Search

Laura Brambilla,1 Valeria Frangione,2 Marisa Meloni1 1VitroScreen, in vitro Research Laboratory, Milan, Italy; 2IBSA Institut Biochimique SA, Pambio-Noranco, SwitzerlandCorrespondence: Marisa Meloni, VitroScreen, Via Mosè Bianchi, 103, Milan, 20149, Italy, Tel +39 02 89077608, Email marisa.meloni@vitroscreen.comPurpose: Medical Device Regulation (EU) 2017/745 requires the principal mode of action (MoA) to be demonstrated by experimental data. The MoA of Ialuril® Prefill (combined as HA+CS+CaCl2: sodium hyaluronate 1.6%, sodium chondroitin sulphate 2% w/v and calcium chloride 0.87%) Class III medical device, indicated for intravesical instillation to reduce urinary tract infections, has been evaluated on a 3D reconstructed human bladder epithelium (HBE).Methods: Three experimental designs; i) E. coli strain selection (DSM 103538, DSM 1103) to investigate the HA+CS+CaCl2 properties in modifying bacterial growth in liquid broth (CFU 4h and 24h) at 80%, 50% and 25% concentrations; ii) evaluation of film forming properties on HBE after 15 min exposure by quantifying caffeine permeation across the epithelium; iii) capacity to counteract E. coli adhesion and biofilm formation on colonized HBE by viable counts and ultrastructural analysis by scanning electron microscopy (SEM) using ciprofloxacin as the reference antimicrobial molecule.Results: No significant differences were observed in bacterial viability for both the E. coli strains. HA+CS+CaCl2 reduced caffeine permeation of 51.7% and 38.1% at 1h and 2h, respectively and determined a significant decrease in caffeine permeation rate at both timepoints supporting HA+CS+CaCl2 capacity to firmly adhere to the bladder epithelium creating a physical barrier on the surface. The viable counts in HBE treated tissues then infected with E. coli resulted not different from the negative control suggesting that the device did not inhibit E. coli growth. SEM images showed homogenous product distribution over the HBE surface and confirmed the capacity of HA+CS+CaCl2 to adhere to the bladder epithelium, counteracting biofilm formation.Conclusion: The results support the capacity of HA+CS+CaCl2 to counteract bacterial invasion by using a physico-mechanical mode of action: this medical device represents a valid alternative to antibiotics in the treatment of recurrent UTIs.Keywords: recurrent cystitis, urinary tract infections, E. coli UPEC, E. coli biofilm, hyaluronic acid, chondroitin sulphate, film forming properties, anti-bacterial adhesion, reconstructed 3D human bladder epithelium

Michela Figorilli,1 Mario Meloni,2 Giuseppe Lanza,3,4 Elisa Casaglia,1 Rosamaria Lecca,1 Francesca Lea Saibene,2 Patrizia Congiu,1 Monica Puligheddu1 1Sleep Disorder Research Center, Department of Medical Sciences and Public Health, University of Cagliari, Cagliari, Italy; 2IRCCS, Fondazione Don Carlo Gnocchi ONLUS, Milan, Italy; 3Department of Surgery and Medical-Surgical Specialties, University of Catania, Catania, Italy; 4Clinical Neurophysiology Research Unit, Oasi Research Institute-IRCCS, Troina, ItalyCorrespondence: Monica Puligheddu, Sleep Disorder Research Center, Department of Medical Sciences and Public Health, SS 554 Km 4500, 09042 Monserrato CA, Cagliari, Italy, 09042, Tel +07051096001, Email puligheddu@unica.itAbstract: Rapid eye movement (REM) sleep behavior disorder (RBD) is the result of the loss of physiological inhibition of muscle tone during REM sleep, characterized by dream-enacting behavior and widely recognized as a prodromal manifestation of alpha-synucleinopathies. Indeed, patients with isolated RBD (iRBD) have an extremely high estimated risk to develop a neurodegenerative disease after a long follow up. Nevertheless, in comparison with PD patients without RBD (PDnoRBD), the occurrence of RBD in the context of PD (PDRBD) seems to identify a unique, more malignant phenotype, characterized by a more severe burden of disease in terms of both motor and non-motor symptoms and increased risk for cognitive decline. However, while some medications (eg, melatonin, clonazepam, etc.) and non-pharmacological options have been found to have some therapeutic benefits on RBD there is no available treatment able to modify the disease course or, at least, slow down the neurodegenerative process underlying phenoconversion. In this scenario, the long prodromal phase may allow an early therapeutic window and, therefore, the identification of multimodal biomarkers of disease onset and progression is becoming increasingly crucial. To date, several clinical (motor, cognitive, olfactory, visual, and autonomic features) neurophysiological, neuroimaging, biological (biofluids or tissue biopsy), and genetic biomarkers have been identified and proposed, also in combination, as possible diagnostic or prognostic markers, along with a potential role for some of them as outcome measures and index of treatment response. In this review, we provide an insight into the present knowledge on both existing and future biomarkers of iRBD and highlight the difference with PDRBD and PDnoRBD, including currently available treatment options.Keywords: Parkinson’s disease, REM sleep behavior disorder, synucleinopathy, biomarkers, neurodegeneration

