Nizar N. Zein

Clinical Significance of Hepatitis C Virus Genotypes

On the basis of phylogenetic analysis of nucleotide sequences, multiple genotypes and subtypes of hepatitis C virus (HCV) have been identified. Characterization of these genetic groups is likely to facilitate and contribute to the development of an effective vaccine against infection with HCV. Differences among HCV genotypes in geographic distributions have provided investigators with an epidemiologic marker that can be used to trace the source of HCV infection in a given population. HCV genotype 1 may represent a more aggressive strain and one that is less likely to respond to interferon treatment than HCV genotype 2 or 3. However, these observations require confirmation before HCV genotyping can be used in clinical settings.

Hepatitis C Virus Genotypes in the United States: Epidemiology, Pathogenicity, and Response to Interferon Therapy

Infection with hepatitis C virus (HCV) has been identified as the major cause of post-transfusion non-A, non-B hepatitis [1]. Chronic liver disease occurs in at least 50% of patients with acute HCV infection, and cirrhosis develops in 20% of these patients [2]. The virus has a single-stranded RNA genome that is approximately 10 Kbp long. A comparison of HCV genomic sequences from around the world has shown substantial heterogeneity of nucleotide sequences within several regions of the viral genome [3]. Hepatitis C virus has been classified into multiple strains or genotypes on the basis of the identification of these genomic differences. It has been suggested that the heterogeneity in sequence seen among HCV genotypes may be associated with variant antigenic and biological properties [4]. In addition, outcome of liver disease and rates of response to interferon therapy may vary according to HCV genotype [5, 6]. Therefore, understanding the distribution and properties of HCV genotypes may have important implications for prognosis and therapy. We evaluated the distribution of HCV genotypes in distinct geographic regions of the United States and determined the clinical characteristics of and response to interferon therapy in patients with one of several HCV genotypes. We used the classification system developed by Simmonds and colleagues [7] because it was recently adopted by consensus at the Second International Conference of HCV and Related Viruses (August 1994, San Diego, California). In this system, HCV genotypes are classified into six major genotypes (1 to 6, ordered according to when they were discovered) and 11 subtypes (1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 4a, 5a, and 6a). Methods Serum Samples We analyzed serum samples of 208 patients who were positive for antibody to HCV and had chronic liver disease. The samples were retrospectively obtained from four tertiary referral centers in the United States (59 consecutive samples from the Mayo Clinic, Rochester, Minnesota; 48 consecutive samples from the University of Vermont, Burlington, Vermont; 49 consecutive samples from the University of Miami, Miami, Florida; and 52 consecutive samples from the University of Washington Virology Laboratory, Seattle, Washington [this last center provided samples from Washington State, Idaho, Utah, Oregon, and California]). Twenty-nine patients were excluded from the study: Nineteen had no detectable products for DNA sequencing, and 10 had ambiguous sequencing results. The remaining 179 samples were the focus of this study. No clinical information was available on the patients whose samples were obtained from the University of Washington Virology Laboratory; thus, these samples were used only to study the geographic distribution of HCV genotypes. Data on interferon treatment were available for 78 patients from the Mayo Clinic and the University of Vermont. Samples from these two institutions were obtained from patients who had agreed to participate in trials of interferon treatment. Reverse Transcriptase and Polymerase Chain Reaction We selected the direct sequencing technique because it remains the gold standard and the only way to definitively identify all HCV genotypes and subtypes. Hepatitis C virus RNA was extracted from 100-L aliquots of serum after the addition of 1 mL of RNAzol B solution (Biotecx Laboratories, Houston, Texas) (2 mol of guanidinium thiocyanate per L, 12.5 mol of sodium citrate per L, 0.25% N-laroylsarcosine, 0.05 mol of 2-mercaptoethanol per L, 100 mmol of sodium acetate per L, and 50% water-saturated phenol). After the addition of 100 L of chloroform, samples were spun for 15 minutes at 14 000 g and the aqueous phase was extracted. Total RNA was precipitated by the addition of isopropanol and 2 L of glycogen and incubation at 4 C for 45 minutes. An RNA pellet was recovered by centrifugation at 14 0006 g, washed in 1 mL of 70% ethanol solution, dried, and resuspended in 10 L of RNAase-free water (Promega, Madison, Wisconsin). Ribonucleic acid was reverse-transcribed into complementary DNA by using reverse transcriptase and an antisense oligonucleotide primer (5-CGCGGAATTCCTGGTCATAGCCTCCGTGAA-3) in the presence of reverse-transcriptase buffer (100 mmol of tris-HCl per L, 500 mmol of KCl per L, 1% Triton X-100, and a pH of 8.6 at 25 C) (Promega) and 3.0 mmol of magnesium per L. Hepatitis C virus complementary DNA was amplified by polymerase chain reaction (PCR) in the presence of the sense oligonucleotide primer (5-TGGGGATCCCGTATGATACCCGCTGCTTTGA-3), PCR