George N. Ioannou

Circulating MicroRNAs in Patients with Chronic Hepatitis C and Non-Alcoholic Fatty Liver Disease

MicroRNAs miR-122, miR-34a, miR-16 and miR-21 are commonly deregulated in liver fibrosis and hepatocellular carcinoma. This study examined whether circulating levels of these miRNAs correlate with hepatic histological disease severity in patients with chronic hepatitis C infection (CHC) or non-alcoholic fatty-liver disease (NAFLD) and can potentially serve as circulating markers for disease stage assessment. We first used an in vitro model of hepatitis C virus (HCV) infection to measure the extracellular levels of these four miRNAs. Whereas miR-21 extracellular levels were unchanged, extracellular levels of miR-122, miR-34a and to a lesser extent miR-16, steadily increased during the course of HCV infection, independently of viral replication and production. Similarly, in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher than in control individuals, while miR-21 levels were unchanged. There was no correlation between the serum levels of any of these microRNAs and HCV viral loads. In contrast, miR-122 and miR-34a levels positively correlated with disease severity. Identical results were obtained in an independent cohort of CHC patients. We extended the study to patients with NAFLD. As observed in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher in NAFLD patients than in controls, while miR-21 levels were unchanged. Again, miR-122 and miR-34a levels positively correlated with disease severity from simple steatosis to steatohepatitis. In both CHC and NAFLD patient groups, serum levels of miR-122 and miR-34a correlated with liver enzymes levels, fibrosis stage and inflammation activity. miR-122 levels also correlated with serum lipids in NAFLD patients. Conclusion: Serum levels of miR-34a and miR-122 may represent novel, noninvasive biomarkers of diagnosis and histological disease severity in patients with CHC or NAFLD.

Predictors of colorectal cancer screening participation in the United States

OBJECTIVE:Our aim was to identify predictors of colorectal cancer screening in the United States and subgroups with particularly low rates of screening.METHODS:The responses to a telephone-administered questionnaire of a nationally representative sample of 61,068 persons aged ≥50 yr were analyzed. Current screening was defined as either sigmoidoscopy/colonoscopy in the preceding 5 years or fecal occult blood testing (FOBT) in the preceding year, or both.RESULTS:Overall, current colorectal cancer screening was reported by 43.4% (sigmoidoscopy/colonoscopy by 22.8%, FOBT by 9.9%, and both by 10.7%). The lowest rates of screening were reported by the following subgroups: those aged 50–54 yr (31.2%), Hispanics (31.2%), Asian/Pacific Islanders (34.8%), those with education less than the ninth grade (34.4%), no health care coverage (20.4%), or coverage by Medicaid (29.2%), those who had no routine doctors visit in the last year (20.3%), and every-day smokers (32.1%). The most important modifiable predictors of current colorectal cancer screening were health care coverage (OR = 1.7, 95% CI = 1.5–1.9) and a routine doctors visit in the last year (OR = 3.5, 95% CI = 3.2–3.8). FOBT was more common in women than in men (OR = 1.8, 95% CI = 1.6–2.0); sigmoidoscopy/colonoscopy was more common in Hispanics (OR = 1.4, 95% CI = 1.1–1.7) and Asian/Pacific Islanders (OR = 2.4, 95%= CI 1.5–3.9) relative to whites, in persons without routine doctors visits in the preceding year (OR = 3.3, 95% CI = 2.8–4), and in persons with poor self-reported health (OR = 1.3, 95% CI = 1.2–1.5).CONCLUSION:Interventions should be developed to improve screening for the subgroups who reported the lowest screening rates. Such interventions may incorporate individual screening strategy preferences.

The prevalence and predictors of elevated serum aminotransferase activity in the United States in 1999-2002.

OBJECTIVES:The presence of elevated serum aminotransferase activity is a sign of possible underlying liver disease. We aimed to describe the prevalence and associations of elevated serum aminotransferase activity in a recent, nationally representative U.S. survey.METHODS:We described the prevalence and predictors of elevated alanine aminotransferase (ALT >43 IU/L) or elevated aspartate aminotransferase (AST >40 IU/L) activity among 6,823 participants of the National Health and Nutrition Examination Survey (NHANES) conducted between 1999 and 2002. We compared our findings to the results already published based on the NHANES conducted between 1988 and 1994.RESULTS:In NHANES 1999–2002, the prevalences of elevated ALT, AST, or either ALT or AST were 8.9%, 4.9%, and 9.8%, respectively, in the entire population and 7.3%, 3.6%, and 8.1%, respectively, after excluding participants who tested positive for hepatitis C virus (HCV) antibody or reported excessive alcohol consumption. Strong predictors of elevated ALT activity included increasing waist circumference and body mass index, alcohol consumption, male sex, Mexican American ethnicity, decreasing age, and presence of HCV antibody. In NHANES 1988–1994, which employed a different assay methodology, the prevalences of elevated aminotransferases were approximately half of the prevalences we describe in NHANES 1999–2002, but the predictors of elevated ALT activity were similar.CONCLUSIONS:The current prevalence of elevated ALT activity in the United States (8.9%) is more than double that of previously available estimates. This prevalence is very high (7.3%) even among persons without viral hepatitis C or excessive alcohol consumption and is strongly associated with risk factors for nonalcoholic fatty liver disease.

