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Background No sequential long-term data exist for Greece on the etiological evolution and incidence of cirrhosis and hepatocellular carcinoma. Therefore, we studied their etiological evolution over a period of 25 years in the island of Crete. Methods We studied 812 cases of cirrhosis (561 male, median age 69 years) and 321 cases of hepatocellular carcinoma (234 male, median age 70 years) from the database of our Center. Cases were classified into five-year periods according to incidence and etiology (hepatitis B, hepatitis C, alcohol, alcohol plus viral, and non-alcoholic fatty liver disease). Results Overall, there was an increase in the incidence of hepatocellular carcinoma. A significant fourfold reduction in the incidence of hepatitis C-related cirrhosis was observed, which was degraded from first to third place as a risk factor for cirrhosis. Alcohol gradually became the first risk factor in cirrhosis (1990-94: 36.1%, 2010-14: 52.3%) and carcinoma, while the steepest increase in incidence of cirrhosis and carcinoma was associated with non-alcoholic fatty liver disease. Conclusions The incidence of cirrhosis remained constant over the years, but the incidence of hepatocellular carcinoma increased during the last decade. Risk factors for cirrhosis and hepatocellular carcinoma have changed over the past 25 years in Crete. The initial high hepatitis C virus association has significantly decreased, with alcohol now ranking first among risk factors. Non-alcoholic fatty liver disease is continually increasing and is a prominent risk factor for cirrhosis and hepatocellular carcinoma. Keywords Hepatocellular carcinoma, cirrhosis, non-alcoholic fatty liver disease, HCV, hepatocellular carcinoma Ann Gastroenterol 2017; 30 (3): 357-363

Summary The CXC chemokines, monokine induced by interferon (IFN)-gamma (MIG) (CXCL9), IFN-gamma-induced protein 10 (IP-10) (CXCL10) and IFN-inducible T cell alpha chemoattractant (I-TAC) (CXCL11), are known to attract CXCR3- (CXCR3A and CXCR3B) T lymphocytes. We investigated MIG, IP-10 and I-TAC mRNAs expression by semi-quantitative multiplex reverse transcription–polymerase chain reaction (RT–PCR) in liver biopsies obtained from patients with a first diagnosis of primary biliary cirrhosis [(PBC) = 20] compared to patients with normal liver biopsy [normal controls (NCs) = 20]. Chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) in serum. Measurements were repeated 6 months after ursodeoxycholic acid (UDCA) treatment in PBC patients. CXCR3A and CXCR3B mRNAs expression was examined in immunomagnetically sorted CD3+ peripheral blood lymphocytes (PBL) pre- and post-treatment by RT–PCR. Flow cytometry was used to evaluate the expression of CXCR3+ PBLs of NCs and PBC patients. A marked mRNA expression of MIG and IP-10 was found in PBC patients. I-TAC mRNA was not detected. In serum of PBC patients there was a significant increase of MIG and IP-10 compared to NCs. Interestingly, there was a significant reduction of these proteins in patients' serum after UDCA treatment. I-TAC was not statistically different between groups. CXCR3A mRNA expression was found in PBLs from PBC patients as well as in NCs. CXCR3B mRNA was expressed in four of 20 (19%) NCs and 20 of 20 PBC patients. Flow cytometry revealed a significantly lower CXCR3 expression in NCs (13·5%) than in PBC (37·2%), which was reduced (28·1%, P

Eur J Clin Invest 2012 Abstract Background Activin-A is a molecule of the TGF superfamily, implicated in liver fibrosis, regeneration and stem cell differentiation. However, data on activins in liver diseases are few. We therefore studied serum levels of activin-A in chronic liver diseases. To identify the origin of activin-A, levels in the hepatic vein were also estimated. Materials and methods Nineteen controls and 162 patients participated in the study: 39 with hepatocellular carcinoma (HCC: 19 viral associated and 20 alcohol associated), 18 with chronic hepatitis C (CHC), 47 with primary biliary cirrhosis (26 PBC stage I–II and 21 stage IV), 22 with alcoholic cirrhosis (AC, hepatic vein blood available in 16), 20 with HCV cirrhosis (hepatic vein blood available in 18) and 16 patients with alcoholic fatty liver with mild to moderate fibrosis but no cirrhosis. Results Activin-A levels were significantly increased (P

