The effect of the electromagnetic field on metabolic-active bacterial biofilm experimentallyinduced on titanium dental implants.

Microbial biofilm is of paramount importance in the development of mucositis or peri-implantitis in patients with dental implants. This study was designed to investigate whether an electromagnetic field at high frequency waves directly applied on 33 titanium implants could remove experimentally-induced Enterococcus faecalis bacterial biofilm. A specially designed device (X-IMPLANT) was used to generate the electromagnetic field, with output power of 8 W, supply frequency (action/pause) 3/2s, and an output frequency of 625±5% kHz in plastic devices containing the biofilm-covered implants immersed in sterile saline. The bacterial biofilm on both treated and untreated control implants was quantitatively measured by phenol red-based Bio-Timer-Assay reagent. The kinetic analysis of the curves showed that the electrical treatment generated by the X-IMPLANT device completely removed the bacterial biofilm after 30 minutes of treatment (p<0.01). Elimination of the biofilm was also confirmed by chromatic observation in the macro-method. Our data seem to indicate that the procedure could be considered for clinical application in peri-implantitis to counteract bacterial biofilm on dental implants.

Disinfection of Root Canals with Laser-Activated Irrigation, Photoactivated Disinfection, and Combined Laser Techniques: An Ex Vivo Preliminary Study.

Objective: This study aimed to evaluate the efficacy of laser-activated irrigation using photon-induced photoacoustic streaming (PIPS®) and photoactivated disinfection (PAD) techniques and their combination to improve penetration and activation of toluidine blue in the endodontic space of teeth experimentally infected with Enterococcus faecalis. Materials and methods: Twenty-seven extracted single-root teeth were instrumented, sterilized, and infected with E. faecalis and divided into seven groups of three teeth each: Group A [sodium hypochlorite (NaClO) 5% hand irrigation], Group B [NaClO 5% hand irrigation+ethylenediaminetetraacetic acid (EDTA)+NaClO 5% activated by PIPS], Group C (EDTA+NaClO 5% activated by PIPS), Group D (toluidine blue activated by PAD), Group E (toluidine blue activated by PIPS and PAD), Group F (NaClO 5% hand irrigation+toluidine blue activated by PAD), and Group G (NaClO 5% hand irrigation+toluidine blue activated by PIPS and PAD). Finally, positive and negative group controls were prepared. The presence of biofilms after the treatments was assessed by the BioTimer assay. PIPS was performed with an Er:YAG laser (2940 nm, LightWalker, Fotona® d.o.o., Slovenia) at 20 mJ, 15 Hz, 0.3 W, and 50-µs pulse duration. PAD was performed with a 635 nm diode laser (Smart M, Lasotronix®, Poland) at 400 mW in continuous wave (CW). Results: When NaClO was used, significant decontamination (p ≤ 0.05) was obtained in all experimental groups with respect to the positive control, other than Group G. Irrigation with EDTA+NaClO activated by PIPS produced a higher level of decontamination than Group A (p ≤ 0.05). Significant results in reducing biofilm load compared with the control and Group A were observed when NaClO was coupled with toluidine blue activated by PAD (p ≤ 0.05). Conclusions: Disinfection of root canals can be obtained using a combination of different irrigants, photosensitizers, and activation protocols. EDTA+NaClO using the PIPS protocol and toluidine blue activated by PAD (both preceded by NaClO irrigation) can be considered effective tools. The possibility of replacing NaClO with toluidine blue, whatever the method of activation, should be further investigated.

Microbiological and FE-SEM Assessment of d-PTFE Membrane Exposed to Oral Environment after Alveolar Socket Preservation Managed with Granular nc-HA.

AIM: The aim of this study was to analyze, by the aid of microbiological analysis and the field emission scanning electron microscopical (FE-SEM) analysis, the role of high-density polytetrafluoroethylene (d-PTFE) membranes in avoiding the microbial colonization of a nanocrystalline hydroxyapatite (nc-HA) bone graft and the involvement of this colonization in the healing process. MATERIALS AND METHODS: Six patients underwent extraction of unrecoverable teeth, and a socket preservation technique was carried out with nc-HA synthetic bone graft and then covered with a d-PTFE membrane. After 28 days from surgery, FE-SEM analysis and BioTimer assay technique to assess the microbiological count of streptococci species were carried out. Data were collected and analyzed by the Student's t test (confidence interval: 95%). RESULTS: The mean amount of bacteria measured on the upper side of the membrane was 6.52 ± 0.50 CFU, while on the lower side, it was 6.59 ± 0.40 CFU. Significant differences were not found between the two sides of the membrane or between the different sectors (p > 0.05). The FE-SEM analysis revealed structured biofilms on both sides of the membrane: species of cocci, bacilli, and fusobacteria were recognizable in occasional settled vegetations. CONCLUSION: Since the amount of bacteria found was low, the improved impermeability of the d-PTFE membrane permitted the healing process to proceed uneventful and without signs of infection or inflammation. CLINICAL RELEVANCE: The infection of the graft site could lead to a failure of the socket preservation technique which could delay or compromise the rehabilitation following procedures. The use of d-PTFE can improve the bone regeneration thanks to its antimicrobial properties.

