STUDIO DELLE CARATTERISTICHE FENOTIPICHE E FUNZIONALI DELLE CELLULE DENDRITICHE CIRCOLANTI IN PAZIENTI AFFETTI DA CARCINOMA COLON-RETTALE

Background. Tra le patologie tumorali, il carcinoma del colon-retto (CRC) è una delle principali cause di morte in tutto il mondo. Il tasso di sopravvivenza a 5 anni dopo resezione chirurgica è del 25-40%, nonostante i regimi di chemioterapia adiuvante. Il sistema immunitario dei pazienti affetti da cancro è estremamente compromesso e incapace di esercitare una risposta anti-tumorale efficace. Alcuni meccanismi di tumor-escape coinvolgono le cellule dendritiche (DC), le più importanti cellule presentanti l’antigene, con un ruolo centrale nell’immunità sia innata che adattativa. Le cellule dendritiche rappresentano solo lo 0.1-1% delle cellule mononucleate del sangue periferico (PBMC), e, nel loro stato immaturo, sono distribuite in maniera ubiquitaria in tutti i tessuti, dove svolgono la funzione di uptake e processazione di antigeni riconosciuti come non-self. Una volta catturato, le DC vanno incontro a maturazione, e migrano agli organi linfoidi secondari, dove presentano l’antigene ai linfociti T, generando una risposta immunitaria antigene-specifica. In pazienti affetti da cancro alla prostata, alla mammella, e al fegato è stato osservato che diversi fattori prodotti dal tumore, quali VEGF, IL-6, IL-10, inducono difetti quantitativi e funzionali delle DC, che promuovono uno stato di tolerogenicità verso il tumore stesso. Nei pazienti affetti da CRC sono stati eseguiti diversi studi circa il grado di infiltrazione intra-tumorale delle cellule del sistema immunitario, con risultati discordanti. Invece, studi concernenti lo status di queste cellule a livello sistemico sono ancora scarsi, nonostante la notevole importanza delle DC circolanti e di quelle derivate in vitro dai monociti (MoDC) al fine di ottenere una risposta anti-tumorale efficace, e di identificare nuovi approcci immunoterapici maggiormente efficienti e personalizzati per ogni singolo individuo. Scopo dello studio. L’obiettivo di questo lavoro consiste nello studio dello status delle DC nei pazienti affetti da CRC al fine di contribuire a spiegare i meccanismi di tumor-escape e di identificare potenziali parametri immunologici utili per la stadiazione, la prognosi e la terapia. Inoltre la caratterizzazione delle DC in vitro potrebbe fornire utili criteri biologici per la valutazione delle DC come possibili candidati per l’approccio immunoterapico. Metodi. 26 pazienti affetti da CRC sono stati inclusi nello studio, prima della resezione chirurgica, e caratterizzati per l’enumerazione ex vivo delle DC circolanti mieloidi (mDC) e plasmacitoidi (pDC), e per la valutazione in vitro della capacità dei monociti di differenziare in DC, confrontando i risultati con i controlli sani (HC). Inoltre le stesse analisi sono state eseguite a diversi time-point fino ad un massimo di 12 mesi dopo l’operazione chirurgica, al fine di definire lo status delle DC dopo trattamenti convenzionali, quali la chirurgia e/o la chemioterapia. Le DC circolanti dei controlli e dei pazienti con CRC sono enumerate direttamente sul campione sangue periferico in toto raccolto in EDTA attraverso analisi citofluorimetrica multiparametrica. Le cellule dendritiche sono identificate come cellule CD14-CD16-CD85K+ e CD33+ o CD123+ per il subset mieloide e plasmacitoide, rispettivamente. Per la generazione delle DC in vitro, le cellule mononucleate del sangue periferico sono isolate tramite gradiente di densità Ficoll-Hypaque da campioni dei controlli e dei pazienti raccolti in eparina. Le cellule CD14+ sono purificate dalle PBMCs tramite selezione immunomagnetica e coltivate alla concentrazione di 1x106 cell/ml in terreno RPMI 1640 completo supplementato con GM-CSF e IL-4 (both 50 ng/ml). Dopo 6 giorni di coltura, è aggiunto alla coltura LPS (100 ng/ml) per ulteriori 24 ore di incubazione, al fine di indurre la maturazione delle DC. Le analisi fenotipiche e funzionali sono eseguite tramite citofluorimetria sulle cellule coltivate non aderenti. Al giorno +6 la generazione delle MoDC è valutata tramite l’analisi dell’espressione della molecola CD14, e la funzionalità come capacità di fagocitare la sostanza FITC-destrano. Al giorno +7, la maturazione delle DC è determinata fenotipicamente tramite la