PurposeTo characterize the effect of hepatic vessel flow using 4-dimensional (4D) flow magnetic resonance (MR) imaging and correlate their effect on microwave ablation volumes in an in vivo non-cirrhotic porcine liver model.Materials and methodsMicrowave ablation antennas were placed under ultrasound guidance in each liver lobe of swine (n = 3 in each animal) for a total of 9 ablations. Pre- and post-ablation 4D flow MR imaging was acquired to quantify flow changes in the hepatic vasculature. Flow measurements, along with encompassed vessel size and vessel-antenna spacing, were then correlated with final ablation volume from segmented MR images.ResultsThe linear regression model demonstrated that the preablation measurement of encompassed hepatic vein size (β = -0.80 ± 0.25, 95% confidence interval [CI] -1.15 to -0.22; P = .02) was significantly correlated to final ablation zone volume. The addition of hepatic vein flow rate found via 4D flow MRI (β = -0.83 ± 0.65, 95% CI -2.50 to 0.84; P = .26), and distance from antenna to hepatic vein (β = 0.26 ± 0.26, 95% CI -0.40 to 0.92; P = .36) improved the model accuracy but not significantly so (multivariate adjusted R2 = 0.70 vs univariate (vessel size) adjusted R2 = 0.63, P = .24).ConclusionsHepatic vein size in an encompassed ablation zone was found to be significantly correlated with final ablation zone volume. Although the univariate 4D flow MR imaging-acquired measurements alone were not found to be statistically significant, its addition to hepatic vein size improved the accuracy of the ablation volume regression model. Pre-ablation 4D flow MR imaging of the liver may assist in prospectively optimizing thermal ablation treatment.

A digital mobile card-matching game called eFlip was developed to assist second-year undergraduate medical science students to learn core content and understand key associations in physiology. Our team developed customized content of core physiological associations, of increasing difficulty, for upload on a generic card-matching platform. The generic game was extended with add-ons coded to allow identification and access of student usage data for students who consented to have their game usage linked to academic performance such as final course marks and grades. Here, we describe the development of the eFlip game content, the student usage profile, and the game's impact on learning. Students were invited to use eFlip prior to and during the final examination period. Of those who used eFlip, 152 students consented to participate in a study of game use on learning performance outcomes. Within the students who consented, 74 played the game and 78 did not play the game (nonplayers). The mean course mark of the students who played the game [69.57%; 95% confidence level (CI) (67.22, 71.92)] was higher than that of the nonplayers [65.33%; 95% CI (62.67, 67.99)] (P = 0.02). Playing eFlip was also associated with reduced failure rates in students who played the game (1% failure rate) compared with the students who did not play the game (10% failure rate; P = 0.02). The number of games played by students peaked just prior to the course final examination. Overall, students who chose to play eFlip demonstrated improved grades that were associated with a higher probability of passing the physiology course.

