Effect of bovine lactoferrin on recurrent urinary tract infections: in vitro and in vivo evidences.

Uropathogenic Escherichia coli (UPEC) strains are the primary cause of urinary tract infections (UTIs). UPEC strains are able to invade, multiply and persisting in host cells. Therefore, UPEC strains are associated to recurrent UTIs requiring long-term antibiotic therapy. However, this therapy is suboptimal due to the increase of multidrug-resistant UPEC. The use of non-antibiotic treatments for managing UTIs is required. Among these, bovine lactoferrin (bLf), a multifunctional cationic glycoprotein, could be a promising tool because inhibits the entry into the host cells of several intracellular bacteria. Here, we demonstrate that 100 µg/ml bLf hinders the invasion of 2.0 ± 0.5 × 104 CFU/ml E. coli CFT073, prototype of UPEC, infecting 2.0 ± 0.5 × 105 cells/ml urinary bladder T24 epithelial cells. The highest protection (100%) is due to the bLf binding with host surface components even if an additional binding to bacterial surface components cannot be excluded. Of note, in the absence of bLf, UPEC survives and multiplies, while bLf significantly decreases bacterial intracellular survival. After these encouraging results, an observational survey on thirty-three patients affected by recurrent cystitis was performed. The treatment consisted in the oral administration of bLf alone or in combination with antibiotics and/or probiotics. After the observation period, a marked reduction of cystitis episodes was observed (p < 0.001) in all patients compared to the episodes occurred during the 6 months preceding the bLf-treatment. Twenty-nine patients did not report cystitis episodes (87.9%) whereas the remaining four (12.1%) experienced only one episode, indicating that bLf could be a worthwhile and safe treatment in counteracting recurrent cystitis.

Influence of oral administration mode on the efficacy of commercial bovine Lactoferrin against iron and inflammatory homeostasis disorders.

Milk derivative bovine Lactoferrin (bLf), a multifunctional glycoprotein available in large quantities and recognized as safe, possesses high homology and identical functions with human Lactoferrin. There are numerous food supplements containing bLf which, however, can vary in its purity, integrity and, consequently, functionality. Here, we report on a clinical trial where bLf (100 mg two times/day) was orally administered before (Arm A) or during meals (Arm B) to pregnant women with hereditary thrombophilia and suffering from anemia of inflammation. A significant increase of the number of red blood cells (RBCs), hemoglobin (Hb), total serum iron (TSI) and serum ferritin (sFtn) levels, along with a significant decrease of interleukin-6 were detected after 30 days in Arm A, but not in Arm B, thus letting us to hypothesize that bLf inefficacy could be related to its degradation by digestive proteases. To verify this hypothesis, bLf was incubated in gastric juice collected before or after meals. An undigested or a digested profile was observed when bLf was incubated in gastric juice sampled before or after meals, respectively. These results can explain the beneficial effect observed when bLf is administered under fasting conditions, i.e. in the absence of active proteases.

Lactoferrin in Aseptic and Septic Inflammation.

Lactoferrin (Lf), a cationic glycoprotein able to chelate two ferric irons per molecule, is synthesized by exocrine glands and neutrophils. Since the first anti-microbial function attributed to Lf, several activities have been discovered, including the relevant anti-inflammatory one, especially associated to the down-regulation of pro-inflammatory cytokines, as IL-6. As high levels of IL-6 are involved in iron homeostasis disorders, Lf is emerging as a potent regulator of iron and inflammatory homeostasis. Here, the role of Lf against aseptic and septic inflammation has been reviewed. In particular, in the context of aseptic inflammation, as anemia of inflammation, preterm delivery, Alzheimer's disease and type 2 diabetes, Lf administration reduces local and/or systemic inflammation. Moreover, Lf oral administration, by decreasing serum IL-6, reverts iron homeostasis disorders. Regarding septic inflammation occurring in Chlamydia trachomatis infection, cystic fibrosis and inflammatory bowel disease, Lf, besides the anti-inflammatory activity, exerts a significant activity against bacterial adhesion, invasion and colonization. Lastly, a critical analysis of literature in vitro data reporting contradictory results on the Lf role in inflammatory processes, ranging from pro- to anti-inflammatory activity, highlighted that they depend on cell models, cell metabolic status, stimulatory or infecting agents as well as on Lf iron saturation degree, integrity and purity.

Physico-chemical properties influence the functions and efficacy of commercial bovine lactoferrins.

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.

Effect of bovine lactoferrin on Chlamydia trachomatis infection and inflammation.