Introduction: Circulating tumor cells (CTCs) are considered as a “liquid biopsy” that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers, including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab. Methods: CTCs were isolated based on their size using the ISET platform from 27 patients before treatment, after 1 cycle and 3 cycles. CTCs were detected with Giemsa staining and immunofluorescence (IF) experiments, using either pancytokeratin (A45-B/B3) (CK7)/PD-1/CD45 or (A45-B/B3)(CK7)/PD-L1/CD45 combination of antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC cell lines: H460, H1299, HCC827 and SKMES in normal blood were used to evaluate the detection method. Results: Giemsa evaluation in Nivolumab-treated patients at baseline (25 evaluable samples), after the 1st (9 evaluable samples) and the 3rd (8 evaluable patients) cycle of treatment showed that CTCs could be detected in 48% (12/25), 33.3% (3/9) and 50% (4/8) of patients, respectively. IF could also reveal the presence of CK-positive cells in 44.4% (12/27), 22% (2/9) and 75% (6/8) patients, respectively. PD-1 (+) CTCs were detected in 33.3% (4/12) of patients at baseline, in, 0% after the 1 and 16.7% (1/6) of patients after the 3rd cycle. The same percentages were identified for PD-L1 expression in the same cohort of patients. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Multivariate analysis revealed that the presence of CK-positive cells is an independent prognostic factor for OS (p=0.028) Conclusion: Nivolumab reduced the number of PD-1- and PD-L1-expressing CTCs in advanced NSCLC patients. Furthermore the expression of both markers at baseline is associated with the clinical outcome. Citation Format: Galatea Kallergi, Anastasios Koutsopoulos, Eleni Lagoudaki, Despoina Agouraki, Eleni-Kiriaki Vetsika, Anastasia Voumvouraki, Stuart S. Martin, Filippos Koinis, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Detection of PD-L1 and PD-1 positive circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1587.

A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y-chromosome instability. We studied 17 clinically well-characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y-chromosome specific-sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y-chromosome loci hosting genes other than SRY, and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y-chromosome instability.

