Luigi Rosa

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.

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.

Perturbation methods in torsion of thin hollow Saint-Venant cylinders

In this paper we propose a perturbation method for calculating the fields appearing in Saint-Venant torsion theory in terms of a parameter (e) characterizing the thickness of the cross section (CS) of Saint-Venant cylinders (SVC). We recover all the classical formulas found by Bredt [1] (see also Vlasov, [2]) as terms of first order in e.

Numerical investigation of a particle system compared with first and second gradient continua: Deformation and fracture phenomena*

A discrete system constituted of particles interacting by means of a centroid-based law is numerically investigated. The elements of the system move in the plane, and the range of the interaction can be varied from a more local form (first-neighbours interaction) up to a generalized nth order interaction. The aim of the model is to reproduce the behaviour of deformable bodies with standard (Cauchy model) or generalized (second gradient) deformation energy density. The numerical results suggest that the considered discrete system can effectively reproduce the behaviour of first and second gradient continua. Moreover, a fracture algorithm is introduced and some comparison between first- and second-neighbour simulations are provided.

Lactoferrin efficiently counteracts the inflammation-induced changes of the iron homeostasis system in macrophages

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 (Fpn), membrane-bound ceruloplasmin (Cp), cytosolic ferritin (Ftn), 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 upregulation of cytosolic Ftn and downregulation of Fpn, membrane-bound Cp, 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 antiinflammatory 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 Fpn, membrane-bound Cp, cytosolic Ftn, 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.

Aerosolized bovine lactoferrin reduces neutrophils and pro-inflammatory cytokines in mouse models of Pseudomonas aeruginosa lung infections

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.

Nanoscale quantification of intracellular element concentration by X-ray fluorescence microscopy combined with X-ray phase contrast nanotomography

We present here a correlative X-ray microscopy approach for quantitative single cell imaging of molar concentrations. By combining the elemental content provided by X-ray fluorescence microscopy and the morphology information extracted from X-ray phase nanotomography, we determine the intracellular molarity distributions. This correlative method was demonstrated on a freeze-dried human phagocytic cell to obtain the absolute elemental concentration maps of K, P, and Fe. The cell morphology results showed a very good agreement with atomic-force microscopy measurements. This work opens the way for non-destructive single cell chemical analysis down to the sub-cellular level using exclusively synchrotron radiation techniques. It will be of high interest in the case where it is difficult to access the morphology using atomic-force microscopy, for example, on frozen-hydrated cells or tissues.

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.

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.

Role of lactoferrin and its receptors on biliary epithelium

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.