Sylvia Benzaken

Chondrex (YKL-40), a potential new serum fibrosis marker in patients with alcoholic liver disease

Objectives Chondrex (YKL‐40) is a mammalian member of a protein family that includes bacterial chitinases. The pattern of its expression in certain tissues such as human liver or cartilage suggests a function in remodelling or degradation of extracellular matrix. The purpose of this study was to assess whether circulating YKL‐40 might be a serum fibrosis marker in alcoholics. Methods Plasma YKL‐40 was determined in 146 consecutive heavy drinkers (106 men, 40 women; mean age, 49.2 ± 9.0 years). Liver biochemical parameters and serum fibrosis markers such as hyaluronate were also measured. Fibrosis and inflammation in liver biopsy were evaluated using a semi‐quantitative scoring system. Results Plasma YKL‐40 increased in parallel with the severity of fibrosis (P < 0.00001). YKL‐40 also increased in the presence of hepatic inflammation (P < 0.01). Receiver operating characteristic curves of Chondrex revealed that a threshold of 330 μg/E gave a specificity of 88.5%; however, the sensitivity was only 50.8%. Only 11.5% of patients without severe fibrosis displayed a Chondrex plasma level above this threshold. A positive correlation was found between Chondrex and hyaluronate (r = 0.40, P < 0.0001), and a negative correlation was shown between Chondrex and the prothrombin index (r = −0.37, P < 0.0001). Conclusions The severity of liver fibrosis is associated with elevated circulating Chondrex levels. The overlap in YKL‐40 values prevents use of Chondrex in a screening programme. High levels of Chondrex (above 330 μg/E) are predictive of severe liver fibrosis. Increased plasma YKL‐40 may reflect the remodelling of liver fibrosis in alcoholics. Eur J Gastroenterol Hepatol 12:989‐993

Effect of ondansetron, a 5-HT3 receptor antagonist, on fatigue in chronic hepatitis C: a randomised, double blind, placebo controlled study

Background and aims: There are no available effective therapies for fatigue associated with chronic hepatitis C (CHC). The serotonin antagonist ondansetron has been shown to be effective in the chronic fatigue syndrome. In this randomised, placebo controlled, double blind trial, we investigated the effect of orally administered ondansetron on fatigue in CHC. Methods: Thirty six patients with CHC were included if fatigue was their predominant symptom and they scored more than 4 on a visual analogue scale (0–10). During the study, fatigue and depression were measured on days 0, 15, 30, and 60 using a validated self report questionnaire (fatigue impact scale and Beck depression inventory). Patients were randomised to receive ondansetron tablets 4 mg twice daily or placebo for one month followed by an additional four weeks of observation. Results: Fatigue score was 85.4 (28.2) and 98.2 (26.9) in the ondansetron and placebo groups, respectively (NS). Ondansetron significantly reduced the fatigue score with more than 30% improvement on day 15 (57.1 (38.9); p<0.01), day 30 (54.5 (37.6); p<0.01), and day 60 (60.8 (37.3); p<0.01) whereas placebo did not. Overall, the reduction in fatigue was significantly higher with ondansetron compared with placebo (ANOVA for repeated measurements) for the whole follow up period (p = 0.03) or for the treatment period only (p = 0.04). Ondansetron also significantly reduced depression scores. Conclusions: The 5-hydroxytryptamine receptor type 3 antagonist ondansetron had a significant positive effect on fatigue in CHC. These observations support the concept that fatigue involves serotoninergic pathways and may encourage further evaluations of the efficacy of ondansetron on fatigue in chronic liver diseases.

Diagnostic accuracy of a rapid urine-screening test (Multistix8SG) in cirrhotic patients with spontaneous bacterial peritonitis.

Objective To assess the diagnostic accuracy of a rapid urine-screening test (Multistix8SG) for spontaneous bacterial peritonitis (SBP) in cirrhotic patients. Methods Seventy-two consecutive patients (44 males, 28 females; mean age 61.6 years) with cirrhosis and ascites were included in the study. A diagnostic paracentesis was performed on hospital admission in all patients and 2 days after antibiotic treatment in the case of SBP (polymorphonuclear [PMN] count over 250/mm3 in ascitic fluid). Each fresh sample of ascitic fluid was also tested using the Multistix8SG urine test, and the results were scored as negative, trace or positive. Results Nine of the 72 patients had SBP and the Multistix8SG urine test was positive. After 48 h of antibiotic therapy, the PMN count of three of these nine patients was still above 250/mm3 and the Multistix8SG test remained positive. In three other patients with SBP, the PMN count dropped below 250/mm3 and the Multistix8SG test result had become negative. Two of the nine SBP patients died before 48 h, and paracentesis was not performed in the ninth case. In the other 63 patients, the PMN count in ascitic fluid was below 250/mm3; the Multistix8SG test revealed 17 trace results and 46 negative results. At the threshold of 250 PMN/mm3 in ascitic fluid, this test had a sensitivity and a specificity of 100%. Conclusion A positive Multistix8SG urine test result in ascitic fluid appears to be an indication for antibiotic treatment.

Fatigue is associated with high circulating leptin levels in chronic hepatitis C.