Η ηπατική ίνωση είναι ένα δυναμικό φαινόμενο η πρόοδος του οποίου εξαρτάται από την ενεργοποίηση των αστεροειδών κυττάρων του ήπατος. Η παραγωγή θεμέλιας ουσίας υπόκειται στην επίδραση πολλών κυτταροκινών ενώ η κλινική παρακολούθηση ασθενών με χρόνια ηπατοπάθεια επιβάλλει την παρακολούθηση της εξέλιξης της ίνωσης. Μέχρι σήμερα χρυσός κανόνας παραμένει η βιοψία ήπατος, μία μέθοδος αιματηρή με βαθμό νοσηρότητας. Επιπλέον, παράγοντες πού χρησιμοποιούνται θεραπευτικά είναι δυνατόν να επηρεάσουν αρνητικά την εξέλιξη της ίνωσης με απρόβλεπτα αποτελέσματα. Η Οκτρεοτίδη (συνθετική σωματοστατίνη) χρησιμοποιείται ευρέως για τον έλεγχο της κιρσορραγίας και τη θεραπεία του ηπατοκυτταρικού καρκινώματος. Η επίδραση της στην διαδικασία της ίνωσης δεν έχει μελετηθεί. ΣΚΟΠΟΣ: Μελετήθηκε η επίδραση της Οκτρεοτίδης σε παραμέτρους του ορρού σχετιζόμενους με την ίνωση ασθενών με χρόνια ηπατοπάθεια. ΜΕΘΟΔΟΙ: Επιλέχθηκαν πρωτεΐνες του ορρού πού έχει αποδειχθεί ότι σχετίζονται με την ίνωση. Μελετήθηκαν 4 ομάδες πρωτεϊνών: α) Οι πρωτεΐνες λεπτίνη, λαμινίνη, κολλαγόνο-IV και το υαλουρονικό οξύ. Μετρήθηκαν στο περιφερικό αίμα ασθενών με χρόνιες ηπατοπάθειες όπως και στο αίμα των ηπατικών φλεβών κατόπιν καθετηριασμού κιρρωτικών ασθενών. Επιδιώχθηκε με την δημιουργία περιοχών κάτω από την καμπύλη (Area Under the Curve,AUC), να διερευνηθεί η δυνατότητα διαχωρισμού ασθενών με χρόνια ιογενή ηπατίτιδα από ασθενείς με κίρρωση, όπως και η δυνατότητα διαχωρισμού ασθενών με πρώιμα στάδια πρωτογενούς χολικής κιρρώσεως από το κιρρωτικό στάδιο της νόσου. Μελετήθηκε επίσης η επίδραση της Οκτρεοτίδης στους παράγοντες αυτούς. Οι μετρήσεις έγιναν με εμπορικά διαθέσιμες ELISA. β) Στους ίδιους ασθενείς μελετήθηκε βιοχημικά (ELISA), η ομάδα των πρωτεϊνών TGFβ, δηλαδή οι TGFβ1, TGFβ2, TGFβ3 και η επίδραση της Οκτρεοτίδης στα επίπεδά τους. Επί πλέον, στο μέρος αυτό μελετήθηκε ιστοχημικά (μέθοδοι αλκαλικής φωσφατάσης και ανοσοφθορισμού) η εντόπιση στον ηπατικό ιστό των πρωτεϊνών αυτών. Επίσης, μελετήθηκε σε περιορισμένο αριθμό ηπατικών ιστών η έκφραση του mRNA της πρωτεΐνης FoxP3 χαρακτηριστικής των Treg κυττάρων, δεδομένου ότι δυνητικά θα μπορούσε να επηρεάζεται από τα επίπεδα των ισομορφών του TGFβ. γ) Σε άλλη ομάδα ασθενών μελετήθηκε η πρωτεΐνη Ακτιβίνη Α (ELISA), πού επίσης σχετίζεται με την ίνωση αλλά και την αναγέννηση των ηπατοκυττάρων και η επίδραση της Οκτρεοτίδης στα επίπεδα της. Στο μέρος αυτό μελετήθηκε και η εντόπιση της πρωτεΐνης δεδομένου ότι υπάρχουν λίγες πληροφορίες σχετικά με αυτό. Απομονώθηκαν κύτταρα Kupffer και αστεροειδή κύτταρα ήπατος αρουραίου και μελετήθηκε η έκφραση της Ακτιβίνης Α και η επίδραση της Οκτρεοτίδης με ημιποσοτική PCR. δ) Τέλος μελετήθηκαν τα επίπεδα των μεταλλοπρωτεασών του ορρού και ο αναστολέας αυτών TIMP1 (ELISA), όπως και η επίδραση της Οκτρεοτίδης σε αυτούς. ..................

