Biomedical subjects

Francesca Berlutti

Publications and source records attributed to Francesca Berlutti.

11 recordsLinked to original sources

PubMed2006

Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strains.

Intestinal epithelial cells are able to differentially interact with commensal or pathogenic microorganisms, triggering a physiological or destructive inflammation, respectively. To mimic commensal-enteroinvasive bacteria-host cell interaction, we infected Caco-2 cells with noninvasive Escherichia coli HB101 and with recombinant invasive E. coli HB101(pRI203). Using DNA microarray mRNA profiling and ELISA assays, we studied the expression of several cytokine and cytokine-related genes in infected Caco-2 cells in the absence or presence of bovine lactoferrin (bLf). Infection of Caco-2 cells with the noninvasive strain induced a slight increase in the expression of interleukin 8 (IL-8), whereas infection with invasive E. coli HB101(pRI203) induced a significant increase in the expression of IL-8 as well as other pro-inflammatory cytokines. The addition of bLf, in native- or holo-form, did not influence expression of cytokine genes by uninfected Caco-2 cells, but it decreased expression of IL-8 by cells infected with E.coli HB101. Moreover, except for IL-8, bLfs dramatically downregulated pro-inflammatory cytokines upexpressed by Caco-2 cells infected with the invasive strain. Although IL-8 was decreased by bLfs, it remained upregulated, suggesting that it could be a signal of persistence of intracellular bacteria. The bLf ability to reduce expression of some pro-inflammatory cytokines, which appears independent of its iron saturation, might represent an important natural mechanism in regulating epithelial cell responses to pathogenic bacteria and in limiting cell damage and the spread of infections.

Animals↗

PubMed2006

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.

Administration, Oral↗

PubMed2005

Ala160 and His116 residues are involved in activity and specificity of apyrase, an ATP-hydrolysing enzyme produced by enteroinvasive Escherichia coli.

The virulence plasmid-carried apy (phoN2) gene of Shigella and related enteroinvasive Escherichia coli (EIEC) encodes apyrase, an ATP-diphosphohydrolase belonging to class A of the non-specific acid phosphatases (A-NSAPs). Apyrase and A-NSAPs share three domains of conserved amino acids (domains D1-D3) containing residues forming the putative active site of apyrase. In spite of their similarity, apyrase and A-NSAPs show different substrate specificity, apyrase being able to hydrolyse nucleotide tri- and diphosphates, but not monophosphates, as well as p-nitrophenyl phosphate (pNPP), while A-NSAPs are also active towards monophosphates and pNPP. In this paper, to get further insights into the structure-function relationship of apyrase, a random and site-directed mutagenesis of the apy gene of EIEC strain HN280 was conducted. Results indicate that amino acids located within the D2 and D3 conserved domains (Ser157 and Arg192, respectively) as well as residues located in the N-terminal (Ser97) and C-terminal (Glu233) domains are required for enzyme activity. Surprisingly, Ala160, located near the D2 domain and considered to be important for enzyme specificity, is required for enzyme activity, as its substitution with Thr led to the inactivation of enzyme activity. Furthermore, residue His116 is involved in apyrase specificity, since the H116L apyrase mutant shows substrate specificity resembling that of A-NSAPs.

Adenosine Triphosphate↗

PubMed2004

Effect on bovine lactoferrin on the activation of the enteroinvasive bacterial type III secretion system.

