Adhesion of human gingival fibroblasts/Streptococcus mitis co-culture on the nanocomposite system Chitlac-nAg

10noComposite materials are increasingly used as dental restoration. In the field of biomaterials, infections remain the main reason of dental devices failure. Silver, in the form of nanoparticles (AgNPs), ions and salt, well known for its antimicrobial properties, is used in several medical applications in order to avoid bacterial infection. To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized AgNPs with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a coculture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release raised up to 20 %. Moreover the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces with the co-culture system even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKCα translocated into nuclei. These data confirm that Chitlac-nAg have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.openopenCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, SilviaCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, Silvi

Pyridoxal 5′-Phosphate-Dependent Enzymes at the Crossroads of Host–Microbe Tryptophan Metabolism

The chemical processes taking place in humans intersects the myriad of metabolic pathways occurring in commensal microorganisms that colonize the body to generate a complex biochemical network that regulates multiple aspects of human life. The role of tryptophan (Trp) metabolism at the intersection between the host and microbes is increasingly being recognized, and multiple pathways of Trp utilization in either direction have been identified with the production of a wide range of bioactive products. It comes that a dysregulation of Trp metabolism in either the host or the microbes may unbalance the production of metabolites with potential pathological consequences. The ability to redirect the Trp flux to restore a homeostatic production of Trp metabolites may represent a valid therapeutic strategy for a variety of pathological conditions, but identifying metabolic checkpoints that could be exploited to manipulate the Trp metabolic network is still an unmet need. In this review, we put forward the hypothesis that pyridoxal 5\u27-phosphate (PLP)-dependent enzymes, which regulate multiple pathways of Trp metabolism in both the host and in microbes, might represent critical nodes and that modulating the levels of vitamin B6, from which PLP is derived, might represent a metabolic checkpoint to re-orienteer Trp flux for therapeutic purposes

Effect of Chitlac-nAg on Streptococcus mitis internalization into human gingival fibroblasts

The surfaces of the oral cavity are always exposed to a broad variety of microor- ganisms able to form biofilms (Filoche et al, 2010) characterized by microbial com- munities that are organized as a network of cell-to-cell interactions. Streptococci are the predominant bacterial population of the oral environment and S.mitis in particular is the first colonizer of the oral biofilm (Di Giulio et al, 2013). Silver-based medical products have been proven to be effective in retarding and preventing bacterial growth. In order to prevent silver nanoparticles aggregation, a lactose-modified chitosan has been set up and resulted effective in stabilizing colloidal solution of nanoparticles (Chitlac-nAg) (Travan et al, 2009). Since many bacteria are able to internalize into eukaryotic cells, in our study we have investigated both the intracellular signaling governing S. mitis internalization into HGFs and the biological effect of ChitlacnAg on eukaryotic and prokaryotic cells in a co-culture model system. The internalization of S. mitis into HGFs is due to F-actin cytoskeleton reorganization and reduced expression within the cell. Immunofluorescence shows actin polymerization at invasion sites along with vinculin increased expression and spot organization. Vinculin is an adaptor protein that regulates the adhesion of integrin receptors to actin cytoskeleton. In presence of S. mitis an increment of integrin β1 and FAK expression, responsible for the entrance of the microorganism in HGFs is consistent, as revealed by electron microscopy analysis. This adhesion and uptake proteins profile is the same in the presence of saliva as well as bacteria uptake. When Chitlac-nAg is administred to cell culture the expression of all four proteins decreases and Ag nano- particles are recognized within the cells. Further, in presence of Ag nanoparticles the low amount of FAK is almost localized at nuclear level. In presence of Ag and S.mitis, the expression of all four proteins is increased, with respect to control, and F-actin cytoskeleton rearranged, while a raised number of bacteria is shown. This effect is mit- igated by the presence of saliva in cell culture, which probably prevents bacteria entry into the cell. These results let us hypothesize that Chitlac-nAg, developing its bacteri- cidal action could represent a good component of tooth paste and mouthwash

Sexuality education in Italy 2016-2020: a national survey investigating coverage, content and evaluation of school-based educational activities

Comprehensive sexuality education is an important means of promoting sexual well-being amongst young people and is key to preventing sexually transmitted infections (STIs). However, sexuality education is not currently included in the formal curriculum in Italian schools. The aim of this study was to develop an inventory of schoolbased sexuality education (SBSE) activities carried out by external providers and implemented in Italy from 2016 to 2020. A desk review and survey were carried out. In the desk review online documents on STI prevention were analysed. The survey investigated the providers, objectives, content and methods used to implement SBSE activities in secondary schools. Findings revealed a highly heterogeneous situation in terms of geographical coverage, service providers, objectives and evaluation. Some SBSE activities were classified as adopting a comprehensive approach to sexuality education, while the majority focused on STI prevention, and many were single-session activities. Although most activities were said to have been evaluated no results were available. The data showed that SBSE is not systematically and equally delivered across Italy. Action is needed to provide young people with evidence-based, age-appropriate and accurate education about sexual and reproductive health and wellbeing

Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface

Potential Antibacterial Activity of Carvacrol-Loaded Poly(DL-lactide-co-glycolide) (PLGA) Nanoparticles against Microbial Biofilm

The ability to form biofilms contributes significantly to the pathogenesis of many microbial infections, including a variety of ocular diseases often associated with the biofilm formation on foreign materials. Carvacrol (Car.) is an important component of essential oils and recently has attracted much attention pursuant to its ability to promote microbial biofilm disruption. In the present study Car. has been encapsulated in poly(dl-lactide-co-glycolide (PLGA) nanocapsules in order to obtain a suitable drug delivery system that could represent a starting point for developing new therapeutic strategies against biofilm-associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier

A Triple and Quadruple Therapy with Doxycycline and Bismuth for First-Line Treatment of Helicobacter pylori Infection: A Pilot Study

Background: Tetracycline-containing triple therapy has been suggested as an alternative first-line therapy for H. pylori infection. Aim: To evaluate the effect of two dosages of doxycycline (DOX) associated with amoxicillin and esomeprazole with and without bismuth subcitrate as first-line treatment of H. pylori infection. Methods: Helicobacter pylori-positive patients underwent a 10-day therapy randomized into four groups: Group A received esomeprazole, amoxicillin, and DOX-100 mg b.i.d. (EAD-100), Group B a quadruple therapy with esomeprazole, amoxicillin, DOX-100 mg b.i.d. and bismuth subcitrate (EADB-100), Group C a triple therapy with esomeprazole, amoxicillin, and DOX-200 mg b.i.d. (EAD-200) and Group D a quadruple therapy with esomeprazole, amoxicillin, DOX-200 mg b.i.d., and bismuth subcitrate (EADB-200). Success was accessed by 13C urea breath test 2 months after the end of treatment. The number of patients to be recruited for each group had to be at least 50 subjects. Treatment success of 80% or less was considered unacceptable. Stopping rules therefore were anytime six failures had occurred. Results: In the EAD-100 group and in EAD-200 group, the recruitment was stopped at the 14th and 15th patient, respectively. Fifty-two patients entered in the EADB-100 group and 51 in the EADB-200 group. Intention to treat eradication was in EADB-100 group 46/52 (88.5%, 95% CI 76.6-95.6); in the EADB-200 group 47/51 (92.1%, 95% CI: 81.1-97.8) (n.s.). Side effects were absent. Conclusion: The adjunction of bismuth subcitrate to a triple therapy that includes esomeprazole, amoxicillin, and DOX in patients who are treated for the first time for the H. pylori infection potentiates the therapeutic effect. This regimen, however, deserves to be optimized in terms of duration and dose of DOX