Graeme J. M. Alexander

Genetic variation in IL28B and spontaneous clearance of hepatitis C virus

Hepatitis C virus (HCV) infection is the most common blood-borne infection in the United States, with estimates of 4 million HCV-infected individuals in the United States and 170 million worldwide. Most (70–80%) HCV infections persist and about 30% of individuals with persistent infection develop chronic liver disease, including cirrhosis and hepatocellular carcinoma. Epidemiological, viral and host factors have been associated with the differences in HCV clearance or persistence, and studies have demonstrated that a strong host immune response against HCV favours viral clearance. Thus, variation in genes involved in the immune response may contribute to the ability to clear the virus. In a recent genome-wide association study, a single nucleotide polymorphism (rs12979860) 3 kilobases upstream of the IL28B gene, which encodes the type III interferon IFN-λ3, was shown to associate strongly with more than a twofold difference in response to HCV drug treatment. To determine the potential effect of rs12979860 variation on outcome to HCV infection in a natural history setting, we genotyped this variant in HCV cohorts comprised of individuals who spontaneously cleared the virus (n = 388) or had persistent infection (n = 620). We show that the C/C genotype strongly enhances resolution of HCV infection among individuals of both European and African ancestry. To our knowledge, this is the strongest and most significant genetic effect associated with natural clearance of HCV, and these results implicate a primary role for IL28B in resolution of HCV infection.

Early indicators of prognosis in fulminant hepatic failure

The successful use of orthotopic liver transplantation in fulminant hepatic failure has created a need for early prognostic indicators to select the patients most likely to benefit at a time when liver transplantation is still feasible. Univariate and multivariate analysis was performed on 588 patients with acute liver failure managed medically during 1973-1985, to identify the factors most likely to indicate a poor prognosis. In acetaminophen-induced fulminant hepatic failure, survival correlated with arterial blood pH, peak prothrombin time, and serum creatinine--a pH less than 7.30, prothrombin time greater than 100 s, and creatinine greater than 300 mumol/L indicating a poor prognosis. In patients with viral hepatitis and drug reactions three static variables [etiology (non A, non B hepatitis or drug reactions), age less than 11 and greater than 40 yr, duration of jaundice before the onset of encephalopathy greater than 7 days] and two dynamic variables (serum bilirubin greater than 300 mumol/L and prothrombin time greater than 50 s) indicated a poor prognosis. The value of these indicators in determining outcome was tested retrospectively in a further 175 patients admitted during 1986-1987, leading to the construction of models for the selection of patients for liver transplantation.

Liver Transplantation in European Patients with the Hepatitis B Surface Antigen

BACKGROUND The role of liver transplantation in patients positive for the hepatitis B surface antigen (HBsAg) is controversial because of the high rate of recurrent hepatitis B virus (HBV) infection. It has not been determined whether this risk is greater for certain patients and whether the administration of anti-hepatitis B surface antigen (anti-HBs) immune globulin is beneficial. METHODS We conducted a retrospective study at 17 European centers of 372 consecutive HBsAg-positive patients who underwent liver transplantation between 1977 and 1990. Recurrence of HBV infection was defined as the reappearance of HBsAg in serum. RESULTS For all 334 patients with follow-up data, the mean (+/- SE) three-year actuarial risk of recurrence of HBV was 50 +/- 3 percent. The risk was 67 +/- 4 percent among 163 patients with HBV-related cirrhosis, 32 +/- 5 percent among 110 patients with cirrhosis related to hepatitis delta virus, 40 +/- 16 percent among 14 patients with fulminant hepatitis delta infection, and 17 +/- 7 percent among 39 patients with fulminant HBV infection (P < 0.001). Among the patients with HBV-related cirrhosis, the risk of HBV recurrence was greatest (83 +/- 6 percent) in those who were seropositive for HBV DNA at the transplantation and lowest (58 +/- 7 percent) in those with neither HBV DNA nor hepatitis B e antigen detectable in serum. With respect to the use of passive prophylaxis with anti-HBs immune globulin, the risk of HBV recurrence was 75 +/- 6 percent among the 67 patients given no immunoprophylaxis, 74 +/- 5 percent among the 83 treated for two months, and 36 +/- 4 percent among the 209 treated for six months or longer (P < 0.001). In a multivariate analysis the predictors of a lower risk of HBV recurrence were the long-term administration of the immune globulin, hepatitis delta virus superinfection, and acute liver disease. For the entire study cohort, survival was 75 percent at one year and 63 percent at three years, but for those in whom HBV infection recurred, survival was 68 percent at one year and 44 percent at three years. CONCLUSIONS In this retrospective study of HBsAg-positive patients, liver transplantation had better results in those who had fulminant hepatitis or delta virus superinfection. An absence of viral replication at the time of transplantation and long-term immunoprophylaxis were associated with a reduced risk of recurrent HBV infection and reduced mortality.

