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ITMI20130513A1 - DIAGNOSTIC METHOD FOR AUTOIMMUNE PATHOLOGIES OF THE LIVER - Google Patents

DIAGNOSTIC METHOD FOR AUTOIMMUNE PATHOLOGIES OF THE LIVER Download PDF

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ITMI20130513A1
ITMI20130513A1 IT000513A ITMI20130513A ITMI20130513A1 IT MI20130513 A1 ITMI20130513 A1 IT MI20130513A1 IT 000513 A IT000513 A IT 000513A IT MI20130513 A ITMI20130513 A IT MI20130513A IT MI20130513 A1 ITMI20130513 A1 IT MI20130513A1
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autoimmune
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liver diseases
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Cristina Zanini
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Euroclone S P A
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    • G01N33/5067—Liver cells
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

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Description

Titolo: “Metodo diagnostico per patologie autoimmuni del fegato” Title: "Diagnostic method for autoimmune liver diseases"

DESCRIZIONE DESCRIPTION

Campo tecnico dell’invenzione Technical field of the invention

La presente invenzione concerne un metodo per la diagnosi di malattie autoimmuni che comprende l’uso di cellule come substrato in sostituzione dei substrati rappresentati da tessuti animali comunemente impiegati. The present invention relates to a method for the diagnosis of autoimmune diseases which includes the use of cells as a substrate to replace the substrates represented by commonly used animal tissues.

Descrizione dell’arte nota Description of the known art

Le malattie autoimmuni costituiscono un insieme di patologie caratterizzate da un'alterazione del sistema immunitario che comporta lo sviluppo di risposte cellulari e umorali dirette contro componenti dell'organismo (self). In un individuo sano il sistema immunitario distingue gli antigeni propri dell’organismo, denominati self, da quelli esogeni (non-self) grazie a un processo noto come “tolleranza immunologica”. Questa prevede che i linfociti che si attivano contro gli antigeni del self, e che si definiscono autoreattivi, vengano eliminati negli organi linfoidi primari (tolleranza centrale) oppure che vengano eliminati o almeno resi non-responsivi (anergici) negli organi linfoidi secondari (tolleranza periferica) [Lampropoulou V e coll, 2008]. Nell’individuo affetto da malattia autoimmunitaria si manifesta, invece, una condizione denominata “rottura della tolleranza”; questa è dovuta ad un’alterata selezione e/o regolazione dei cloni autoreattivi, nonché ad anomalie nella presentazione dell’antigene. Sulla superficie delle cellule presentanti l’antigene (APC) si ritrova il complesso maggiore di istocompatibilità di tipo II (MHC II), responsabile della presentazione dell’antigene ai linfociti T. Nel caso di rottura della tolleranza centrale, il complesso MHC II presenta troppo debolmente l’antigene perché si abbia l’attivazione dei cloni autoreattivi, ma in modo sufficientemente perché si attui la selezione positiva [Benson R.A e coll, 2010]. Un ruolo importate nelle malattie autoimmuni è giocato anche dalla componente genetica, principalmente dall’associazione con lo human leukocyte antigen (HLA, l’equivalente dell’ MHC per l’uomo). Un esempio di controllo genetico sulla rottura della tolleranza è costituito dal gene Autoimmune Regulator (AIRE), posto sul cromosoma 21q22.3. Mutazioni nel gene AIRE riducono significativamente il repertorio di antigeni self contro i quali il sistema immunitario non reagisce; pertanto, non appena i linfociti T vengono liberati dal timo nel torrente circolatorio, hanno luogo numerosi processi di sensibilizzazione del sistema immunitario contro componenti del self [Matsumoto M., 2009]. Autoimmune diseases are a set of pathologies characterized by an alteration of the immune system that involves the development of cellular and humoral responses directed against components of the organism (self). In a healthy individual, the immune system distinguishes the body's own antigens, called self, from exogenous (non-self) ones thanks to a process known as "immunological tolerance". This provides that the lymphocytes that are activated against the self antigens, and which are defined as autoreactive, are eliminated in the primary lymphoid organs (central tolerance) or that they are eliminated or at least rendered non-responsive (anergic) in the secondary lymphoid organs (peripheral tolerance) ) [Lampropoulou V et al, 2008]. On the other hand, in the individual suffering from autoimmune disease, a condition called "breakdown of tolerance" occurs; this is due to an altered selection and / or regulation of the self-reactive clones, as well as to anomalies in the presentation of the antigen. The major histocompatibility complex type II (MHC II) is found on the surface of antigen presenting cells (APC), which is responsible for antigen presentation to T lymphocytes. weakly the antigen for the activation of the autoreactive clones, but sufficiently for the positive selection to take place [Benson R.A and coll, 2010]. An important role in autoimmune diseases is also played by the genetic component, mainly by the association with the human leukocyte antigen (HLA, the equivalent of MHC for humans). An example of genetic control over tolerance breakdown is the Autoimmune Regulator (AIRE) gene, located on chromosome 21q22.3. Mutations in the AIRE gene significantly reduce the repertoire of self antigens against which the immune system does not react; therefore, as soon as the T lymphocytes are released from the thymus into the bloodstream, numerous sensitization processes of the immune system against components of the self take place [Matsumoto M., 2009].

