Liver stiffness measurement and biochemical markers in Senegalese chronic hepatitis B patients with normal ALT and high viral load

Background and Aims

Despite the high prevalence of chronic hepatitis B (CHB) in Africa, few studies have been performed among African patients. We sought to evaluate liver stiffness measurement by FibroScan® (LSM) and two biochemical scores (FibroTest®, Fibrometer®) to diagnose liver fibrosis in Senegalese CHB patients with HBV plasma DNA load ≥3.2 log₁₀ IU/mL and normal alanine aminotransferase (ALT) values.

Methods

LSM and liver fibrosis biochemical markers were performed on 225 consecutive HBV infected Senegalese patients with high viral load. Patients with an LSM range between 7 and 13 kPa underwent liver biopsy (LB). Two experienced liver pathologists performed histological grading using Metavir and Ishak scoring.

Results

225 patients were evaluated (84% male) and LB was performed in 69 patients, showing F2 and F3 fibrosis in 17% and 10% respectively. In these patients with a 7–13 kPa range of LSM, accuracy for diagnosis of significant fibrosis according to LB was unsatisfactory for all non-invasive markers with AUROCs below 0.70. For patients with LSM values below 7 kPa, FibroTest® (FT), and Fibrometer® (FM) using the cut-offs recommended by the test promoters suggested a fibrosis in 18% of cases for FT (8% severe fibrosis) and 8% for FM. For patients with LSM values greater than 13 kPa, FT, FM suggested a possible fibrosis in 73% and 70%, respectively.

Conclusion

In highly replicative HBV-infected African patients with normal ALT and LSM value below 13 kPa, FibroScan®, FibroTest® or Fibrometer® were unsuitable to predict the histological liver status of fibrosis.     

Non-Invasive Evaluation of Cystic Fibrosis Related Liver Disease in Adults with ARFI, Transient Elastography and Different Fibrosis Scores

Background

Cystic fibrosis-related liver disease (CFLD) is present in up to 30% of cystic fibrosis patients and can result in progressive liver failure. Diagnosis of CFLD is challenging. Non-invasive methods for staging of liver fibrosis display an interesting diagnostic approach for CFLD detection.

Aim

We evaluated transient elastography (TE), acoustic radiation force impulse imaging (ARFI), and fibrosis indices for CFLD detection.

Methods

TE and ARFI were performed in 55 adult CF patients. In addition, AST/Platelets-Ratio-Index (APRI), and Forns'' score were calculated. Healthy probands and patients with alcoholic liver cirrhosis served as controls.

Results

Fourteen CF patients met CFLD criteria, six had liver cirrhosis. Elastography acquisition was successful in >89% of cases. Non-cirrhotic CFLD individuals showed elastography values similar to CF patients without liver involvement. Cases with liver cirrhosis differed significantly from other CFLD patients (ARFI: 1.49 vs. 1.13 m/s; p = 0.031; TE: 7.95 vs. 4.16 kPa; p = 0.020) and had significantly lower results than individuals with alcoholic liver cirrhosis (ARFI: 1.49 vs. 2.99 m/s; p = 0.002). APRI showed the best diagnostic performance for CFLD detection (AUROC 0.815; sensitivity 85.7%, specificity 70.7%).

Conclusions

ARFI, TE, and laboratory based fibrosis indices correlate with each other and reliably detect CFLD related liver cirrhosis in adult CF patients. CF specific cut-off values for cirrhosis in adults are lower than in alcoholic cirrhosis.     

High Hepatitis B Surface Antigen Levels Predict Insignificant Fibrosis in Hepatitis B e Antigen Positive Chronic Hepatitis B

Introduction

There is no data on the relationship between hepatitis B surface antigen (HBsAg) levels and liver fibrosis in hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B (CHB).

Methods

Serum HBsAg and HBV DNA levels in HBeAg-positive CHB patients with liver biopsies were analyzed. The upper limit of normal (ULN) of alanine aminotransferase (ALT) was 30 and 19 U/L for men and women respectively. Histologic assessment was based on Ishak fibrosis staging for fibrosis and Knodell histologic activity index (HAI) for necroinflammation.