Activin-A is a molecule of the TGF superfamily, implicated in liver fibrosis, regeneration and stem cell differentiation. However, data on activins in liver diseases are few. We therefore studied serum levels of activin-A in chronic liver diseases. To identify the origin of activin-A, levels in the hepatic vein were also estimated.Nineteen controls and 162 patients participated in the study: 39 with hepatocellular carcinoma (HCC: 19 viral associated and 20 alcohol associated), 18 with chronic hepatitis C (CHC), 47 with primary biliary cirrhosis (26 PBC stage I-II and 21 stage IV), 22 with alcoholic cirrhosis (AC, hepatic vein blood available in 16), 20 with HCV cirrhosis (hepatic vein blood available in 18) and 16 patients with alcoholic fatty liver with mild to moderate fibrosis but no cirrhosis.Activin-A levels were significantly increased (P0·001) in serum of patients with AC (median 673 pg/mL, range 449-3279), compared with either controls (149 pg/mL, 91-193) or patients with viral cirrhosis (189 pg/mL, 81-480), CHC (142 pg/mL, 65-559) PBC stage I-II (100 pg/mL, 59-597) and PBC stage IV (104 pg/mL, 81-579). Only patients with AC-associated HCC had significantly increased levels of activin-A (2403 pg/mL, 1561-7220 pg/mL). Activin-A serum levels could accurately discriminate AC from cirrhosis of other aetiologies and noncirrhotic alcoholic fatty liver with fibrosis.Increased serum levels of activin-A only in patients with alcohol-related cirrhosis or HCC suggest a possible role of this molecule in the pathophysiology of AC. Further research is warranted to elucidate its role during the profibrotic process and its possible clinical applications.

Background Primary biliary cirrhosis (PBC) is an organ specific autoimmune disease of still unidentified genetic etiology. We have shown that endothelins (ETs), produced by the liver endothelial cells are increased in PBC and may play a major pathogenetic role. Aims To study gene polymorphisms related to the endothelial cells ( eNOS , EDN-1 genes) and, to investigate whether the previously reported association of CTLA4 gene polymorphisms is replicated in a genetically homogeneous Greek population. Patients and methods Genomic DNA was extracted from 100 PBC patients (83 females, 93% AMA+, 74/100 Ludwig stage I–II) and 158 healthy controls. eNOS , CTLA4 and ET1 polymorphisms were determined by PCR–RFLPs analysis. Results Both eNOS intron4 VNTR and eNOS exon7 G894T SNP were significantly associated with increased risk in PBC. EDN-11 rs2071942 “A” and rs5370 “T” alleles appeared a tendency for association with disease progression. No association was found between PBC and the CTLA4 SNPs analyzed. Conclusions We demonstrated that eNOS , a gene related to the liver endothelium function is associated with PBC. Contrarily, the important in adaptive immunity gene CTLA4 was not associated with the disease in the homogeneous population analyzed. These results are compatible partially with our previous hypothesis that defects of the liver endothelial system, leading to endothelin overproduction, may be a fundamental early pathogenetic mechanism in PBC.

Background and PurposezzThe potential association between the severity of autonomic dys- function and peripheral neuropathy has not been extensively investigated, with the few studies yielding inconsistent results. We evaluated the relationship between autonomic dysfunction and peripheral neuropathy in chronic hemodialysis patients in a cross-sectional study. MethodszzCardiovascular autonomic function was assessed in 42 consecutive patients with ch- ronic renal failure treated by hemodialysis, using a standardized battery of 5 cardiovascular reflex tests. Symptoms of autonomic dysfunction and of peripheral neuropathy were evaluated using the Autonomic Neuropathy Symptom Score (ANSS) and the Neuropathy Symptoms Score. Neurolo- gical deficits were assessed using the Neuropathy Disability Score. Conduction velocities along the sensory and motor fibers of the sural and peroneal nerves were measured. Thermal thresholds were documented using a standardized psychophysical technique. ResultszzParasympathetic and sympathetic dysfunction was prevalent in 50% and 28% of cases, respectively. Peripheral neuropathy was identified in 25 cases (60%). The prevalence of periph- eral neuropathy did not differ between patients with impaired (55%) and normal (75%) auto- nomic function (p=0.297; Fisher's exact test). The electrophysiological parameters for peripheral nerve function, neuropathic symptoms, abnormal thermal thresholds, age, gender, and duration of dialysis did not differ significantly between patients with and without autonomic dysfunction. Patients with autonomic dysfunction were more likely to have an abnormal ANSS (p=0.048). The severity of autonomic dysfunction on electrophysiological testing was positively correlated with ANSS (r=0.213, p=0.036). ConclusionszzThe present data indicate that although cardiovascular autonomic dysfunction is prevalent among patients with chronic renal failure, it is not associated with the incidence of pe- ripheral neuropathy. J Clin Neurol 2010;6:143-147

