Scienze mediche
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- PublicationCharacterization of cancer behaviour after laser biostimulation at cellular and molecular level(Università degli studi di Trieste, 2015-03-26)
;Ottaviani, Giulia ;Biasotto, MatteoZacchigna, SerenaThe main aim of this project is to provide an answer to the following issues: - on the one hand the definition of the cellular and molecular mechanisms of action of the laser therapy and its interaction with tissues - on the other hand the safety of laser therapy and is potential consequences on cancer behaviour We created a mouse model of oral carcinogenesis to assess potential differences in tumour angiogenesis in tissues treated with laser therapy compared to the control ones, by using both histological analysis and injection of Nano FluoSpheres®. A chemical carcinogen (4-NQO) dissolved in their drinking water was administered to C57BL/6 female mice (n = 50), 8-week old, since this compound is able to induce the formation of multiple oral tumours. Among these, 25 mice underwent to 4 session of laser therapy on consecutive days employing the HPLT-1 protocol, while the remaining mice were used as controls. During the 21st week, 15 animals per group were sacrificed to perform an accurate histological analysis of their tongue, while 10 were subjected to a quantitative assessment of angiogenesis through a 3D reconstruction of the tumour vascular network after the in vivo perfusion with Nano FluoSpheres®. Any increase concerning neither the number/extension of dysplastic and neoplastic areas nor tumour angiogenesis was registered in the treated group. Moreover, treated animals showed a tendency to border and to isolate tumour areas. The laser seemed to normalize tumour vessels, promoting their covering by smooth muscle cells, thus reducing ectasia and vascular permeability, as assessed by reduced Nano FluoSpheres® leakiness. The histological analysis performed on the oral carcinogenesis mice model was compared to the images of the same tumours acquired by Narrow Band Imaging. Three raters experienced in the use of this technology analyzed the images, classifying all visible lesions according to different pathological grades; the obtained results were than compared with the histological analysis, used as reference standard. The statistical analysis revealed both high sensitivity (96%) and specificity (99%) for this technology. Supported by other studies, the Narrow Band Imaging is expected to hold great potential for the clinical evaluation of tumour angiogenesis, as well as for the early detection of potentially malignant lesions of the oral cavity. The important clinical outcome in term of wound healing and our interest in the analysis of cell behaviour after laser therapy were the starting point for the evaluation of the effect of laser therapy on different cell lines: Human Skin Fibroblasts, Human Umbilical Vein Endothelial Cells, Human Coronary Artery Smooth Muscle Cells, Neonatal Rat Ventricular Myocytes, Human Bone Osteosarcoma Epithelial Cells and Mouse B16F10 Melanoma Cells. We set different powers, energies and wavelengths, and we performed the evaluations at different experimental times (6, 24, 48 and 96 hours after the irradiation). In general, laser irradiation resulted in an increase of both cell metabolism (ATPlite) and proliferation (cell count, AlamarBlu, BrdU incorporation), albeit with different timing and intensity in the various cell lines. Consistent with published results, we observed a clear increase in cancer cell metabolism upon laser irradiation; to evaluate the cancer behaviour in vivo, the same melanoma cells were implanted in C57BL/6 female mice (n = 16), 6-week old, at the dorsal subcutaneous level. As soon as the masses were visible to the naked eye (approximately on day 10), mice were homogeneously divided into 4 groups according to tumour size: 3 groups were subjected to different laser protocols (LPLT-6; MPLT-13 and HPLT-7) for 4 consecutive days (days 11 to 14), while the fourth group was used as control. On day 15, all animals were euthanized to measure the tumour volume and weight. A deep histological analysis on tumour invasion and cancer immune response (CD1a, CD4, CD8, CD25, CD68 kp1 and Melan-A) was performed, as well as the analysis of the expression levels of cytokines involved in the immune system activation (TNFα, IFNα and IFNγ). Laser therapy did not foster tumour growth or invasiveness (CD68 kp1 and Melan-A), but rather seemed to contain its extension. Moreover, in the laser groups, tumour infiltration by immune cells was much more higher compared to the control ones (CD4+, CD8+, CD25+ cells), consistent with the increased expression of IFNγ. Of notice, CD1a