pneumoniae planktonic cells. The mechanism of damage was also investigated, as well as the impact of these peptides on the transcription levels of genes known to encode important virulence factors. S. pneumoniae planktonic cells were treated with bLF, LFcin17–30, LFampin265–284 and LFchimera at different time points. The viability of treated planktonic cells was assessed by dilution and plating (in CFU/ml). The interaction between LF and LF-peptides coupled to fluorescein was visualized using a confocal microscope and flow cytometry, whereas the damage at structural levels was observed by electron microscopy. Damage to bacterial membranes was further evaluated by membrane permeabilization by use of propidium iodide and flow cytometry, and finally, the expression of pneumococcal genes was evaluated by qRT-PCR. bLF and LFchimera were the best bactericidal agents. bLF and peptides interacted with bacteria causing changes in the shape and size of the cell and membrane permeabilization. Moreover, the luxS gene was down-regulated in bacteria treated with LF. In conclusion, LF and LFchimera have a bactericidal effect, and LF down-regulates genes involved in the pathogenicity of pneumococcus, thus demonstrating potential as new agents for the treatment of pneumococcal infections.
Keywords: Lactoferrin; Peptides; Bactericidal effect; Planktonic cells; Pneumococcus

Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl derivative also slightly affects PC. Tryptophan fluorescence of selective peptide R-DIM-P-LF11-322 revealed specific peptide penetration into the PS membrane interface and circular dichroism showed change of its secondary structure by increase of proportion of β-sheets just in the presence of the cancer mimic. Data correlated with in vitro studies with cell lines of human melanomas, their metastases and melanocytes, revealing R-DIM-P-LF11-322 to exhibit strongly increased specificity for cancer cells. This indicates the need of high affinity to the target PS, a minimum length and net positive charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity.
Keywords: Peptides; Cancer therapy; Liposomes; Melanoma; Membrane biophysics; Lactoferricin derivatives; Cell membrane permeabilization; Phosphatidylserine

Safety and efficacy of lactoferrin versus ferrous sulphate in curing iron deficiency and iron deficiency anaemia in hereditary thrombophilia pregnant women: an interventional study by Rosalba Paesano; Enrica Pacifici; Samanta Benedetti; Francesca Berlutti; Alessandra Frioni; Antonella Polimeni; Piera Valenti (999-1006).
Objective Evaluate the safety and efficacy of bovine lactoferrin (bLf) versus the ferrous sulphate standard intervention in curing iron deficiency (ID) and ID anaemia (IDA) in pregnant women affected by hereditary thrombophilia (HT). Design Interventional study. Setting Secondary-level hospital for complicated pregnancies in Rome, Italy. Population 295 HT pregnant women (≥18 years) suffering from ID/IDA. Methods Women were enrolled in Arm A or B in accordance with their personal choice. In Arm A, 156 women received oral administration of 100 mg of bLf twice a day; in Arm B, 139 women received 520 mg of ferrous sulphate once a day. Therapies lasted until delivery. Main outcome measures Red blood cells, haemoglobin, total serum iron, serum ferritin (haematological parameters) were assayed before and every 30 days during therapy until delivery. Serum IL-6, key factor in inflammatory and iron homeostasis disorders, was detected at enrolment and after therapy at delivery. Possible maternal, foetal, and neonatal adverse effects were assessed. Results Haematological parameters were significantly higher in Arm A than in Arm B pregnant women (P ≤ 0.0001). Serum IL-6 significantly decreased in bLf-treated women and increased in ferrous sulphate-treated women. BLf did not exert any adverse effect. Adverse effects in 16.5 % of ferrous sulphate-treated women were recorded. Arm A women experienced no miscarriage compared to five miscarriages in Arm B women. Conclusions Differently from ferrous sulphate, bLf is safe and effective in curing ID/IDA associated with a consistent decrease of serum IL-6. The absence of miscarriage among bLf-treated women provided an unexpected benefit. Trial registration: Identifier NCT01221844.
Keywords: Lactoferrin; Anaemia; Iron deficiency; Pregnancy; Hereditary thrombophilia