buffer (500 mmol of KCl per L, 100 mmol of tris-HCl per L, and a pH of 8.3) (Perkins-Elmer-Cetus, Norwalk, New Jersey), 2.0 mmol of magnesium per L, and Amplitaq DNA polymerase (Perkins-Elmer-Cetus). The PCR assay was done in a DNA thermal cycler for 50 cycles (94 C for 1 minute, 58 C for 1 minute, and 72 C for 5 minutes). Products of the PCR assay were analyzed by gel electrophoresis in 3% agarose gel that was stained with ethidium bromide. The appearance of a band 401-base pair was considered a positive result. To avoid and monitor for possible contamination with exogenous sequences during extraction or amplification, extraction of nucleic acid and genomic amplification steps were done in separate laboratories. Ribonucleic acid samples from at least one negative and one positive sample were extracted, subjected to reverse transcription, and amplified in each batch of samples tested by PCR. No false-positive results were obtained in any of the negative controls. Sequencing and Genotyping Each fragment of the PCR product, which was approximately 401 base pairs long, was desalted before undergoing sequencing with a direct column-purification method (Wizard PCR Preps DNA Purification System, Promega). Automated sequencing was done by using a standard Sanger procedure, which involved the incorporation of fluorescein-labeled dideoxynucleotides and detection on an acrylamide gel (ABI model 373 A, Applied Biosystems, Hercules, California). Nucleotide sequences were aligned and compiled with the previously reported sequences by using the Pileup program (Wisconsin Genetic Computer Group, Madison, Wisconsin)[8]. Cluster analysis was done by using the unweighted-pair group mean average, which was included in the program. These methods allowed comparison of a 222-base pair fragment of DNA that was homologous to nucleotide positions 7975 to 8196 in the NS5 region of the prototype virus. Collection of Epidemiologic Data We studied the geographic distribution of the HCV genotypes identified in the blood samples. Data from all samples were combined to define the prevalence of the HCV genotypes in patients with chronic hepatitis C in the United States. When available, age, sex, risk factors for HCV acquisition, and liver histologic findings at the time of presentation were recorded for each patient. Risk factors for acquiring HCV included history of blood transfusion, history of injection drug use, and employment at a health care facility. Liver histologic findings were classified into three groups: mildly active hepatitis (portal inflammation without substantial hepatocyte necrosis), moderately active hepatitis (inflammation with hepatocyte necrosis), and liver cirrhosis. Accurate history of alcohol consumption was not available for many of these patients and thus was not included in the analysis. The investigator who did the genotyping was blinded to the clinical data of patients at the time of analysis. Liver biopsy specimens were independently interpreted at each center. Pathogenicity of Hepatitis C Virus Genotypes To study the possible differences in the pathogenicity of HCV genotypes, we divided patients into two groups: patients with mild hepatitis and patients with severe hepatitis. Mild hepatitis was defined as 1) pretreatment alanine aminotransferase levels that were less than three times the normal level and 2) no cirrhosis seen during examination of the liver biopsy specimen obtained before treatment. Severe hepatitis was defined as pretreatment alanine aminotransferase levels greater than three times the normal level or the presence of liver cirrhosis on pretreatment biopsy. Response to Interferon Seventy-eight patients received an average dose of 3 million U of interferon (interferon- or consensus interferon) for 6 months. Response to interferon was defined as the normalization of alanine aminotransferase levels at the end of therapy. Partial response to interferon (defined as decreased but not completely normal alanine aminotransferase levels) was considered to be a treatment failure. Sustained biochemical response was defined as a normal alanine aminotransferase level 6 months after the discontinuation of interferon treatment. Statistical Analysis We used the rank-sum and Kruskal-Wallis tests to compare continuous variables (such as age) between groups, and we used the Fisher exact test to assess associations in tabular data. Because few patients had genotype 2a, 3, or 4, all tests of association between genotype and other factors are based on data that were collapsed into four groups: genotype 1a, genotype 1b, genotypes 2a and 2b, and genotypes 3 and 4. Logistic regression was used to evaluate the association between response to interferon and the combined predictors of cirrhosis and genotype. We used the SAS statistical analysis package (SAS Institute, Cary, North Carolina) for all calculations. Results Geographic Distribution of Hepatitis C Virus Genotypes Hepatitis C virus genotype 1a was present in 104 of 179 (58%) patients with chronic HCV infection; genotype 1b was the second most common genotype encountered (38 of 179 patients [21%]). Genotype 2b was present in 23 patients (13%), and genotype 3a was present in 8 patients (5%). Four patients (2%) had HCV genotype 2a, and 2 (1%) had genotype 4a. Geographic region and distribution of genotypes were not significantly associated (P = 0.18). However, samples obtained from the western United States conta