Laura Ceriotti,1 Paolo Buratti,1 Enrico Stefano Corazziari,2 Marisa Meloni1 1In vitro Innovation Center, VitroScreen srl, Milan, Italy; 2Department of Gastroenterology, IRCCS Humanitas Research Hospital, Rozzano (Milan), ItalyCorrespondence: Laura Ceriotti, VitroScreen, in vitro Innovation Center, Via Mosè Bianchi, 103, Milan, 20149, Italy, Email laura.ceriotti@vitroscreen.comPurpose: A novel experimental design based on a human-reconstructed oesophageal epithelium (HO2E) model has been applied to quantitively assess the properties of a set of liquid formulations, Device A (Gerdoff® Protection), Device B (Esoxx® One), and Device C (Marial® gel) developed to form a temporary physical barrier on the oesophageal epithelium and modify epithelial permeability so to protect the oesophageal mucosa from refluxate components.Methods: The formulations were applied to a prewetted HO2E model for 15 min. Then, a 0.5% caffeine solution was applied, and its penetration kinetics was assessed at 1 h and 2 h in acidic environments (pH= 3.3) to mirror exposure of the oesophageal mucosa to acidic reflux in GORD patients. Caffeine permeated into the basolateral compartment (evaluated by HPLC-UV) and Lucifer yellow (LY) permeability were quantified 15 min after application of the caffeine in acidic environments.Results: At the 15 min timepoint, Device A reduced caffeine permeation by 77.2% and LY flux by 30.4% compared to the untreated control and with a faster mode of action than that of the other liquid formulations. Transepithelial caffeine flux was reduced, albeit with different timing and efficiency, by all three compounds up to the end of the 2 hour experiment. At 1 h, Device A reduced the caffeine flux by 79.2%; Device B, by 67.2%; and Device C, by 37%.Conclusion: These results confirm the ability of the medical devices tested to interact with the oesophageal epithelium and create a temporary physical protective film for up to 2 hours after their application. The results underline differences in the mechanism of action of the three medical devices, with Device A performing faster than the other formulations. The overall results support the relevance of the reconstructed mucosal model to investigate oesophageal epithelium–product interactions and precisely differentiate liquid formulation performance.Keywords: gastro-oesophageal reflux disease, film-forming properties, caffeine permeation, epithelial permeability, epithelial protection, Lucifer yellow assay

Purpose: Pilates-based programs can help pregnant women deal with the physical and psychological burden derived by major changes in their body. This study was designed to primarily test, in pregnant women, the dose-response and health effects of both pre-partum and post-partum, online Pilates-based program on weight control, low back pain (LBP) severity, sleep disturbances, mood and depression levels. Methods: A total of 136 pregnant women were screened for eligibility (low-risk pregnancy; aged 18–45 years; single pregnancy; 20–28-week gestational age, normal BMI). Participants completed an online Pilates-based program during pregnancy and after delivery, through an online platform guided by a Pilates, Yoga and Lagree certified instructor. Main outcomes (weight control, LBP disability, sleep quality, mood, and mental health) were assessed at baseline and at the completion of pre- and post-partum programs. Results: Regarding pre-partum, significant gains in weight were observed only in low- and intermediate-amount exercisers. Only high-amount exercisers did not display significant increase in LBP-related disability (+42.7%; p =.21) unlike low (+12.2%; p <.0001) and intermediate exercisers (+9.6%; p <.0001). Sleep disturbances increased significantly in low- (+24.3%; p =.005) but not in intermediate- (+4.6%; p =.50) and high-amount exercisers (−0.1%; p =.91). Regardless of the amount of exercise, depression scores improved in all groups. Following post-partum intervention (n = 40), only intermediate-amount exercisers showed significant reductions in sleep disturbances (−24.1%; p =.003) and depression (15.9%; p =.04). Conclusions: Approximately 270 min/week of an online Pilates-based program were needed to prevent LBP worsening, and sleep deterioration in pregnant women. A different pattern was outlined for those women resuming the Pilates-based intervention at post-partum, with 150 min/week emerging as the "dose" of exercise capable of inducing the largest improvements in LBP, sleep, and mood disturbances. [ABSTRACT FROM AUTHOR]