Elevated serum alanine aminotransferase activity and calculated risk of coronary heart disease in the United States

In the United States, elevated serum alanine aminotransferase (ALT) activity in the absence of viral hepatitis or excessive alcohol consumption is most commonly attributed to nonalcoholic fatty liver disease (NAFLD). NAFLD is related to predictors of coronary heart disease (CHD) such as insulin resistance and central obesity. We examined the association between elevated serum ALT activity and the 10‐year risk of CHD as estimated using the Framingham risk score (FRS). We performed a cross‐sectional analysis comparing participants in the Third National Health and Nutrition Examination Survey with normal and elevated ALT activity (>43 IU/L), examining the mean levels of FRS. Among participants without viral hepatitis or excessive alcohol consumption, those with elevated ALT activity (n = 267) had a higher FRS than those with normal ALT activity (n = 7,259), both among men (mean difference in FRS 0.25, 95% CI 0.07‐0.4; hazard ratio for CHD 1.28, 95% CI 1.07‐1.5) and women (mean difference in FRS 0.76, 95% CI 0.4‐1.1; hazard ratio for CHD 2.14, 95% CI 1.5‐3.0). The ALT threshold for increased risk of CHD was higher in men (>43 IU/L) than in women (>30 IU/L). Elevated ALT activity was not associated with higher FRS among nonobese participants with viral hepatitis or excessive alcohol consumption. In conclusion, individuals with elevated serum ALT activity in the absence of viral hepatitis or excessive alcohol consumption, most of whom have NAFLD, have an increased calculated risk of CHD. This association is more prominent in women. (HEPATOLOGY 2006;43:1145–1151.)

Hepatic Free Cholesterol Accumulates in Obese, Diabetic Mice and Causes Nonalcoholic Steatohepatitis

BACKGROUND & AIMS Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH. METHODS Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice. RESULTS Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis. CONCLUSIONS In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice.

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR−/−) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR−/− mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR−/− mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second “hit” that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.

Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis

The majority of patients with nonalcoholic fatty liver disease (NAFLD) have “simple steatosis,” which is defined by hepatic steatosis in the absence of substantial inflammation or fibrosis and is considered to be benign. However, 10%‐30% of patients with NAFLD progress to fibrosing nonalcoholic steatohepatitis (NASH), which is characterized by varying degrees of hepatic inflammation and fibrosis, in addition to hepatic steatosis, and can lead to cirrhosis. The cause(s) of progression to fibrosing steatohepatitis are unclear. We aimed to test the relative contributions of dietary fat and dietary cholesterol and their interaction on the development of NASH. We assigned C57BL/6J mice to four diets for 30 weeks: control (4% fat and 0% cholesterol); high cholesterol (HC; 4% fat and 1% cholesterol); high fat (HF; 15% fat and 0% cholesterol); and high fat, high cholesterol (HFHC; 15% fat and 1% cholesterol). The HF and HC diets led to increased hepatic fat deposition with little inflammation and no fibrosis (i.e., simple hepatic steatosis). However, the HFHC diet led to significantly more profound hepatic steatosis, substantial inflammation, and perisinusoidal fibrosis (i.e., steatohepatitis), associated with adipose tissue inflammation and a reduction in plasma adiponectin levels. In addition, the HFHC diet led to other features of human NASH, including hypercholesterolemia and obesity. Hepatic and metabolic effects induced by dietary fat and cholesterol together were more than twice as great as the sum of the separate effects of each dietary component alone, demonstrating significant positive interaction. Conclusion: Dietary fat and dietary cholesterol interact synergistically to induce the metabolic and hepatic features of NASH, whereas neither factor alone is sufficient to cause NASH in mice. (HEPATOLOGY 2013)

The prevalence of cirrhosis and hepatocellular carcinoma in patients with human immunodeficiency virus infection

