A novel flow cytometry tool for fibrosis scoring through hepatic stellate cell differentiation.

Hepatic stellate cells (HSCs) are a central fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. Peripheral blood lymphocytes from HCV patients are phagocytized by HSCs and induce their differentiation. This study aimed to characterize HSCs differentiation using a flow cytometry tool for fibrosis scoring. NK cells from healthy donors and from patients with chronic HCV with various severities of fibrosis were co-cultured with a human HSC line (LX2). LX2 phagocytosis of NK cells were stained for NK cells (CD45/CD56/CD3) and NK activation marker (CD107a) as well as INF-γ, apoptosis (Annexin-V) and α-smooth-muscle-actin (αSMA, as a marker of LX2 activation). In addition, reactive oxygen species (ROS) and the senescence marker P15 were analyzed prior to flow cytometry analysis. LX2 mono-cultures demonstrated a homogenous cell-population according to size (forward-scattered; FSC), granularity and αSMA expressions. However, on their co-culture with NK cells, the HSCs formed four subpopulations, which were stratified by αSMA intensities and cell size. NK cells isolated from heathy donors did not activate LX2-cells. In contrast, HCV exposed to NK cells from both F1 and F4 fibrosis grade patients, showed elevated CD107a and INF-γ levels and increased αSMA intensities in two of the four cell populations, with fibrosis scoring showing a linear correlation with αSMA intensities and NK phagocytosis. The αSMAintermediate /SizeLow HSCs sub-population showed higher proliferation following F4-NK cells with higher phagocytosis ability, suggesting an active/regulatory population. The αSMAhigh /Sizehigh subpopulations showed low proliferation and phagocytosis capacity, and were correlated with higher apoptosis, increased ROS and P15 intensities, suggesting senescing cells. Taken together, NK cells lead to heterogeneous differentiation of HSCs. Flow-cytometry may provide a novel means of characterizing HSCs in relation to the severity of liver fibrosis. © 2017 International Society for Advancement of Cytometry.

The association between adipocytokines and biomarkers for nonalcoholic fatty liver disease-induced liver injury: a study in the general population.

BACKGROUND AND AIM: Leptin and adiponectin have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). However, the usefulness of adipocytokines as a screening tool for nonalcoholic steatohepatitis (NASH) and fibrosis could not be evaluated in the general population due to the invasive nature of liver biopsy. The aim was to evaluate the association between adipocytokines and presumed liver injury in the general population using noninvasive biomarkers. METHODS: A cross-sectional study of 375 individuals, sampled from the National Health Survey was conducted. The exclusion criterion was any known secondary etiology for liver disease. Anthropometrics, serum leptin, adiponectin, insulin, lipids, and FibroMax were measured. RESULTS: Three hundred and thirty-eight individuals met the inclusion criteria and had valid FibroMax. Fibrosis diagnosed by the FibroTest was found in 25.7% of the patients, of whom 12.8% had significant fibrosis. Steatohepatitis was diagnosed by the NASH test in 0.9% and borderline NASH in 31.4% of the patients. Adiponectin was an independent negative correlate of borderline NASH [odds ratio (OR): 0.92; 95% confidence interval (CI): 0.86-0.98/1 µg/ml] together with high-density lipoprotein, and leptin was a positive correlate (OR: 1.03; CI: 1.01-1.06/1 ng/ml), together with abdominal obesity, serum triglycerides, and HbA1C. The OR for borderline NASH was 20.7 (CI: 7.5-57.5) when both high leptin (upper quartile) and suboptimal adiponectin were present, adjusting for age and sex. The FibroTest was not associated with leptin and adiponectin. The strongest predictors for fibrosis were age, sex, abdominal obesity, and insulin. CONCLUSION: Low adiponectin and high leptin and the combination of both have a strong independent association with presumed early-stage NASH. However, early-stage fibrosis cannot be predicted by these adipocytokines.

Fibrotest-Actitest: the biochemical marker of liver fibrosis--the Israeli experience.