Chlamydia trachomatis is an obligate, intracellular pathogen responsible for the most common sexually transmitted bacterial disease worldwide, causing acute and chronic infections. The acute infection is susceptible to antibiotics, whereas the chronic one needs prolonged therapies, thus increasing the risk of developing antibiotic resistance. Novel alternative therapies are needed. The intracellular development of C. trachomatis requires essential nutrients, including iron. Iron-chelating drugs inhibit C. trachomatis developmental cycle. Lactoferrin (Lf), a pleiotropic iron binding glycoprotein, could be a promising candidate against C. trachomatis infection. Similarly to the efficacy against other intracellular pathogens, bovine Lf (bLf) could both interfere with C. trachomatis entry into epithelial cells and exert an anti-inflammatory activity. In vitro and in vivo effects of bLf against C. trachomatis infectious and inflammatory process has been investigated. BLf inhibits C. trachomatis entry into host cells when incubated with cell monolayers before or at the moment of the infection and down-regulates IL-6/IL-8 synthesized by infected cells. Six out of 7 pregnant women asymptomatically infected by C. trachomatis, after 30 days of bLf intravaginal administration, were negative for C. trachomatis and showed a decrease of cervical IL-6 levels. This is the first time that the bLf protective effect against C. trachomatis infection has been demonstrated.

Lactoferrin: A Natural Glycoprotein Involved in Iron and Inflammatory Homeostasis.

Human lactoferrin (hLf), an iron-binding multifunctional cationic glycoprotein secreted by exocrine glands and by neutrophils, is a key element of host defenses. HLf and bovine Lf (bLf), possessing high sequence homology and identical functions, inhibit bacterial growth and biofilm dependently from iron binding ability while, independently, bacterial adhesion to and the entry into cells. In infected/inflamed host cells, bLf exerts an anti-inflammatory activity against interleukin-6 (IL-6), thus up-regulating ferroportin (Fpn) and transferrin receptor 1 (TfR1) and down-regulating ferritin (Ftn), pivotal actors of iron and inflammatory homeostasis (IIH). Consequently, bLf inhibits intracellular iron overload, an unsafe condition enhancing in vivo susceptibility to infections, as well as anemia of inflammation (AI), re-establishing IIH. In pregnant women, affected by AI, bLf oral administration decreases IL-6 and increases hematological parameters. This surprising effect is unrelated to iron supplementation by bLf (80 µg instead of 1-2 mg/day), but to its role on IIH. AI is unrelated to the lack of iron, but to iron delocalization: cellular/tissue overload and blood deficiency. BLf cures AI by restoring iron from cells to blood through Fpn up-expression. Indeed, anti-inflammatory activity of oral and intravaginal bLf prevents preterm delivery. Promising bLf treatments can prevent/cure transitory inflammation/anemia/oral pathologies in athletes.

Safety and efficacy of lactoferrin versus ferrous sulphate in curing iron deficiency and iron deficiency anaemia in hereditary thrombophilia pregnant women: an interventional study.

UNLABELLED: 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: ClinicalTrials.gov Identifier NCT01221844.

Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases.

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.

Bovine lactoferrin in preventing preterm delivery associated with sterile inflammation.

Preterm delivery (PTD) occurs before the 37th week of gestation. Iron deficiency anemia and inflammatory processes either related to infection or sterile inflammatory response represent risk factors for PTD. Bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, can represent a new therapeutic approach for PTD treatment. Here an open-label cohort and subcohort study is reported. The cohort was designed to assess the effect of bLf oral administration on iron and inflammatory homeostasis in anemic pregnant women. The subcohort including women of the cohort with PTD threat was additionally treated with bLf intravaginal administration. A significant improvement of hematological parameters was observed in the women's cohort together with a consistent decrease of serum interleukin-6 (IL-6) levels. Combined administration of oral and intravaginal bLf to the women's subcohort with PTD threat decreased IL-6 in both serum and cervicovaginal fluids, cervicovaginal prostaglandin F(2α), and suppressed uterine contractility. BLf administration blocked further shortening of cervical length and the increase of fetal fibronectin thus prolonging the length of pregnancy. The deliveries occurred between the 37th and 38th week of gestation. These results provide strong evidence for a role of bLf in PTD treatment, thus extending the therapeutic potential of this multifunctional natural protein.

Antiviral properties of lactoferrin--a natural immunity molecule.