valutazione dell’espressione delle molecole CD40, CD80, CD83 e HLA-DR, mentre a livello funzionale è valutata sia la capacità delle DC di indurre la proliferazione di linfociti T naïve, tramite il saggio di reazione linfocitaria mista (MLR), sia la secrezione di citochine in coltura da parte delle DC (IL-6, IL-10, IL-12, TNF-α e TGF-β1) tramite analisi ELISA. L’analisi statistica è effettuata utilizzando il software GraphPadPrism. I risultati sono espressi come valore medio ± errore standard della media (SEM). Differenze significative nel confronto tra i controlli sani e i pazienti sono determinate con il test di Student a due code o il test di Mann-Whitney, a seconda che i valori seguano una distribuzione normale o meno, rispettivamente. Valori di p<0.05 sono considerati statisticamente significativi. Risultati. Al primo time-point, il numero assoluto di pDC nei pazienti è significativamente inferiore rispetto ai controlli, e ciò può essere principalmente attribuito ai pazienti in stadio III-IV. Il numero assoluto di mDC invece non mostra differenze rispetto ai controlli; il numero totale di DC circolanti nei pazienti in stadio III-IV è significativamente ridotto rispetto ai soggetti sani. Ai prelievi successivi, il numero di DC circolanti non mostra alcuna differenza rispetto ai controlli, suggerendo un recupero quantitativo dopo la rimozione del tumore. Prima delle resezione chirurgica del tumore, i pazienti con CRC mostrano una compromessa capacità di generare DC in vitro, e di ottenere DC pienamente mature e funzionali, come mostrato dalla riduzione dell’espressione delle molecole co-stimolatorie e dell’antigene CD83. Inoltre, la secrezione di IL-10 da parte delle DC mature è significativamente superiore nei pazienti in stadio III-IV rispetto ai controlli e ai pazienti in stadio I, mentre la produzione di IL-12 e TNF-α è ridotta in maniera significativa nei pazienti in tutti gli stadi rispetto ai soggetti sani. Anche la capacità di stimolare la proliferazione di linfociti T allogenici appare significativamente inferiore nei pazienti di tutti gli stadi rispetto agli HC. Ai time-point successivi si osserva un recupero della capacità differenziativa, mentre quella maturativa permane significativamente compromessa, sia dopo resezione chirurgica, sia dopo trattamento chemioterapico, come mostrato dalle caratterizzazioni fenotipiche e funzionali. La compromissione delle DC sembra persistere fino a 12 mesi dopo l’operazione chirurgica, in particolare nei pazienti sottoposti a chemioterapia, anche se la coorte di pazienti deve essere incrementata per poter effettuare valutazioni statistiche. Conclusioni. La compromissione del numero di DC circolanti e della capacità di generare in vitro DC pienamente mature e funzionali potrebbe rappresentare uno dei meccanismi di tumor-escape che si verifica nei pazienti con CRC. Queste alterazioni sono correlate alla progressione tumorale, dimostrando un peggioramento dello status del sistema immunitario non solo locale ma anche sistemico, con l’avanzamento del tumore. Inoltre, la capacità dei monociti di generare in vitro DC pienamente mature permane parzialmente compromessa anche dopo l’allontanamento del tumore e dopo trattamento chemioterapico, fino a 12 mesi dopo la resezione chirurgica. Questa osservazione richiede ulteriori investigazioni con una coorte di pazienti più numerosa e un follow-up più lungo, al fine di determinare il comportamento delle DC durante la malattia e la remissione. I nostri risultati inoltre orientano verso il non utilizzo di DC autologhe in pazienti affetti da cancro colon-rettale, in particolare di quelli in stadio avanzato di malattia, per approcci immunoterapici concernenti l’uso di vaccini basati sulle DC, in quanto esse risultano incapaci di indurre una efficiente proliferazione e attivazione dei linfociti T, e quindi una efficace risposta immunitaria anti-tumorale. Considerando i numerosi tentativi di elaborare nuovi criteri di classificazione dei pazienti con cancro basati sul grado di reazione immunitaria a livello locale, che abbiano un valore prognostico superiore rispetto a quelli attuali, e quindi conseguenze importanti nella pratica clinica, si ritiene che lo studio delle proprietà fenotipiche e funzionali delle DC debba essere considerato come un argomento di estrema importanza

