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The purpose of the work performed during the preparation of this thesis was to provide a better knowledge on fish mucosal immunity and immune responses in a marine fish model (European sea bass) against a pathogenic virus (Betanodavirus), with a view of developing possible strategies of vaccination. The thesis is developed in seven chapters, the basic terminology has been introduced and defined in Chapter 1. The general aspects of Teleost immunology, basic information of the animal model (European sea bass) and viral encephalopathy and retinopathy (VER) disease were also described. To increase the knowledge of mucosal immunity, some basic cellular aspects typical of T cells like in vitro induction of proliferation by lectins and expression of T cell-related genes during proliferation were investigated (Chapter 2). GIALT leukocytes were able to proliferate, as measured by CFSE incorporation, in response to ConA and PHA. The proliferation was higher at 48 h and PHA induced higher proliferation with respect to ConA. The mean percentages of cells recognized by both monoclonal antibodies against pan-Tcells (mAb DLT15) and CD45 cells (mAb DLT22) doubled after 48 h of stimulation by lectins. T cells were founded in the epithelium of branchial filaments and transcription was particularly high for TRβ, TRγ and IL-10 followed by CD8, CD45 and CD4, thus confirming that proliferating cells contained T cells. In addition, the cellular characterization of a new polyclonal antibody against sea bass IgT-specific leukocytes was dealt (Chapter 3). IgT-specific leukocytes were labelled by IIF and detected by flow cytometry and fluorescence microscopy. These cells presented dimensions and morphology of leukocytes-type cells and were more abundant in the head kidney than in the spleen or gills. Moreover, a very poor content of soluble IgT was obtained in serum and a high content in mucus. Finally, the immunoprecipitation of IgT-specific leukocytes from head kidney with Dynabeads allowed us to get two fractions, one of them rich in IgT-specific leukocytes and the other rich in IgM-specific leukocytes as verified by RT-PCR. On the other hand, the effects of Betanodavirus immunization on immune responses in sea bass were investigated. First, a capture-based ELISA system that can be used as a tool to investigate and quantify the presence of VERv in biological samples of adult or juveniles sea bass was developed (Chapter 4). In fact, VERv was detected and quantified in brain homogenates of in vivo infected sea bass. This capture-based ELISA system was able to detect different RGNNV strains and can be speculated that is able to detect other genotypes because of the conservation of capsid protein residues and the similarities between genotypes. Subsequently, was performed an experimental approach that consisted of investigate the possibilites of immunizing young sea bass against VERv by means of mucosal (immersion) vaccination employing a formylated Betanodavirus preparation. (Chapter 5). The results of this round of immunisations showed that very few serum antigen-specific IgM can be detected by Indirect ELISA; no in vitro VERv-induced gills leukocyte proliferation can be induced; a modulation in transcription levels of genes coding for Mx and ISG-12 was observed after 24 hours and VERv antigens were detected in the gills by immunohistochemistry. These results shown that a single immersion vaccination without boosting induces a weakly systemic IgM-based immunity in sea bass. The following was to evaluate immune response of sea bass after intraperitoneal and immersion immunization with different vaccines preparations inactivated by using formylation, BPL and temperature (Chapter 6). Interestingly, 30 days post-intraperitoneal immunization with formalin-inactivated VERv a significant antigen-specific IgM titer was detected, at difference with other inactivation protocols. Moreover, the quantitative expression of antiviral genes in gut and head kidney was analyzed and a challenge experiments using live VERv in the IP formalin inactivated group was performed. Mx gene showed a modulation of transcripts on gut after 48 hours and on head kidney after 24 hours post injection. Instead