Marisa Meloni, Paolo Buratti, Francesco Carriero, Laura Ceriotti VitroScreen, In Vitro Innovation Center, Milan, 20149, ItalyCorrespondence: Laura Ceriotti Via Mosè Bianchi, 103, Milan, 20149, ItalyTel +39 02 89077608Email laura.ceriotti@vitroscreen.comPurpose: A novel experimental model based on a 3D reconstructed human oesophageal epithelium model (HO2E) has been developed to investigate the structural and functional changes of the oesophageal epithelium following exposure to a solution of HCl 0.1 N (pH = 1.2) mirroring GERD microenvironment condition.Methods: The barrier structure modification after the exposure to the acid solution on HO2E tissues was investigated immediately after damage induction and after 1 hour post incubation and compared to HO2E tissues exposed to phosphate buffered saline solution. Immunofluorescence (IF) was applied to quantify the expression and localization of barrier function proteins: Claudin-1 (CLDN-1), Claudin-4 (CLDN-4), Zonulin-1 (ZO-1), E-Cadherin and Mucin-1 (MUC1). Barrier functionality was measured by TEER.Results: In the acidic microenvironment, TEER measurement has shown some limitations and results were not applicable, whereas the evaluation of protein localization and quantification provided clear and robust evidence of the damage which occurred to the epithelium barrier structure. CLDN-4 expression significantly decreased after exposure to acid. ZO-1 protein appeared upregulated immediately after exposure to HCl and was mainly localized in the cytoplasm and not on the cell membrane. This different localization was also observed for CLND-1. CLDN-1, MUC1 and, to a lower extent, ZO-1 expression increased during the post-incubation period.Conclusion: The relevant tissue biomarkers identified, CLDN-1 and MUC1, can be used to monitor TJ structure and epithelial barrier recovery after acid-induced damage which, in our experimental conditions, were non-destructive and suitable for recovery studies. The established model can be useful to investigate the mechanism of action of formulations acting on this specific pathophysiological condition and/or designed to potentiate the physiological defense mechanisms of oesophageal mucosa.Keywords: gastro-oesophageal reflux disease, GERD, 3D reconstructed human oesophageal epithelium, Claudin-1, Claudin-4, E-Cadherin, Zonulin-1, Mucin-1, TEER measurement

Francesca Rescigno, Laura Ceriotti, Marisa Meloni VitroScreen, In Vitro Innovation Center, Milan, ItalyCorrespondence: Francesca RescignoVitroScreen, In Vitro Innovation Center, via Mosè Bianchi, 103, Milan, 20149, ItalyEmail francesca.rescigno@vitroscreen.comObjective: Dermis spheroids from different donors (40 and 50 years old) were developed from primary fibroblasts to demonstrate their capacity to synthetize and organize the main dermal structural components when cultured in 3D microenvironment, forming endogenous de novo ECM according to their potential metabolic activity.Methods: Dermis spheroids were produced from primary human dermal fibroblasts at early passages in hanging drop culture system. Dermis models were characterized in terms of spheroid diameter, PICP release, collagen III and CD44 expression.Results: An increase of collagen III synthesis (101%) was found in the young donor compared to the old donor (23.5%) after seven days of culture by immunofluorescence. The progressive ECM assembly over the time and dermis maturation was showed by Masson’s trichrome staining and by immunofluorescence for collagen III and CD44; both molecules significantly accumulated in the dermal compartment from day seven to day 10 of culture with a global decrease for both spheroid models after 21 days of culture.Conclusion: Our results showed that specific culture conditions in the 3D scaffold-free microenvironment allowed the physiological and progressive ECM assembly of miniaturized dermis models reflecting phenotypic profile features of “young” and “old” native tissue from which cells were isolated with a potential application to personalized care approaches in dermatological research on aging processes and medicine.Keywords: 3D culture, dermis, scaffold-free spheroids, intrinsic aging, extracellular matrix, tissue remodeling

Barbara De Servi,1 Marisa Meloni,1 Amina Saaid,2 Josip Culig3 1In Vitro Research Laboratories, VitroScreen SrL, Milan, Italy; 2Department of R&D and Innovation, Laboratoire Fumouze, Levallois-Perret, France; 3Department of Pharmacology and Clinical Pharmacology, University of Applied Health Sciences, Zagreb, CroatiaCorrespondence: Barbara De ServiIn Vitro Research Laboratories, VitroScreen Srl, Via Mosè Bianchi 103, Milan 20149, ItalyTel +39-02-89077608Fax +39-02-45478817Email barbara.deservi@vitroscreen.comBackground: Nasal irrigation is often used for managing sinonasal conditions and maintaining nasal hygiene, which is critical to overall nasal health and to provide protection against airborne contaminants and pathogens. However, studies comparing efficacies of different solutions are needed.Purpose: This in vitro study evaluated the ionic balance of an isotonic diluted seawater solution (Stérimar Nasal Hygiene, SNH) and its safety and efficacy for regular nasal hygiene in comparison to electrodialyzed seawater (EDS).Materials and Methods: Ionic balance of SNH, EDS and pure seawater was measured by mass spectrometry and chromatography to be compared to the ionic balance of human plasma as reported in the literature. Safety was measured through cytotoxicity (lactate dehydrogenase release) and pro-inflammation (interleukin-8 secretion) assays using a 3D-reconstituted human nasal epithelium model. For efficacy, adenosine 5′-triphosphate (ATP) release assays, and histological (alcian blue) and immunohistochemical (aquaporin 3) stainings were performed on tissues under hypotonic challenge where saline solution was used as the negative control.Results: Compared to EDS, the ionic balance of SNH was more similar to human plasma and pure seawater. SNH reduced hypotonic stress-associated ATP release and maintained tissue morphology more effectively and lastingly compared to EDS. Both solutions were safe to use on nasal epithelium, as neither of them caused cytotoxicity or induced (pro-) inflammation.Conclusion: In comparison to EDS, this study confirms the safety and efficacy of SNH in maintaining good nasal hygiene consistent with its benefits reported in clinical trials.Keywords: nasal irrigation, electrolyte balance, homeostasis, medical device, nasal mucosa

