Françoise Roudot-Thoraval

Extrahepatic Immunologic Manifestations in Chronic Hepatitis C and Hepatitis C Virus Serotypes

Extrahepatic immunologic abnormalities have been shown to occur frequently in patients with chronic hepatitis C virus (HCV) infection. Hepatitis C virus now appears to cause those cases of mixed cryoglobulinemia that were previously considered essential [1-5]. Indeed, HCV RNA has been detected in the serum specimens of about 90% of patients with essential mixed cryoglobulinemia [1, 2, 4]. In addition, cryoglobulin is found, usually at low levels, in the serum specimens of one third to one half of patients with chronic hepatitis C [6, 7]; rheumatoid factor, which may play a role in cryoglobulinemia, is present in the serum specimens of about 70% of patients [6]. Various autoantibodies have been seen in the serum of 40% to 50% of patients with chronic HCV infection [6, 8], and HCV has been associated with cases of autoimmune thyroiditis [9]. Salivary gland lesions, characterized by lymphocytic capillaritis, are seen in about half of patients and are sometimes associated with lymphocytic sialadenitis resembling that of the Sjogren syndrome [6]. Finally, HCV may cause the chronic liver disease frequently associated with lichen planus [10]. Recently, sequences of different HCV variants were classified into different genotypes on the basis of overall sequence similarity [11-22]. A consensus nomenclature for HCV genotypes has been proposed [23], in which the six HCV genotypes identified so far are numbered in the order of their discovery. Within each genotype, subtypes have been identified by lower case letters, which are also given in order of discovery [23]. Correspondence among the classifications reported so far is presented in Table 1. Different techniques for determining HCV genotype have been developed in recent months. Currently, in addition to sequencing the genome, investigators can use three techniques based on the polymerase chain reaction (PCR). The technique described by Okamoto and colleagues [24, 25] is based on a nested PCR amplification of the HCV genome and uses primers located in the core region: The first round of PCR uses a pair of universal (non-type-specific) primers and the second uses a pair of type-specific primers. The method of McOmish and colleagues [17, 18] is based on PCR amplification of the 5 noncoding region of the genome done with a pair of universal primers, followed by enzymatic digestion of the amplified products and analysis of their restriction fragment length polymorphism. Stuyver and colleagues described a line probe assay for the determination of HCV genotypes [22], in which a PCR amplification is done using universal primers located in the 5 noncoding region of the genome. This is followed by hybridization of the amplified products to oligonucleotide probes attached as parallel bands on nitrocellulose strips. On the other hand, a serotyping immunoenzymatic assay to detect genotype-specific antibodies directed to epitopes encoded by the NS4 region of the HCV genome has been developed [26]. This technique, in its present form, allows the differentiation of HCV serotypes 1, 2, and 3, which correspond to HCV genotypes 1, 2, and 3 in the consensus nomenclature [23]. Table 1. Correspondence between the Major Published Classification Systems for Hepatitis C Virus Genotypes* Several studies indicate that particular HCV genotypes are associated with more severe liver disease and poorer response to interferon- therapy [27-30]. The factors determining immunologic abnormalities in patients with chronic hepatitis C are largely unknown. We used a serotyping assay to study whether the occurrence of extrahepatic immunologic abnormalities in patients with chronic hepatitis C is serotype dependent. Methods Patients Fifty-nine consecutive patients with chronic hepatitis C were prospectively studied. Thirty-four were men and 25 were women; their mean age was 52 years (range, 18 to 77 years). In all cases, the diagnosis of chronic hepatitis C was based on long-term elevation of serum alanine aminotransferase levels in the blood, positive serologic markers of HCV infection (found using second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay, Ortho Diagnostic Systems, Raritan, New Jersey), and the absence of any other cause of chronic liver disease. Specimens obtained by percutaneous liver biopsy showed chronic active hepatitis in all 56 patients tested and associated cirrhosis in 15 of the 56 (27%). Before any treatment was given, serum specimens were tested for cryoglobulin, rheumatoid factor, and many antitissue antibodies, and biopsy of labial salivary glands was done. Hepatitis C virus serotype was determined in all patients by immunoenzymatic assay. Study Methods Detection of Cryoglobulinemia Venous blood (20 mL) was taken from fasting patients in a room at 37 C, allowed to clot at this temperature, and then separated by centrifugation. After centrifugation, the supernatant was removed from the serum, incubated