Attualmente, la diagnosi di malattie autoimmuni impiega due principali tecniche mediante determinazioni: Currently, the diagnosis of autoimmune diseases employs two main techniques by means of determinations:

- ELISA; - ELISA;

- IFA (immunofluorescenza indiretta). - IFA (indirect immunofluorescence).

In particolare, nel campo dell'immunofluorescenza indiretta si utilizzano, per gli anticorpi antitessuto, sezioni criostatiche di animali. Per alcune determinazioni sono per ora indispensabili tessuti di primati, come il terzo distale dell'esofago oppure il cervelletto di scimmia. In particular, in the field of indirect immunofluorescence cryostatic sections of animals are used for anti-tissue antibodies. For some determinations, primate tissues, such as the distal third of the esophagus or the monkey cerebellum, are indispensable for now.

L’uso di tessuti animali è tuttavia soggetto a non pochi problemi di tipo etico. However, the use of animal tissues is subject to many ethical problems.

Nell’ambito delle patologie autoimmuni, le epatopatie rappresentano un insieme di patologie caratterizzate da un campo diagnostico molto complesso, dovuto alla numerosità dei sottotipi della malattia (epatopatia autoimmune di tipo I, II e III sclerosi biliare primaria, lupus,..) e dalla differente specificità dei prodotti commerciali oggi disponibili per uso diagnostico, oltre che dalla difficoltà interpretativa del risultato clinico. In the context of autoimmune diseases, hepatopathies represent a set of pathologies characterized by a very complex diagnostic field, due to the number of subtypes of the disease (autoimmune liver disease type I, II and III primary biliary sclerosis, lupus, ...) and by the different specificity of commercial products available today for diagnostic use, as well as the difficulty in interpreting the clinical result.

E’ riconosciuto pertanto il bisogno di sviluppare metodi per la diagnosi di patologie autoimmuni del fegato che garantiscano una buona attendibilità dei risultati diagnostici ottenuti. The need to develop methods for the diagnosis of autoimmune liver diseases that ensure good reliability of the diagnostic results obtained is therefore recognized.

Inoltre, è ricercato un metodo che impieghi materiali alternativi rispetto ai tessuti animali. In addition, a method is being sought that uses alternative materials to animal tissues.

Riassunto dell’invenzione Summary of the invention

La presente invenzione sfrutta le potenzialità delle cellule derivanti da epatoma umano, eventualmente differenziate e manipolate ad hoc, in sostituzione dei tessuti animali e delle tecnologie delle sezioni al criostato. Rispetto al sacrificio di animali per congelarne i tessuti e allestire sezioni criostatiche da impiegare come substrati diagnostici, la coltura di cellule risulta una tecnica meno cruenta, più flessibile e facilmente applicabile alla preparazione di kit diagnostici. The present invention exploits the potential of cells deriving from human hepatoma, possibly differentiated and manipulated ad hoc, to replace animal tissues and cryostat section technologies. Compared to the sacrifice of animals to freeze their tissues and set up cryostatic sections to be used as diagnostic substrates, cell culture is a less bloody technique, more flexible and easily applicable to the preparation of diagnostic kits.