Lactoferrin for prevention of neonatal sepsis by Christie G. Turin; Alonso Zea-Vera; Alonso Pezo; Karen Cruz; Jaime Zegarra; Sicilia Bellomo; Luis Cam; Raul Llanos; Anne Castañeda; Lourdes Tucto; Theresa J. Ochoa (1007-1016).
Preterm neonates are at risk to acquire infections. In addition to the high mortality associated with sepsis, these patients are at risk for long-term disabilities, particularly neurodevelopment impairment. Several interventions have been evaluated to reduce rates of infections in neonates but have not proven efficacy. Lactoferrin (LF), a milk glycoprotein with anti-inflammatory, immunomodulatory and anti-microbial properties, has the potential to prevent infections in young children. We performed a review of current and ongoing clinical trials of LF for prevention of neonatal sepsis, and found eleven registered clinical trials that include more than 6,000 subjects. Few of these trials have finished; despite their small sample size, the preliminary results show a trend towards a positive protective effect of LF on neonatal infections. Larger trials are underway to confirm the findings of these initial studies. This information will help to define LF’s role in clinical settings and, if proven effective, would profoundly affect the treatment of low birth weight neonates as a cost-effective intervention worldwide.
Keywords: Lactoferrin; Bovine lactoferrin; Recombinant human lactoferrin; Neonatal sepsis; Prevention; Clinical trial

Inhibition of intestinal polyp growth by oral ingestion of bovine lactoferrin and immune cells in the large intestine by Masaaki Iigo; David B. Alexander; Jiegou Xu; Mitsuru Futakuchi; Masumi Suzui; Takahiro Kozu; Takayuki Akasu; Daizo Saito; Tadao Kakizoe; Koji Yamauchi; Fumiaki Abe; Mitsunori Takase; Kazunori Sekine; Hiroyuki Tsuda (1017-1029).
Studies using animal models have demonstrated that ingestion of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs of experimental animals. As a result of these studies, a blinded, randomized, controlled clinical trial was conducted in the National Cancer Center Hospital, Tokyo, Japan to determine whether ingestion of bLF had an effect on the growth of colorectal polyps in humans. Patients with colorectal polyps ≤5 mm diameter and likely to be adenomas ingested 0, 1.5, or 3.0 g bLF daily for 1 year. Ingestion of 3.0 g bLF suppressed the growth of colorectal polyps and increased the level of serum human lactoferrin in trial participants 63 years old or younger. The purpose of the present study was to investigate correlations between immune parameters and changes in polyp size. Trial participants with regressing polyps had increased NK cell activity, increased serum hLF levels (indicating increased neutrophil activity), and increased numbers of CD4+ cells in the polyps. These findings are consistent with a correlation between higher immune activity and suppression of colorectal polyps. In addition, participants with regressing polyps had lower numbers of PMNs and increased numbers of S100A8+ cells in the polyps, consistent with a correlation between lower inflammatory potential in the colon and suppression of colorectal polyps. Trial participants ingesting bLF had increased serum hLF levels, a possible increase in systemic NK cell activity, and increased numbers of CD4+ and CD161+ cells in the polyps. Taken together, our findings suggest that bLF suppressed colorectal polyps by enhancing immune responsiveness.
Keywords: Ingestion of bovine lactoferrin; Immune function; Human intestine; Ancillary study of a human clinical trial