at 4 C for 8 days, and examined daily for cryoprecipitation. Detection of Rheumatoid Factor Rheumatoid factor was measured using a nephelometer analyzer (BNA, Behring, Marburg, Germany); polystyrene particles coated with human globulin were agglutinated when mixed with samples containing rheumatoid factor. Normal values were those less than 18 IU/mL. Detection of Autoantibodies Antinuclear, anti-smooth muscle, type 1 anti-liver-kidney microsomal (anti-LKM1), and antimitochondrial antibodies were detected by indirect immunofluorescence using air-dried cryostat sections from rat or mouse livers and kidneys and HEp-2 cells (Kallestad, Chaska, Minnesota) as substrates. Antithyroid microsomal antibodies were detected by indirect immunofluorescence using surgical specimens of human thyrotoxic thyroid as substrate. In all cases, the classic Weller and Coon indirect immunofluorescence method was used with fluorescein-labeled goat immunoglobulin directed to IgG, IgA, and IgM (Pasteur Diagnostics, Marnes la Coquette, France) as a second layer [31]. The serum specimens were tested undiluted for anti-DNA antibodies, at a 1/5 dilution for antimicrosomal antibodies, and at a 1/10 dilution for other antibodies. The titers were established using increasing dilutions up to 1/2560. Antithyroglobulin antibodies were detected using an hemagglutination kit (Thymune-T, Wellcome Diagnostics, Dartford, United Kingdom). Labial Salivary Gland Examination All biopsies were done in macroscopically normal mucosa. The samples were fixed in Bouin fluid, embedded in paraffin, and stained with hematoxylin-eosin-safranin. All sections were examined blind by two pathologists and graded according to the Chisholm and Mason classification system [32]. Determination of Serotypes The 59 serum specimens in our study were tested for the presence of serotype-specific antibodies using the recently developed enzyme immunoassay [26]. A series of eight branched peptides, synthesized from two antigenic regions of HCV genotypes 1, 2, and 3, were used to coat polypropylene microtiter wells overnight at 4 C. After washing, the wells were blocked with 150 L of blocking solution (phosphate-buffered saline, 0.1% Tween 20, and 2% bovine serum albumin) for 1 hour at room temperature. Blocking assays were done using mixes of type-specific peptides at a final concentration of 1 mg/mL (for example, 100:1 excess over that used to coat the wells). Plasma specimens from the 59 patients were diluted in the blocking solution and 100 L were added to antigen-coated and blocked wells. The first incubation was done overnight at 4 C. Plates were washed four times in phosphate-buffered saline and 0.1% Tween 20 and then incubated with horseradish peroxidase-conjugated anti-human IgG (1/20 000 in phosphate-buffered saline and 0.1% Tween 20 for 1 hour at room temperature). The plates were finally washed four times in phosphate-buffered saline and 0.1% Tween 20 and incubated with substrate (50 g of O-phenylenediamine per milliliter and 0.1% H2O2 [30 volumes] for 30 minutes in the dark at room temperature). Optical densities were read at 490 nm; values ranged from 100 to 2000 mU. Results Prevalence of Immunologic Abnormalities Our results are presented in Table 2. Cryoglobulin was found in the serum specimens of 20 of the 56 patients tested (36%), and rheumatoid factor was present at abnormal levels in 42 of the 59 patients (71%). Table 2. Prevalence of the Different Immunologic Abnormalities according to Hepatitis C Virus Serotype At least one type of antitissue antibody was detected in the serum specimens of 24 of the 59 patients (41%). Thirteen patients (22%) had serum antinuclear antibodies and 13 (22%) had anti-smooth muscle antibodies at a significant titer (greater than 1/40). Anti-LKM1 antibodies were found in 3 patients (5%) and antithyroid antibodies were found in 5 (8%); 4 of these 5 had antithyroglobulin and 1 had antithyroid microsomes. No antimitochondrial antibodies were found. Labial salivary gland biopsies were done in those 49 of the 59 patients who had no contraindication and who gave informed consent; lesions were found in 24 of them (49%). In all patients, these lesions were characterized by lymphocytic capillaritis, as previously described [6]. In 7 patients (14%), they were associated with more severe lesions, grades 3 and 4 by the Chisholm and Mason classification (lymphocytic sialadenitis) [32], and resembled the lymphocytic sialadenitis of the Sjogren syndrome (14%). Only one of the patients with salivary gland lesions had a mild case of the ocular sicca syndrome shown by the Schirmer test. The prevalences of the different immunologic abnormalities did not vary significantly according to the presence of cirrhotic findings in liver specimens. Hepatitis C Virus Serotypes Thirty-five of the 59 patients (59%) were infected with HCV serotype 1, 6 (10%) were infected with serotype 2, and 7 (12%) were infected with serotype 3 (serotypes are here described using the proposed consensus nomenclature [23]). Two

