Georgios N. Dalekos

Safety and efficacy of lamivudine in patients with severe acute or fulminant hepatitis B, a multicenter experience

Summary.  Acute hepatitis B progresses to liver failure with the need of liver transplantation in about 1% of cases. We treated patients with severe acute or fulminant hepatitis B with lamivudine in an attempt to prevent hepatitis B virus (HBV) reinfection after potential liver transplantation. Since September 2000, 17 patients with severe acute or fulminant HBV infection were treated with 100 or 150 mg lamivudine daily once we had evidence for a severe course as indicated by an INR >2.0. These were compared to a historic control from our unit and to external patients. Fourteen of the 17 patients (82.4%) survived with full recovery without liver transplantation. All these 14 individuals cleared HBsAg on lamivudine within less than 6 months. Twelve patients recovered quickly as indicated by a normalized prothrombin time within 1 week while two patients had a more prolonged course. None of the patients showed an adverse event. Three patients requiring transplantation despite lamivudine therapy had more advanced disease on admission, of whom one had additionally ingested paracetamol (acetaminophen) while the second was already HBV‐DNA negative by polymerase chain reaction on admission. The lamivudine treated patients had significant higher frequency of survival without liver transplantation 82.4 vs 20% (4/20) in the historic control (P < 0.001). Similar data were derived from external centres using lamivudine (15/20, 75%). Lamivudine is safe in patients with severe acute or fulminant hepatitis B, leading to fast recovery with the potential to prevent liver failure and liver transplantation when administered early enough.

Value of anti-HBc screening of blood donorsfor prevention of HBV infection: results of a 3-year prospective study in Northwestern Greece

BACKGROUND: The risk of infection with transfusion‐transmitted viruses has been reduced remarkably. However, a zero‐risk blood supply remains a popular goal. Some authorities have introduced the screening for antibody to HBc (anti‐HBc) as a surrogate test for the presence of several infectious agents. A 3‐year prospective study was conducted in the Epirus region of Greece to determine the prevalence of several blood‐borne viruses. One component of the study was the prevalence of HBV infection markers and the potential value of anti‐HBc testing of donors in this area.

Autoantibodies and defined target autoantigens in autoimmune hepatitis: an overview

Autoimmune hepatitis (AIH) is a disease of unknown aetiology characterised by hypergammaglobulinaemia, non-organ and liver-related autoantibodies, association with HLA-DR3 or DR4 and a favourable response to immunosuppression. The current classification of AIH and the several autoantibodies/target autoantigens found in this disease are reported. The importance of these markers in the differential diagnosis and the study of pathogenesis of AIH is also given. AIH is subdivided into two major types: AIH type 1 (AIH-1) and AIH type 2 (AIH-2). AIH-1 is characterised by the detection of smooth muscle autoantibodies (SMA) and/or antinuclear antibodies (ANA). Antineutrophil cytoplasmic autoantibodies (ANCA), in most cases of perinuclear pattern (p-ANCA), by the indirect immunofluorescence assay, antibodies against the asialoglycoprotein receptor (anti-ASGP-R) and antibodies to soluble liver antigens or liver-pancreas (anti-SLA/LP) may be useful for the identification of individuals who are seronegative for ANA/SMA. AIH-2 is characterised by the presence of specific autoantibodies against liver and kidney microsomal antigens (anti-LKM type 1 or infrequently anti-LKM type 3) and/or autoantibodies against liver cytosol 1 antigen (anti-LC1). Anti-LKM-1 and anti-LKM-3 autoantibodies are also detected in some patients with chronic hepatitis C (HCV) and chronic hepatitis D (HDV). For these reasons, the distinction between AIH and chronic viral hepatitis is of particular importance. Cytochrome P450 2D6 (CYP2D6) is the major target autoantigen of anti-LKM-1 autoantibodies in both conditions (AIH-2 and HCV infection). Recent data have demonstrated the expression of CYP2D6 on the surface of hepatocytes, suggesting a pathogenetic role of anti-LKM-1 autoantibodies in liver injury. Family 1 of UDP-glycuronosyltransferases has been identified as the target autoantigen of anti-LKM-3. The molecular target of anti-SLA/LP autoantibodies has been identified recently as a 50 kDa protein with unknown structure and function. A liver-specific enzyme, the formiminotransferase cyclodeaminase, was identified as the target autoantigen of anti-LC1 autoantibodies. Anti-ASGP-R and anti-LC1 autoantibodies appear to correlate better with the severity of AIH and the response to treatment. The latter may suggest a pathogenic role of these autoantibodies in the hepatocellular damage in AIH. In general, however, autoantibodies should not be used to monitor treatment or to predict AIH activity or outcome. Finally, current knowledge concerning a specific form of AIH that may develop in some patients with a rare genetic syndrome, the autoimmune polyglandular syndrome type-1 (APS-1), is also discussed. Autoantibodies against liver microsomes (anti-LM) are the specific autoantibodies found in AIH as a disease component of APS-1. However, anti-LM autoantibodies have also been described in cases of dihydralazine-induced hepatitis. Cytochrome P450 1A2 has been identified as the target autoantigen of anti-LM autoantibodies in both disease entities.