Introduction Lamivudine has been largely replaced for the treatment of chronic HBV infection by new (and more expensive) antivirals with a high viral resistance barrier. It has been reported that lamivudine resistance can be as high as 20% after one year and up to 80% at five years. We therefore assessed a cohort of our patients for the development of Lamivudine resistance over time. Method 100 chronic HBV hepatitis patients, histologically diagnosed (male 67, female 33), median age 59 years (range 20–75), initially received lamivudine and were followed up for at least 5 years. All were HBeAg negative, HbeAb positive. Genotype D was found in all 23 patients genotyped. All were non-cirrhotic on liver biopsy before treatment (66% Ishak fibrosis stage 2–3, 34% Ishak stage 4–5). Median HBV DNA at the commencement of treatment was 450000 IU/ml. Only 12 patients had a viral load over 1000000 IU/ml. Lamivudine resistance was considered when an increase of 2 logs in the viral load, followed by an aminotransferase increase after an initial response was noted on follow up. Results All patients responded to lamivudine with normalisation of aminotransferases and subsequent reduction of HBV DNA to less than 100 IU/ml within 6 months from treatment initiation. Lamivudine resistance was 5% at one year, 10% at 2 years, 17% at 4 years and 21% at 5 years Conclusion The risk for lamivudine resistance development in our chronic HBV hepatitis patients is well below the reported in the literature. Whether this is due to the relative lower levels of viral load or to the lack of cirrhotics in our cohort remains to be further examined. Nonetheless we believe that in view of the low cost, lamivudine use should possibly be reconsidered as a first line treatment In HBV infection for non-cirrhotic patients with a viral DNA below 1000000 IU/ml. Disclosure of interest None Declared.

A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y-chromosome instability. We studied 17 clinically well-characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y-chromosome specific-sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y-chromosome loci hosting genes other than SRY, and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y-chromosome instability. (c) 2005 Wiley-Liss, Inc.

Cytogenetic and FISH analysis was performed in 139 patients to detect the pathognomonic of Di George/ Velocardiofacial syndrome (DGS/VFCS) deletion 22q11.2. An abnormal karyotype was revealed in 2/139 cases (47, XXY and 46, XX, 2p+). A deletion was found in 17/139 (12.2%) patients (14 males/ 3 females), inherited in 3 (2 maternal and 1 paternal). Patients with 22q11.2 deletion exhibited facial dysmorphic features (82%), congenital heart defects (70%), immunological problems (47%), multiple congenital anomalies (64%), hypocalcemia (47%), mental retardation/learning difficulties (35%), cleft palate/velopharyngeal insufficiency (23.5%), seizures/hypotonia (23%) and growth retardation (12%). Among 56/139 patients with detailed available clinical data, the 22q11.2 deletion was confirmed in all cases with hypocalcemia and in over half of the cases with multiple congenital anomalies, immunological problems and hypotonia/seizures (70%, 60% and 57%, respectively). Genetic reevaluation of 39 patients without the 22q11.2 deletion contributed to the classification of 14 (37%) under different syndromes, emphasizing the need for stricter referral criteria.