Results

140 patients (65% male, median age 32.7 years) were recruited. 56 (40%) had ALT ≤2×ULN. 72 (51.4%) and 42 (30%) had fibrosis score ≤1 and necroinflammation grading ≤4 respectively. Patients with fibrosis score ≤1, when compared to patients with fibrosis score >1, had significantly higher median HBsAg levels (50,320 and 7,820 IU/mL respectively, p<0.001). Among patients with ALT ≤2×ULN, serum HBsAg levels achieved an area under receiver operating characteristic curve of 0.869 in predicting fibrosis score ≤1. HBsAg levels did not accurately predict necroinflammation score. HBsAg ≥25,000 IU/mL was independently associated with fibrosis score ≤1 (p = 0.025, odds ratio 9.042).Using this cut-off HBsAg level in patients with ALT ≤2×ULN, positive and negative predictive values for predicting fibrosis score ≤1 were 92.7% and 60.0% respectively. HBV DNA levels had no association with liver histology.

Conclusion

Among HBeAg-positive patients with ALT ≤2×ULN, high serum HBsAg levels can accurately predict fibrosis score ≤1, and could potentially influence decisions concerning treatment commencement and reduce the need for liver biopsy.     

The CXCR3(+)CD56Bright phenotype characterizes a distinct NK cell subset with anti-fibrotic potential that shows dys-regulated activity in hepatitis C

Background

In mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C.

Methods

Circulating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(−) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15).

Results

We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis.

Conclusion

We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis.     

Soluble serum CD81 is elevated in patients with chronic hepatitis C and correlates with alanine aminotransferase serum activity

Aim

Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity.

Patients and Methods

Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry.

Results

Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027).

Conclusion

CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis.     

Mutational Analysis of ATP8B1 in Patients with Chronic Pancreatitis

Background

Mutations in genes encoding cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) are associated with chronic pancreatitis. However, in many patients with a familial chronic pancreatitis pattern suggesting a genetic cause, no mutations in either of these genes can be found, indicating that other, still unknown, associated genes exist. In this respect ATP8B1 is an interesting candidate due to its strong expression in the pancreas, its supposed general function in membrane organization and the higher incidence of pancreatitis in patients with ATP8B1 deficiency.

Methods

We analyzed all 27 ATP8B1 coding exons and adjacent non-coding sequences of 507 chronic pancreatitis patients by direct sequencing. Exons that harbored possible relevant variations were subsequently sequenced in 1,027 healthy controls.

Results

In the exonic regions, 5 novel non-synonymous alterations were detected as well as 14 previously described alterations of which some were associated with ATP8B1 deficiency. However, allele frequencies for any of these variations did not significantly differ between patients and controls. Furthermore, several non-synonymous variants were exclusively detected in control subjects and multiple variants in the non-coding sequence were identified with similar frequencies in both groups.

Conclusions

We did not find an association between heterozygous ATP8B1 variants and chronic pancreatitis in our cohort of patients with hereditary and idiopathic chronic pancreatitis.     

A large population histology study showing the lack of association between ALT elevation and significant fibrosis in chronic hepatitis B

Objective

We determined the association between various clinical parameters and significant liver injury in both hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients.

Methods

From 1994 to 2008, liver biopsy was performed on 319 treatment-naïve CHB patients. Histologic assessment was based on the Knodell histologic activity index for necroinflammation and the Ishak fibrosis staging for fibrosis.

Results

211 HBeAg-positive and 108 HBeAg-negative patients were recruited, with a median age of 31 and 46 years respectively. 9 out of 40 (22.5%) HBeAg-positive patients with normal ALT had significant histologic abnormalities (necroinflammation grading ≥7 or fibrosis score ≥3). There was a significant difference in fibrosis scores among HBeAg-positive patients with an ALT level within the Prati criteria (30 U/L for men, 19 U/L for women) and patients with a normal ALT but exceeding the Prati criteria (p = 0.024). Age, aspartate aminotransferase and platelet count were independent predictors of significant fibrosis in HBeAg-positive patients with an elevated ALT by multivariate analysis (p = 0.007, 0.047 and 0.045 respectively). HBV DNA and platelet count were predictors of significant fibrosis in HBeAg-negative disease (p = 0.020 and 0.015 respectively). An elevated ALT was not predictive of significant fibrosis for HBeAg-positive (p = 0.345) and -negative (p = 0.544) disease. There was no significant difference in fibrosis staging among ALT 1–2×upper limit of normal (ULN) and >×2 ULN for both HBeAg-positive (p = 0.098) and -negative (p = 0.838) disease.

Conclusion

An elevated ALT does not accurately predict significant liver injury. Decisions on commencing antiviral therapy should not be heavily based on a particular ALT threshold.     

Reproductive status is associated with the severity of fibrosis in women with hepatitis C

Introduction

Chronic hepatitis C is the main cause of death in patients with end-stage liver disease. Prognosis depends on the increase of fibrosis, whose progression is twice as rapid in men as in women. Aim of the study was to evaluate the effects of reproductive stage on fibrosis severity in women and to compare these findings with age-matched men.