Increased Hepatic and Circulating Interleukin-6 Levels in Human Nonalcoholic Steatohepatitis

BACKGROUND:Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) are growing public health problems, and are strongly associated. The link between the two conditions remains poorly understood. Hepatic interleukin-6 (IL-6), a major proinflammatory cytokine, expression is increased in animal models of NAFLD, while in mice, selective sustained upregulation of IL-6 in the liver results in systemic insulin resistance. The extent and clinical significance of hepatic IL-6 expression in human NAFLD, as well as potential mechanisms by which steatosis may increase IL-6 production in the liver, have not been examined.AIMS:To ascertain the occurrence and significance of IL-6 expression in the liver in human NAFLD.PATIENTS AND METHODS: Plasma was obtained at time of liver biopsy from 50 consecutive patients with suspected NAFLD. Histology was assessed blindly. Hepatic IL-6 expression was assessed by immunohistochemistry, while plasma IL-6 levels were determined by an enzyme-linked immunosorbent assay.RESULTS:IL-6 expression was markedly increased in the livers of patients with nonalcoholic steatohepatitis (NASH) as compared to patients with simple steatosis (P < 0.005) or normal biopsies (P < 0.010), confirming the presence of hepatic IL-6 expression in human NASH. A positive correlation was observed between hepatocyte IL-6 expression and degree of inflammation and stage of fibrosis. Furthermore, liver IL-6 expression positively correlated with plasma IL-6 levels and degree of systemic insulin resistance. Culture of liver cells with saturated, but not mono- or polyunsaturated, FFA resulted in a significant increase in IL-6 messenger RNA (mRNA) and protein expression.CONCLUSION:Collectively, these data suggest that increased hepatic IL-6 production may play an important role in NASH development, as well as in systemic insulin resistance and diabetes.

Prevalence of diabetes mellitus in patients with end-stage liver cirrhosis due to hepatitis C, alcohol, or cholestatic disease

BACKGROUND/AIMS The aims were to study: 1) the prevalence of diabetes mellitus in patients with end-stage liver cirrhosis due to hepatitis C, alcohol, or cholestatic liver disease, 2) viral and host immunogenetic factors that may predispose to diabetes, and 3) liver transplantation outcome in patients with or without diabetes. METHODS Fasting blood glucose values of patients who underwent liver transplantation because of hepatitis C-related cirrhosis (73 patients) were compared with those of patients with cirrhosis due to cholestatic (78 patients) or alcoholic liver disease (53 patients) and to a general population. Data on diabetes prevalence in a population without liver cirrhosis was based on the prevalence of diabetes in Olmsted County, Minnesota, residents. HLA was determined using serologic assays. Hepatitis C virus genotypes were determined with polymerase chain reaction amplification and direct sequencing. Hepatitis G RNA was detected with polymerase chain reaction. Liver transplantation outcome in patients with or without diabetes was determined with rejection, retransplantation, or death at 1 year after transplantation as end points. RESULTS Of 64 patients with hepatitis C alone, 16 (25%) had diabetes before transplantation compared with 1 of 78 (1.3%) with cholestatic liver disease (p= 0.0001) and 10 of 53 (19%) with alcoholic liver disease (p=0.36). Nine patients had hepatitis C plus cholestatic liver disease; one of these (11%) had diabetes. The prevalence of diabetes in patients with cholestatic liver cirrhosis was not different from that of the general population. The frequency of hepatitis G virus coinfection, HLA-DR3, or HLA-DR4 in hepatitis C and diabetes was not different from that of hepatitis C alone. The distribution of hepatitis C virus genotype was similar in those with and those without diabetes. Diabetes was not associated with increased risk of rejection, retransplantation, or death at 1 year after transplantation, and had no impact on overall survival after transplantation. CONCLUSIONS 1) The risk of diabetes is not increased in patients with liver cirrhosis due to cholestatic liver disease but is in patients with liver cirrhosis due to hepatitis C or alcoholic liver disease; 2) cofactors (age, sex, body mass index, hepatitis G virus coinfection, hepatitis C virus genotype, or HLA-DR3/DR4) did not explain the increased risk of diabetes in patients with hepatitis C; 3) diabetes before liver transplantation did not change the outcome at 1 year after transplantation or survival.