Gaia Pellegatta,1 Marco Spadaccini,1 Laura Lamonaca,1 Vincenzo Craviotto,1 Ferdinando D’Amico,1 Laura Ceriotti,2 Marisa Meloni,2 Alessandro Repici1 1Humanitas Clinical and Research Center and Humanitas University, Digestive Endoscopy Unit, Division of Gastroenterology, Rozzano, MI, Italy; 2VitroScreen, Milano, ItalyCorrespondence: Gaia PellegattaDigestive Endoscopy Unit, Division of Gastroenterology, Humanitas Research Hospital, Via Manzoni 56, Rozzano, MI 20089, ItalyTel +390282247091Email gaia.pellegatta@humanitas.itPurpose: New medical devices that contain hyaluronic acid (HA) and chondroitin sulphate (CS), with or without antacid components, have been developed for the treatment of gastroesophageal reflux disease (GERD) with the aim of improving oesophageal mucosal defences by creating a film on the oesophageal mucosa and acting as a mechanical barrier against the noxious components of refluxate, both acidic and basic.Methods: The film-forming and protective efficacy of medical device A based on HA and CS plus aluminium hydroxide, device B combining HA and CS with magnesium trisilicate and device C with only the combination of HA and CS was tested on a reconstructed human oesophageal epithelium (HO2E/S/5) as a biological model in 2 different pH environments, neutral and acidic, to mimic realistic conditions. Caffeine penetration kinetics and Lucifer yellow (LY) permeability modifications induced by these products were compared to those induced by a negative control series (saline solution, code NC) and positive control series (white Vaseline, code V) under neutral and acidic pH conditions.Results: Under neutral and acidic pH conditions, compared to the negative control, all the products tested reduced (> 80% and 85– 90%, respectively) the caffeine passage, and no significant difference was observed among the products tested. Under neutral and acidic conditions, the LY permeabilities registered with device A and device C were not different from that registered with the negative control, while an LY flux% increase was calculated after 2 hrs of treatment (21.1%) with device B under acidic conditions.Conclusion: These results confirm the ability of the products tested to interact with the oesophageal epithelium in order to adhere and create a stable protective film for at least 2 hours after their homogeneous distribution on the epithelium surface. Further clinical studies are needed to test these devices in the topical treatment of gastroesophageal reflux symptoms.Keywords: hyaluronic acid, chondroitin sulphate, gastroesophageal reflux disease, antacid, caffeine, Lucifer yellow