Cirrhosis is a leading cause of death among patients infected with human immunodeficiency virus (HIV). We sought to determine risk factors for and time trends in the prevalence of cirrhosis, decompensated cirrhosis, and hepatocellular carcinoma (HCC) among patients diagnosed with HIV who received care in the Veterans Affairs (VA) health care system nationally between 1996 and 2009 (n = 24,040 in 2009). Among patients coinfected with HIV and hepatitis C virus (HCV), there was a dramatic increase in the prevalence of cirrhosis (3.5%‐13.2%), decompensated cirrhosis (1.9%‐5.8%), and HCC (0.07%‐1.6%). Little increase was observed among patients without HCV coinfection in the prevalence of cirrhosis (1.7%‐2.2%), decompensated cirrhosis (1.1%‐1.2%), and HCC (0.03%‐0.13%). In 2009, HCV infection was present in the majority of patients with HIV who had cirrhosis (66%), decompensated cirrhosis (62%), and HCC (80%). Independent risk factors for cirrhosis included HCV infection (adjusted odds ratio [AOR], 5.82; 95% confidence interval [CI], 5.0‐6.7), hepatitis B virus (HBV) infection (AOR, 2.40; 95% CI, 2.0‐2.9), age (AOR, 1.03; 95% CI, 1.02‐1.04), Hispanic ethnicity (AOR, 1.76; 95% CI, 1.4‐2.2), diabetes (AOR, 1.79; 95% CI, 1.6‐2.1), and alcohol abuse (AOR, 1.78; 95% CI, 1.5‐2.1), whereas black race (AOR, 0.56; 95% CI, 0.48‐0.64) and successful eradication of HCV (AOR, 0.61; 95% CI, 0.4‐0.9) were protective. Independent risk factors for HCC included HCV infection (AOR, 10.0; 95% CI, 6.1‐16.4), HBV infection (AOR, 2.82; 95% CI, 1.7‐4.7), age (AOR, 1.05; 95% CI, 1.03‐1.08), and low CD4+ cell count (AOR, 2.36; 95% CI, 1.3‐4.2). Among 5999 HIV/HCV‐coinfected patients, 994 (18%) had ever received HCV antiviral treatment, of whom 165 (17%) achieved sustained virologic response. Conclusion: The prevalence of cirrhosis and HCC has increased dramatically among HIV‐infected patients driven primarily by the HCV epidemic. Potentially modifiable risk factors include HCV infection, HBV infection, diabetes, alcohol abuse, and low CD4+ cell count. (HEPATOLOGY 2013)

Hepatitis B Virus in the United States: Infection, Exposure, and Immunity Rates in a Nationally Representative Survey

BACKGROUND Up-to-date estimates of the prevalence of hepatitis B virus (HBV) infection, exposure, and immunity are necessary to assess the effectiveness of ongoing programs aimed at preventing HBV transmission. OBJECTIVE To determine the prevalence and associations of chronic HBV infection, past exposure, and immunity in the United States from 1999 to 2008. DESIGN Nationally representative, cross-sectional household survey. SETTING U.S. civilian, noninstitutionalized population. PARTICIPANTS 39 787 participants in the National Health and Nutrition Examination Survey (1999 to 2008) aged 2 years or older. MEASUREMENTS Chronic HBV infection was defined by presence of serum HBV surface antigen and past exposure by serum antibody to hepatitis B core antigen among persons aged 6 years or older. Infant immunity was defined by presence of serum antibody to hepatitis B surface antigen among children aged 2 years. RESULTS Among persons aged 6 years or older, 0.27% (95% CI, 0.20% to 0.34%) had chronic HBV infection (corresponding to approximately 704 000 persons nationwide), and 4.6% (CI, 4.1% to 5.0%) had been exposed to HBV (approximately 11 993 000 persons). These estimates are lower (P < 0.001) than estimates of HBV infection (0.42%) and exposure (5.1%) in the United States reported from 1988 to 1994. Infection and past exposure were very uncommon among persons aged 6 to 19 years. Children aged 2 years have high rates of immunity (68.6% [CI, 64.1% to 73.2%]). Adults, including those at high risk for infection, have much lower rates of immunity. LIMITATIONS Incarcerated and homeless persons were not sampled. Categorization of race or ethnicity did not identify high-risk groups, such as persons of Asian and Pacific Islander descent. CONCLUSION A cohort of children and adolescents is growing up in the United States with high rates of immunity against HBV and very low rates of infection. Vaccination of high-risk adults should continue to be emphasized. PRIMARY FUNDING SOURCE The Veterans Affairs Research Enhancement Award Program.

Is obesity associated with anemia of chronic disease? A population-based study.

Obesity is characterized by chronic, low‐grade, systemic inflammation, which, in turn, has been associated with anemia of chronic disease. We hypothesized that obesity may be associated with the features of anemia of chronic disease, including low hemoglobin concentration, low serum iron and transferrin saturation (TS), and elevated serum ferritin. We compared normal‐weight to overweight and obese adult participants of the third National Health and Nutrition Examination Survey with respect to hemoglobin concentration and levels of serum iron, TS, and ferritin. Measured BMI was used to categorize participants into normal weight (BMI < 25 kg/m2, n = 6,059), overweight (BMI 25 to <30 kg/m2, n = 5,108), mildly obese (BMI 30 to <35 kg/m2, n = 2,366), moderately obese (BMI 35 to <40 kg/m2, n = 850), and severely obese (BMI ≥ 40 kg/m2, n = 465). After adjustment for age, gender, menstruation, race/ethnicity, education, alcohol consumption, smoking, blood donation, and dietary iron intake, serum ferritin was progressively higher with increasing BMI category, whereas serum iron and TS were progressively lower. However, compared to normal‐weight persons, those in all other higher BMI categories did not have a significant change in hemoglobin concentration after adjustment for the above‐mentioned confounders. Overweight and obesity were associated with changes in serum iron, TS, and ferritin that would be expected to occur in the setting of chronic, systemic inflammation. However, overweight and obese persons were not more likely to be anemic compared with normal‐weight persons.