Lactoferrin, a multifunctional iron binding glycoprotein, plays an important role in immune regulation and defence mechanisms against bacteria, fungi and viruses. Lactoferrin's iron withholding ability is related to inhibition of microbial growth as well as to modulation of motility, aggregation and biofilm formation of pathogenic bacteria. Independently of iron binding capability, lactoferrin interacts with microbial, viral and cell surfaces thus inhibiting microbial and viral adhesion and entry into host cells. Lactoferrin can be considered not only a primary defense factor against mucosal infections, but also a polyvalent regulator which interacts in viral infectious processes. Its antiviral activity, demonstrated against both enveloped and naked viruses, lies in the early phase of infection, thus preventing entry of virus in the host cell. This activity is exerted by binding to heparan sulphate glycosaminoglycan cell receptors, or viral particles or both. Despite the antiviral effect of lactoferrin, widely demonstrated in vitro studies, few clinical trials have been carried out and the related mechanism of action is still under debate. The nuclear localization of lactoferrin in different epithelial human cells suggests that lactoferrin exerts its antiviral effect not only in the early phase of surface interaction virus-cell, but also intracellularly. The capability of lactoferrin to exert a potent antiviral activity, through its binding to host cells and/or viral particles, and its nuclear localization strengthens the idea that lactoferrin is an important brick in the mucosal wall, effective against viral attacks and it could be usefully applied as novel strategy for treatment of viral infections.

Lactoferrin efficacy versus ferrous sulfate in curing iron deficiency and iron deficiency anemia in pregnant women.

Iron deficiency (ID) and iron deficiency anemia (IDA) are the most common iron disorders throughout the world. ID and IDA, particularly caused by increased iron requirements during pregnancy, represent a high risk for preterm delivery, fetal growth retardation, low birth weight, and inferior neonatal health. Oral administration of ferrous sulfate to cure ID and IDA in pregnancy often fails to increase hematological parameters, causes adverse effects and increases inflammation. Recently, we have demonstrated safety and efficacy of oral administration of 30% iron saturated bovine lactoferrin (bLf) in pregnant women suffering from ID and IDA. Oral administration of bLf significantly increases the number of red blood cells, hemoglobin, total serum iron and serum ferritin already after 30 days of the treatment. The increasing of hematological values by bLf is related to the decrease of serum IL-6 and the increase of serum hepcidin, detected as prohepcidin, whereas ferrous sulfate increases IL-6 and fails to increase hematological parameters and prohepcidin. bLf is a more effective and safer alternative than ferrous sulfate for treating ID and IDA in pregnant women.

Efficacy of Lactoferrin Oral Administration in the Treatment of Anemia and Anemia of Inflammation in Pregnant and Non-pregnant Women: An Interventional Study.

The discovery of the ferroportin-hepcidin complex has led to a critical review on the treatment of anemia and anemia of inflammation (AI). Ferroportin, the only known mammalian iron exporter from cells to blood, is negatively regulated by hepcidin, a hormone peptide able to bind to ferroportin, leading to its degradation. Therefore, new efficient therapeutic interventions acting on hepcidin and ferroportin are imperative to manage anemia and AI. Bovine milk derivative lactoferrin (bLf), a glycoprotein able to chelate two ferric ions per molecule, is emerging as a natural anti-inflammatory substance able to modulate hepcidin and ferroportin synthesis through the down-regulation of interleukin-6 (IL-6). Here, an interventional study (ClinicalTrials.gov Identifier: NCT01221844) was conducted by orally administering 100 mg of 20-30% iron-saturated bLf (corresponding to 70-84 µg of elemental iron) twice a day. This treatment was compared with the Italian standard therapy, consisting in the oral administration of 329.7 mg of ferrous sulfate once a day (corresponding to 105 mg of elemental iron). Treatments were carried out on 29 anemic women with minor ß-thalassemia (20 pregnant and 9 non-pregnant), 149 women with hereditary thrombophilia (HT) (70 pregnant and 79 non-pregnant) affected by AI and 20 anemic pregnant women suffering from various pathologies. In anemic pregnant and non-pregnant women with minor ß-thalassemia, presenting undetectable hepcidin levels, differently from ferrous sulfate management, bLf decreased IL-6 (from 25 ± 8 to 6 ± 3 pg/ml) and increased total serum iron (TSI) (from 54 ± 17 to 80 ± 9 µg/dl). BLf was also more efficient than ferrous sulfate in AI treatment in HT pregnant and non-pregnant women by decreasing both serum IL-6 (from 89 ± 8 to 58 ± 6 pg/ml) and hepcidin (from 115 ± 23 to 65 ± 10 ng/ml), thus increasing hematological parameters, such as the number of red blood cells (RBCs), the concentration of hemoglobin, TSI and serum ferritin. BLf was also efficient in treating anemia in other pathological pregnancies. Taken together all the results, bLf, showing a greater benefit and efficacy than the standard ferrous sulfate management, can be considered as a promising compound in treating anemia and AI through its ability to down-regulate IL-6, thus restoring ferroportin-mediated iron export from cells to blood in a hepcidin-dependent or independent way.