Incidence of inflammatory bowel disease in north west Greece: rarity of Crohn's disease in an area where ulcerative colitis is common.

Ulcerative colitis (UC) and Crohns disease (CD) are generally regarded as diseases of affluent societies of the Western World, although their frequency in less affluent areas is not well established. This retrospective study therefore, assesses the incidence of UC and CD in a semirural area of north west Greece during the 10 year period 1982-1991. By the 31 December 1991, 61 patients had met standard diagnostic criteria for UC (annual incidence 4.0/10(5), 95% confidence intervals 3.0 to 5.0/10(5)) and only five patients met the diagnostic criteria for CD (annual incidence 0.3/10(5), 95% confidence intervals 0.1 to 0.8/10(5)) in this area of 157,214 inhabitants. UC incidence was lowest in the first three years at 1.8/10(5) per annum and subsequently increased to 4.8 and 5.1/10(5) per annum for the successive four and three year periods respectively. UC incidence was slightly higher in men. A third of all cases of UC had pancolitis while a quarter had only proctitis. More than one half were categorised as having moderate or severe colitis. Three quarters of the patients resided in urban areas. The incidence of CD was a twelfth of the UC incidence, which is in considerable contrast with most Western countries where the incidence of CD is usually no less than a third that for UC. The rarity of CD points to the absence of aetiological environmental factors specific for CD.

Increased incidence of anti-cardiolipin antibodies in patients with hepatitis C is not associated with aetiopathogenetic link to anti-phospholipid syndrome

Objective Chronic infection with hepatitis C virus (HCV) has been found to be associated with various diseases known as extra‐hepatic manifestations of HCV. Recently, HCV has been implicated as a cause of the anti‐phospholipid syndrome (APLS). We conducted a study in a well‐characterized area for epidemiological and prospective studies in the north‐western part of Greece in order to address whether an aetiopathogenesis exists between HCV and APLS. Design Seventy‐five patients with chronic hepatitis C were investigated for the presence of anti‐cardiolipin antibodies (anti‐CL) and for a past medical history supportive to the diagnosis of APLS. In addition, 24 patients with well‐defined APLS (primary or secondary) and 12 patients with systemic lupus erythematosus (SLE) were tested for the presence of markers of HCV infection (anti‐HCV and HCV RNA). The SLE patients were anti‐CL‐positive but none of them had developed any of the known clinical features of APLS. In addition, 267 healthy subjects were investigated for the presence of anti‐CL. Methods IgG and IgM anti‐CL were determined by a quantitative isotype‐specific solid phase enzyme‐linked immunosorbent assay set up in our laboratory. Anti‐HCV was determined using a third‐generation enzyme immunoassay and a confirmatory third‐generation recombinant immunoblot assay. Active virus replication was defined by the detection of HCV RNA using a combination assay based on a reverse transcriptase polymerase chain reaction and a DNA enzyme immunoassay. Results Of the HCV patients, 37.3% had IgG and/or IgM anti‐CL (P < 0.00005 compared to healthy controls (2.25%)). However, the mean titres of each specific isotype were significantly lower in HCV patients compared with those found in the APLS patients (P < 0.05 for IgM and P < 0.001 for IgG isotypes). The mean titres of IgG anti‐CL were also significantly lower in HCV patients compared with those found in the SLE patients (P < 0.01). All patients with APLS or SLE (n = 36) tested negative for HCV infection markers. In addition, neither thrombotic events nor thrombocytopenia were associated with a positive anti‐CL test in HCV patients. Conclusions A significant proportion of HCV patients (37.3%) had detectable anti‐CL of low titre. However, this finding was not associated with the development of APLS. On the other hand, none of the APLS patients was positive for HCV. Taken together, our data rather failed to reveal an aetiopathogenetic link between HCV and APLS. For this reason, testing for HCV in patients with APLS or follow‐up for the possibility of the development of APLS in HCV patients cannot be suggested, at least in Greek patients. More prospective studies of longer duration are required in order to address whether HCV is involved or not in the aetiopathogenesis of APLS. Eur J Gastroenterol Hepatol 12:67 ‐ 74

