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Lactoferrin (Lf) is a cationic glycoprotein synthetized by exocrine glands and is present in all human secretions. It is also secreted by neutrophils in infection and inflammation sites. This glycoprotein possesses antimicrobial activity due to its capability to chelate two ferric ions per molecule, as well as to interact with bacterial and viral anionic surface components. The cationic features of Lf bind to cells, protecting the host from bacterial and viral injuries. Its anti-inflammatory activity is mediated by the ability to enter inside the nucleus of host cells, thus inhibiting the synthesis of proinflammatory cytokine genes. In particular, Lf down-regulates the synthesis of IL-6, which is involved in iron homeostasis disorders and leads to intracellular iron overload, favoring viral replication and infection. The well-known antiviral activity of Lf has been demonstrated against DNA, RNA, and enveloped and naked viruses and, therefore, Lf could be efficient in counteracting also SARS-CoV-2 infection. For this purpose, we performed in vitro assays, proving that Lf exerts an antiviral activity against SARS-COV-2 through direct attachment to both SARS-CoV-2 and cell surface components. This activity varied according to concentration (100/500 μg/ml), multiplicity of infection (0.1/0.01), and cell type (Vero E6/Caco-2 cells). Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between Lf and the spike S glycoprotein, which can thus hinder viral entry into the cells. These in vitro observations led us to speculate a potential supplementary role of Lf in the management of COVID-19 patients.

Lactoferrin (Lf), a multifunctional cationic glycoprotein synthesized by exocrine glands and neutrophils, possesses an in vitro antiviral activity against SARS-CoV-2. Thus, we conducted an in vivo preliminary study to investigate the antiviral effect of oral and intranasal liposomal bovine Lf (bLf) in asymptomatic and mild-to-moderate COVID-19 patients. From April 2020 to June 2020, a total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided into three groups. Thirty-two patients (14 hospitalized and 18 in home-based isolation) received only oral and intranasal liposomal bLf, 32 hospitalized patients were treated only with standard of care (SOC) treatment, and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, untreated, healthy subjects were added for ancillary analysis. Liposomal bLf-treated COVID-19 patients obtained an earlier and significant (p &lt, 0.0001) SARS-CoV-2 RNA negative conversion compared to the SOC-treated and untreated COVID-19 patients (14.25 vs. 27.13 vs. 32.61 days, respectively). Liposomal bLf-treated COVID-19 patients showed fast clinical symptoms recovery compared to the SOC-treated COVID-19 patients. In bLf-treated patients, a significant decrease in serum ferritin, IL-6, and D-dimers levels was observed. No adverse events were reported. These observations led us to speculate a potential role of bLf in the management of mild-to-moderate and asymptomatic COVID-19 patients.

Lactoferrin, a multifunctional cationic glycoprotein, secreted by exocrine glands and neutrophils, possesses an antiviral activity extendable to SARS-CoV-2.We performed in vitro assays proving lactoferrin antiviral activity through direct attachment to both virus and cell surface components. This activity varied according to concentration (100/500μg/ml), multiplicity of infection (0.1/0.01) and cell type (Vero E6/Caco-2 cells).Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between the lactoferrin and the Spike S glycoprotein, thus hindering the viral entry into the cells.Hence, we conducted a clinical trial to investigate effect and tolerability of a liposomal lactoferrin formulation as a supplementary nutraceutical agent in mild-to-moderate and asymptomatic COVID-19 patients.A total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided in 3 groups according to the administered regimen. Thirty-two patients, 14 hospitalised and 18 in home-based insolation received oral and intranasal liposomal bovine lactoferrin (bLf), 32 hospitalised patients were treated with standard of care treatment (hydroxychloroquine, azitromicin and lopinavir/darunavir), and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, not-treated, healthy subjects were added as a control group for ancillary analysis.bLf-supplemented COVID-19 patients obtained an earlier and significant (p < 0,0001.) median rRT-PCR SARS-COV-2 RNA negative conversion than standard of care-treated and non-treated COVID-19 patients (14.25 vs 27.13 vs 32.61 days, respectively).In addition, bLf-supplemented COVID-19 patients showed significant fast clinical symptoms recovery than standard of care-treated and non-treated COVID-19 patients. Moreover, in bLf-supplemented patients, a significant decrease of either serum ferritin or IL-6 levels or host iron overload, all parameters characterizing inflammatory processes, were observed. Serum D-dimers was also found significantly decreased following bLf supplement. No adverse events were reported.These in vitro and in vivo observations led us to speculate a potential and safe supplementary role of Blf in the management of mild-to-moderate and asymptomatic COVID-19 patients.