ISG-12 gene was quantitatively expressed after 48 hours on gut and head kidney. Finally, a significant increase (81.9 %) in the relative percentage survival of immunizated fish with respect to un-immunized fish was observed. This result suggest that intraperitoneally vaccination with formalin-inactivated VERv could be a possible candidate vaccine for viral encephalopathy and retinopathy of sea bass. In Chapter 7, features of sea bass mucosal immunity focusing in proliferating mucosal T cells and IgT-specific leukocytes are examined. Also immune responses after mucosal and systemic immunization of sea bass against Betanodavirus are discussed. Lo scopo di questo lavoro di tesi è stato quello di fornire una migliore conoscenza sull'immunità mucosale nei Vertebrati utilizzando pesci Teleostei come modello, tramite lo studio delle risposte immunitarie in un modello di pesci marini (la spigola Europea) nei confronti di un virus patogeno (Betanodavirus), al fine di sviluppare possibili strategie di vaccinazione. L’elaborato è costituito da 7 capitoli, dove, inizialmente viene introdotta e definita la terminologia di base utilizzata (Chapter 1). Sono stati anche descritti gli aspetti generali dell'immunologia dei Teleostei, le informazioni di base del modello animale (spigola europea) e la malattia dell'encefalopatia virale e della retinopatia (VER). Per aumentare la conoscenza dell'immunità mucosale sono stati studiati alcuni aspetti cellulari di base tipici delle cellule T e l'espressione dei geni correlati alle cellule T durante la proliferazione in vitro indotta da lectine (Chapter 2). I leucociti delle branchie, in risposta alle lectine ConA e PHA, sono stati in grado di proliferare, come misurato dall'incorporazione di un colorante vitale. La proliferazione è stata superiore misurata a 48 ore e la PHA ha indotto una maggiore proliferazione rispetto alla ConA. Le percentuali medie di cellule riconosciute da entrambi gli anticorpi monoclonali contro le cellule pan-T (mAb DLT15) e le cellule CD45 (mAb DLT22) sono raddoppiate dopo 48 ore di stimolazione da parte di lectine. Le cellule T sono state indiciduate tramite immunoistochimica nell'epitelio dei filamenti branchiali e la trascrizione è stata particolarmente elevata per TRβ, TRγ e IL-10 seguita da CD8, CD45 e CD4, confermando così che le cellule che proliferano sono cellule T. Inoltre è stata effettuata la caratterizzazione cellulare di un nuovo anticorpo policlonale contro i leucociti IgT-specifici della spigola (Chapter 3). I leucociti IgT-specifici sono stati marcati mediante IIF ed rilevati mediante citometria di flusso e microscopia a fluorescenza. Queste cellule presentano le dimensioni e la morfologia tipiche dei leucociti e sono risultate più abbondanti a livello renale piuttosto che nella milza o nelle branchie. Inoltre, mentre nel siero è stato rilevato un basso contenuto di IgT solubili, nel muco è risultato elevato. Infine, l'immunoprecipitazione di leucociti IgT-specifici a livello renale con Dynabeads ci ha permesso di ottenere due frazioni, una delle quali ricca di leucociti IgT-specifici e l'altra ricca di leucociti IgM specifici, come è stato verificato mediante RT-PCR. Parallelamente, sono stati studiati gli effetti dell'immunizzazione del Betanodavirusv (VERv) sulle risposte immunitarie nelle spigole. Innanzitutto è stato sviluppato un sistema capture-based ELISA che può essere utilizzato come strumento per indagare e quantificare la presenza di VERv in campioni biologici di spigole adulte o giovani (Chapter 4). VERv è stato rilevato e quantificato in omogenati di cervello di spigola infettata in vivo. Questo sistema ELISA è stato in grado di rilevare diversi ceppi RGNNV e si può ipotizzare che sia in grado di rilevare altri genotipi a causa della conservazione dei residui proteici del capside e le somiglianze tra genotipi. Successivamente, è stato eseguito un approccio sperimentale che consiste nell'esaminare le possibilità di immunizzazione della spigola giovane contro VERv attraverso vaccinazione mucosale (immersione), impiegando un preparato di Betanodavirus inattivato con formalina. (Chapter 5). I risultati di questo ciclo di vaccinazioni hanno mostrato che pochissime IgM specifiche possono essere rilevate mediante