Introduction, background and aim Activin A is a molecule of the TGF superfamily, implicated in liver fibrosis, regeneration and stem cell differentiation. However, data on activins in liver diseases are very few. The authors therefore studied serum levels of Activin A in chronic liver diseases. To identify the origin of Activin A, levels in the hepatic vein were estimated and expression of Activin A was studied in isolated rat Kupffer and stellate cells. Methods 162 patients participated in the study: 39 with Hepatocellular Carcinoma (HCC, 19 viral cirrhosis associated, 20 alcoholic cirrhosis associated), 18 with Chronic hepatitis C (CHC), 47 with Primary Biliary Cirrhosis (PBC, 26 stage I–II and 21 stage IV), 22 with Alcoholic cirrhosis (hepatic vein blood available in 16), 20 with HCV cirrhosis (hepatic vein blood available in 18) and 16 patients with alcoholic fatty liver with mild to moderate fibrosis but no cirrhosis. 19 normal controls were also included. A commercially available ELISA was used for serum determinations and a semiquantitative PCR for Activin A expression in isolated rat Kupffer and Stellate cells. Results Activin-A levels were significantly increased (p Conclusions Serum levels of Activin A are increased in patients with alcohol related cirrhosis or HCC and can discriminate these patients from cirrhotics of other aetiologies.

Introduction Conflicting data for the role that cigarette smoking may play in Primary Biliary Cirrhosis (PBC) have been reported. Some studies have suggested an association of smoking with a more advanced fibrotic stage. The aim of the present study therefore was to assess the association between smoking and a) the severity of histological findings at the time of diagnosis, b) the immunological features of a genetically homogeneous and geographically defined population of PBC patients. Methods Smoking history data were collected from 171 PBC patients of Cretan origin (163 female) using a standardised questionnaire. Diagnosis was based on standard biochemical, Immunological and histological criteria. Liver biopsy was performed in 148 patients at diagnosis. Liver fibrosis and histological inflammatory activity were semi-quantified according to a METAVIR-based classification system. Odds ratios (OR) were assessed using logistic regression analysis. Results Smoking history prior to diagnosis was reported in 56 patients (32,7%%). Twenty-six patients (15,2%) were active smokers at diagnosis. Male gender (AOR 8.19, 95% CI: 3.014–11.937), alcohol intake >20 g/d (AOR, 2.20, 95% CI: 1.029–4.099), severe steatosis (AOR, 5.31, 95% CI: 2.019–9.919)), and F3–F4 fibrosis stage (AOR 1.21 95% CI: 1.015–3.031), but not piecemeal necrosis grade, bile duct paucity and cholangitis, or immunological laboratory data, were associated with smoking history. Multiple logistic regression analysis identified smoking intensity, years of passive smoking and significant necroinflammatory histological activity as independent risk factors of advanced liver fibrosis (F3–F4 stage) at diagnosis, adjusted for age, gender, BMI and alcohol consumption. For every pack-year increase in smoking intensity there was a 3.2 times higher likelihood of advanced fibrosis (95% CI: 2.018–6.294). Conclusion Our study results confirm the previously reported link between smoking history and the risk of advanced liver fibrosis at diagnosis in PBC. The mechanism by which smoking may accelerate the histological progression of PBC is unknown and larger studies are needed to define it. Disclosure of Interest None Declared.