Role of Lactobacilli and Lactoferrin in the Mucosal Cervicovaginal Defense.

The innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. Vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. Lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. Several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal pH, modulation of immunity, and production of bioactive compounds. Among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. Lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. Lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by Neisseria gonorrheae, Chlamydia trachomatis, and Trichomonas vaginalis infections promoting both innate and adaptive immune responses. In vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. Then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. Considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis.

Lactobacilli-lactoferrin interplay in Chlamydia trachomatis infection.

In the cervicovaginal microenvironment, lactobacilli are known to protect against genital infections and, amongst the host defence compounds, lactoferrin has recently acquired importance for its anti-microbial and anti-inflammatory properties. An abnormal genital microenvironment facilitates the acquisition of pathogens like Chlamydia trachomatis, the leading cause of bacterial sexually transmitted infections worldwide. The aim of our study is to investigate the effects of Lactobacillus crispatus, Lactobacillus brevis and bovine lactoferrin on chlamydial infection, in order to shed light on the complex interplay between host defence mechanisms and C. trachomatis. We have also evaluated the effect of these defence factors to modulate the chlamydia-mediated inflammatory state. To this purpose, we have determined the infectivity and progeny production of C. trachomatis as well as interleukin-8 and interleukin-6 synthesis. The main result of our study is that the combination of L. brevis and bovine lactoferrin is the most effective in inhibiting the early phases (adhesion and invasion) of C. trachomatis infection of cervical epithelial cells and in decreasing the levels of both cytokines. In conclusion, the interaction between L. brevis and lactoferrin seems to play a role in the protection against C. trachomatis, reducing the infection and regulating the immunomodulatory activity, thus decreasing the risk of severe complications.

The influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia.

Iron is a fundamental element for humans as it represents an essential component of many proteins and enzymes. However, this element can also be toxic when present in excess because of its ability to generate reactive oxygen species. This dual nature imposes a tight regulation of iron concentration in the body. In humans, systemic iron homeostasis is mainly regulated at the level of intestinal absorption and, until now, no regulated pathways for the excretion of iron have been found. The regulation and maintenance of systemic iron homeostasis is critical to human health. Excessive iron absorption leads to iron-overload in parenchyma, while low iron absorption leads to plasma iron deficiency, which manifests as hypoferremia (iron deficiency, ID) and ID anaemia (IDA). ID and IDA are still a major health problem in pregnant women. To cure ID and IDA, iron supplements are routinely prescribed. The preferred treatment of ID/IDA, consisting in oral administration of iron as ferrous sulphate, often fails to exert significant effects on hypoferremia and may also cause adverse effects. Lactoferrin (Lf), an iron-binding glycoprotein abundantly found in exocrine secretions of mammals, is emerging as an important regulator of systemic iron homeostasis. Recent data suggest that this natural compound, capable of interacting with the most important components of iron homeostasis, may represent a valuable alternative to iron supplements in the prevention and cure of pregnancy-associated ID and IDA. In this review, recent advances in the molecular circuits involved in the complex cellular and systemic iron homeostasis will be summarised. The role of Lf in curing ID and IDA in pregnancy and in the maintenance of iron homeostasis will also be discussed. Understanding these mechanisms will provide the rationale for the development of novel therapeutic alternatives to ferrous sulphate oral administration in the prevention and cure of ID and IDA.

Oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women.

Iron deficiency anemia (IDA) during pregnancy continues to be of world-wide concern. IDA is a risk factor for preterm delivery and subsequent low birth weight, and possibly for poor neonatal health. Iron supplementation in pregnancy is a widely recommended practice, yet intervention programs have met with many controversies. In our study, 300 women at different trimesters of pregnancy were enrolled in a trial of oral administration of ferrous sulfate (520 mg once a day) or 30% iron-saturated bovine lactoferrin (bLf) (100 mg twice a day). Pregnant women refusing treatment represented the control group. In this group hemoglobin and total serum iron values measured after 30 d without treatment decreased significantly, especially in women at 18-31 weeks of pregnancy. In contrast, after 30 d of oral administration of bLf, hemoglobin and total serum iron values increased and to a greater extent than those observed in women treated orally for 30 d with ferrous sulfate, independently of the trimester of pregnancy. Unlike ferrous sulfate, bLf did not result in any side effects. These findings lead us to hypothesize that lactoferrin could influence iron homeostasis directly or through other proteins involved in iron transport out of the intestinal cells into the blood.