Improvement by Acetylcysteine of Hemodynamics and Oxygen Transport in Fulminant Hepatic Failure

BACKGROUND When administered early after an overdose of acetaminophen, intravenous acetylcysteine prevents hepatic necrosis by replenishing reduced stores of glutathione. How acetylcysteine improves the survival of patients with established liver damage induced by acetaminophen, however, is unknown. This study was undertaken to determine whether the beneficial effect of acetylcysteine under such circumstances could be due to enhancement of oxygen delivery and consumption. METHODS We studied the effect of acetylcysteine on systemic hemodynamics and oxygen transport in 12 patients with acetaminophen-induced fulminant hepatic failure and 8 patients with acute liver failure from other causes. The acetylcysteine was given in a dose of 150 mg per kilogram of body weight in 250 ml of 5 percent dextrose over a period of 15 minutes and then in a dose of 50 mg per kilogram in 500 ml of 5 percent dextrose over a period of 4 hours; measurements were made before treatment began and after 30 minutes of the regimen. RESULTS In the patients with acetaminophen-induced liver failure, the infusion of acetylcysteine resulted in an increase in mean oxygen delivery from 856 to 975 ml per minute per square meter of body-surface area (P = 0.0036), due to an increase in the cardiac index from 5.6 to 6.7 liters per minute per square meter (P = 0.0021). Mean arterial pressure rose from 88 to 95 mm Hg (P = 0.0054) despite a decrease in systemic vascular resistance from 1296 to 1113 dyn.sec.cm-5 per square meter (P = 0.027). There was an increase in oxygen consumption from 127 to 184 ml per minute per square meter (P = 0.0007) associated with an increase in the oxygen-extraction ratio from 16 to 21 percent (P = 0.022). The effects in the patients with acute liver failure from other causes were similar. CONCLUSIONS The increase in oxygen delivery and consumption in response to acetylcysteine may account for its beneficial effect on survival in patients with fulminant hepatic failure induced by acetaminophen.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α1-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α1-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells

Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.

Increased Plasma Tumor Necrosis Factor in Severe Alcoholic Hepatitis

STUDY OBJECTIVE To determine whether elevated tumor necrosis factor levels contribute to the clinical manifestations and complications of severe acute alcoholic hepatitis and to evaluate the relation between tumor necrosis factor and plasma levels of endotoxin and interleukin-1 beta. DESIGN Prospective, controlled study. SETTING The liver unit of a university teaching hospital. PATIENTS We studied 21 patients with acute severe alcoholic hepatitis. There were four control groups: patients with inactive alcoholic cirrhosis, alcoholic persons without liver disease, patients with impaired renal function, and normal subjects. MEASUREMENTS AND MAIN RESULTS With one exception, patients with alcoholic hepatitis had higher tumor necrosis factor levels (mean, 26.3 ng/L; 95% CI, 21.7 to 30.9) than normal subjects (6.4 ng/L; CI, 5.4 to 7.4). Patients who subsequently died had a higher tumor necrosis factor level (34.7 ng/L; CI, 27.8 to 41.6) than survivors (16.6 ng/L; CI, 14.0 to 19.2). In patients with alcoholic hepatitis, tumor necrosis factor levels correlated positively with serum bilirubin (r = 0.74; P = 0.0009) and serum creatinine (r = 0.81; P = 0.0003). Patients with alcoholic hepatitis had higher tumor necrosis factor levels than patients with inactive alcoholic cirrhosis (11.1 ng/L; CI, 8.9 to 13.3) and severely alcoholic persons without liver disease (6.4 ng/L; CI, 5.0 to 7.8). Patients with abnormal renal function had lower tumor necrosis factor levels (14.1 ng/L; CI, 5.4 to 22.8) than patients with alcoholic hepatitis. Serial samples obtained during a 1-week period from patients with alcoholic hepatitis showed no significant change in tumor necrosis factor when patients who died were compared with survivors. No correlation was found between tumor necrosis factor and plasma endotoxin. Levels of interleukin-1 beta did not exceed 20 ng/L. CONCLUSIONS Elevations in tumor necrosis factor in alcoholic hepatitis are most marked in severe cases, suggesting that tumor necrosis factor plays a role in the pathogenesis.