Introduction It is well known that polymorphisms of the TPMT gene (coding for thiopourine methyl-transferase), influence response to treatment with azathioprine. Polymorphisms of the ABCB1 gene (coding for p-glycoprotein 170) has been associated with IBD and resistance to treatment but results are conflicting. The aim of this study was to determine the frequencies of TPMT and ABCB1 gene polymorphisms in IBD patients from Crete, a population genetically homogeneous, and how these polymorphisms might influence response to treatment and disease behaviour. Methods A total of 222 IBD patients records were reviewed for intake of azathioprine, possible adverse reactions, response to treatment and need for colectomy. All patients were genotyped for TPMT gene polymorphisms, that have been related to intolerance to azathioprine (G238C, G460A and A719C) as well as ABCB1 gene polymorphisms (G2677T/A and C3435T), using a PCR-RFLP method. The same polymorphisms were also determined in 119 age and sex healthy controls. Results Allele frequencies of TPMT gene in our study population were found to be in concordance with those reported in other Caucasian populations. 76 IBD patients were identified receiving azathioprine, of whom 16 were discontinued (10 CD, 6 UC) due to adverse reaction. 2 of them were found to carry the G460A and A719G alleles (TPMT 3A genotype) (12.5%). For the ABCB1 gene, G2677T/A allele frequencies were found to be similar to those reported in the literature. There was no association of G2677T/A or C3435T with clinical phenotype, or resistance to treatment. However, 77.3% of 22/222 patients who did not respond to therapy and required surgery, where found to carry both the C3434T and the G2677T mutation. Conclusion Our study was conducted in a genetically homogenous population in the island of Crete. No correlation of any single SNP was found with either clinical activity or response to treatment. However, most patients who carried both the G2677T and C3435T mutations were refractory to treatment, a finding which implies that resistance to treatment in IBD patients is a more complex issue, which requires the presence of a genetic locus rather than a single SNP. Disclosure of Interest None Declared.

Causes of chromosomal nondisjunction is one of the remaining unanswered questions in human genetics. In order to increase our understanding of the mechanisms underlying nondisjunction we have performed a molecular study on trisomy 8 and trisomy 8 mosaicism. We report the results on analyses of 26 probands (and parents) using 19 microsatellite DNA markers mapping along the length of chromosome 8. The 26 cases represented 20 live births, four spontaneous abortions, and two prenatal diagnoses (CVS). The results of the nondisjunction studies show that 20 cases (13 maternal, 7 paternal) were probably due to mitotic (postzygotic) duplication as reduction to homozygosity of all informative markers was observed and as no third allele was ever detected. Only two cases from spontaneous abortions were due to maternal meiotic nondisjunction. In four cases we were not able to detect the extra chromosome due to a low level of mosaicism. These results are in contrast to the common autosomal trisomies (including mosaics), where the majority of cases are due to errors in maternal meiosis.

AIM: To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis (PBC). METHODS: Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B (HBV) virus, 3 patients with hepatitis C virus (HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis (AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targeted by autoantibodies present in the patients sera was performed by indirect immunofluorescence (IIF) analysis using paraffin-embedded liver sections as a substrate. Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy. RESULTS: In total, 18/21 (85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21 (47.6%) in lymphocytes, 8/21 (38%) in cholangiocytes and 7/21 (33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G (IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining (20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody, positive staining was observed in 75% of lymphocytes, 62.5% of cholangiocytes, 37.5% of hepatocytes and 50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections, weak positive staining of cholangiocytes was observed in 3/21 (14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients. CONCLUSION: In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

We present a study of a mentally retarded and mildly dysmorphic female in whom initial cytogenetic studies identified the karyotype 46,X, + mar. Further characterisation of the structurally abnormal chromosome by fluorescence in situ hybridisation (FISH) showed that it is composed of both X and Y chromosome material with a centromere originating from the Y chromosome. The presence of the DMD gene and the absence of the XIST gene was shown by FISH using locus specific probes. The Y segment included the SRY and ZFY genes. Based on these findings, the karyotype was defined as 46, X,der(Y)t(X;Y) (p21.1;q11). This case illustrates male to female sex reversal owing to a partial duplication of the short arm of the X chromosome in the presence of SRY.

Summary We present a case of a female patient with monosomy of X chromosome in peripheral lymphocytes and skin fibroblasts, normal ovarian function and associated multiple congenital abnormalities of the aorta: bicuspid aortic valve, dilatation of the ascending aorta and multiple cystic structures of the aortic wall, complicated by endarteritis. We review the literature on fertile women with 45,X karyotype and the possible pathogenetic mechanisms of the aortic defects described as 'cystic medial necrosis of the aorta'.