Cryo-nanoimaging of Single Human Macrophage Cells: 3D Structural and Chemical Quantification.

X-ray microscopy is increasingly used in biology, but in most cases only in a qualitative way. We present here a 3D correlative cryo X-ray microscopy approach suited for the quantification of molar concentrations and structure in native samples at nanometer scale. The multimodal approach combines X-ray fluorescence and X-ray holographic nanotomography on "thick" frozen-hydrated cells. The quantitativeness of the X-ray fluorescence reconstruction is improved by estimating the self-attenuation from the 3D holography reconstruction. Applied to complex macrophage cells, we extract the quantification of major and minor elements heavier than phosphorus, as well as the density, in the different organelles. The intracellular landscape shows remarkable elemental differences. This novel analytical microscopy approach will be of particular interest to investigate complex biological and chemical systems in their native environment.

Bovine Lactoferrin Pre-Treatment Induces Intracellular Killing of AIEC LF82 and Reduces Bacteria-Induced DNA Damage in Differentiated Human Enterocytes.

LF82, a prototype of adherent-invasive E. coli (AIEC), is able to adhere to, invade, survive and replicate into intestinal epithelial cells. LF82 is able to enhance either its adhesion and invasion by up-regulating carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM-6), the main cell surface molecule for bacterial adhesion, and its intracellular survival by inducing host DNA damage, thus blocking the cellular cycle. Lactoferrin (Lf) is a multifunctional cationic glycoprotein of natural immunity, exerting an anti-invasive activity against LF82 when added to Caco-2 cells at the moment of infection. Here, the infection of 12 h Lf pre-treated Caco-2 cells was carried out at a time of 0 or 3 or 10 h after Lf removal from culture medium. The effect of Lf pre-treatment on LF82 invasiveness, survival, cell DNA damage, CEACAM-6 expression, apoptosis induction, as well as on Lf subcellular localization, has been evaluated. Lf, even if removed from culture medium, reduced LF82 invasion and survival as well as bacteria-induced DNA damage in Caco-2 cells independently from induction of apoptosis, modulation of CEACAM-6 expression and Lf sub-cellular localization. At our knowledge, this is the first study showing that the sole Lf pre-treatment can activate protective intracellular pathways, reducing LF82 invasiveness, intracellular survival and cell-DNA damages.

Aerosolized Bovine Lactoferrin Counteracts Infection, Inflammation and Iron Dysbalance in A Cystic Fibrosis Mouse Model of Pseudomonas aeruginosa Chronic Lung Infection.

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.

BioTimer assay as complementary method to vortex-sonication-vortex technique for the microbiological diagnosis of implant associated infections.

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.

Biotimer assay: A reliable and rapid method for the evaluation of central venous catheter microbial colonization.

Adherent bacteria and biofilm frequently colonize central venous catheters (CVCs). CVC colonization is correlated to infections and particularly to bloodstream ones. The classical microbiological methods to determine of CVC colonization are not fully reliable and are time-consuming. BioTimer Assay (BTA) is a biological method already used to count bacteria adherent to abiotic surfaces and biofilm without sample manipulation. BTA employs specific reagents whose color changed according to bacterial metabolism. BTA is based on the principle that a metabolic reaction will be faster when more bacteria are present in the sample. Therefore, the time required for color changes of BTA reagents determines the number of bacteria present in the sample through a correlation line. Here, for the first time, we applied BTA and a specifically developed laboratory procedure to evaluate CVC colonization in comparison with the routine microbiological method (RMM). 125 CVCs removed from patients for suspected catheter-related bloodstream infection (CRBSI) or at hospital discharge were examined. BTA was reliable in assessing sterility and CVC colonization (100% agreement with RMM) and in recognizing the presence of fermenting or non-fermenting bacteria (97.1% agreement with RMM) shortening the analytical time by between 2- and 3-fold. Moreover, the reliability of BTA as early alert of CRBSI was evaluated. The sensitivity, specificity, positive, and negative predictive values for BTA as an early alert of CRBSI were 100, 40.0, 88.8 and 100%, respectively. In conclusion, BTA and the related laboratory procedure should be incorporated into routine microbiological methods since it can be considered a reliable tool to evaluate CVC colonization in a very short time and a rapid alert for CRBSIs.