A prospective evaluation of dermatological side-effects during alpha-interferon therapy for chronic viral hepatitis.

OBJECTIVE Alpha-interferon therapy may occasionally account for immune-mediated phenomena. This study was conducted in an attempt to investigate the incidence of the development of immune-mediated dermatological diseases during alpha-interferon therapy in patients with chronic viral hepatitis. The latter has not been evaluated prospectively, whereas most of the previous studies examined small numbers of interferon treated patients or consisted of case reports. DESIGN A prospective case-control study. SETTING A tertiary referral centre. PARTICIPANTS One hundred and twenty consecutive patients with chronic viral hepatitis (67 with hepatitis B, 45 with hepatitis C, six with both hepatitis viruses, and two with delta hepatitis) were evaluated during a course of alpha-interferon therapy. In addition, 120 consecutive patients with chronic liver diseases (disease control group), who had never received alpha-interferon therapy, were evaluated during the period of the study (at least for 12 months). INTERVENTIONS Recombinant alpha-interferon at a dose of 4.5 or 5 million units subcutaneously (s.c.) three times per week for 6 to 12 months was administered to patients with hepatitis B. The patients with chronic hepatitis C were treated with 3 million units s.c. three times per week for 12 to 18 months. The patients with chronic hepatitis B and C infections received 4.5 million units for 6 months, and then 3 million units for an additional 6 to 12 months. Finally, the patients with chronic delta hepatitis received 5 million units for 1 year or more. MAIN OUTCOME MEASURES To assess prospectively the incidence of these dermatological disorders during alpha-interferon therapy and to estimate if there is any relationship between their development and the clinical, laboratory or other characteristics of the patients with chronic hepatitis. RESULTS Three to 6 months after the initiation of alpha-interferon three patients with chronic viral hepatitis (two with hepatitis C and one with hepatitis B) developed lichen planus, whereas one patient with hepatitis C developed relapsing aphthous stomatitis. The development of these disorders was significantly associated only with the presence of antinuclear antibodies before the initiation of alpha-interferon (P=0.000000). None of the patients from the disease control group had such a manifestation during the follow-up. Lichen planus resolved after the end of therapy in all of them. In contrast, therapy was discontinued in the patient who developed aphthous stomatitis, owing to the painful lesions. CONCLUSIONS This study demonstrated that alpha-interferon may rarely (3.3%) induce immune-mediated dermatological disorders, especially lichen planus. The development of these disorders may reflect a subclinical or covert autoimmune background of patients, as suggested by the presence, although in low titres, of antinuclear antibodies. However, when lichen planus developed, it was mild, did not require the discontinuation of therapy and resolved after alpha-interferon administration had ceased.

Soluble interleukin-2 receptors, antineutrophil cytoplasmic antibodies, and other autoantibodies in patients with ulcerative colitis.