Worsening of steatosis is an independent factor of fibrosis progression in untreated patients with chronic hepatitis C and paired liver biopsies

Background and aims: Steatosis, a frequent histological finding in patients with chronic hepatitis C (CHC), has been suggested to influence liver fibrosis progression. The aim of the present study was to evaluate in patients with CHC and paired liver biopsies the relationship between the evolution of steatosis and that of fibrosis between the two biopsies. Methods: Ninety six patients were selected according to the following criteria: absence of treatment; absence of cirrhosis at initial biopsy; and serum hepatitis B surface antigen and human immunodeficiency virus antibody negativity. Degrees of necroinflammatory activity, fibrosis, and steatosis grades were assessed in the two biopsies. In addition to histological lesions, parameters studied included the source of infection, duration of infection, body mass index, alcohol intake, alanine aminotransferase levels, hepatitis C virus genotype, and viral load. Results: The mean interval between the two biopsies was 48 (32) months. Steatosis was found in 54% of patients at first biopsy, and was severe in 9%. Worsening of steatosis was observed in 34% of patients, stability in 50%, and improvement in 16%. Worsening of steatosis was significantly associated with hepatic fibrosis progression in patients with (p=0.03) or without (p<0.03) steatosis at diagnosis. Overall, fibrosis progression was observed in 31% of patients and stability in 69%. In a univariate analysis, fibrosis progression was associated with male sex (p=0.05), worsening of histological activity (p=0.04), and worsening of steatosis (p=0.0003). In a multivariate analysis, the only factor independently associated with fibrosis progression was worsening of steatosis (worsening v improvement/stability: odds ratio 4.7 (95% confidence interval 1.3–10.8); p=0.0001). Conclusions: Our results suggest that in untreated patients with CHC and serial liver biopsies, fibrosis progression is strongly associated with worsening of steatosis.

Impact of pretransplantation transarterial chemoembolization on survival and recurrence after liver transplantation for hepatocellular carcinoma.

The actual impact of transarterial chemoembolization before liver transplantation (LT) for hepatocellular carcinoma (HCC) on patient survival and HCC recurrence is not known. Between 1985 and 1998, 479 patients with HCC in 14 French centers were evaluated for LT. Among these 479 patients, this case‐control study included 100 patients who received transarterial chemoembolization before LT (TACE group) and 100 control patients who did not receive chemoembolization (no‐TACE group). Patients and controls were matched for the pre‐LT tumor characteristics, the period of transplantation, the time spent on the waiting list, and pre‐ and posttransplantation treatments. Kaplan‐Meier estimates were calculated 5 years after LT and were compared with the log‐rank test. The mean waiting time before LT was 4.2 ± 3.2 months in the TACE group and 4.3 ± 4.4 months in the no‐TACE group. The median number of TACE procedures was 1 (range: 1‐12). Demographic data, median alpha‐fetoprotein level (21.6 ng/mL and 22.0 ng/mL, respectively), and pre‐ and post‐LT morphologic characteristics of the tumors did not differ in the TACE and no‐TACE groups. Overall 5‐year survival was 59.4% with TACE and 59.3% without TACE (ns). Survival rates did not differ significantly between the two groups with respect to the time on the waiting list, the tumor diameter, or the type of TACE (selective or nonselective). In the TACE group, 30 patients had tumor necrosis ≥80% on the liver explant with a 5‐year survival rate of 63.2%, compared with 54.2% among their matched controls (P = 0.9). In conclusion, with a mean waiting period of 4.2 months and 1 TACE procedure, pre‐LT TACE does not influence post‐LT overall survival and disease‐free survival. (Liver Transpl 2005;11:767–775.)