positive dendritic cells were particularly abundant in the dermis in the control group, while they migrated to "wrap" the tumour in laser groups. Based on these results, we applied the same laser protocols on primary mouse bone marrow dendritic cells, with and without lipopolysaccharide stimulation. These cells did not enhance cell metabolism upon laser treatment, but reduced TNFα and increased IFNγ expression. Finally, CD-1 female mice (n = 30), age 6-7 weeks old, were used to assess the expression of different cytokines (Collagen I, Collagen III, Collagen IV, FSP1, IL-2, IL-6, IL-10, IFNα, IFNβ, IFNγ, MMP-9, PDGFβ, TGFβ, TNFα) after the laser therapy at the dorsal level with and without the presence of a skin wound. The analysis confirmed an increase in the IFNγ expression; a similar trend was registered concerning IL-2, IL-6, IFNα, IFNβ, Collagen I, Collagen III, MMP-9, PDGFβ expression in the laser treated mice compared to controls. From both in vitro and in vivo analysis we can state that the laser therapy is effective in stimulating cell metabolism and proliferation, and in boosting a potent immune response in vivo. We can therefore foresee that the treatment of cutaneous and mucosal lesions in oncological patients can be safely performed even in potentially dysplastic or neoplastic areas.851 621 - PublicationDevelopment of a new nano-engineered biopolymer-based dental adhesive system(Università degli studi di Trieste, 2015-03-26)
;Diolosà, MarinaCadenaro, MilenaLo scopo di questa tesi di dottorato è stato studiare l'effetto sulla durabilità dell’interfaccia adesiva del chitosano, modificato con gruppi metacrilici, con l’obiettivo di migliorare l'affidabilità clinica dei restauri dentali. Attualmente, la ricerca in questo settore mira ad aumentare la durata nel tempo del legame resina-dentina. Questa ricerca propone un nuovo concetto di sistema adesivo bi-funzionale basato su un biopolimero naturale in grado di creare doppia reticolazione covalente. Il chitosano è un polimero biocompatibile e non tossico. In questo studio è stato ipotizzato che il chitosano modificato con gruppi metacrilici sia in grado di legarsi covalentemente alla resina dei restauri dentale e, grazie alla presenza di cariche positive residue sul polisaccaride, di interagire elettrostaticamente con la dentina demineralizzata. Il Chitosano metacrilato è stato introdotto nel primer di un sistema adesivo sperimentale etch&rinse a tre passaggi per valutarne la forza di legame alla dentina. Le prove d’invecchiamento sono state condotte utilizzando due metodiche: invecchiamento statico e dinamico. Il secondo metodo simula la presenza del sistema adesivo nell’ambiente orale, sottoposto sia a carico masticatorio, che a stress termici. Le metodiche d’invecchiamento sono necessarie per valutare la stabilità nel tempo dei restauri dentali. L'introduzione del chitosano modificato con gruppi metacrilici nel primer di un sistema adesivo etch&rinse ha determinato: buoni valori di adesione dopo 24 ore (T0 μTBS); (2) buona stabilità dello strato ibrido quando sottoposto a simulazione meccanica della masticazione e stress termici (TCS μTBS). In sintesi, la prima parte di questa tesi di dottorato ha avuto lo scopo di esaminare lo stato dell'arte dei materiali dentali attualmente utilizzati in odontoiatria e il problema più importante in odontoiatria restaurativa: la degradazione dell'interfaccia adesiva. La seconda fase di questa ricerca è stata dedicata al trovare la formulazione ottimale di un sistema adesivo contenente chitosano modificato con gruppi metacrilici. Diverse formulazioni sono state testate e il sistema sperimentale identificato come A3* è stato considerato la migliore formulazione utilizzabile come sistema adesivo. Questa formulazione è stata poi nominata Chit-MA70. Sono stati eseguiti diversi test per valutare le caratteristiche dell’adesivo sperimentale, compresi test meccanici (test di microtensile). L'espressione del nanoleakage è stata valutata utilizzando la microscopia ottica e la microscopia elettronica a scansione (SEM). Per valutare la sostantività del chitosano sullo strato adesivo, il polimero è stato marcato con fluoresceina e i campioni sono stati osservati al microscopio confocale a scansione laser (CLSM). Nella fase finale di questo studio sono stati condotti test di citotossicità per valutare l'effetto del sistema adesivo Chit-MA70 sulla vitalità cellulare. Le prove eseguite su fibroblasti gengivali hanno rivelato una tossicità ridotta, se non praticamente assente, del sistema adesivo contenente chitosano metacrilato. Considerando i buoni risultati di questo studio, il chitosano modificato con gruppi metacrilici è stato proposto come componente di un sistema adesivo sperimentale. I risultati ottenuti utilizzando questo nuovo sistema adesivo sono stati rivendicati in un brevetto (febbraio 2014) e pubblicati in Biomacromolecules nel mese di ottobre (2014).799 575 - PublicationThe influence of resin-based dental materials on oral biofilms developement(Università degli studi di Trieste, 2015-03-26)
