Amany Zekry

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.

Real‐time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real‐time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA‐binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real‐time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low‐level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.

Historical epidemiology of hepatitis C virus (HCV) in selected countries

Chronic infection with hepatitis C virus (HCV) is a leading indicator for liver disease. New treatment options are becoming available, and there is a need to characterize the epidemiology and disease burden of HCV. Data for prevalence, viremia, genotype, diagnosis and treatment were obtained through literature searches and expert consensus for 16 countries. For some countries, data from centralized registries were used to estimate diagnosis and treatment rates. Data for the number of liver transplants and the proportion attributable to HCV were obtained from centralized databases. Viremic prevalence estimates varied widely between countries, ranging from 0.3% in Austria, England and Germany to 8.5% in Egypt. The largest viremic populations were in Egypt, with 6 358 000 cases in 2008 and Brazil with 2 106 000 cases in 2007. The age distribution of cases differed between countries. In most countries, prevalence rates were higher among males, reflecting higher rates of injection drug use. Diagnosis, treatment and transplant levels also differed considerably between countries. Reliable estimates characterizing HCV‐infected populations are critical for addressing HCV‐related morbidity and mortality. There is a need to quantify the burden of chronic HCV infection at the national level.

Strategies to manage hepatitis C virus (HCV) disease burden

The number of hepatitis C virus (HCV) infections is projected to decline while those with advanced liver disease will increase. A modeling approach was used to forecast two treatment scenarios: (i) the impact of increased treatment efficacy while keeping the number of treated patients constant and (ii) increasing efficacy and treatment rate. This analysis suggests that successful diagnosis and treatment of a small proportion of patients can contribute significantly to the reduction of disease burden in the countries studied. The largest reduction in HCV‐related morbidity and mortality occurs when increased treatment is combined with higher efficacy therapies, generally in combination with increased diagnosis. With a treatment rate of approximately 10%, this analysis suggests it is possible to achieve elimination of HCV (defined as a >90% decline in total infections by 2030). However, for most countries presented, this will require a 3–5 fold increase in diagnosis and/or treatment. Thus, building the public health and clinical provider capacity for improved diagnosis and treatment will be critical.

Chronic Hepatitis C Is Associated With Peripheral Rather Than Hepatic Insulin Resistance

BACKGROUND & AIMS Chronic hepatitis C (CHC) is associated with insulin resistance (IR), liver steatosis (genotype 3), and increased diabetes risk. The site and mechanisms of IR are unclear. METHODS We compared cross-sectionally 29 nonobese, normoglycemic males with CHC (genotypes 1 and 3) to 15 adiposity and age-matched controls using a 2-step hyperinsulinemic-euglycemic clamp with [6,6-(2)H(2)] glucose to assess insulin sensitivity in liver and peripheral tissues and (1)H-magnetic resonance spectroscopy to evaluate liver and intramyocellular lipid. Insulin secretion was assessed after intravenous glucose. RESULTS Insulin secretion was not impaired in CHC. Peripheral insulin sensitivity was 35% higher in controls vs CHC (P < .001) during high-dose (264.3 +/- 25 [standard error] mU/L) insulin (P < .001); this was negatively associated with viral load (R(2) = .12; P = .05) and subcutaneous fat (R(2) = .41; P < .001). IR was similar in both genotypes despite 3-fold increased hepatic fat in genotype 3 (P < .001). Hepatic glucose production (P = .25) and nonesterified free fatty acid (P = .84) suppression with insulin were not different between CHC and controls inferring no adipocyte IR, and suggesting IR is mainly in muscle. In CHC, intramyocellular lipid was nonsignificantly increased but levels of glucagon (73.8 +/- 3.6 vs 52.8 +/- 3.1 ng/mL; P < .001), soluble tumor necrosis factor receptor 2 (3.1 +/- 0.1 vs 2.3 +/- 0.1 ng/mL; P < .001), and Lipocalin-2 (36.4 +/- 2.9 vs 19.6 +/- 1.6 ng/mL; P < .001) were elevated. CONCLUSIONS CHC represents a unique infective/inflammatory model of IR, which is predominantly in muscle, correlates with subcutaneous, not visceral, adiposity, and is independent of liver fat.

A prospective cross-over study comparing the effect of Mycophenolate versus azathioprine on allograft function and viral load in liver transplant recipients with recurrent chronic HCV infection

It has been suggested that Mycophenolate Mofetil (MMF) may have an antiviral effect in patients with recurrent HCV infection post‐liver transplantation. We conducted a prospective cross‐over study in liver transplant recipients with recurrent chronic HCV infection to examine whether substitution of azathioprine (AZA) with MMF would reduce HCV load and improve allograft function. Thirteen long standing HCV liver transplant recipients receiving AZA were enrolled in a 9‐month prospective cross‐over study. In the initial 3 months lead‐in period, baseline viral loads and biochemistry were recorded. Following this, MMF was substituted for AZA at a dose of 1 gm twice/day for a period of 3 months after which patients were switched back to AZA and observed for a further 3 months. Viral loads, biochemical allograft function, and adverse effects were closely monitored during the study period. Thirteen patients (12 males and 1 female) were enrolled. The mean age was 54 (±8) years and the mean time from transplantation was 68 (±35) months. Baseline mean viral load was 0.74 × 106(±0.47 × 106) messenger RNA (mRNA) copies/ml. By the end of the MMF treatment period, the mean viral load increased to a level of 1.64 × 106 (±1.3 × 106) mRNA copies/ml (P = 0.026) compared to baseline. The increase in viral load however was not associated with an increase in ALT level. In a cohort of 13 HCV liver transplant recipients with recurrent chronic HCV infection, substitution of azathioprine with MMF did not lead to a decrease in viral load. (Liver Transpl 2004;10:52–57.)