Background and aims: Fatigue is a frequent and disabling symptom reported by patients with chronic hepatitis C (CHC). Its mechanism is poorly understood. Recent attention has focused on the role of leptin and energy expenditure in CHC. Our aims were to analyse fatigue in CHC and to determine its relationship with disease activity, resting energy expenditure (REE), circulating leptin, and tumour necrosis factor α (TNF-α). Methods: Seventy eight CHC patients, 22 healthy controls, and 13 primary biliary cirrhosis (PBC) patients underwent measurements of REE, body composition, leptin, and TNF-α. All subjects completed the fatigue impact scale (FIS) questionnaire. A liver biopsy and viral load measurements were performed in all patients. Results: Thirty eight of 78 CHC patients considered fatigue the worst or initial symptom of their disease. The fatigue score of patients was significantly higher than that of controls (53.2 (40.1) v 17.7 (16.9); p<0.0001) and was more pronounced in females (p=0.003). Leptin was increased significantly in CHC patients compared with controls (15.4 (20.7) v 6.4 (4.1) ng/ml; p<0.05). In CHC patients, the fatigue score correlated significantly with leptin corrected for fat mass (r=0.30, p=0.01). This correlation increased when the physical domain of fatigue was included (r=0.39, p=0.0009). Furthermore, a similar positive correlation was found in PBC patients (r=0.56, p=0.04). No correlation was found between fatigue and age, REE, liver function tests, viral load, or the METAVIR score in CHC patients. Conclusions: Fatigue is present in CHC patients and is more pronounced in females. The FIS questionnaire is clinically relevant and may be useful for future therapeutic trials aimed at reducing fatigue. Fatigue may be partly mediated by leptin.

Characterization of centromere alterations in liposarcomas

Supernumerary ring and large marker chromosomes are a characteristic of atypical lipomas and well‐differentiated liposarcomas (ALP‐WDLPS) and are composed of amplified 12q14–15 sequences in association with variable segments from other chromosomes. Although stably transmitted, these chromosomes contain centromeric alterations, showing no detectable alpha‐satellite sequences. We performed C‐banding, fluorescence in situ hybridization, and immunostaining with anti‐centromere antibodies in 8 cases of liposarcomas with supernumerary rings and large markers, including 5 ALP‐WDLPS and 3 dedifferentiated‐LPS and high‐grade LPS. Our results with alpha‐satellite probes and anti‐CENPB antibodies confirm the lack of detectable alpha‐satellite sequences in the five ALP‐WDLPS supernumerary chromosomes, whereas centromeric activity was proved by the detection of kinetochores by using anti‐CENPC antibodies. In contrast, the high grade and dedifferentiated liposarcomas showed a different pattern. In 2 cases, amplified chromosome 12 sequences, including amplification of alpha‐satellite 12 sequences in 1 case, were present on chromosomes with typical centromeres. In another case, the rings were similar to WDLPS‐ALP rings, but a large marker contained a chromosome 5 centromere and amplified alpha‐satellite sequences from chromosome 8. ALP‐WDLPS is the first example of a tumor class for which the presence of stable analphoid chromosomes is a constant and specific abnormality. Formation of newly derived centromeres, so‐called neocentromeres, could be an original and effective way to maintain a selective advantage in neoplastic cells by conferring stability to the supernumerary chromosomes of ALP‐WDLPS. The activation of normally non‐centromeric sequences might be obtained by an epigenetic mechanism due to the peculiar chromatin conformation of these highly complex chromosomes.

Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy

The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR-restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFR<45 ml/min per 1.73 m(2) at end of follow-up). High anti-PLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis.

Prediction of membranous nephropathy recurrence after transplantation by monitoring of anti-PLA2R1 (M-type phospholipase A2 receptor) autoantibodies: a case series of 15 patients

BACKGROUND The predictive value of anti-M-type phospholipase A2 receptor (PLA2R1) autoantibodies for membranous nephropathy (MN) recurrence after renal transplantation remains controversial. METHODS Our aim was to monitor anti-PLA2R1 IgG4 activity using a sensitive enzyme-linked immunosorbent assay in 15 kidney transplant recipients with MN, and to test the correlation between antibody titres and MN recurrence. RESULTS Five patients never exhibited anti-PLA2R1 antibodies, and one of them relapsed. Ten patients (67%) had IgG4 anti-PLA2R1 antibodies at the time of transplantation and during follow-up. The presence of IgG4 anti-PLA2R1 antibodies at the time of kidney transplantation does not imply MN recurrence (P = 0.600, n = 15). However, a positive IgG4 anti-PLA2R1 activity during follow-up (>Month 6) was a significant risk factor for MN relapse (P = 0.0048, n = 10). Indeed, four patients had persistent IgG4 anti-PLA2R1 activity after transplantation and relapsed. Among them, one was successfully treated with rituximab. Another had persistently high IgG4 anti-PLA2R1 activity and exhibited a histological relapse but no proteinuria while on treatment with renin-angiotensin system inhibitors. In contrast, the six other patients who did not relapse exhibited a decrease of their IgG4 anti-PLA2R1 activity following transplant immunosuppression, including two with proteinuria due to biopsy-proven differential diagnoses. A weak transplant immunosuppressive regimen was also a risk factor of MN recurrence (P = 0.0048, n = 10). Indeed, the six patients who received both an induction therapy and a combined treatment with calcineurin inhibitors/mycophenolate exhibited a decrease of IgG4 anti-PLA2R1 activity and did not relapse, while the four patients who did not receive this strong immunosuppressive treatment association had persistently high IgG4 anti-PLA2R1 activity and relapsed. CONCLUSION The monitoring of IgG4 anti-PLA2R1 titres during follow-up helps to predict MN recurrence, and a strong immunosuppressive treatment of anti-PLA2R1 positive patients may prevent recurrence.