Breve descrizione delle Figure Brief description of the Figures

La Figura 1 mostra il risultato del saggio condotto su cellule da epatoma umano per anticorpi antimitocondrio; la Figura 2 mostra il risultato del saggio su cellule epatoma umano per autoanticorpi ASMA-proteine del citoscheletro (cerchiati); le Figure 3 e 4 mostrano i risultati dei saggi su cellule da epatoma umano per auto-anticorpi mitocondriali in pazienti affetti da cirrosi primaria biliare. Figure 1 shows the result of the assay conducted on human hepatoma cells for antimitochondrial antibodies; Figure 2 shows the result of the assay on human hepatoma cells for ASMA autoantibodies to cytoskeletal proteins (circled); Figures 3 and 4 show the results of human hepatoma cell assays for mitochondrial autoantibodies in patients with primary biliary cirrhosis.

Oggetto dell’invenzione Object of the invention

Un primo oggetto dell’invenzione è rappresentato da un metodo in vitro per la diagnosi e/o la prognosi di patologie autoimmuni del fegato che comprende l’impiego di cellule ottenute da carcinoma epatocellulare (epatoma). A first object of the invention is an in vitro method for the diagnosis and / or prognosis of autoimmune liver diseases which includes the use of cells obtained from hepatocellular carcinoma (hepatoma).

Un ulteriore oggetto dell’invenzione è rappresentato da un kit per la diagnosi e/o la prognosi di malattie autoimmuni del fegato. A further object of the invention is represented by a kit for the diagnosis and / or prognosis of autoimmune liver diseases.

Descrizione dettagliata dell’invenzione Secondo un primo oggetto, la presente invenzione mette a disposizione un metodo per diagnostica malattie autoimmuni del fegato. Detailed description of the invention According to a first object, the present invention provides a method for diagnosing autoimmune liver diseases.

Per tali scopi sono impiegate cellule da carcinoma epatocellulare. For these purposes, hepatocellular carcinoma cells are used.

Più in particolare, sono impiegate cellule HepaRG® (Biopredict, Francia), le quali rappresentano un tipico modello in vitro di epatociti umani con caratteristiche uniche riconducibili alle cellule epatiche primarie e che esprimono vari enzimi epatici come quelli del citocromo P450 (CYPs1A2*, 2C9, 2C19, 2D6, 2E1, 3A4*), enzimi di fase 2 (UGT1A1, GSTA1, GSTA4,GSTM1), recettori nucleari AhR, PXR, CAR, PPARa), alcuni dei quali sono presenti in sieri di pazienti affetti da epatopatia autoimmune (Autoantibodies Shoenfeld et al, 2007). More specifically, HepaRG® cells (Biopredict, France) are used, which represent a typical in vitro model of human hepatocytes with unique characteristics attributable to primary liver cells and which express various liver enzymes such as those of cytochrome P450 (CYPs1A2 *, 2C9 , 2C19, 2D6, 2E1, 3A4 *), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), nuclear receptors AhR, PXR, CAR, PPARa), some of which are present in sera from patients with autoimmune liver disease ( Autoantibodies Shoenfeld et al, 2007).

Le cellule HepaRG® sono in grado di formare una cocoltura di epatociti e di cellule epiteliali fenotipicamente simili a quelle dei dotti biliari. Inoltre, presentano caratteristiche riconducibili alle cellule staminali come la plasticità, cioè la divisione di cellule immature e la capacità differenziativa in epatociti maturi. HepaRG® cells are capable of forming a coculture of hepatocytes and epithelial cells phenotypically similar to those of the bile ducts. Furthermore, they have characteristics attributable to stem cells such as plasticity, i.e. the division of immature cells and the differentiation capacity in mature hepatocytes.

Le colonie che derivano da questa differenziazione sono facilmente individuabili grazie a due marcatori: la falloidina e il CYP3A4, che consentono di verificare con certezza il tipo di differenziamento occorso. The colonies that derive from this differentiation are easily identifiable thanks to two markers: phalloidin and CYP3A4, which allow us to verify with certainty the type of differentiation that has occurred.

In particolare, la falloidina è specifica per l’antigene dell’F-actina e, quindi, tale colorazione evidenzia la differenziazione in senso epatocitario. La presenza dell’anticorpo per l’enzima specifico della catena respiratoria CYP3A4, invece, permette di evidenziare la componente cellulare dei dotti biliari e, quindi, la differenziazione in canalicoli biliari. In particular, phalloidin is specific for the F-actin antigen and, therefore, this coloring highlights the differentiation in the hepatocytic sense. The presence of the antibody for the specific enzyme of the respiratory chain CYP3A4, on the other hand, allows to highlight the cellular component of the bile ducts and, therefore, the differentiation into biliary canaliculi.