Insulin resistance and steatosis in hepatitis C virus infection

The relationship between hepatitis C virus (HCV), steatosis, and insulin resistance is genotype specific, and steatosis and insulin resistance are closely linked to the progression of liver disease in HCV infected patients.

A novel role for adiponectin in regulating the immune responses in chronic hepatitis C virus infection

Adipose tissue releases pro‐inflammatory and anti‐inflammatory mediators, including adiponectin, which elicit a broad range of metabolic and immunological effects. The study aim was to determine in subjects infected with chronic hepatitis C virus (HCV) the effects of total adiponectin and its high‐molecular‐weight (HMW) and low‐molecular‐weight isoforms on HCV‐specific immune responses. Serum levels of total adiponectin and its isoforms were determined by immunoassay. The ex vivo effect of adiponectin on the HCV‐specific T‐cell response was examined by interferon gamma (IFN‐γ) enzyme‐linked immunosorbent spot and enzyme‐linked immunosorbent assay cytokine assays. The role of the mitogen‐activated protein kinase (MAPK) signaling pathway in mediating the adiponectin effect on T cells was also evaluated. We found that serum levels of total and HMW adiponectin were significantly decreased in subjects with chronic HCV and increased body mass index (BMI) compared with HCV‐infected lean subjects. The presence of an anti‐HCV specific immune response was strongly associated with lower BMI (P = 0.004) and higher serum total (P = 0.01) and HMW (P = 0.02) adiponectin. In ex vivo assays, total adiponectin and the HMW adiponectin isoform enhanced HCV‐specific IFN‐γ production (P = 0.02 and 0.03, respectively). Adiponectin‐R1 receptors were expressed on T cells and monocytes. In depletion experiments, the IFN‐γ response to adiponectin was entirely dependent on the simultaneous presence of both CD4 and CD8 T cells, and to a lesser extent, natural killer cells. Selective inhibition of p38MAPK activity by SB203580 abrogated the IFN‐γ response to adiponectin, whereas extracellular signal‐regulated kinase 1/2 inhibition by PD98059 did not affect the response. Conclusion: In chronic HCV, a reciprocal association exists between BMI, adiponectin, and the anti‐HCV immune responses, emphasizing the important role played by adiposity in regulating the immune response in HCV infection. (HEPATOLOGY 2008;48:374–384.)

Serum proteomic analysis focused on fibrosis in patients with hepatitis C virus infection

BackgroundDespite its widespread use to assess fibrosis, liver biopsy has several important drawbacks, including that is it semi-quantitative, invasive, and limited by sampling and observer variability. Non-invasive serum biomarkers may more accurately reflect the fibrogenetic process. To identify potential biomarkers of fibrosis, we compared serum protein expression profiles in patients with chronic hepatitis C (CHC) virus infection and fibrosis.MethodsTwenty-one patients with no or mild fibrosis (METAVIR stage F0, F1) and 23 with advanced fibrosis (F3, F4) were retrospectively identified from a pedigreed database of 1600 CHC patients. All samples were carefully phenotyped and matched for age, gender, race, body mass index, genotype, duration of infection, alcohol use, and viral load. Expression profiling was performed in a blinded fashion using a 2D polyacrylamide gel electrophoresis/LC-MS/MS platform. Partial least squares discriminant analysis and likelihood ratio statistics were used to rank individual differences in protein expression between the 2 groups.ResultsSeven individual protein spots were identified as either significantly increased (α2-macroglobulin, haptoglobin, albumin) or decreased (complement C-4, serum retinol binding protein, apolipoprotein A-1, and two isoforms of apolipoprotein A-IV) with advanced fibrosis. Three individual proteins, haptoglobin, apolipoprotein A-1, and α2-macroglobulin, are included in existing non-invasive serum marker panels.ConclusionBiomarkers identified through expression profiling may facilitate the development of more accurate marker algorithms to better quantitate hepatic fibrosis and monitor disease progression.

Enhanced antiviral treatment efficacy and uptake in preventing the rising burden of hepatitis C‐related liver disease and costs in Australia

Chronic hepatitis C virus (HCV) infection is an important cause of advanced liver disease and liver‐related deaths in Australia. Our aim was to describe the burden of HCV infection and consider treatment strategies to reduce HCV‐related morbidity and mortality.

Effects of immunosuppression and organ transplantation on the natural history and immunopathogenesis of hepatitis C virus infection

The hepatitis C virus (HCV) is recognized as the leading cause for parenterally transmitted hepatitis. It is characterized by a high propensity to chronicity. Several efforts have been directed towards understanding the natural history of chronic HCV infection and the immunopathogenic pathways involved in mediating liver injury in the non‐immunosuppressed and immunosuppressed states. In the non‐immunosuppressed setting, liver damage seems to be largely immune mediated. In contrast, in the non‐immunosuppressed state, there are several other factors that may modify the natural course of the infection and play a role in mediating liver injury. In this review we will address the natural history, virological and immunological aspects of HCV infection. Also, the role played by immunosuppression and organ transplantation in modifying the course of the infection and the pathogenesis of liver injury will be discussed.( Note )