Lactoferrin is part of the immune system and multiple tissues including the gastrointestinal (GI) tract, liver, and lung contain receptors for lactoferrin. Lactoferrin has many functions, including antimicrobial, immunomodulatory, and iron binding. Additionally, lactoferrin inhibits the migration of eosinophils, which are constitutively present in the GI tract, and increase during inflammation. Lactoferrin suppresses eosinophil infiltration into the lungs and eosinophil migration in -vitro. Healthy pigs have a large population of eosinophils in their small intestine and like humans, pigs have small intestinal lactoferrin receptors (LFR); thus, pigs were chosen to investigate the effects of consumption of milk containing recombinant human lactoferrin (rhLF-milk) on small intestinal eosinophils and expression of eosinophilic cytokines. In addition, LFR localization was analyzed in duodenum and circulating eosinophils to determine if the LFR could play a role in lactoferrin’s ability to inhibit eosinophil migration. In the duodenum there were significantly fewer eosinophils/unit area in pigs fed rhLF-milk compared to pigs fed control milk (p = 0.025); this was not seen in the ileum (p = 0.669). In the duodenum, no differences were observed in expression of the LFR, or any eosinophil migratory cytokines, and the amount of LFR protein was not different (p = 0.386). Immunohistochemistry (IHC) showed that within the duodenum the LFR localized on the brush border of villi, crypts, and within the lamina propria. Circulating eosinophils also contained LFRs, which may be a mechanism allowing lactoferrin to directly inhibit eosinophil migration.
Keywords: Eosinophils; Gastrointestinal tract; Human lactoferrin; Inflammation; Lactoferrin receptor; Transgenic

Behavioral effects of bovine lactoferrin administration during postnatal development of rats by Jason Shumake; Douglas W. Barrett; Michelle A. Lane; Anja J. Wittke (1039-1055).
We tested the hypothesis that rats consuming bovine lactoferrin (bLf) during postnatal development would show better performance of stressful tasks during adolescence. In the first study, we orally administered bLf (750 mg/kg) once daily between postnatal days 16–34. Rats then underwent a battery of behavioral tests: open field (forced exploration of risky environment), light–dark emergence (voluntary exploration of risky environment), baited holeboard (working and reference memory), food neophobia (preference for familiar versus novel food), forced swim (test for antidepressant efficacy), and shuttle-box escape (learning to escape footshock). bLf-supplemented rats showed less exploration of the risky environment, greater preference for the familiar food odor, and faster escape responses. The effect of bLf on forced-swim behavior depended on sex: immobility increased for males and decreased for females. In the next study, we replaced the forced-swim test with an escape-swim test in which rats learned to use a visual cue to locate an escape platform, and we tested the dose response of bLf on this and the shuttle-box escape test, with subjects receiving vehicle or bLf at 500, 1,000, or 2,000 mg/kg. Under this modified testing battery, improvement of escape from footshock was not observed at any dose. However, males, but not females, showed a significant dose-dependent effect of bLf on acquisition of the water-escape task. On average, males receiving a higher dose mastered the task 20–25 % sooner than rats receiving a lower dose or vehicle. These results offer preliminary evidence that bLf supplementation during development can improve subsequent cognitive performance during stress.
Keywords: Bovine lactoferrin; Transferrin; Rat; Behavior; Stress; Learning and memory

Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia by Hsiao-Ling Chen; Chih-Ching Yen; Shih-Ming Wang; Tung-Chou Tsai; Zi-Lun Lai; Jheng-Yue Sun; Willei Lin; Wu-Huei Hsu; Chuan-Mu Chen (1057-1068).
This study investigated the ability of aerosolized bovine lactoferrin (bLF) to protect the lungs from injury induced by chronic hyperoxia. Female CD-1 mice were exposed to hyperoxia (FiO2 = 80 %) for 7 days to induce lung injury and fibrosis. The therapeutic effects of bLF, administered via an aerosol delivery system, on the chronic lung injury induced by this period of hyperoxia were measured by bronchoalveolar lavage, lung histology, cell apoptosis, and inflammatory cytokines in the lung tissues. After exposure to hyperoxia for 7 days, the survival of the mice was significantly decreased to 20 %. The protective effects of bLF against hyperoxia were further confirmed by significant reductions in lung edema, total cell numbers in bronchoalveolar lavage fluid, inflammatory cytokines (IL-1β and IL-6), pulmonary fibrosis, and apoptotic DNA fragmentation. The aerosolized bLF protected the mice from oxygen toxicity and increased the survival fraction to 66.7 % in the hyperoxic model. The results support the use of an aerosol therapy with bLF in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure or chronic obstructive pulmonary disease.
Keywords: Hyperoxic lung injury; Lung fibrosis; Aerosol therapy; Bovine lactoferrin; Oxygen toxicity