Laura Ceriotti,1 Silvia Balzaretti,1 Salvatore Barone,2 Marisa Meloni1 1VitroScreen, Milan, Italy; 2NTC S.r.l., Milan, ItalyCorrespondence: Laura CeriottiVitroScreen, Via Mosè Bianchi 103, Milan 20149, ItalyTel +39 02 89077608Email laura.ceriotti@vitroscreen.comObjective: To assess the eye tolerability of a buffered ophthalmic solution containing microglycine (sodium hydroxymethylglycinate, mwaterTM) in an in vitro model.Materials and Methods: A multiple endpoint analysis (MEA) approach was applied to the reconstructed human corneal epithelium (HCE) model. Sodium hydroxymethylglycinate solution (0.04%) and two ophthalmic ointments containing microglycine (Protectorial, containing 0.02% of sodium hydroxymethylglycinate, and Edenight, containing 0.04% of sodium hydroxymethylglycinate) were investigated. The buffered solution and the ointments were tested on HCE after acute (one application in 24 hrs, followed or not by 16 hrs of recovery) or repeated (one application per day for three consecutive days) exposures; benzalkonium chloride (BAK) 0.01% and saline isotonic solution were used as positive and negative controls, respectively. Cellular viability, trans-epithelial electrical resistance (TEER), lactate dehydrogenase (LDH) release and histo-morphology were evaluated.Results: BAK 0.01% toxicity in HCE was confirmed for the 24+16 hrs acute and repeated exposure protocols, while, after 24–hours acute treatment, only modifications of the superficial cell layer were visible compared with the negative control. Sodium hydroxymethylglycinate had a very good tolerability profile and a neutral impact on the corneal surface after acute or repeated exposure. The Protectorial and Edenight ointments preserved cell viability in the different exposure protocols, suggesting a good local tolerability profile. Modifications of the superficial layers were observed on histo-morphological analysis and confirmed by increased release of LDH after 24+16 hrs acute exposure (+65% and +76% for Protectorial and Edenight, respectively) and TEER values after 24+16 hrs and 72 hrs exposure protocols. These results were dependent on the ointments’ accumulation on the corneal epithelium due to their physical form (semi-solid) and lipophilic properties.Conclusion: Sodium hydroxymethylglycinate, alone or as part of eye ointments, was found to be non-toxic after acute or repeated exposure in the reconstructed HCE model.Keywords: BAK, biocompatibility, delayed toxicity, HCE, microglycine

Mario Ganau,1 Sibel Huet,1 Nikolaos Syrmos,2 Marco Meloni,3 Jayaratnam Jayamohan1 1Department of Neurosurgery, Oxford University Hospitals NHS Foundation Trust, Oxford, UK; 2Department of Neurosurgery, School of Medicine, Aristotle University of Thessaloniki, Macedonia, Greece; 3Department of Medicine and Surgery, Bicocca University, Milan, ItalyCorrespondence: Mario GanauDepartment of Neurosurgery, Oxford University Hospitals NHS Foundation Trust, Oxford, UKEmail Mario.Ganau@alumni.harvard.eduAbstract: Septo-optic dysplasia (SOD), also known as de Morsier syndrome, is a rare congenital disorder belonging to the group of mid-line brain malformations. Despite the highly variable phenotypic penetration, its classical triad include a) optic nerve hypoplasia (ONH), b) agenesis of septum pellucidum and corpus callosum, and c) hypoplasia of the hypothalamo-pituitary axis. SOD has stringent diagnostic criteria requiring 2 or more features of the classic triad, therefore it represents a separate entity from other conditions such as ONH and achiasmia syndromes which share only some of these aspects, or SOD plus syndrome which is characterized by additional cortical abnormalities. Starting from its etiology and epidemiology, this narrative review focuses on the management of SOD patients, including their diagnosis, treatment and follow-up. To date, SOD is not curable; nonetheless, many of its symptoms can be improved through a tailored approach, consisting of hormonal replacement, corrective ophthalmological surgery and neuropsychological support.Keywords: septo-optic dysplasia, optic nerve hypoplasia, hypopituitarism, achiasmia, hydrocephalus, congenital visual loss

Song Huang,1 Samuel Constant,1 Barbara De Servi,2 Marisa Meloni,2 Josip Culig,3 Marco Bertini,4 Amina Saaid5 1Epithelix, Geneva, Switzerland; 2Department of in Vitro Research, VitroScreen, Milan, 20149, Italy; 3Department of Pharmacology, University of Applied Health Sciences, Zagreb, 10000, Croatia; 4R&D Department, Laboratori Baldacci SpA, Pisa, Italy; 5Department of R&D and Innovation, Laboratoire Fumouze, Levallois-Perret, 92686, FranceCorrespondence: Marco BertiniLaboratori Baldacci SpA, Via S. Michele degli Scalzi, 73, 56124 Pisa, ItalyTel +39 5 031 3271Fax +39 5 057 0170Email bertini@baldaccilab.comBackground: The common cold is a viral infectious disease with symptoms such as runny nose, sore throat, and mainly, nasal congestion. State-of-the-art therapeutic approaches focus on alleviating the symptoms of this disease by non-invasive and simple-to-use methods. Nasal irrigation is one of the most accepted approaches to ease nasal congestion which, if left untreated, has a negative impact on the quality of life of patients.Purpose: In this study, the safety and efficacy of a novel hypertonic seawater solution for nasal lavage enriched with hyaluronic acids, eucalyptus oil, copper, and manganese salts (Stérimar Stop & Protect Cold and Flu; SSPCF) have been investigated in vitro.Methods: An in vitro 3D reconstituted human nasal epithelium tissue model, MucilAir™, has been used in this study to investigate the safety of SSPCF on nasal epithelium by measuring transepithelial electrical resistance (TEER), lactate dehydrogenase (LDH), and interleukin-8 (IL-8) secretion. The efficacy of SSPCF was measured by mucociliary clearance (MCC), ATP release, Alcian blue and aquaporin (AQP3) stainings.Results: SSPCF treatment respected nasal epithelium tissue integrity and enhanced barrier function without inducing a cytotoxic response. Secreted LDH and IL-8 levels were similar to untreated controls. MCC rate was increased 2.5-fold and ATP release decreased 87% upon SSPCF treatment, indicating improved decongestion activity. SSPCF treatment after hypotonic stress helped recover cellular organization, as shown by Alcian blue and AQP3 staining assays.Conclusion: SSPCF appears as a safe and effective nasal irrigation formula that may alleviate the symptoms associated with common cold such as nasal congestion.Keywords: common cold, hypertonic seawater, nasal irrigation, pathogens, ATP release, decongestion