Tecniche di Classificazione di Traffico IPsec basate su SVM

Uno dei meccanismi di protezione sviluppati che permette di cifrare ed autenticare i dati e nascondere l’applicazione usata dall’utente, è IPsec, un’estensione del protocollo IP. I metodi classici di classificazione non sono utili quando i dati sono cifrati, poichè si basano sull'analisi del payload o sulla porta utilizzata a livello di trasporto. Per la classificazione di traffico IPsec, sono state quindi sfruttate delle tecniche che utilizzano i comportamenti statistici dei parametri IP del traffico cifrato. Questa tesi ha l’obiettivo di mostrare com’è possibile riconoscere il tipo di applicazione utilizzata da utenti che proteggono il loro traffico di Rete con IPsec, avvalendosi esclusivamente delle informazioni del livello IP, come la lunghezza dei pacchetti che compongono il flusso di dati, il tempo di arrivo o la direzione del flusso dei pacchetti. Analizzando queste informazioni statisticamente, è possibile costruire un modello di classificazione dei protocolli più comunemente utilizzati, sfruttando le tecniche Support Vector Machines

Regenerative medicine in hearing recovery

Hearing loss, or deafness, affects 360 million people worldwide of which about 32 million are children. Deafness is irreversible when it involves sensory hair cell death because the regenerative ability of these cells is lost in mammals after embryo development. The therapeutic strategies for deafness include hearing aids and/or implantable devices. However, not all patients are eligible or truly benefit from these medical devices. Regenerative medicine based on stem cell application could play a role in both improvement of extant medical devices and in vivo recovery of auditory function by regeneration of inner ear cells and neurons. A review of recent literature on the subject indicates that two promising approaches to renewal and differentiation of cochlear tissues are transplantation of stem cells and in situ administration of growth factors. Rather than directly regenerating dead cells, these procedures apparently induce, through various pathways, differentiation of resident cochlear cells. More studies on the possible adverse effects of transplanted cells and the recovery of tonotopic sensorineural activity or required. To date, no reliable clinical results have been obtained in the field of cochlear regeneration. \ua9 2017 International Society for Cellular Therapy

Multidisciplinary evaluation of the remineralization potential of three fluoride-based toothpastes on natural white spot lesions.

OBJECTIVES This in vitro study aimed assessing the remineralization potential of three commercial fluoride-based toothpastes in permanent teeth with natural white spot lesions (WSLs). A multidisciplinary approach based on Raman microspectroscopy (RMS), Scanning electron microscopy (SEM), Energy-dispersive x-ray spectroscopy (EDS), and Vickers microhardness (VMH) was exploited. METHODS N = 12 human molars with natural WSLs in the proximal-vestibular zone were selected and divided into 4 groups (n = 3) according to the different treatments: HAF (hydroxyapatite with fluoride ions); SMF (sodium monofluorophosphate with arginine); SF (sodium fluoride with enzymes), and CTRL (untreated group). All toothpastes tested contained 1450 ppm of fluoride. Teeth samples were submitted to the following protocol: a 7-day pH cycling treatment, with two daily exposures (2 min each time) to the commercial toothpastes described above. The surface micromorphology (SEM), the chemical/elemental composition (RMS and EDS), and the Vickers microhardness (VMH) were evaluated. Statistical analysis was performed. RESULTS A remarkable remineralization of WSLs in SEM images was observed in all treated groups compared to CTRL. In particular, HAF and SF displayed higher values of VMH, phosphates amount (I960), crystallinity (FWHM960), and lower ones of C/P (I1070/I960) with respect to CTRL. Intermediate values were found in SMF, higher than CTRL but lower with respect to HAF and SF. As regards the Ca/P ratio, statistically significant differences (p < 0.05) were found between SF and the other groups. CONCLUSIONS All the tested dentifrices have shown to remineralize the WSLs. SF and HAF have comparable capability in hardness recovery and crystallinity; however, SF shows the best remineralizing potential according to both micromorphological and chemical analyses. Clinical relevance The daily use of toothpastes containing hydroxyapatite partially replaced with fluoride, sodium monofluorophosphate with arginine and sodium fluoride toothpaste associated with enzymes represents a preventive, therapeutic, effective, and non-invasive tool for remineralize WSLs