Impact of UCSF criteria according to pre‐ and post‐OLT tumor features: Analysis of 479 patients listed for HCC with a short waiting time

Orthotopic liver transplantation (OLT) indication for hepatocellular carcinoma (HCC) is currently based on the Milan criteria. The University of California, San Francisco (UCSF) recently proposed an expansion of the selection criteria according to tumors characteristics on the explanted liver. This study: 1) assessed the validity of these criteria in an independent large series and 2) tested for the usefulness of these criteria when applied to pre‐OLT tumor evaluation. Between 1985 and 1998, 479 patients were listed for liver transplantation (LT) for HCC and 467 were transplanted. According to pre‐OLT (imaging at date of listing) or post‐OLT (explanted liver) tumor characteristics, patients were retrospectively classified according to both the Milan and UCSF criteria. The 5‐yr survival statistics were assessed by the Kaplan‐Meier method and compared by the log‐rank test. Pre‐OLT UCSF criteria were analyzed according to an intention‐to‐treat principle. Based on the pre‐OLT evaluation, 279 patients were Milan+, 44 patients were UCSF+ but Milan− (subgroup of patients that might benefit from the expansion), and 145 patients were UCSF− and Milan−. With a short median waiting time of 4 months, 5‐yr survival was 60.1 ± 3.0%, 45.6 ± 7.8%, and 34.7 ± 4.0%, respectively (P < 0.001). The 5‐yr survival was arithmetically lower in UCSF+ Milan− patients compared to Milan+ but this difference was not significant (P = 0.10). Based on pathological features of the explanted liver, 5‐yr survival was 70.4 ± 3.4%, 63.6 ± 7.8%, and 34.1 ± 3.1%, in Milan+ patients (n = 184), UCSF+ Milan− patients (n = 39), and UCSF− Milan− patients (n = 238), respectively (P < 0.001). However, the 5‐yr survival did not differ between Milan+ and UCSF+ Milan− patients (P = 0.33). In conclusion, these results show that when applied to pre‐OLT evaluation, the UCSF criteria are associated with a 5‐yr survival below 50%. Their applicability is therefore limited, despite similar survival rates compared to the Milan criteria, when the explanted liver is taken into account. Liver Transpl 12:1761‐1769, 2006.

Comparative Efficacy of Interferon Alfa in Cirrhotic and Noncirrhotic Patients With Non-A, Non-B, C Hepatitis

BACKGROUND/AIMS Because the effects of interferon in the presence and absence of cirrhosis are still debated in chronic active hepatitis type non-A, non-B, C (NANB/C), the aim of this study was to determine to what extent the presence of cirrhosis influences the response to interferon. METHODS We compared the response to interferon alfa in 108 patients with chronic active hepatitis NANB/C with or without cirrhosis. The patients were randomly assigned to one of the two regimens: one group received 6 months of interferon 3 MU 3 times weekly, while the second group received a 12-month course, 3 MU three times weekly during the first 6 months, 2 MU for the following 3 months, and 1 MU for the last 3 months. RESULTS In both regimens, the proportion of patients with normal alanine aminotransferase at the end of treatment was significantly lower in the cirrhotic than in the noncirrhotic patients. In males, abnormal serum alkaline phosphatase levels and gamma glutamyl transpeptidase were also related to a lesser response independent of cirrhosis. The response at the end of treatment was not significantly different between the two regimens in either the cirrhotic or the noncirrhotic patients. However, the 12-month regimen gave a significantly higher rate of sustained response 6 months after the end of treatment in patients without cirrhosis. CONCLUSIONS It is suggested that the presence of cirrhosis markedly reduces the rate of response to interferon and that in noncirrhotic patients, a 1-year treatment regimen could improve the beneficial effect of interferon.