Evidence for a link between hepatitis C virus infection and diabetes mellitus in a cirrhotic population

Abnormalities of carbohydrate metabolism, including hyperinsulinaemia and insulin resistance, are well recognised complications of cirrhosis. While diabetes mellitus can be explained in many instances on the basis of coincident pancreatic disease, in most the characteristic glucose intolerance of cirrhosis is not readily explicable. A previous clinical observation that hepatitis C virus infection and diabetes mellitus appeared to be associated was formally tested by a retrospective review of 100 consecutive adult patients with cirrhosis undergoing assessment for liver transplantation. Hepatitis C virus was diagnosed by conventional serological and histological criteria. Twenty-three patients had diabetes mellitus, of whom 18 were being treated with insulin. Of the 34 patients with hepatitis C virus-related cirrhosis, 17 (50%) had diabetes mellitus, in contrast to just six (9%) of the 66 patients with cirrhosis unrelated to hepatitis C virus (chi2 = 19.1, p < 0.0001) with an odds ratio for hepatitis C virus by diabetes mellitus status 10.0 (95% confidence interval 3.4 to 29.3). Hierarchical loglinear model analysis of those factors of potential relevance to the development of diabetes mellitus revealed that only hepatitis C virus interacted significantly with diabetes mellitus while the relation between diabetes mellitus and origin, sex, body mass index and severity of cirrhosis was conditional. By multiple logistic regression analysis of diabetes mellitus status in relation to the same variables, only hepatitis C virus status was statistically significant (p < 0.0001). Origin, sex, severity of cirrhosis, body mass index and therapy were not significantly associated.(ABSTRACT TRUNCATED AT 250 WORDS)

European collaborative study on factors influencing outcome after liver transplantation for hepatitis C

BACKGROUND & AIMS Liver transplantation for hepatitis C virus (HCV)-related liver disease is characterized by frequent graft infection by HCV. The prognosis and risk factors for morbidity and mortality in this condition were determined. METHODS A retrospective study of 652 consecutive anti-HCV-positive patients undergoing liver transplantation between 1984 and 1995 in 15 European centers was conducted; 102 patients coinfected with hepatitis B virus (HBV) received immunoglobulin prophylaxis for antibody to hepatitis B surface antigen. RESULTS Overall, 5-year survival was 72%. Five-year actuarial rates of hepatitis and cirrhosis were 80% and 10%. Genotypes 1b, 1a, and 2 were detected in 214 (80%), 24 (9%), and 24 (9%) of 268 patients analyzed. The only discriminant factor for patient or graft survival was hepatocellular carcinoma as primary indication. Independent risk factors for recurrent hepatitis included the absence of HBV coinfection before transplantation (relative risk [RR], 1.7; 95% confidence interval [CI], 1.2-2.6; P = 0.005), genotype 1b (RR, 2; 95% CI, 1.3-2.9; P = 0.01), and age > 49 years (RR, 1.4; 95% CI, 1.1-1.8; P = 0.01). CONCLUSIONS The results of transplantation for HCV-related disease are compromised by a significant risk of cirrhosis, although 5-year survival is satisfactory. Genotype 1b, age, and absence of pretransplantation coinfection by HBV are risk factors for recurrent HCV.

Hepatitis B virus reinfection after orthotopic liver transplantation: Serological and clinical implications

The implications of hepatitis B virus (HBV) reinfection after liver transplantation were studied in 29 patients followed for 1.7-15 years. Of 20 patients with HBV infection alone, nine were HBeAg and HBV DNA seronegative and 11 had evidence of HBV replication as measured by HBeAg or HBV DNA seropositivity. Nine patients had co-existing HBV and delta virus (HDV) infection. Five patients became HBsAg seronegative after transplantation (four immediately and one after an hepatitic episode). Of the 20 patients with HBV infection alone, 17 had evidence of viral replication after transplantation with markedly increased HBV DNA levels. Five patients with HDV infection had HBV DNA in serum, but in significantly lower amounts than in those with HBV infection alone. Twenty-five episodes of graft dysfunction attributed to recurrent HBV infection occurred in 19 patients (65.5%). Thirteen episodes (in 12 patients) were self-resolving acute hepatitic illnesses. Six patients had a rapidly progressive illness leading to graft loss within 6 weeks, with the distinctive histological features termed fibrosing cholestatic hepatitis (FCH). Liver function tests in these patients showed markedly abnormal serum bilirubin and prothrombin times, but only modest increases in serum transaminase levels. An additional six patients lost their graft as a consequence of HBV recurrence through various pathogenetic mechanisms including possible (but unproven) FCH, chronic active hepatitis or late-onset hepatic failure. Co-existing HDV infection appeared to confer some medium-term protection from graft loss.