Shigella and enteroinvasive Escherichia coli (EIEC) strains secrete virulence proteins by a complex machinery called the type III secretion (TTS) apparatus. Secretion of virulence proteins is a tightly-regulated phenomenon such that the TTS system is weakly active when bacteria are grown in common laboratory media. Activation of the TTS system is triggered by contact with eukaryotic cells, or can be artificially stimulated by the addition of Congo red dye to the growth medium. Exploiting the ability of bovine lactoferrin (bLf) to bind iron we have found that the TTS of EIEC strain HN280 seems to be activated in conditions of low-iron availability, obtained by incubation of bacteria with bLf enclosed within a dialysis bag. Activation of secretion was assessed by measuring the release of IpaB and C, chosen as reporters of secreted virulence proteins. The contribution of small bLf-derived components, diffusing across the dialysis membrane, in the release of Ipa proteins has also been determined. Activation of secretion was not due to bLf-induced damage of the HN280 outer membrane and was not associated with increased transcription of the mxi operon. Thus, low-iron availability might be an environmental signal perceived by enteroinvasive micro-organisms in order to modulate secretion of virulence proteins.

Animals↗

PubMed2004

Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans.

Streptococcus mutans, a gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries. Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear. The data reported in this study indicates that iron-deprived saliva (Fe3+ < 0.1 microM) increases S. mutans aggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+ from 0.1 to 1 microM), while iron-loaded saliva (Fe3+ > 1 microM) inhibits both phenomena. Our findings are consistent with the hypothesis that S. mutans aggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 microg/ml) in the apo- or iron-saturated form. Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases. On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S. mutans. Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S. mutans counteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity. In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S. mutans free, aggregated or biofilm on abiotic surfaces. Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S. mutans pathogenicity.

Animals↗

PubMed2004

Lactoferrin functions: current status and perspectives.

Lactoferrin, an iron-binding glycoprotein synthesized by neutrophils and exocrine glands, plays an important role in human innate defense mechanisms against bacteria, fungi, and viruses. First, a bacteriostatic activity of lactoferrin, depending on iron withholding to bacteria, and successively a bactericidal iron-independent effect, related to its binding on bacterial surfaces, was recognized. Many other functions have been ascribed to this cationic protein, including the inhibiting action toward bacterial adhesion and invasion of target host cells. Recent research also reported the lactoferrin influence on bacterial aggregation and biofilm development of Pseudomonas aeruginosa and Streptococcus mutans. The different lactoferrin functions can be justified by different physicochemical properties of the molecule, which include the iron-binding capability, the binding to anionic cell surfaces and molecules, and serine protease activity.

Anti-Bacterial Agents↗

PubMed2003

Quantitative evaluation of bacteria adherent to polyelectrolyte HEMA-based hydrogels.

The use of adhesive poly(HEMA)-based hydrogels is standard practice in dental restorative procedures. Microorganisms, which potentially can cause oral pathologies, may colonize these polymers. In the present work, bacterial adhesion to polymers prepared with 2-hydroxyethyl methacrylate (HEMA) and to different molar ratios of 2-acrylamido-2-methylpropane-sulfonic acid (AMPS) and/or to 2-methacryloyloxyethyl-tri-methyl-ammonium chloride (METAC) co-monomers were tested. A colorimetric assay system that utilizes the Microbo revelation medium (Microbo srl, Rome, Italy) for microbial counts is shown to be capable of counting the number of adherent bacterial cells without removing them from polymer surfaces. In conditions that mimic those present in the oral cavity, similar bacterial adhesion percentages on the same polymer were observed with the different bacteria belonging to both gram-positive and gram-negative genera, such as Streptococcus sobrinus and Streptococcus oralis (resident microorganisms in the oral cavity) and Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa (transient microorganisms in the oral cavity). It is determined that the physico-chemical characteristics of poly(HEMA)-based hydrogels are the major factors promoting bacterial adhesion, which increased with increasing water content in the swollen polymers, reaching maximal values on the cationic polymers.

Bacteria↗

PubMed2003

The molecular class C acid phosphatase of Chryseobacterium meningosepticum (OlpA) is a broad-spectrum nucleotidase with preferential activity on 5'-nucleotides.