Carriage of GB Virus C/Hepatitis G Virus RNA Is Associated with a Slower Immunologic, Virologic, and Clinical Progression of Human Immunodeficiency Virus Disease in Coinfected Persons

The prevalence of GB virus C (GBV-C) infection is high in human immunodeficiency virus (HIV)-infected persons. However, the long-term consequences of coinfection are unknown. HIV-positive persons with a well-defined duration of infection were screened on the basis of their GBV-C/hepatitis G virus (HGV) RNA status and studied. GBV-C/HGV viremia was observed in 23, who carried the virus over a mean of 7.7 years. All parameters (survival, CDC stage B/C, HIV RNA load, CD4 T cell count) showed significant differences in terms of the cumulative progression rate between persons positive and negative for GBV-C/HGV RNA. When GBV-C/HGV RNA-positive and -unexposed subjects were matched by age, sex, baseline HIV RNA load, and baseline CD4 T cell count, HIV disease progression appeared worse in GBV-C/HGV RNA-negative subjects. The carriage of GBV-C/HGV RNA is associated with a slower progression of HIV disease in coinfected persons.

Effect of antiviral treatment on evolution of liver steatosis in patients with chronic hepatitis C: indirect evidence of a role of hepatitis C virus genotype 3 in steatosis

Background and aim: Recent studies suggest that liver steatosis in chronic hepatitis C may be the expression of a direct cytopathic effect of hepatitis C virus (HCV), particularly in patients infected with genotype 3. To investigate this hypothesis, we studied the relationship between steatosis evolution and HCV clearance after antiviral treatment in patients with chronic hepatitis C and paired liver biopsies. Methods: A total of 151 patients (37 with HCV genotype 3; 114 with HCV non-3 genotypes) were selected according to the following criteria: presence of steatosis at initial biopsy; no antiviral treatment prior to the first biopsy; antiviral treatment received between the two biopsies; body mass index (BMI) <28 kg/m2; absence of excessive alcohol intake; no serum hepatitis B surface antigen or human immunodeficiency virus antibodies; and absence of diabetes mellitus. Evolution of steatosis was examined by comparing steatosis grades between the two biopsies. Results: Twenty five patients (16.5%) were sustained virological responders (SVR) to antiviral treatment. Steatosis evolution after antiviral treatment was as follows: improvement in 36% of cases; stability in 51%; and worsening in 13%. Steatosis improvement was significantly more frequent in SVR than in non-responders (NR) (64% v 31%; p<0.004). This significant difference occurred in patients infected with genotype 3 (91% v 19%; p<0.0001) but not in those infected with non-3 genotypes (43% v 34%; NS). Among the 25 SVR, improvement in steatosis was significantly more frequent in patients infected with genotype 3 than in those infected with non-3 genotypes (91% v 43%; p<0.04) whereas in NR, improvement in steatosis did not differ between those infected with genotype 3 and non-3 genotypes (19% v 34%; NS). In multivariate analysis, four factors were independently associated with steatosis improvement: sustained virological response to antiviral therapy (odds ratio (OR) 6.06 (95% confidence interval (CI) 1.61–22.9); p = 0.01), severe steatosis (OR 5.50 (95% CI 1.54–19.6); p = 0.01), HCV genotype 3 (OR 2.90 (95% CI 0.85–10.0); p = 0.07), and BMI >25 kg/m2 (OR 0.24 (95% CI 0.08–0.73); p = 0.02). Conclusions: Our results showed significant improvement in steatosis in patients infected with HCV genotype 3, who achieved sustained viral clearance. This provides further evidence for direct involvement of HCV genotype 3 in the pathogenesis of hepatic steatosis.

Increased incidence of oropharyngeal squamous cell carcinomas after liver transplantation for alcoholic cirrhosis.

BACKGROUND THE aim of this study was to describe the features of posttransplantation tumors observed in a series of liver transplant recipients with special reference to patients receiving a transplant for alcoholic cirrhosis. METHODS Among 171 consecutive liver transplant recipients, 90 patients who had received a first liver allograft for cirrhosis were studied. After liver transplantation, detection of de novo malignancies was prospectively undertaken and the characteristics of the patients in whom tumors occurred were compared with those in whom tumors did not develop. RESULTS With a follow-up of 45.2+/-21.2 months, 11 tumors were observed in 90 patients (overall incidence of 12.2%). The incidence of tumors was higher in patients receiving a transplant for alcoholic cirrhosis than in patients receiving a transplant for nonalcoholic cirrhosis (26.7% vs. 5.0%, P<0.01). Squamous cell carcinoma (SCC) of the oropharynx or esophagus and posttransplant lymphoproliferative disorders were mainly observed. SCC (uvula in two cases, tongue in one case, esophagus in one case, pharynx in one case) occurred exclusively in patients transplanted for alcoholic cirrhosis (16.7% vs. 0%, P=0.001). The incidence of posttransplant lymphoproliferative disorders was similar in alcoholics and nonalcoholics (6.7% vs. 5%, NS). Survival was not influenced by the occurrence of SCC. CONCLUSION The incidence of oropharyngeal SCC could be high in patients receiving a transplant for alcoholic cirrhosis. This could be due to an additional effect of posttransplantation immunosuppression in patients exposed to alcohol and tobacco before transplant. Careful posttransplantation screening of oropharyngeal SCC is warranted after liver transplantation for alcoholic cirrhosis.