Prevention and management of gastroesophageal varices and variceal hemorrhage in cirrhosis

Prevention and Management of Gastroesophageal Varices and Variceal Hemorrhage in Cirrhosis Guadalupe Garcia-Tsao, M.D.,1 Arun J. Sanyal, M.D.,2 Norman D. Grace, M.D., FACG,3 William D. Carey, M.D., MACG,4 the Practice Guidelines Committee of the American Association for the Study of Liver Diseases and the Practice Parameters Committee of the American College of Gastroenterology 1Section of Digestive Diseases, Yale University School of Medicine and VA-CT Healthcare System, New Haven, Connecticut; 2Division of Gastroenterology, Virginia Commonwealth University Medical Center, Richmond, Virginia; 3Division of Gastroenterology, Brigham and Women’s Hospital, Boston, Massachusetts; 4The Cleveland Clinic, Cleveland, Ohio

Polymorphisms in the interleukin-10, tumor necrosis factor-α, and transforming growth factor-β1 genes in chronic hepatitis C patients treated with interferon and ribavirin

BACKGROUND/AIMS In hepatitis C infection, the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect the response to antiviral therapy. Additionally, polymorphisms in the cytokine genes may affect the production of the cytokines. In this study, we determined the frequency of the genotypes associated with polymorphisms of the interleukin-10 and tumor necrosis factor-alpha gene promoters, and transforming growth factor-beta 1 gene leader sequence, and investigated their association with clinical features and the response to interferon-alpha and ribavirin therapy in chronic hepatitis C infection. METHODS Genomic DNA from 80 patients and 37 racially matched healthy controls was studied by polymerase chain reaction and direct automated sequencing. RESULTS The interleukin-10 -1082 G/G genotype was identified more frequently in patients than in controls (P=0.048). The transforming-growth factor-beta 1 +29 (codon 10) C/C genotype was associated with resistance to the therapy (P=0.029). After adjusting for potential confounding variables, patients exhibiting the C/C genotype were less likely to respond to treatment than patients with the T/T or T/C genotypes. CONCLUSIONS These results suggest that inheritance of the interleukin-10 -1082 G/G and the transforming growth factor-beta 1 +29 C/C genotypes, which appear to affect the cytokine production, may be associated with susceptibility to chronic hepatitis C infection and resistance to combined antiviral therapy.

Relationship between hepatitis C genotype and severity of recurrent hepatitis C after liver transplantation

BACKGROUND Recurrence of hepatitis C virus (HCV) infection after liver transplantation is universal, but the relationship between hepatitis C genotype and posttransplant outcome has been controversial. The aim of this study was to assess the relationship between hepatitis C genotype on posttransplant frequency of recurrent hepatitis, histologic severity of recurrence, and progression to cirrhosis. METHODS We studied 42 HCV RNA positive patients who received transplants between 1985 and 1994. Sera were tested for HCV RNA and protocol liver biopsies were in obtained the posttransplant period. Biopsies were scored according to the histologic activity index (HAI) and staged in a blinded fashion. RESULTS The distribution of hepatitis C genotypes distribution was as follows: 1a, 19 (45%); 1b, 17 (40%); 2b, 3 (7%); and 1 each of 2a, 3a, and 4a. There was histologic evidence of hepatitis in 38 of 42 (90.4%) of patients. Hepatitis C was mild, moderate, or severe (HAI>3) in 38% of grafts and minimal (HAI 0-3) in 62%. Overall HAI scores and histologic stage were higher in the genotype 1b group. Six of 17 (35%) genotype 1b patients had cirrhosis compared with 2 of 25 (8%) in the non-1b genotype group. CONCLUSIONS (1) Histologic evidence of recurrent hepatitis C is seen in 90% of liver allografts; (2) Histologic hepatitis C recurs with similar frequency in genotype 1b and non-1b recipients; (3) Genotype 1b is associated with more severe histologic disease recurrence than non-1b genotypes; (4) Genotype 1b appears to be associated with a higher degree of posttransplant fibrosis and cirrhosis than non-1b genotypes.