Recently, the world has been dealing with a devastating global pandemic coronavirus infection, with more than 12 million infected worldwide and over 300,000 deaths as of May 15th 2020, related to a novel coronavirus (2019-nCoV), characterized by a spherical morphology and identified through next-generation sequencing. Although the respiratory tract is the primary portal of entry of SARS-CoV-2, gastrointestinal involvement associated with nausea, vomiting and diarrhoea may also occur. No drug or vaccine has been approved due to the absence of evidence deriving from rigorous clinical trials. Increasing interest has been highlighted on the possible preventative role and adjunct treatment of lactoferrin, glycoprotein of human secretions part of a non-specific defensive system, known to play a crucial role against microbial and viral infections and exerting anti-inflammatory effects on different mucosal surfaces and able to regulate iron metabolism. In this review, analysing lactoferrin properties, we propose designing a clinical trial to evaluate and verify its effect using a dual combination treatment with local, solubilized intranasal spray formulation and oral administration. Lactoferrin could counteract the coronavirus infection and inflammation, acting either as natural barrier of both respiratory and intestinal mucosa or reverting the iron disorders related to the viral colonization.

The oral cavity is a non-uniform, extraordinary environment characterized by mucosal, epithelial, abiotic surfaces and secretions as saliva. Aerobic and anaerobic commensal and pathogenic microorganisms colonize the tongue, teeth, jowl, gingiva, and periodontium. Commensals exert an important role in host defenses, while pathogenic microorganisms can nullify this protective function causing oral and systemic diseases. Every day, 750–1000 mL of saliva, containing several host defense constituents including lactoferrin (Lf), are secreted and swallowed. Lf is a multifunctional iron-chelating cationic glycoprotein of innate immunity. Depending on, or regardless of its iron-binding ability, Lf exerts bacteriostatic, bactericidal, antibiofilm, antioxidant, antiadhesive, anti-invasive, and anti-inflammatory activities. Here, we report the protective role of Lf in different oral pathologies, such as xerostomia, halitosis, alveolar or maxillary bone damage, gingivitis, periodontitis, and black stain. Unlike antibiotic therapy, which is ineffective against bacteria that are within a biofilm, adherent, or intracellular, the topical administration of Lf, through its simultaneous activity against microbial replication, biofilms, adhesion, and invasiveness, as well as inflammation, has been proven to be efficient in the treatment of all known oral pathologies without any adverse effects.

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.

Despite recent advances in cancer therapy, current treatments, including radiotherapy, chemotherapy, and immunotherapy, although beneficial, present attendant side effects and long-term sequelae, usually more or less affecting quality of life of the patients. Indeed, except for most of the immunotherapeutic agents, the complete lack of selectivity between normal and cancer cells for radio- and chemotherapy can make them potential antagonists of the host anti-cancer self-defense over time. Recently, the use of nutraceuticals as natural compounds corroborating anti-cancer standard therapy is emerging as a promising tool for their relative abundance, bioavailability, safety, low-cost effectiveness, and immuno-compatibility with the host. In this review, we outlined the anti-cancer properties of Lactoferrin (Lf), an iron-binding glycoprotein of the innate immune defense. Lf shows high bioavailability after oral administration, high selectivity toward cancer cells, and a wide range of molecular targets controlling tumor proliferation, survival, migration, invasion, and metastasization. Of note, Lf is able to promote or inhibit cell proliferation and migration depending on whether it acts upon normal or cancerous cells, respectively. Importantly, Lf administration is highly tolerated and does not present significant adverse effects. Moreover, Lf can prevent development or inhibit cancer growth by boosting adaptive immune response. Finally, Lf was recently found to be an ideal carrier for chemotherapeutics, even for the treatment of brain tumors due to its ability to cross the blood–brain barrier, thus globally appearing as a promising tool for cancer prevention and treatment, especially in combination therapies.