Impact of smoking on histological liver lesions in chronic hepatitis C

Aims and methods: To examine the association between smoking and histological liver lesions in chronic hepatitis C, we studied 244 consecutive patients (152 men, 92 women; mean age 45.9 (12.6) years) with histologically proven chronic hepatitis C. Daily tobacco consumption during the six months preceding liver biopsy was recorded as the number of cigarettes smoked daily. Total lifetime tobacco consumption was recorded as the number of cigarette packs smoked per year (packs-years). Liver biopsy specimens were graded for histological activity and fibrosis according to the METAVIR scoring system. Results: The proportion of patients with moderate (A2) or marked (A3) activity increased gradually from 62.0% in non-smokers to 81.7% in patients who smoked more than 15 cigarettes per day (p<0.009). A similar relationship was observed with total lifetime tobacco consumption: 59.0% of patients who had never smoked had grade A2 or A3 disease activity compared with 84.6% of patients who smoked more than 20 packs per year (p<0.002). Multivariate analysis showed that age over 50 years (odds ratio (OR) 5.4), alcohol intake exceeding 20 g/day (OR 2.75), and tobacco consumption of more than 15 cigarettes/day (OR 3.6) were independently related to the histological activity score. No relationship was found between the severity of fibrosis and either daily tobacco consumption or total lifetime tobacco consumption. Multivariate analysis showed that only age over 50 years (OR 8.8), daily alcohol intake exceeding 30 g/day (OR 3.4), and histological activity score (OR 7.9) were independently related to the fibrosis score. Conclusion: This study suggests that smoking, independent of alcohol, could aggravate the histological activity of chronic hepatitis C and that patients with chronic hepatitis C virus infection should be advised to reduce or stop smoking.

Pulmonary Hypertension and Cor Pulmonale during Severe Acute Chest Syndrome in Sickle Cell Disease

RATIONALE Steady-state mild pulmonary hypertension is a risk factor for death in adults with sickle cell disease. Acute pulmonary hypertension has been reported during exercise and vasoocclusive pain crisis in these patients. OBJECTIVES The aim of the present study was to evaluate changes in pulmonary pressures and cardiac biomarkers during severe acute chest syndrome and their associations with mortality. METHODS We prospectively evaluated 70 consecutive adults who received standardized treatment in our intensive care unit for a total of 84 episodes. At admission, cardiac biomarkers were measured. Transthoracic echocardiography was performed for pulmonary hypertension assessment via measurement of tricuspid regurgitant jet velocity and was repeated when possible after resolution. MEASUREMENTS AND MAIN RESULTS Tricuspid jet velocity was less than 2.5 m/second in 34 (40%) of the 84 episodes, 2.5 to 2.9 m/second in 19 (23%), and 3 m/second or greater in 31 episodes (37%). Cor pulmonale occurred in 11 (13%) episodes. Tricuspid jet velocity showed significant positive correlations with B-type natriuretic peptide (rho = 0.54, P < 0.01) and cardiac troponin I (rho = 0.42, P < 0.01). Pulmonary pressures increased compared with steady state then decreased after resolution. All five patients who required invasive ventilation and all four patients who died during the immediate hospital course had jet velocity values of 3 m/second or greater. Overall mortality was 12.9% (9 patients) and survival was significantly lower in patients whose jet velocity was 3 m/second or greater during at least one episode, compared with the other patients (P < 0.01). CONCLUSIONS Pulmonary pressures increase during severe acute chest syndrome, and pulmonary hypertension is associated with cardiac biomarker elevation and a higher risk of death.