BACKGROUND: The Fibrotest-Actitest is a six-parameter scoring system that allows quantification of liver fibrosis and inflammation. This test has been validated by several studies in hepatitis B and C viruses and alcoholic liver disease, with a high correlation between the liver biopsy and the results of the FT-AT (AUROC between 0.78 and 0.95). The FT-AT was introduced in Israel (Rambam Laboratory) in March 2005. OBJECTIVES: To assess the results of HCV patients who underwent the test during the period March 2005 to February 2006. METHODS: Serum was taken and brought to the central laboratory performing the tests within 4 hours. Six parameters were evaluated using commercial kits approved by the designer of the test (Biopredictive): total bilirubin, gamma-glutamyltransferase, alpha-2 macroglobulin, haptoglobin, alanine aminotransferase, and apolipoprotein-A1. The results were sent to the website of Biopredictive (France), which provided the FT-AT score online using a patented formula. RESULTS: Of the 325 patients tested, only 4 were not interpretable because of hemolysis. Patients' age ranged from 7 to 72 years (median 42); 54% were female. Liver biopsy was performed in 81 patients and was compared with the results of the Fibrotest. Findings were as follows: 27% of the patients were F0, 19% F1, 20% F2, 17% F3 and 17% F4; 18% were A0, 32% A1, 28% A2 and 22% A3. The AUROC curve comparing the Fibrotest with liver biopsy with a cutoff point at F2 and A2 for significant fibrosis and inflammation was 0.85 and 0.79 respectively. CONCLUSION: Fibrotest is a simple and effective method to assess liver fibrosis and inflammation and can be considered an alternative to liver biopsy in most patients with HCV.

Lamivudine prophylaxis for hepatitis B virus infection after lung transplantation.

BACKGROUND: Several reports have shown the efficacy of prophylactic lamivudine treatment for hepatitis B virus (HBV) infection in liver and renal transplantations. No data are available, however, after lung transplantation. We report our experience with prophylactic lamivudine treatment in lung transplant recipients with HBV infection or when the donor was HBc antibody positive. METHODS: All our 120 lung transplant recipients and their donors were routinely screened for HBV markers. All recipients who tested positive for hepatitis B surface antigen and negative for HBV-DNA, or had organs from donors who tested positive for hepatitis B core antibody, were treated prophylactically with lamivudine for 12 months after lung transplantation. Patients whose liver functions became abnormal during follow-up were tested for HBV serology and HBV-DNA. RESULTS: Eleven of 120 lung transplant recipients (9.2%) were treated with prophylaxis lamivudine. Four recipients were hepatitis B surface antigen positive, and 7 recipients received organs from donors positive for HBc antibodies. Median follow-up after treatment was 24 months. All patients had normal alanine transaminase and undetectable levels of HBV-DNA before treatment. No side effects of lamivudine therapy were reported by any of the patients. Reactivation with alanine transaminase elevation and high HBV-DNA levels occurred in 2 patients. Both of them were recipients positive for hepatitis B surface antigen. In the first patient, lamivudine-resistant strain was detected and adefovir dipivoxil was started. In the other, reactivation developed 2 months after the end of lamivudine treatment. Lamivudine treatment was resumed, with rapid normalization of the HBV-DNA. CONCLUSIONS: Use of lamivudine is considered safe for suppressing HBV infection after lung transplantation.

Hepatic production of insulin-like growth factors in normal and diseased liver.

BACKGROUND/AIMS: IGF-I levels are reduced in cirrhotic patients. However, it is not known whether this decreased level is the result of reduced hepatic production or modified bioavailability secondary to decreased binding proteins. We determined the hepatic production of IGF-I and IGF-II and their receptors in normal and diseased liver. METHODOLOGY: Twenty-five patients included, 11 controls with normal liver and 14 with either chronic hepatitis or cirrhosis. mRNA for IGF-1, IGF-II and their receptors were measured. Immunohistochemical staining was performed to localize the IGF-producing cells. RESULTS: In 11 normal livers, the IGF-I mRNA levels were 4.95 +/- 1.8; in the 14 diseased livers, the levels were 1.22 +/- 0.69 (p < 0.001). IGF-II mRNA levels were 3.78 +/- 1.45 for the control and 5.11 +/- 2.15 in the diseased livers (NS). IGF-I receptor levels were 1.15 +/- 0.83 in the normal and 0.31 +/- 0.22 in the liver disease group (p < 0.05). There was no statistical difference between the two groups for IGF-II receptor. CONCLUSIONS: Patients with chronic liver disease have a significant reduction in their hepatic production of IGF-I, whereas IGF-II tends to be elevated. Treatment with recombinant IGF-I in patients with metabolic or endocrine complications of cirrhosis might prove useful.