Cholangiocarcinoma (CCA) represents the second most common primary hepatic malignancy and originates from the neoplastic transformation of the biliary cells. The intrahepatic subtype includes two morpho-molecular forms: large-duct type intrahepatic CCA (iCCA) and small-duct type iCCA. Iron is fundamental for the cellular processes, contributing in tumor development and progression. The aim of this study was to evaluate iron uptake, storage, and efflux proteins in both lipopolysaccharide-inflamed small and large cholangiocytes as well as in different iCCA subtypes. Our results show that, despite an increase in interleukin-6 production by both small and large cholangiocytes, ferroportin (Fpn) was decreased only in small cholangiocytes, whereas transferrin receptor-1 (TfR1) and ferritin (Ftn) did not show any change. Differently from in vitro models, Fpn expression was increased in malignant cholangiocytes of small-duct type iCCA in comparison to large-duct type iCCA and peritumoral tissues. TfR1, Ftn and hepcidin were enhanced, even if at different extent, in both malignant cholangiocytes in comparison to the surrounding samples. Lactoferrin was higher in large-duct type iCCA in respect to small-duct type iCCA and peritumoral tissues. These findings show a different iron handling by inflamed small and large cholangiocytes, and small and large-duct type iCCA. The difference in iron homeostasis by the iCCA subtypes may have implications for the tumor management.

SARS-CoV-2, an enveloped, single-stranded RNA virus causing COVID-19, exerts morbidity and mortality especially in elderly, obese individuals and those suffering from chronic conditions. In addition to the availability of vaccines and the limited efficacy of the first dose of vaccine against SARS-CoV-2 variants, there is an urgent requirement for the discovery and development of supplementary antiviral agents. Lactoferrin (Lf), a pleiotropic cationic glycoprotein of innate immunity, has been proposed as a safe treatment combined with other therapies in COVID-19 patients. Here, we present a small retrospective study on asymptomatic, paucisymptomatic, and moderate symptomatic COVID-19 Lf-treated versus Lf-untreated patients. The time required to achieve SARS-CoV-2 RNA negativization in Lf-treated patients (n = 82) was significantly lower (p &lt, 0.001) compared to that observed in Lf-untreated ones (n = 39) (15 versus 24 days). A link among reduction in symptoms, age, and Lf treatment was found. The Lf antiviral activity could be explained through the interaction with SARS-CoV-2 spike, the binding with heparan sulfate proteoglycans of cells, and the anti-inflammatory activity associated with the restoration of iron homeostasis disorders, which favor viral infection/replication. Lf could be an important supplementary treatment in counteracting SARS-CoV-2 infection, as it is also safe and well-tolerated by all treated patients.

Lactoferrin (Lf), an iron-chelating glycoprotein of innate immunity, produced by exocrine glands and neutrophils in infection/inflammation sites, is one of the most abundant defence molecules in airway secretions. Lf, a pleiotropic molecule, exhibits antibacterial and anti-inflammatory functions. These properties may play a relevant role in airway infections characterized by exaggerated inflammatory response, as in Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) subjects. To verify the Lf role in Pseudomonas aeruginosa lung infection, we evaluated the efficacy of aerosolized bovine Lf (bLf) in mouse models of P. aeruginosa acute and chronic lung infections. C57BL/6NCrl mice were challenged with 106 CFUs of P. aeruginosa PAO1 (acute infection) or MDR-RP73 strain (chronic infection) by intra-tracheal administration. In both acute and chronic infections, aerosolized bLf resulted in nonsignificant reduction of bacterial load but significant decrease of the neutrophil recruitment and pro-inflammatory cytokine levels. Moreover, in chronic infection the bLf-treated mice recovered body weight faster and to a higher extent than the control mice. These findings add new insights into the benefits of bLf as a mediator of general health and its potential therapeutic applications.