Luigi Uccioli,1 Marco Meloni,1 Valentina Izzo,1 Laura Giurato,1 Stefano Merolla,2 Roberto Gandini2 1Department of Systems Medicine, 2Department of Interventional Radiology, Università di Tor Vergata Roma, Rome, Italy Abstract: Critical limb ischemia (CLI) is considered the most severe pattern of peripheral artery disease. It is defined by the presence of chronic ischemic rest pain, ulceration or gangrene attributable to the occlusion of peripheral arterial vessels. It is associated with a high risk of major amputation, cardiovascular events and death. In this review, we presented a complete overview about physiopathology, diagnosis and holistic management of CLI. Revascularization is the first-line treatment, but several challenging cases are not treatable by conventional techniques. Unconventional techniques for the treatment of complex below-the-knee arterial disease are described. Furthermore, the state-of-the-art on gene and cell therapy for the treatment of no-option patients is reported. Keywords: peripheral arterial disease, critical limb ischemia, medical therapy, revascularization, cell therapy

Marisa Meloni,1 Giampiero Cattaneo,2 Barbara De Servi11VitroScreen In Vitro Research Laboratories, 2Thea Farma, Milan, ItalyBackground: By using a biologically relevant and sensitive three-dimensional model of human corneal epithelium and multiple endpoint analysis, assessment of the potential for eye irritation and long-term compatibility of four registered ophthalmological preparations, ie, Timolabak®, Timoptol®, Nyogel®, and Timogel®, was performed. This approach enables classification of the potential for irritation, discriminating between mildly irritant and non-irritant ocular substances.Methods: The exposure protocol included two time periods, ie, 24 hours (acute application) and 72 hours (repeated applications twice daily). This approach allows assessment of not only the acute reaction but also possible recovery, as well as mimicking the potential cumulative effects associated with long-term application. Using benzalkonium chloride (BAK) 0.01% as a positive control, the following parameters were quantified: cellular viability by MTT test, histological analysis by hematoxylin and eosin staining, passive release of interleukin-1a by enzyme-linked immunosorbent assay, and OCLN gene expression by quantitative real-time polymerase chain reaction.Results: Cell viability was reduced to under the 50% cutoff value after acute exposure (24 hours) to BAK 0.01%, and after repeated application (72 hours) of Timoptol and Nyogel. Histological analysis after acute exposure showed signs of superficial damage with all formulations, and severe changes after repeated applications of Timoptol, BAK 0.01%, and Nyogel. Timolabak and Timogel did not significantly alter the morphology of the human corneal epithelial cells after the different exposure times. Interleukin-1α release was greater than that for the negative control (>20 pg/mL) and the positive control (BAK 0.01%), Nyogel, and Timoptol treatments and not different after treatment with Timolabak and Timogel. Expression of OCLN, a sign of epithelial barrier impairment, was only significantly upregulated at 24 hours by BAK 0.01%, suggesting a toxic reaction at the ocular surface. OCLN was also overexpressed after repeated application of Nyogel and Timogel.Conclusion: Overall, the multiple endpoint analysis approach allows classification of these products according to decreasing order of irritation potential as follows: BAK 0.01%, Timoptol, Nyogel, Timogel, and Timolabak.Keywords: occludin, gene expression, interleukin-1α, eye compatibility, human corneal epithelium