Ulcerative colitis (UC) is an inflammatory bowel disease of unknown aetiology. In this study, serum samples from 80 patients with UC were studied for the presence of various autoantibodies and soluble interleukin-2 receptor molecules (sIL-2Rs) in an attempt to determine the degree of activation of the immune system in this disease process. Autoantibodies detected included rheumatoid factors (in 5% of patients), antinuclear antibodies (in 51.3%), anti-Ro(SSA) (in 1.3%), anticardiolipin antibodies (IgG and/or IgM classes in 26.3%), anti-double stranded DNA (IgG or IgM classes in 45%), and antineutrophil cytoplasmic antibodies (ANCAs, in 30%). The ANCAs had a perinuclear pattern (p-ANCA) in 95.8%, without anti-myeloperoxidase activity, at least in an enzyme linked immunosorbent assay (ELISA) system. Raised concentrations of sIL-2R were found in 32.5% of patients (26/80, 18 with active and eight with inactive UC). The mean (SD) sIL-2R concentrations were significantly higher in patients with active UC (595 (219) u/ml v 406 (162) u/ml, p = 0.0001) and in patients with ANCAs (584 (177) u/ml in ANCA positive v 447 (212) u/ml in ANCA negative patients, p < 0.01). The sIL-2R concentrations were correlated with increased serum concentrations of C3c (r = 0.23, p < 0.05) or C4 (r = 0.4, p < 0.001) components of the complement system and erythrocyte sedimentation rate (ESR, r = 0.44, p = 0.0001). Platelets, ESR, and C3c were not associated with disease activity (p = 0.06, 0.33 and 0.86) whereas mean (SD) serum concentrations of C4 were higher in active disease (37.4 (11.9) mg/dl v 32.3 (10.3) mg/dl, p < 0.05). The sIL-2Rs had 53% sensitivity and 82.6% specificity for disease activity whereas platelet counts had 53% sensitivity and 58.7% specificity. To conclude, UC is accompanied by an autoimmune response that results in the production of several autoantibodies and cellular immune activation, as shown by the high sIL-2R concentration, is also present. The identification of the target antigen(s) of p-ANCA would possibly act as an indicator of disease activity if this distinct subset of ANCAs can be attributed to the pathogenesis of UC. The sIL-2R concentrations seem to be a useful laboratory marker for assessing activity of the disease.

Occult hepatitis B virus infection in Greek patients with chronic hepatitis C and in patients with diverse nonviral hepatic diseases

Summary.  Occult hepatitis B virus (HBV) infection has been reported in patients with chronic hepatitis C who are negative for HBV surface antigen (HBsAg). However, the significance of ‘silent’ HBV in hepatitis C virus (HCV) infection is unknown. We investigated 540 subjects for the presence of occult HBV in Greek HCV patients, patients with nonviral liver diseases and healthy donors in an attempt to determine the frequency and importance of this phenomenon. One hundred and eighty‐seven anti‐HCV(+)/HBsAg(−) patients’ sera were investigated for the presence of HBV‐DNA by polymerase chain reaction. Two hundred and eighty‐two selected blood donors (positive for antibodies to HBV core antigen) and 71 patients with various nonviral hepatic diseases consisted the control groups [both controls were anti‐HCV(−)/HBsAg(−)]. HBV‐DNA was detected in 26.2% of HCV‐infected patients vs 8.5% of patients with nonviral diseases (P = 0.003) and 0/282 of donors (P = 0.0000). HBV‐DNA was neither associated with HBV markers, nor with the clinical status of HCV and nonHCV patients. Neither epidemiological, histologic and virologic data nor the response to therapy were associated with the HBV‐DNA detection. Hence one quarter of HCV‐infected patients had occult HBV infection. Similar findings were not found in both control groups. Occult HBV infection in Greek patients with chronic hepatitis C does not seem to modify the progression of chronic liver disease. Further studies of longer duration are needed in order to clarify the role of ‘silent’ HBV infection in HCV‐infected patients.

Increased incidence of anti-LKM autoantibodies in a consecutive cohort of hepatitis C patients from central Greece.