PEERS’ ANATOMY: teaching among students as an original approach to learning anatomy – a project made in Alma Mater

Anatomy is an ancient discipline which benefited greatly from technological improvements. Those very same technologies, along with a shift towards more clinically relevant topics, might be endangering anatomy’s central role as a cornerstone of medical education. A review of local realities showed anatomy to be quite uniformly taught by means of lectures alone. On the basis of our own experience, and comforted by references in literature (Day et al. [1]), we propose a revaluation of the teaching approach to anatomy, which includes an integration between different available resources and, above all, introduces peer-to-peer activity. Over the course of the last decade, based on a proposal from the Anatomy Department, more and more medical students took part to gross anatomy workshops abroad, thus consolidating what today is a large group of tutors. Working in parallel with the lectures, back home these students organize activities where notions are not a mere tool to pass the exam, but are aimed at giving younger students solid foundations on which to build their future competence. On the grounds of the experiences acquired in these years, we managed to divide the tutoring activities into six areas: surface and topographic anatomy, muscles and skeleton, heart and thorax, neuroanatomy, abdomen and pelvis, didactic coordination. Each group is based on the equal division of tasks and on respecting each tutor’s expertise, attitudes and skills. These workshops have proved to be highly effective both for students, as a chance to experience anatomy “hands-on”, and for tutors, as an opportunity of mastering the subject. Being appointed a tutor outlines a shift from a deductive learning method, typical of the pre-exam phase, to an inductive one, more useful in the future as medicine doctors. To sum up, the availability of adequate facilities for cadaveric dissection certainly enhances the teaching of anatomy; what our experience shows, however, is that only direct involvement of students as tutors brings out the full potential of these activities. We therefore propose abandoning a pure “learning-to-do” approach, in favour of a more effective “learning-by- doing” strategy

A novel DAG-dependent mechanism links PKCα and cyclin B1 regulating the G2/M progression of cell cycle

Protein kinase C α has been reported to regulate cell cycle in several cell lines. Most of the reports describe a role for PKC α in G1/S transition but little is known about its possible involvement in G2/M progression. Our studies on the effects of PKC inhibitors, PKC α silencing and overexpression demonstrated a novel and positive role for PKC α in cyclin B1 regulation in human erythroleukemia cell line, K562. On the other hand, using PKC inhibitors and a PKC α inactive mutant, we could report that PKC α activity was not necessary for cyclin B1 regulation. Moreover, immunoprecipitation and immunocytochemistry experiments showed that these two proteins could physically interact each other and enter into the nuclei during G2/M progression. In order to better understand this mechanism, we investigated how PKC α could be attracted into the nuclei. We found a high increase of nuclear DAG during the G2/M phase. Then, using PMA and PLC inhibitors, we showed that PKC α translocation was due to the increase in nuclear DAG. Surprisingly, we saw the same effect on cyclin B1. Finally, in order to discover which PLC was involved, we silenced the nuclear localized PLCβ1 founding a decrease in PKC α and cyclin B1 nuclear amount. Taken together, our data demonstrate the existence of a novel DAG dependent mechanism linking PKC α and cyclin B1 which can regulate their entry into the nuclei during the G2/M phase of cell cycle

Efficacy of an All-Natural Polyherbal Mouthwash in Patients With Periodontitis: A Single-Blind Randomized Controlled Trial