To enumerate bacteria adherent to medical devices, Vortex-Sonication-Vortex Method (VSVM) and BioTimer Assay (BTA) have been applied. VSVM counts detached microorganisms whereas BTA enumerates adherent microorganisms through microbial metabolism. However, the limitation of VSVM consists in incomplete detachment of adherent microorganisms while BTA is unable to identify microbial genera and species. Herein, the combined use of VSVM and BTA for the diagnosis and enumeration of adherent microorganisms causing implant-associated-infections (IAIs) is reported. Over 2016–2018, 46 patients with IAIs were enrolled and their 82 explanted devices were submitted firstly to VSVM and then to BTA. VSVM plus BTA detected microorganisms in 39/46 patients (84.7%) compared with 32/46 (69.5%) and 31/46 (67.3%) by VSVM and BTA alone, respectively. Likely, combined methods led to microorganism detection in 54/82 devices (65.9%) compared with each method alone [43/82 (52.4%), 44/82 (53.6%) for VSVM and BTA, respectively]. The combination of both methods (concordance 75.6%) raised the sensitivity of microbial analysis in IAIs compared with either VSVM or BTA alone, thus representing a simple and accurate way for the identification and enumeration of microorganisms adherent on devices. Moreover, BTA reagent applied in a new apparatus allowed also the enumeration of the microorganisms adherent on different segments of cardiac electrodes, thus contributing to define IAIs pathogenesis.

Cystic fibrosis (CF) is a genetic disorder affecting several organs including airways. Bacterial infection, inflammation and iron dysbalance play a major role in the chronicity and severity of the lung pathology. The aim of this study was to investigate the effect of lactoferrin (Lf), a multifunctional iron-chelating glycoprotein of innate immunity, in a CF murine model of Pseudomonas aeruginosa chronic lung infection. To induce chronic lung infection, C57BL/6 mice, either cystic fibrosis transmembrane conductance regulator (CFTR)-deficient (Cftrtm1UNCTgN(FABPCFTR)#Jaw) or wild-type (WT), were intra-tracheally inoculated with multidrug-resistant MDR-RP73 P. aeruginosa embedded in agar beads. Treatments with aerosolized bovine Lf (bLf) or saline were started five minutes after infection and repeated daily for six days. Our results demonstrated that aerosolized bLf was effective in significantly reducing both pulmonary bacterial load and infiltrated leukocytes in infected CF mice. Furthermore, for the first time, we showed that bLf reduced pulmonary iron overload, in both WT and CF mice. In particular, at molecular level, a significant decrease of both the iron exporter ferroportin and iron storage ferritin, as well as luminal iron content was observed. Overall, bLf acts as a potent multi-targeting agent able to break the vicious cycle induced by P. aeruginosa, inflammation and iron dysbalance, thus mitigating the severity of CF-related pathology and sequelae.

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.

The liver is a frontline immune site specifically designed to check and detect potential pathogens from the bloodstream to maintain a general state of immune hyporesponsiveness. One of the main functions of the liver is the regulation of iron homeostasis. The liver detects changes in systemic iron requirements and can regulate its concentration. Pathological states lead to the dysregulation of iron homeostasis which, in turn, can promote infectious and inflammatory processes. In this context, hepatic viruses deviate hepatocytes’ iron metabolism in order to better replicate. Indeed, some viruses are able to alter the expression of iron-related proteins or exploit host receptors to enter inside host cells. Lactoferrin (Lf), a multifunctional iron-binding glycoprotein belonging to the innate immunity, is endowed with potent antiviral activity, mainly related to its ability to block viral entry into host cells by interacting with viral and/or cell surface receptors. Moreover, Lf can act as an iron scavenger by both direct iron-chelation or the modulation of the main iron-related proteins. In this review, the complex interplay between viral hepatitis, iron homeostasis, and inflammation as well as the role of Lf are outlined.