Fecal lactoferrin and intestinal permeability are effective non-invasive markers in the diagnostic work-up of chronic diarrhea by Roberta Caccaro; Renata D’Incà; Matteo Martinato; Elisabetta Dal Pont; Surajit Pathak; Anna Chiara Frigo; Giacomo Carlo Sturniolo (1069-1076).
Non-invasive markers able to identify patients with chronic diarrhea at risk of organic disease are missing. Aim of the study was to assess the diagnostic ability of intestinal permeability (IP) test and fecal lactoferrin (FL) in distinguishing functional from organic disease in patients with chronic diarrhea. We retrospectively enrolled patients referring to the gastroenterology outpatient clinic for chronic diarrhea. Among the 103 patients included, 40 % had an organic disease, with IP and FL levels significantly higher compared to those with a functional disorder (p < 0.0001). Sensitivity, specificity, positive and negative likelihood ratios, area under ROC curves of FL were superior to those of IP in discriminating functional and organic disease (FL: 87.8 and 93.6 %, 13.61 and 0.13, 0.9375; IP: 61.0 and 90.3 %, 6.3 and 0.43, 0.7691). When combining the two tests, the diagnostic ability of FL did not improve. In subgroup analysis, IP confirmed its ability to detect small bowel alterations, while FL could identify both small bowel and colonic alterations. In conclusion, FL is valid to detect inflammation in the gastrointestinal tract, while IP can effectively identify small bowel damage in chronic diarrhea patients. Together these tests could recognize both the presence of intestinal damage and its site.
Keywords: Chronic diarrhea; Fecal marker; Lactoferrin; Sensitivity; Specificity; Sugar probes

Correlation between lactoferrin and beneficial microbiota in breast milk and infant’s feces by Paola Mastromarino; Daniela Capobianco; Giuseppe Campagna; Nicola Laforgia; Pietro Drimaco; Alessandra Dileone; Maria E. Baldassarre (1077-1086).
Lactoferrin (LF) is a natural component of human milk with antimicrobial, immunostimulatory and immunomodulatory properties. Several in vitro studies suggest that LF could promote an environment in the gut of neonates that favors colonization with beneficial bacteria. However, clinical studies on the correlation between the concentration of LF in breast milk and feces of infants and the gut microbiota in infants are lacking. In our study we analyzed the content of LF and the microbiota of breast milk and feces of infants of 48 mother–infant pairs (34 full-term and 14 pre-term infants) at birth and 30 days after delivery. In the term group, a significant decrease of mean LF concentration between colostrum (7.0 ± 5.1 mg/ml) and mature milk (2.3 ± 0.4 mg/ml) was observed. In pre-term group, breast milk LF levels were similar to those observed in full-term group. Fecal LF concentration of healthy infants was extremely high both in term and pre-term infants, higher than the amount reported in healthy children and adults. In term infants mean fecal LF levels significantly increased from birth (994 ± 1,828 μg/ml) to 1 month of age (3,052 ± 4,323 μg/ml). The amount of LF in the feces of 30 day-old term infants was significantly associated with maternal mature milk LF concentration (p = 0.030) confirming that breast milk represents the main source of LF found in the gut of infants. A linear positive correlation between colostrum and mature milk LF concentration was observed (p = 0.008) indicating that milk LF levels reflect individual characteristics. In pre-term infants higher mean concentrations of fecal LF at birth (1,631 ± 2,206 μg/ml) and 30 days after delivery (7,633 ± 9,960 μg/ml) were observed in comparison to full-term infants. The amount of fecal bifidobacteria and lactobacilli resulted associated with the concentration of fecal LF 3 days after delivery (p = 0.017 and p = 0.026, respectively). These results suggest that high levels of fecal LF in neonates, particularly in the first days of life, could represent an important factor in the initiation, development and/or composition of the neonatal gut microbiota. Since early host–microbe interaction is a crucial component of healthy immune and metabolic programming, high levels of fecal LF in neonates may beneficially contribute to the immunologic maturation and well-being of the newborn, especially in pre-term infants.
Keywords: Lactoferrin; Newborn; Beneficial microbiota; Feces; Breast milk