Introduction Background and Aim : The CXC chemokines, MIG, IP-10 and I-TAC, are known to attract CXCR3+ T lymphocytes. CXCR3 gene generates two distinct mRNAs, CXCR3A and CXCR3B, by alternative splicing. In Primary Biliary Cirrhosis (PBC), intrahepatic bile ductules are destroyed by T lymphocytes. We investigated therefore the CXC chemokine axis in this disease. Methods Mig, IP-10 and I-TAC mRNAs expression was studied by RT-PCR in 20 liver biopsies from PBC patients (Ludwig stage II/III) and compared with normal biopsies (NCs = 20). Serum chemokines were assessed by ELISA in 44 PBC patients (Ludwig stage II/III) and 20 normals. Measurements were repeated six months after Ursodeoxycholate (UDCA) treatment. CXCR3A and CXCR3B mRNAs expression were examined in immunomagnetically sorted CD3+ peripheral blood lymphocytes (PBL) by RT-PCR, pre and post treatment. Flow cytometry evaluated the expression of CXCR3+ in PBL of NC and patients. Results A marked mRNA expression of MIG and IP10, but not of I-TAC, was found in PBC patients. Serum MIG (72.86 pg/ml) and IP-10 (660.1 pg/ml) were significantly increased in PBC, compared to NC (33.47 pg/ml and 37.58 pg/ml, respectively). There was a significant reduction of these proteins after treatment with UDCA (40.95 pg/ml for MIG and 289.2 pg/ml for IP-10). I-TAC was not different between groups. CXCR3A mRNA expression was found in PBLs from PBC patients and NCs. CXCR3B mRNA was expressed in 4/20 (19%) NCs and 20/20 PBC patients. Flow cytometry revealed a significantly lower CXCR3 expression in NCs (13.5%) than in PBC (37.2%) which was reduced (28.1%, p Conclusion These data suggest a possible role of CXCR3-binding chemokines and their receptor in the aetiopathogenetic recruitment of lymphocytes in PBC and a new mechanism of action of UDCA.

AIM: To investigate the transforming growth factor-β (TGF-β) isoforms in the peripheral and hepatic venous blood of primary biliary cirrhosis (PBC) patients. METHODS: We examined TGF-β1, TGF-β2 and TGF-β3 (enzyme-linked immunosorbent assay), in 27 stage IV PBC patients (27 peripheral and 15 hepatic vein sera), 35 early (I-II) PBC and 60 healthy controls. As disease controls 28 hepatitis C virus (HCV) cirrhosis (28 peripheral and 17 hepatic vein serum), 44 chronic HCV hepatitis and 38 HCV-related hepatocellular carcinomas were included. We also tested liver tissue by immunohistochemistry to identify localization of TGF isoforms. RESULTS: TGF-β1 was significantly decreased in all cirrhotics (PBC III-IV: median 13.4 ng/mL; range, 7.4-26.2, HCV cirrhosis: 11.6 ng/mL; range, 5.0-33.8), compared to controls (30.9 ng/mL; range, 20.9-37.8). TGF-β2 was increased in viral cirrhosis but not in PBC and chronic hepatitis. TGF-β3 (47.2 pg/mL; range, 27.0-79.7 in healthy controls) was increased in early and late PBC (I-II: 94.3 pg/mL; range, 41.5-358.6; III-IV: 152.8 pg/mL; range, 60.4-361.2; P < 0.001) and decreased in viral cirrhosis (37.4 pg/mL; range, 13.3-84.0; P < 0.05). Hepatic vein TGF-β levels were analogous to those in peripheral blood. Immunohistochemistry identified all isoforms in portal tract lymphocytes, sinusoidal cells and cholangiocytes. TGF-β3 was additionally overexpressed in hepatocytes in PBC patients. CONCLUSION: The serum profile of TGF-β isoforms is different in cirrhotics. Increased TGF-β3 is characteristic of PBC. These findings may be related to the immunological abnormalities of PBC.