Secondo un aspetto particolare, il metodo dell’invenzione comprende l’impiego di cellule da derivate da epatoma umano completamente differenziate e, in particolare, in cellule dei canalicoli biliari oppure in epatociti. According to a particular aspect, the method of the invention comprises the use of cells derived from completely differentiated human hepatoma and, in particular, in cells of the biliary canaliculi or in hepatocytes.

In accordo con un altro aspetto dell’invenzione, invece, le cellule derivate da epatoma umano sono nello stato proliferativo oppure nello stato staminal-like. In accordance with another aspect of the invention, however, the cells derived from human hepatoma are in the proliferative state or in the stem-like state.

Le patologie autoimmuni del fegato che possono essere diagnosticate con il metodo dell’invenzione comprendono numerose malattie fra le quali l’epatopatia autoimmune, la sindrome degli anticorpi antifosfolipidi e, in particolare, la cirrosi biliare primaria (CBP). Autoimmune liver diseases that can be diagnosed with the method of the invention include numerous diseases including autoimmune liver disease, antiphospholipid antibody syndrome and, in particular, primary biliary cirrhosis (CBP).

Quest’ultima, in particolare è una patologia epatica cronica caratterizzata dalla distruzione dei piccoli dotti biliari intraepatici. The latter, in particular, is a chronic liver disease characterized by the destruction of the small intrahepatic bile ducts.

La caratteristica sierologica della cirrosi biliari primaria è rappresentata dalla presenza di anticorpi anti-mitocondriali rilevata nel 90-95% dei casi. The serological characteristic of primary biliary cirrhosis is represented by the presence of anti-mitochondrial antibodies detected in 90-95% of cases.

Più in dettaglio, il metodo comprende la determinazione della presenza di specifici anticorpi in un campione di siero di un soggetto sul quale si vuole effettuare la diagnosi. More in detail, the method comprises the determination of the presence of specific antibodies in a serum sample of a subject on which the diagnosis is to be made.

In particolare, si procede ponendo a contatto il campione di siero con una preparazione di cellule secondo la presente invenzione per un tempo sufficiente a consentire la formazione di complessi antigene-anticorpo specifici. In particular, the serum sample is brought into contact with a preparation of cells according to the present invention for a time sufficient to allow the formation of specific antigen-antibody complexes.

A tale proposito, l’incubazione può essere protratta per almeno 10-60 minuti, preferibilmente 20-40 minuti e generalmente 30 minuti, periodo dopo il quale si effettua un lavaggio allo scopo di rimuovere tutti i componenti non legati. In this regard, the incubation can be continued for at least 10-60 minutes, preferably 20-40 minutes and generally 30 minutes, a period after which a wash is carried out in order to remove all unbound components.

La presenza di anticorpi è in seguito determinata per immunofluorescenza indiretta. The presence of antibodies is then determined by indirect immunofluorescence.

Infatti, dopo il lavaggio viene aggiunto un anticorpo fluorescente. In fact, a fluorescent antibody is added after washing.

Per gli scopi della presente invenzione le cellule sono impiegate nella forma di una preparazione immobilizzata su di un supporto solido e a tale scopo può essere impiegato un comune vetrino da laboratorio. For the purposes of the present invention the cells are used in the form of a preparation immobilized on a solid support and for this purpose a common laboratory slide can be used.

In un secondo oggetto, il metodo dell’invenzione può essere impiegato anche per la prognosi delle stesse patologie autoimmuni, intendendosi con ciò che il metodo fornisce utili informazioni per valutare e predire se il soggetto svilupperà la patologia autoimmune nel tempo. In a second object, the method of the invention can also be used for the prognosis of the same autoimmune diseases, meaning that the method provides useful information for evaluating and predicting if the subject will develop the autoimmune disease over time.

In accordo con un ulteriore oggetto dell’invenzione, è descritto un kit per la diagnosi e/o la prognosi di malattie autoimmuni, che comprende una preparazione di cellule ottenute da epatoma umano. In accordance with a further object of the invention, a kit for the diagnosis and / or prognosis of autoimmune diseases is described, which includes a preparation of cells obtained from human hepatoma.

Per tale scopo, possono essere impiegate le cellule HepaRG® sopra menzionate. For this purpose, the above-mentioned HepaRG® cells can be used.

Tali cellule possono essere completamente differenziate in epatociti o in cellule dei dotti biliari. These cells can be completely differentiated into hepatocytes or bile duct cells.