Glioblastoma, the most lethal form of brain cancer, is characterized by fast growth, migration and invasion of the surrounding parenchyma, with epithelial-to-mesenchymal transition (EMT)-like process being mostly responsible for tumour spreading and dissemination. A number of actors, including cadherins, vimentin, transcriptional factors such as SNAIL, play critical roles in the EMT process. The interleukin (IL)-6/STAT3 axis has been related to enhanced glioblastoma's migration and invasion abilities as well. Here, we present data on the differential effects of native and iron-saturated bovine lactoferrin (bLf), an iron-chelating glycoprotein of the innate immune response, in inhibiting migration in a human glioblastoma cell line. Through a wound healing assay, we found that bLf was able to partially or completely hinder cell migration, depending on its iron saturation rate. At a molecular level, bLf down-regulated both SNAIL and vimentin expression, while inducing a notable increase in cadherins' levels and inhibiting IL-6/STAT3 axis. Again, these effects positively correlated to bLf iron-saturation state, with the Holo-form resulting more efficient than the native one. Overall, our data suggest that bLf could represent a novel and efficient adjuvant treatment for glioblastoma's standard therapeutic approaches.

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 β-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 β-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.

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.

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.

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.

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.

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.

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.

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.

Human lactoferrin (hLf), an 80 kDa multifunctional iron binding cationic glycoprotein, is constitutively secreted by exocrine glands and by neutrophils during inflammation. HLf is recognized as a key element in the host immune defense system. The in vitro and in vivo experiments are carried out with bovine Lf (bLf), which shares high sequence homology and identical functions with hLf including anti-inflammatory activity. Here, in ‘pure’ M1 human macrophages, obtained by stimulation with a mixture of 10 pg/ml LPS and 20 ng/ml IFN-γ, as well as in a more heterogeneous macrophage population, challenged with high-dose of LPS (1 µg/ml), the effect of bLf on the expression of the main proteins involved in iron and inflammatory homeostasis, namely ferroportin, membrane-bound ceruloplasmin, cytosolic ferritin and transferrin receptor 1 and cytokines has been investigated. The increase of IL-6 and IL-1β cytokines, following the inflammatory treatments, is associated with both up-regulation of cytosolic ferritin and down-regulation of ferroportin, membrane-bound ceruloplasmin and transferrin receptor 1. All these changes take part into intracellular iron overload, a very unsafe condition leading in vivo to higher host susceptibility to infections as well as iron deficiency in the blood and anemia of inflammation. It is therefore of utmost importance to counteract the persistence of the inflammatory status to rebalance iron levels between tissues/secretions and blood. Moreover, levels of the anti-inflammatory cytokine IL-10 were increased in cells treated with high doses of LPS. Conversely, IL-10 decreased when the LPS/IFN-γ mix was used, suggesting that only the inflammation triggered by LPS high doses can switch on an anti-inflammatory response in our macrophagic model. Here, we demonstrate that bLf, when included in the culture medium, significantly reduced IL-6 and IL-1β production and efficiently prevented the changes of ferroportin, membrane-bound ceruloplasmin, cytosolic ferritin and transferrin receptor 1 in ‘pure’ M1 macrophages, as well as in the more heterogeneous macrophage population. In addition, the decrease of IL-10 induced by the LPS/IFN-γ mix was counteracted by bovine lactoferrin. Several drugs capable of modulating macrophagic phenotypes are emerging as attractive molecules for treating inflammation and in this sense bovine lactoferrin is no exception.