saggi di ELISA indiretto; in vitro, l’induzione della proliferazione dei linfociti nelle branchie da parte di VERv non può essere indotta; dopo 24 ore è stata osservata una modulazione dei livelli di trascrizione di geni che codificano per Mx e ISG-12 e attraverso l'immunoistochimica sono stati rilevati nelle branchie gli antigeni VERv. Questi risultati hanno dimostrato che una singola immersione della vaccinazione, senza richiamo, induce nella spigola un'immunità sistemica debole. Il passo successivo è stato quello di valutare la risposta immunitaria della spigola Europea dopo dell'immunizzazione intraperitoneale e per immersione con diverse preparazioni di Betanodavirus inattivato in seguito a formilazione, BPL e la temperatura (Chapter 6). È interessante notare che 30 giorni dopo l'immunizzazione intraperitoneale con VERv inattivato con la formalina, è stata rilevata una significativa quantità di IgM specifiche, a differenza degli altri protocolli di inattivazione. Inoltre, è stata analizzata l'espressione quantitativa dei geni antivirali nell'intestino e a livello renale ed è stato fatto un esperimento controllo utilizzando VERv vivo in il gruppo immunizzato intraperitonealmente con virus inattivato con formalina. Il gene Mx ha evidenziato una modulazione nella sua trascrizione nell’intestino dopo 48 ore e a livello renale dopo 24 ore dall'iniezione. Invece, il gene ISG-12 è stato quantitativamente modulato dopo 48 ore, nell'intestino e a livello renale. Infine, è stato osservato un aumento significativo (81,9%) nella sopravvivenza dopo challenge di pesci immunizzati rispetto a pesci non immunizzati. I risultati mostrano che la vaccinazione intraperitoneale con VERv inattivato con formalina potrebbe essere un possibile candidato vaccino per l'encefalopatia virale e la retinopatia della spigola europea. Nel Chapter 7 sono esaminate le caratteristiche dell'immunità mucosale della spigola, focalizzandosi sulla proliferazione mucosale delle cellule T e dei leucociti IgT-specifici. Sono, inoltre, discusse le risposte immunitarie dopo l'immunizzazione mucosale e sistemica della spigola europea contro il Betanodavirus. Dottorato di ricerca in Genetica e biologia cellulare

Objective: to evaluate the role of Epstein-Barr virus (EBV) on the intestinal mucosa in the evolution of inflammatory bowel disease (IBD). The risk factors for EBV infection and the frequency of EBV-associated lymphoproliferative disorders in IBD patients were also investigated.Methods: intestinal biopsies of IBD patients with available EBV status determined by Epstein-Barr-encoding RNA (EBER) in situ hybridization were identified in the Pathology Database of our center. Clinical information, including phenotypic characteristics of IBD, previous treatments, diagnosis of lymphoma and patient outcome were reviewed in all cases.Results: fifty-six patients with IBD (28 Crohn's disease, 27 ulcerative colitis and one unclassified colitis) were included. EBV in intestinal mucosa was positive in 26 patients (46 %) and was associated to a lymphoproliferative syndrome in one case. EBV positivity was associated with severe histological activity (52 % vs 17.2 %; p 0.007), the presence of a lymphoplasmacytic infiltrate (50 % vs 33.3 %; p 0.03) and active steroid treatment (61.5 % vs 33.3 %; p 0.03). Multi-variate analyses only found an association between EBV and lymphoplasmacytosis (p 0.001). Escalation in previous treatment was significantly more frequent in the EBER+ group (53.8 % vs 26.7 %; p 0.038). No cases developed lymphoma during follow-up.Conclusions: EBV on the intestinal mucosa is associated with a poor outcome of IBD and the need for escalation of therapy. Lymphoplasmacytic infiltrate is associated with EBV infection. EBER+ patients used steroids more frequently compared with EBER- patients. No EBER+ patients developed lymphoma during follow-up.

Background: Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization.Results: Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four CH domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (CH2, CH3 and CH4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT+ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma.Conclusions: In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.