Alternativamente, le cellule del kit dell’invenzione possono essere nello stato proliferativo o nello stato staminal-like. Alternatively, the cells of the kit of the invention can be in the proliferative state or in the staminal-like state.

Nell’ambito della presente invenzione, le cellule sono immobilizzare su di un supporto solido opportunamente trattato, ad esempio costituito da un comune vetrino da laboratorio. In the context of the present invention, the cells are immobilized on a suitably treated solid support, for example consisting of a common laboratory slide.

Oltre alla preparazione di cellule, il kit dell’invenzione comprende anticorpi fluorescenti per la determinazione degli autoanticorpi antimitocondrio. In addition to the preparation of cells, the kit of the invention includes fluorescent antibodies for the determination of antimitochondrial autoantibodies.

Parte sperimentale Experimental part

MATERIALI e METODI Materials and methods

Cellule HepaRG® sono state coltivate come da protocollo fornito dal produttore (Biopredic, Francia) su vetrini coattati con Collagene di tipo I (8 well Culture Slide) per facilitarne l’adesione. I vetrini sono stati lavati in PBS, lasciati asciugare per 10' e poi fissati in dimetilchetone freddo (4 C°) per 5 minuti. HepaRG® cells were cultured according to the protocol provided by the manufacturer (Biopredic, France) on slides coated with type I Collagen (8 well Culture Slides) to facilitate their adhesion. The slides were washed in PBS, left to dry for 10 'and then fixed in cold dimethylketone (4 ° C) for 5 minutes.

I sieri di pazienti positivi al test di screening per la cirrosi biliare primaria e con presenza di autoanticorpi anti mitocondrio (come mostrato dal saggio IFA) di pazienti con autoanticorpi anti proteine del citoscheletro e di pazienti con positività citoplasmatica mitocondriale sono stati saggiati sulla preparazione di cellule. Sera from patients positive in the screening test for primary biliary cirrhosis and with the presence of anti mitochondrial autoantibodies (as shown by the IFA assay) of patients with autoantibodies to cytoskeletal proteins and from patients with positive mitochondrial cytoplasmic were tested on the cell preparation .

Abbiamo quindi verificato il pattern di fluorescenza sulla linea cellulare HepaRG® completamente differenziata al giorno sette con il siero di un paziente affetto da cirrosi biliare primaria Risultati We then verified the fluorescence pattern on the fully differentiated HepaRG® cell line on day seven with serum from a patient with primary biliary cirrhosis. Results

I risultati dei saggi sono mostrati nelle figure. The results of the assays are shown in the figures.

In particolare, la Figura 1 mostra il risultato del saggio condotto su cellule HepaRG® al 7° giorno (di differenziazione) per anticorpi anti-mitocondrio. La Figura 2, invece, mostra il risultato del saggio dell’invenzione per valutare la presenza di autoanticorpi ASMA-proteine del citoscheletro (cerchiati) su cellule HepaRG® al 7° giorno di differenziazione. In particular, Figure 1 shows the result of the assay conducted on HepaRG® cells at day 7 (of differentiation) for anti-mitochondrial antibodies. Figure 2, on the other hand, shows the result of the invention assay to evaluate the presence of ASMA autoantibodies-cytoskeletal proteins (circled) on HepaRG® cells on the 7th day of differentiation.

Le Figure 3 e 4 mostrano i risultati di saggi per verificare la presenza di auto-anticorpi mitocondriali in altri due pazienti affetti da cirrosi primaria biliare sempre con cellule HepaRG® differenziate al 7° giorno. Figures 3 and 4 show the results of assays to verify the presence of mitochondrial auto-antibodies in two other patients affected by primary biliary cirrhosis always with differentiated HepaRG® cells on the 7th day.

La descrizione qui sopra riportata ha quindi evidenziato che cellule derivate da epatoma umano completamente differenziate (differenziamento al settimo giorno) oppure non ancora completamente differenziate, come ad esempio cellule nello stato proliferativo oppure nello stato staminal-like) possono essere impiegate come substrato nei metodi di diagnosi e/o prognosi in vitro delle patologie autoimmuni del fegato. The above description has therefore shown that cells derived from human hepatoma completely differentiated (differentiation on the seventh day) or not yet completely differentiated, such as cells in the proliferative state or in the stem-like state) can be used as substrates in the methods of in vitro diagnosis and / or prognosis of autoimmune liver diseases.