;Ionescu, Andrei Cristian ;Breschi, LorenzoCadenaro, MilenaResin-based composites (RBCs) are the most widespread restorative dental materials used nowadays. Nanotechnologies allowed the development and improvement of a new generation of RBCs which nevertheless still present many unsolved issues. The most important is secondary caries, which is the recurrence of dental caries in tissues close to the restoration. Dental caries disease is driven by a dysbiotic biofilm colonizing both natural and artificial surfaces. Many approaches have been developed in order to address this issue, mainly the development of bioactive surfaces with contact-active properties, the optimization of surfaces to obtain anti-adhesive properties and the synthesis of biomimetic materials. The aim of this PhD thesis was to explore the use of nanotechnologies in order to synthesize, evaluate and optimize the formulation of RBCs aimed at successfully controlling oral biofilms development. The experimental part explored all three previously mentioned approaches. Regarding the first approach, a lactose-modified Chitosan carrier for silver nanoparticles (nAg) was developed and used as a coating for RBCs in order to study the antibacterial behavior of a novel material possessing contact-killing properties. The results indicated an antibacterial activity of the coating related to nAg concentration, however rinsing procedures interfered with the coating and deprived nAg of their effect. The surface activation of RBC surfaces prior to coating application showed itself good antibacterial properties that are currently under examination. Considering the second approach, experimental resin-based dental materials differing in their compositions were extensively studied, hypothesizing that biofilm formation on the experimental materials may show a dependency on their surface characteristics and nanotexture. In this sense, the anti-adhesive properties of these materials were evaluated as a possible effective way to control biofilm formation without the need for biocidal agents. The results showed that both hydrophobicity of the resin matrix of RBCs and filler content can influence oral biofilm formation. The lowest values of cariogenic biofilm were reached by less hydrophobic resin and by nanofillers. The third approach evaluated the possibility that biomimetic materials (designed to positively interact with dental hard tissues) may have to control oral biofilms, without the need for specifically biocidal agents. Resin-functionalized nanoparticles of dicalcium phosphate dihydrate (nDCPD) were incorporated into an experimental RBC. Results showed that the RBC filled with functionalized nDCPD showed reduced biofilm formation when compared to a RBC filled with non-functionalized nDCPD. In conclusion, all these three approaches proved to significantly impact oral biofilm formation on RBCs surfaces, however the most interesting result suggests the possibility of influencing biofilm formation without necessarily adding biocidal compounds. In fact, recent studies regarding the human microbiome show that many diseases, including dental caries, are caused by an imbalance between host and biofilms. These diseases may be restored by modifying biofilms composition, without attempting to eradicate biofilms.1220 1064 - PublicationUse of immune-nanoparticles containig chemiotherapeutic agents for the treatment of B-cell malignancies(Università degli studi di Trieste, 2015-03-26)
;Capolla, SaraMacor, PaoloB-cell malignancies are a heterogeneous group of clinical conditions including indolent diseases such as Chronic Lymphocytic Leukemia (CLL) and highly aggressive lymphoproliferative disorders such as Burkitt’s lymphoma. B-cell malignancies treatments take advantage of dose-intensive chemotherapeutic regimens and immunotherapy via monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents and cause several adverse effects. Thus, we propose a novel therapeutic approach for the treatment of CLL and Burkitt’s lymphoma in which high-doses of the association of hydroxychloroquine and chlorambucil (HCQ/CLB) or fludarabine were loaded inside biodegradable nanoparticles (BNPs) coated with an anti-CD20 antibody. First of all, a Burkitt’s lymphoma cell line (BJAB), two CLL cell lines (MEC1 and EHEB) and cells purified from patients’ blood samples were used to confirm CD20 expression and to assess BNPs binding and internalization. These