Aim: This study aimed to evaluate the anti-inflammatory effect and the incidence of adverse effects of an all-natural polyherbal mouthwash in patients with periodontitis, after 3 months of use. These aims were accomplished by using full mouth bleeding score (FMBS), full mouth plaque score (FMPS), probing depth (PD) clinical attachment level (CAL) and a questionnaire recording any adverse events.Methods: The present randomized controlled clinical study considered 40 patients with moderate or severe periodontitis, randomized in two groups: a test group (TG) and a control group (CG). TG was instructed to use a polyherbal mouthwash composed of Propolis resin extract, Plantago lanceolata, Salvia officinalis leaves extract, and 1.75% of essential oils and the CG was given a placebo mouthwash. Both groups were instructed to rinse for 2 min, twice daily after their routine oral home care with the different mouthwashes. Clinical measurements of FMBS, FMPS, PD and CAL were recorded at baseline (T0) and after 3 months (T1). The incidence of adverse outcomes was recorded at every follow-up. Mann–Whitney U test and Wilcoxon signed-rank test were used for the statistical analysis (p &lt; 0.05).Results: The final study sample consisted of 34 healthy individuals, 17 individuals in each of the two groups. TG and CG showed a statistically significant reduction in FMBS (p = 0.001 TG; p = 0.002 CG), FMPS (p = 0.001 TG; p = 0.003 CG), PD (p = 0.001 TG; p = 0.011 CG) and CAL (p &lt; 0.001 TG; p = 0.020 CG) values from baseline to 3 months. The TG showed a statistically significant decrease in FMBS and FMPS compared with the CG. No adverse events or side effects were reported or observed in both groups.Conclusion: The use of polyherbal mouthwash in patients with moderate or severe periodontitis has proved safe and effective in reducing bleeding score and plaque accumulation, after 3 months, compared with placebo, although no difference between the two groups were reported on PD and CAL (both improving at T1)

Distal motor neuropathy associated with novel EMILIN1 mutation

Abstract Elastin microfibril interface-located proteins (EMILINs) are extracellular matrix glycoproteins implicated in elastogenesis and cell proliferation. Recently, a missense mutation in the EMILIN1 gene has been associated with autosomal dominant connective tissue disorder and motor-sensory neuropathy in a single family. We identified by whole exome sequencing a novel heterozygous EMILIN1 mutation c.748C>T [p.R250C] located in the coiled coil forming region of the protein, in four affected members of an autosomal dominant family presenting a distal motor neuropathy phenotype. In affected patient a sensory nerve biopsy showed slight and unspecific changes in the number and morphology of myelinated fibers. Immunofluorescence study of a motor nerve within a muscle biopsy documented the presence of EMILIN-1 in nerve structures. Skin section and skin derived fibroblasts displayed a reduced extracellular deposition of EMILIN-1 protein with a disorganized network of poorly ramified fibers in comparison with controls. Downregulation of emilin1a in zebrafish displayed developmental delay, locomotion defects, and abnormal axonal arborization from spinal cord motor neurons. The phenotype was complemented by wild-type zebrafish emilin1a, and partially the human wild-type EMILIN1 cRNA, but not by the cRNA harboring the novel c.748C>T [p.R250C]. These data suggest a role of EMILIN-1 in the pathogenesis of diseases affecting the peripheral nervous system

Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm

Genetic Pattern, Orthodontic and Surgical Management of Multiple Supplementary Impacted Teeth in a Rare, Cleidocranial Dysplasia Patient: A Case Report

Background: Cleidocranial dysplasia (CCD) is a rare, autosomal dominant skeletal dysplasia with a prevalence of one per million births. The main causes of CCD are mutations in the core-binding factor alpha-1 (CBFA1) or runt-related transcription factor-2 (RUNX2), located at the 6p21 chromosomal region. RUNX2 plays important roles in osteoblast differentiation, chondrocyte proliferation and differentiation, and tooth formation. The disease is characterized by clavicular aplasia or hypoplasia, Wormian bones, delayed closure of cranial suture, brachycephalic head, maxillary deficiency, retention of primary teeth, inclusion of permanent teeth, and multiple supernumerary teeth. Materials and Methods: A 22-year-old girl suffering from cleidocranial dysplasia with short stature, narrow shoulders, craniofacial manifestations (short face, broad forehead, etc.) and dental anomalies (different lower dental elements under eruption, supernumerary and impacted multiple teeth, etc.) was examined at our service (Complex Operative Unit of Odontostomatology of Policlinico of Bari). RX Orthopantomography (OPG) and cone beam computed tomography (CBCT) were requested to better assess the position of the supernumerary teeth and their relationships with others and to evaluate the bone tissue. Results: Under eruption was probably caused by dental interferences with supernumerary teeth; hence, extractions of supernumerary upper canines and lower premolars were performed under general anaesthesia. Surgery outcome was excellent with good tissue healing and improvements in the therapeutic possibilities with future orthodontics. Conclusions: The objective of this article is to give an update about radiological, clinical, and molecular features of CCD and to alert the health team about the importance of establishing an early diagnosis and an appropriate treatment in these patients to prevent impacted teeth complications and to offer them a better quality of life