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-1ß 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-1ß 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.

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.

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.

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.

Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA, the soluble component of the ZnuABC transporter.

BACKGROUND: Under conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake. METHODS: Antibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15Å resolution. RESULTS: Two di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4-chlorophenyl)methyl]-4-phenyl-1H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound. CONCLUSIONS: Two molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)-binding drugs against Salmonella. GENERAL SIGNIFICANCE: The data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria.

Effectiveness of KTP laser versus 980 nm diode laser to kill Enterococcus faecalis in biofilms developed in experimentally infected root canals.

This study aimed to evaluate the antibacterial action of KTP (potassium-titanyl-phosphate) laser irradiations (compared with 980 nm diode laser), associated with conventional endodontic procedures, on Enterococcus faecalis biofilms. Fifty-six dental roots with single canals were prepared with Ni-Ti rotary instruments, autoclaved, inoculated with an E. faecalis suspension and incubated for 72 h. They were randomly allocated to control and treatment groups. Laser parameters were as follows: power 2.5 W, Ton 35 ms, Toff 50 ms (KTP laser); power 2.5 W, Ton 30 ms, Toff 30 ms (980 nm diode laser). To evaluate the residual bacterial load, BioTimer Assay was employed. The chemo-mechanical treatment together with laser irradiations (KTP and 980 nm diode lasers) achieved a considerable reduction of bacterial load (higher than 96% and 93%, respectively). Regarding both laser systems, comparisons with conventional endodontic procedures (mortality rate of about 67%) revealed statistically highly significant differences (P ≤ 0.01). This study confirms that laser systems can provide an additional aid in endodontic disinfection.

Influence of sub-inhibitory antibiotics and flow condition on Staphylococcus aureus ATCC 6538 biofilm development and biofilm growth rate: BioTimer assay as a study model.

Staphylococcus biofilm exhibits high antibiotic resistance and therapeutic doses of antibiotics are often sub-inhibitory. Whereas data are available on the effect of sub-inhibitory antibiotics on matrix formation, little is known on their influence on biofilm population. Here, using BioTimer Assay (BTA), a method developed to quantify biofilm population, the influence of sub-inhibitory gentamicin, ofloxacin and azithromycin on Staphylococcus aureus ATCC 6538 biofilm population in flow with respect to static condition was assessed. Antibiotics and flow condition increased biofilm population even if at different extent, depending on the antibiotic molecule. The greatest bacterial population was found in biofilm developed under flow condition in the presence of azithromycin. A significant increase in biofilm matrix was recorded for biofilm developed in the presence of antibiotics in flow with respect to static condition. The growth rates (GRs) of 24-h biofilm developed under the influence of antibiotics and flow condition were also evaluated using BTA and a specific mathematical model. Antibiotics and flow condition affected the GRs of 24-h biofilm even if at different extent. The lowest GR value was recorded for biofilm developed under flow condition in the presence of ofloxacin. Although further studies are needed, our data indicate that antibiotics and flow condition influenced biofilm development by increasing both bacterial population and matrix formation and affected the GRs of the developed biofilm. To the best of our knowledge, BTA is unique in allowing the calculation of the GRs of biofilm and it may be considered to be a useful study model to evaluate the activity of antibiofilm molecules.

Lactoferrin prevents LPS-induced decrease of the iron exporter ferroportin in human monocytes/macrophages.

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.

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.

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.

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.

LF immunomodulatory strategies: mastering bacterial endotoxin.

Lactoferrin (LF), an iron-binding glycoprotein expressed in most biological fluids, represents a major component of mammalian innate immune system. The multiple activities of LF rely not only on its capacity to bind iron but also to interact with molecular and cellular components of both the host and pathogens. LF can bind and sequester lipopolysaccharide thus preventing proinflammatory pathway activation, sepsis, and tissue damage. However, the interplay between LF and lipopolysaccharide is complex and may lead to different outcomes including both the suppression of inflammatory response and immune activation. Understanding the molecular basis and the functional consequences of this complex interaction is critically relevant in the development of LF-based therapeutic interventions in humans.