The olpA gene of Chryseobacterium meningosepticum, encoding a molecular class C phosphatase, was cloned and expressed in Escherichia coli. The gene encodes a 29-kDa polypeptide containing an amino-terminal signal peptide typical of bacterial membrane lipoproteins. Expression in E. coli results in a functional product that mostly partitions in the outer membrane. A secreted soluble OlpA derivative (sOlpA) lacking the N-terminal cysteine residue for lipid anchoring was produced in E. coli and purified by means of two steps of ion exchange chromatography. Analysis of the kinetic parameters of sOlpA with several organic phosphoesters revealed that the enzyme was able to efficiently hydrolyze nucleotide monophosphates, with a strong preference for 5'-nucleotides and for 3'-AMP. The enzyme was also able to hydrolyze sugar phosphates and beta-glycerol phosphate, although with a lower efficiency, whereas it was apparently inactive against nucleotide di- and triphosphates, diesters, and phytate. OlpA, therefore, can be considered a broad-spectrum nucleotidase with preference for 5'-nucleotides. Its functional behaviour exhibits differences from that of the Haemophilus influenzae OMP P4 lipoprotein, revealing functional heterogeneity among phosphatases of molecular class C.

Acid Phosphatase↗

PubMed2003

Efficacy of anti-retraction devices in preventing bacterial contamination of dental unit water lines.

OBJECTIVE: In this study we examine the efficacy of anti-retraction devices in preventing microbial contamination of dental unit water lines (DUWLs). METHODS: The study was performed on 54 randomly selected DUs in use at private and public institutions for over six months. The selected DUs were all currently commercially available. To evaluate the efficiency of anti-retraction devices, two different methods were employed, mechanical and microbiological. The ADA/ANSI specification #47 (corresponding to a water retraction of less than 40.3 microl) was used for the evaluation of the retraction determined using the mechanical method; the presence/absence of test microorganisms in DUWLs upstream the turbine after the test was used for evaluating the microbiological assay. RESULTS: Both evaluation methods indicated correct amounts of retraction and prevention of DUWL contamination occurred in only two cases (3.7%). Correct retraction was noted in six other dental units (DUs), but their DUWLs became contaminated during testing. Six other produced the opposite results-improper retraction, yet no contamination. The remaining 40 DUs (74.0%) failed both evaluations. CONCLUSION: The results showed that the overwhelming majority of the anti-retraction devices did not prevent retraction when the turbine stopped running, leading to a contamination of the water lines, and to a consequent possible cross-contamination of the patients.

Bacillus↗

PubMed2002

Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression.

In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript.

Apyrase↗

PubMed2001

The Chryseobacterium meningosepticum PafA enzyme: prototype of a new enzyme family of prokaryotic phosphate-irrepressible alkaline phosphatases?

Chryseobacterium meningosepticum is an aerobic Gram-negative rod widely distributed in natural environments. Unlike many bacteria, it produces a phosphate-irrepressible periplasmic alkaline phosphatase (AP). This work describes cloning of the gene encoding that enzyme from C. meningosepticum CCUG 4310 (NCTC 10585), and preliminary characterization of its product. The gene, named pafA, encodes a protein (PafA) of 546 amino acids with a calculated molecular mass of the mature peptide of 58682 Da. PafA exhibits high sequence identity with the PhoV AP of Synechococcus PCC 7942 (49.9% identity) and with the Cda Ca(2+)-dependent ATPase of Myroides odoratus (51.9% identity), while being more distantly related to the PhoD AP of Zymomonas mobilis (22.1% identity) and to the PhoA AP of Escherichia coli (14.0% identity). PafA was partially purified; it exhibits optimal activity at pH 8.5 and is active towards a broad spectrum of substrates including both phosphomonoesters and ATP, with preferential activity for the latter compound. The present findings allow definition of a new family of APs including 60 kDa, periplasmic enzymes whose expression is not influenced by freely available P(i) in the medium. Moreover, PafA can be considered an evolutionary intermediate between Ca(2+)-ATPase of M. odoratus and the APs PhoV of Synechococcus PCC 7942 and PhoD of Z. mobilis.

Alkaline Phosphatase↗