Phenotyping of intrahepatic and peripheral blood lymphocytes in patients with chronic hepatitis C.

The host immune responses have been suggested toplay a role in liver injury occurring in patients withchronic hepatitis C. In order to explore therelationship between the relative proportions ofintrahepatic and peripheral blood lymphocytes (IHL, PBL),the levels of viremia, and the histological hepatitisactivity score, three-color fluorescence-activatedcytometric analysis was performed for 36 patients with chronic hepatitis C and six control subjectswithout chronic hepatitis. The liver biopsy wasperformed before any antiviral therapy. Each liverspecimen was divided into two parts: one forhistological examination and one for immunological analysis.Tricolor CD45 was used to improve“lymphogating.” Fluorescein isothiocyanate-or phycoerythrin-conjugated monoclonal antibodies withspecificity for CD3, CD4, CD8, and CD20 (lymphocyte subpopulations),for CD69 (activated lymphocytes), and for CD16/56(natural killer cells) were used. The livers of patientswith chronic hepatitis C contained a greater proportion of CD4+ lymphocytes that exhibitedmarked expression of CD69 than in control subjects (20.7± 7.3% vs 10.2 ± 4.6%, P = 0.027).Moreover, in patients with chronic hepatitis C, theproportion of CD4+ IHL correlated with the histological hepatitisactivity evaluated by the Knodell score (r = 0.48, P =0.004). No correlation was found between the percentageof CD4+ IHL and the level of viremia ortransaminase activities. Our findings clearly indicate thata cellular immune response does take place inHCV-infected livers and could thus contribute to theoutcome of hepatitis C virus infection.

Case Report: Chronic Hepatitis C and Autoimmunity (Good Response to Immunosuppressive Treatment)

The association between autoimmunity and hepatitis C virus is questioned (1, 2). Markers of autoimmunity (anti-nuclear, anti-smooth muscle, and anti-liver/ kidney microsomal autoantibo dies) occur with high frequency in patients with chronic hepatitis C (3, 4). These autoantibodies are usually present in low titers, and their occurrence should not preclude therapy with interferon, which often improves histological features without exacerbating hepatitis. For the majority of patients with autoimmune hepatitis, the treatment of choice is glucocorticoids (5). This treatment is not recommended for patients with chronic hepatitis C because it increases levels of viremia (6). Similarly interferona is not recommended for patients with autoimmune hepatitis because this cytokine may exacerbate disease activity (7). The choice of treatment is thus very dif® cult for certain rare patients with chronic hepatitis C and features of autoimmune liver diseases. This report describes three patients with chronic hepatitis and combined features of viral and autoimmune liver disease who were successfully treated with azathioprine and prednisone.

Glucose intolerance and hypoadiponectinemia are already present in lean patients with chronic hepatitis C infected with genotype non-3 viruses.

Objectives Steatosis and metabolic abnormalities seem to be frequent and deleterious in chronic hepatitis C. Changes in glucose homeostasis and in adiponectin levels, an adipokine with anti-inflammatory and insulin-sensitive properties, were evaluated in patients with chronic hepatitis C according to steatosis, liver fibrosis and body mass index. Methods Seventy-three patients with chronic hepatitis C (40 men, 33 women) infected with genotypes non-3 and 22 healthy controls (11 men and 11 women) were included in the study and all had a biochemical evaluation, including metabolic parameters, adiponectin measurement, and a liver biopsy. Insulin sensitivity was assessed with the HOMA 1-IR insulin resistance model. Results Steatosis was found in 65.7% of the patients and significant fibrosis (METAVIR F2–F4) was present in 28.7%. The presence of steatosis could only be predicted by fibrosis, whereas significant fibrosis could be predicted by steatosis and age. Adiponectin levels were significantly decreased (−32%) with the severity of the steatosis. Although overweight chronic hepatitis C patients (body mass index≥25 kg/m2) had insulin resistance and hypoadiponectinemia, lean chronic hepatitis C patients (body mass index<25 kg/m2) had already significantly higher glycemia and lower adiponectin levels than in controls. Conclusions This study confirms the high incidence of steatosis in patients infected by hepatitis C virus genotypes non-3, well linked to the development of fibrosis and metabolic abnormalities. Importantly, the present findings put emphasis on the early development of these metabolic abnormalities as they were already found in lean patients with chronic hepatitis C. The direct implication of hepatitis C virus is thus further stressed in the development of steatosis and insulin resistance, with or without involvement of host factors.