Claims (10)

RIVENDICAZIONI 1. Metodo in vitro per la diagnosi e/o la prognosi di patologie autoimmuni del fegato in un soggetto comprendente l’impiego di cellule ottenute da carcinoma epatocellulare. CLAIMS 1. In vitro method for the diagnosis and / or prognosis of autoimmune liver diseases in a subject including the use of cells obtained from hepatocellular carcinoma. 2. Metodo in vitro per la diagnosi e/o la prognosi di patologie autoimmuni secondo la rivendicazione precedente, in cui dette cellule ottenute da carcinoma epatocellulare sono completamente differenziate. 2. In vitro method for the diagnosis and / or prognosis of autoimmune pathologies according to the preceding claim, in which said cells obtained from hepatocellular carcinoma are completely differentiated. 3. Metodo in vitro per la diagnosi e/o la prognosi di patologie autoimmuni secondo la rivendicazione precedente, in cui dette cellule ottenute da carcinoma epatocellulare sono nello stato proliferativo. 3. In vitro method for the diagnosis and / or prognosis of autoimmune pathologies according to the preceding claim, in which said cells obtained from hepatocellular carcinoma are in the proliferative state. 4. Metodo in vitro per la diagnosi e/o la prognosi di patologie autoimmuni secondo la rivendicazione 1 o 2, in cui dette cellule ottenute da carcinoma epatocellulare sono cellule nello stato staminal-like. 4. In vitro method for the diagnosis and / or prognosis of autoimmune pathologies according to claim 1 or 2, in which said cells obtained from hepatocellular carcinoma are cells in the stem-like state. 5. Metodo in vitro per la diagnosi e/o la prognosi di epatopatie autoimmuni secondo una qualsiasi delle rivendicazioni precedenti, in cui dette patologie autoimmuni del fegato sono rappresentate da epatopatie autoimmuni, dalla sindrome degli anticorpi antifosfolipidi, cirrosi biliare primaria. 5. In vitro method for the diagnosis and / or prognosis of autoimmune liver diseases according to any one of the preceding claims, in which said autoimmune liver diseases are represented by autoimmune liver diseases, antiphospholipid antibody syndrome, primary biliary cirrhosis. 6. Metodo in vitro per la diagnosi e/o la prognosi di epatopatie autoimmuni secondo una qualsiasi delle rivendicazioni precedenti, comprendente la fase di porre a contatto per un tempo sufficiente una preparazione di cellule ottenute da carcinoma epatocellulare con un campione di siero. 6. In vitro method for the diagnosis and / or prognosis of autoimmune liver diseases according to any one of the preceding claims, comprising the step of contacting a preparation of cells obtained from hepatocellular carcinoma with a serum sample for a sufficient time. 7. Metodo in vitro per la diagnosi e/o la prognosi di epatopatie autoimmuni secondo la rivendicazione precedente, comprendente la fase ulteriore di determinare e/o quantificare la presenza di autoanticorpi anti-mitocondriali nel campione di serio. 7. In vitro method for the diagnosis and / or prognosis of autoimmune liver diseases according to the preceding claim, comprising the further step of determining and / or quantifying the presence of anti-mitochondrial autoantibodies in the serum sample. 8. Metodo in vitro per la diagnosi e/o la prognosi di epatopatie autoimmuni secondo la rivendicazione precedente, in cui detta determinazione e/o quantificazione avviene mediante immunofluorescenza indiretta. 8. In vitro method for the diagnosis and / or prognosis of autoimmune liver diseases according to the preceding claim, in which said determination and / or quantification takes place by indirect immunofluorescence. 9. Un kit per la diagnosi e/o la prognosi di patologie autoimmuni del fegato comprendente una preparazione di cellule derivanti da epatoma umano, eventualmente immobilizzata su di un supporto solido. 9. A kit for the diagnosis and / or prognosis of autoimmune liver diseases comprising a preparation of cells deriving from human hepatoma, possibly immobilized on a solid support. 10. Il kit per la diagnosi e/o la prognosi di patologie autoimmuni secondo la rivendicazione precedente, in cui detta preparazione è una preparazione di cellule differenziate oppure nello stato proliferativo oppure nello stato staminal-like.The kit for the diagnosis and / or prognosis of autoimmune pathologies according to the preceding claim, wherein said preparation is a preparation of differentiated cells either in the proliferative or in the stem-like state.
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