Objectives In Greece, there are insufficient data regarding the presence of non-organ and liver-related autoantibodies in hepatitis C patients. This study in a consecutive cohort of 39 such patients from central Greece investigates (1) the prevalence of non-organ and liver-related autoantibodies, and (2) the reactivity of anti-liver–kidney microsomal type 1 antibodies (in the case of positivity with at least one of the methods used) against their molecularly defined antigens. Design All serum samples were tested by standard and molecular assays for the presence of anti-nuclear antibodies, smooth muscle antibodies, anti-liver–kidney microsomal type 1 antibodies, antibodies against parietal cells, anti-CYP2A6, anti-CYP1A2 and anti-CYP2D6 autoantibodies. Methods Indirect immunofluorescence, competitive enzyme-linked immunosorbent assays, immunoblotting and novel radioligand assays based on immunoprecipitation of [35S]-methionine labelled recombinant CYP2A6, CYP1A2 and CYP2D6 His-taq fusion proteins produced by in vitro transcription/translation were used. Results Seven out of 39 patients (17.9%) tested positive for smooth muscle antibodies, 2/39 (5.1%) tested positive for anti-nuclear antibodies, 1/39 (2.5%) tested positive for parietal cell antibodies, and 4/39 (10.3%) were found to be anti-liver–kidney microsomal positive (with at least one of the methods used). All sera were negative for anti-CYP2A6 and anti-CYP1A2 autoantibodies. Three out of four anti-liver–kidney microsomal positive samples had the typical liver–kidney microsomal staining pattern shown by indirect immunofluorescence. However, none tested positive for anti-CYP2D6 autoantibodies using the competitive CYP2D6 enzyme-linked immunosorbent assay, the specific CYP2D6 radioligand assay, and western blot using either human microsomes or recombinant CYP2D6. The fourth patient tested negative for anti-liver–kidney autoantibodies by either indirect immunofluorescence or the competitive enzyme-linked immunosorbent assay, but was repeatedly positive for anti-CYP2D6 autoantibodies by the sensitive and specific radioligand assay. Western blot experiments using human microsomes in this patient serum revealed two bands of 50 kDa and 55 kDa that documented as anti-CYP2D6 and anti-uridine triphosphate glucuronosyltransferase autoantibodies when recombinant CYP2D6 and recombinant uridine triphosphate glucuronosyltransferase autoantigens were used for immunoblot, respectively. Conclusions A relatively high incidence of anti-liver–kidney microsomal autoantibodies (10.3%) was found in a consecutive sample of Greek patients with hepatitis C. The expanded panel of assays, however, failed to document CYP2D6 as the target autoantigen of anti-liver–kidney microsomal autoantibodies in most patients. We report for the first time the detection of parietal cell antibodies and both anti-CYP2D6 (anti-liver–kidney microsomal type 1) and anti-uridine triphosphate glucuronosyltransferase (anti-liver–kidney microsomal type 3) autoantibodies in patients who were hepatitis C positive/hepatitis D negative. Further studies are needed to confirm our findings and to determine whether these preliminary results have a clinical importance or not.

Cytochrome P450 2A6: a new hepatic autoantigen in patients with chronic hepatitis C virus infection

BACKGROUND/AIMS Cytochromes P4502A6 (CYP2A6) and P4501A2 (CYP1A2) were described as hepatic autoantigens in the autoimmune polyglandular syndrome type-1 (APS-1). We evaluated the significance of anti-CYP2A6 and anti-CYP1A2 in several hepatic diseases in the absence of APS-1. METHODS A radioligand assay (RLA) based on immunoprecipitation of [(35)S]-methionine-labeled CYP2A6 and CYP1A2 was used. Four hundred and thirty subjects with chronic viral hepatitis (n=185), autoimmune liver diseases (n=181), autoimmune rheumatic diseases (ARD, n=31) and healthy (n=33) were tested. RESULTS Seven out of 366 patients with liver diseases were anti-CYP2A6 positive. Neither healthy nor ARD patients showed anti-CYP2A6. One out of 181 patients with autoimmune liver diseases tested anti-CYP2A6 positive. A significantly higher prevalence of anti-CYP2A6 (P<0.05) was detected with six out of seven patients positive in the viral hepatitis group. The latter were infected by flaviviruses (1 HGV/GBVC, 5 HCV). 4/5 HCV/anti-CYP2A6 positive sera were positive for anti-LKM-1 by immunofluorescence and for anti-CYP2D6 by RLA. None of the 430 sera recognized CYP1A2. CONCLUSIONS For the first time CYP2A6 is reported as a hepatic autoantigen in patients with viral hepatitis caused by flaviviruses and in particular in HCV/anti-LKM-1 positive patients. Multicenter studies are needed in order to investigate the clinical importance of this novel finding. This study further supports that anti-CYP2A6 in the absence of flavivirus is rather limited to APS-1.