Three‐dimensional print of a liver for preoperative planning in living donor liver transplantation

The growing demand for liver transplantation and the concomitant scarcity of cadaveric livers have increased the need for living donor liver transplantation (LDLT). Ensuring the safety of donors and recipients is critical. The preoperative identification of the vascular and biliary tract anatomy with 3‐dimensional (3D) printing may allow better preoperative surgical planning, avert unnecessary surgery in patients with potentially unsuitable anatomy, and thereby decrease the complications of liver transplant surgery. We developed a protocol and successfully 3D‐printed synthetic livers (along with their complex networks of vascular and biliary structures) replicating the native livers of 6 patients: 3 living donors and 3 respective recipients who underwent LDLT. To our knowledge, these are the first complete 3D‐printed livers. Using standardized preoperative, intraoperative, and postoperative assessments, we demonstrated identical anatomical and geometrical landmarks in the 3D‐printed models and native livers. Liver Transpl 19:1304–1310, 2013.

Clinical significance of hepatic iron deposition and serum iron values in patients with chronic hepatitis C infection

OBJECTIVES:To assess the potential association between hepatic iron deposition or serum iron values and hepatic fibrosis and inflammatory activity in patients with chronic hepatitis C virus infection.METHODS:In 100 consecutive patients with hepatitis C virus infection, tissue iron deposition was assessed by quantifying iron stain on liver biopsy specimens. Serum iron, ferritin, and transferrin saturation were determined by standard laboratory procedures. Statistical analyses incorporated potential confounders associated with hepatic fibrosis.RESULTS:Twenty-one patients had no fibrosis (stage 0), 13 had portal fibrosis (stage I), 31 had periportal fibrosis (stage II), 10 had bridging fibrosis (stage III), and 25 had cirrhosis (stage IV). Positive iron stain found in liver biopsy specimens of 19 patients was associated with stage III or IV fibrosis (p= 0.004). No significant difference was found between the iron concentration or the hepatic iron index in patients with stage III or IV fibrosis compared with patients with stage I or II fibrosis. At least 1 of 3 serum iron values assessed was abnormal in 55 patients. In univariate analysis, elevated serum iron (p= 0.01), serum ferritin (p< 0.001), and transferrin saturation (p= 0.002) were associated with stage III or IV fibrosis. In multivariate analysis, the only independent predictive factor of severe hepatic fibrosis was serum ferritin (p< 0.02; odds ratio = 11.35). The serum ferritin value and tissue iron stain had a significant positive correlation (p< 0.001).CONCLUSIONS:Increased hepatic iron deposition may be associated with more advanced hepatic fibrosis in patients with chronic hepatitis C virus infection. The serum ferritin value, an independent predictor of severe hepatic fibrosis in patients with chronic hepatitis C virus infection, may predict hepatic iron deposition and severity of fibrosis.

Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults.

Hepatitis C-associated osteosclerosis (HCAO) is a rare disorder characterized by a marked increase in bone mass during adult life. Despite the rarity of HCAO, understanding the mediator(s) of the skeletal disease is of great interest. The IGFs-I and -II have potent anabolic effects on bone, and alterations in the IGFs and/or IGF-binding proteins (IGFBPs) could be responsible for the increase in bone formation in this disorder. Thus, we assayed sera from seven cases of HCAO for IGF-I, IGF-II, IGF-IIE (an IGF-II precursor), and IGFBPs. The distribution of the serum IGFs and IGFBPs between their ternary ( approximately 150 kD) and binary (approximately 50 kD) complexes was also determined to assess IGF bioavailability. HCAO patients had normal serum levels of IGF-I and -II, but had markedly elevated levels of IGF-IIE. Of the IGFBPs, an increase in IGFBP-2 was unique to these patients and was not found in control hepatitis C or hepatitis B patients. IGF-I and -II in sera from patients with HCAO were carried, as in the case of sera from control subjects, bound to IGFBP-3 in the approximately 150-kD complex, which is retained in the circulation. However, IGF-IIE was predominantly in the approximately 50-kD complex in association with IGFBP-2; this complex can cross the capillary barrier and access target tissues. In vitro, we found that IGF-II enhanced by over threefold IGFBP-2 binding to extracellular matrix produced by human osteoblasts and that in an extracellular matrix-rich environment, the IGF-II/IGFBP-2 complex was as effective as IGF-II alone in stimulating human osteoblast proliferation. Thus, IGFBP-2 may facilitate the targeting of IGFs, and in particular IGF-IIE, to skeletal tissue in HCAO patients, with a subsequent stimulation by IGFs of osteoblast function. Our findings in HCAO suggest a possible means to increase bone mass in patients with osteoporosis.