studies demonstrated BNPs ability to bind malignant B cells and to enter inside cells in a process different from endocytosis. Then, BNPs therapeutic effect was evaluated by MTT test, AnnV/PI assay and western blot to put in evidence apoptosis induction and autophagy inhibition. These experiments demonstrated drugs-loaded BNPs ability to kill malignant B cells with comparable effects than those obtained with free drugs whereas empty BNPs were practically ineffective. In vivo BNPs characterization included the evaluation of their toxicity, biodistribution and therapeutic effect. C57/BL mice were used to evaluate BNPs toxicity which was studied considering survival, loss of body weight and several tissue markers in the blood. Mice receiving 8 injections of free HCQ+CLB died in this experiment whereas animals challenged with the same amount of drugs encapsulated inside BNPs did not show toxic effects suggesting BNPs safety. The importance of antiCD20 antibody in the homing of BNPs was confirmed by in vivo Time-Domain Optical Imaging performed in localized B-cell malignancy-bearing mice. This analysis suggested the ability of antiCD20-conjugated BNPs to specifically target tumor B-cells, with a pick after 24-48 hours. On the contrary, untargeted BNPs localization inside tumor was significantly decreased. In this analysis it was also evident that the liver is the main site of BNPs’ elimination while in the other organs the presence of fluorescent BNPs was very low. Finally, BNPs ability to treat a new xenograft human/SCID leukemia and Burkitt’s lymphoma mouse model was studied. Drugs-loaded BNPs were able to improve HCQ/CLB efficacy in vivo allowing the cure of treated all Burkitt’s lymphoma-bearing mice and 3 out of 7 leukemia-bearing animals. All these data together put the basis for the potential use of BNPs in the treatment of B-cell malignancies.817 765 - PublicationImmunoterapia orale (OIT) nei lattanti: potenzialità terapeutiche e di prevenzione dell'allergia alimentare IgE-mediatanull(Università degli studi di Trieste, 2015-03-02)
;Badina, LauraLongo, GiorgioIntroduzione: A fronte della diagnosi di allergia alimentare IgE-mediata le Linee Guida internazionali International raccomandano una scrupolosa dieta di eliminazione dell’alimento in causa e l’esecuzione di un test di provocazione orale (TPO) non prima dei 9-12 mesi dalla diagnosi, volto a verificare l’eventuale risoluzione spontanea dell’allergia. La sensibilizzazine allergica ad alimenti come il latte e l’uovo inizia nel primo anno di vita e raggiunge il suo massimo nel secondo anno di vita; in seguito in molti soggetti si verifica una riduzione dei livelli di IgE specifiche. Di fatti un elevato numero di bambini rimane a dieta per diversi anni e un numero sempre crescente di allergie alimentari divengono persistenti. D’altro canto numerose evidenze della letteratura evidenziano come il prerequisito fondamentale per l’ecquisizione della tolleranza orale sia la persistente e ripetuta esposizione all’allergene. Obiettivo: verificare l’ipotesi che l’assunzione graduale controllata dell’alimento offendente iniziata subito dopo la diagnosi di allergia alimentare possa accelerare lo sviluppo della tolleranza orale. Metodi: un TPO a basse dosi (10 ml of latte vaccino or 2 ml di emulsione di uovo crudo) è stato effettuato in 211 lattanti (119 maschi; età < 18 mesi) con storia di allergia certa al latte vaccino o all’uovo (reazione immediata da ingestione e provata sensibilizzazine ai test allergometrici) o con allergia potenziale agli stessi alimenti (dermatite atopica and prick-test or IgE sp > VPP 95%). La dose tollerata al TPO è stata assunta giornalmente a domicilio con periodici aumenti di dosaggio fino al raggiungimento della dieta libera per l’alimento in causa. Risultati: Venti lattanti (10%) sono stati esclusi perché già altamente reattivi al primo TPO a basse dosi. Lo studio è stato attualmente completato da 146 bambini e 91% hanno completato il protocollo di desensibilizzazione con successo. In un tempo mediano di 155 giorni (range IQ: 123-217) and 181 giorni (range IQ: 136-275). Nel corso del programma di desensibilizzazione si sono verificate in media 1,1 reazioni avverse per paziente, tutte di grado lieve-moderato. Un aumento significativo delle IgG4 sp e una importante parallela diminuzione delle IgE sp si è verificata alla fine del protocollo di immunoterapia orale (OIT). Conclusions: L’OTI iniziata all’esordio dell’allergia per latte e uovo sembra essere un approccio promettente capace di modificare l’andamento ingravescente della sensibilizzazione allergica nei primi anni di vita. Inoltre appare di più facile e sicura esecuzione rispetto a esperienze analoghe nel bambino più grandicello.1019 5895