Materials and Methods

A retrospective study of 710 consecutive patients with biopsy-proven chronic hepatitis C was conducted, using data from a clinical database of two tertiary Italian care centers. Four age-matched groups of men served as controls. Data about demographics, biochemistry, liver biopsy and ultrasonography were analyzed. Contributing factors were assessed by multivariate logistic regression analysis.

Results

Liver fibrosis was more advanced in the early menopausal than in the fully reproductive (P<0.0001) or premenopausal (P = 0.042) group. Late menopausal women had higher liver fibrosis compared with the other groups (fully reproductive, P<0.0001; premenopausal, P = <0.0001; early menopausal, P = 0.052). Multivariate analyses showed that male sex was independently associated with more severe fibrosis in the groups corresponding to premenopausal (P = 0.048) and early menopausal (P = 0.004) but not late menopausal pairs. In women, estradiol/testosterone ratio decreased markedly in early (vs. reproductive age: P = 0.002 and vs. premenopausal: P<0.0001) and late menopause (vs. reproductive age: P = 0.001; vs. premenopausal: P<0.0001). In men age-matched with menopausal women, estradiol/testosterone ratio instead increased (reproductive age group vs. early: P = 0.002 and vs. late M: P = 0.001).

Conclusions

The severity of fibrosis in women worsens in parallel with increasing estrogen deprivation and estradiol/testosterone ratio decrease. Our data provide evidence why fibrosis progression is discontinuous in women and more linear and severe in men, in whom aging-associated estradiol/testosterone ratio increase occurs too late to noticeably influence the inflammatory process leading to fibrosis.     

Hyperferritinemia is associated with insulin resistance and fatty liver in patients without iron overload

Objective

During the last 10 years we have experienced an increasing number of referrals due to hyperferritinemia. This is probably due to increased awareness of hereditary hemochromatosis, and the availability of a genetic test for this condition. Most of these referred patients were over-weight middle-aged men with elevated ferritin levels, but without the hemochromatosis-predisposing gene mutations. We evaluated the relationship between hyperferritinemia and the metabolic syndrome in 40 patients.

Methods

Forty consecutive patients referred for hyperferritinemia were investigated. The examination programme included medical history, clinical investigation and venous blood samples drawn after an overnight fast. This resulted in 34 patients with unexplained hyperferritinemia, which were further examined. Liver biopsy was successfully performed in 29 subjects. Liver iron stores were assessed morphologically, and by quantitative phlebotomy in 16 patients.

Results

The majority of the patients had markers of the metabolic syndrome, and 18 patients (52%) fulfilled the IDF-criteria for the metabolic syndrome. Mean body mass index was elevated (28,8±4,2), mean diastolic blood pressure was 88,5±10,5 mmHg, and mean fasting insulin C-peptide 1498±539 pmol/l. Liver histology showed steatosis and nuclear glycogen inclusions in most patients (19 out of 29). Only four patients had increased iron stores by histology, of which two could be explained by alcohol consumption. Fourteen of 16 patients normalized ferritin levels after phlebotomy of a cumulative blood amount corresponding to normal iron stores. Ferritin levels were significantly related to insulin C-peptide level (p<0.002) and age (p<0.002).

Conclusion

The present results suggest that liver steatosis and insulin resistance but not increased iron load is frequently seen in patients referred for suspected hemochromatosis on the basis of hyperferritinemia. The ferritin level seems to be positively associated to insulin resistance.     

Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis

Background

Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients.

Methodology

Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets.

Principal Findings

IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro.

Conclusions

IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.     

Prospective validation of FibroTest in comparison with liver stiffness for predicting liver fibrosis in Asian subjects with chronic hepatitis B

Background and Aims

Diagnostic values of FibroTest (FT) for hepatic fibrosis have rarely been assessed in Asian chronic hepatitis B (CHB) patients. We aimed to validate its diagnostic performances in comparison with liver stiffness (LS).

Methods

From 2008 to 2010, 194 CHB patients who underwent liver biopsies along with FT and transient elastography were prospectively enrolled. Fibrosis stage was assessed according to the Batts and Ludwig system.

Results

To predict significant fibrosis (F≥2), advanced fibrosis (F≥3), and cirrhosis (F = 4), areas under receiver operating characteristic curves (AUROCs) of FT were 0.903, 0.907, and 0.866, comparable to those of LS (0.873, 0.897, and 0.910, respectively). Optimized cutoffs of FT to maximize sum of sensitivity and specificity were 0.32, 0.52, and 0.68 for F≥2, F≥3, and F = 4, while those of LS were 8.8, 10.2, and 14.1 kPa, respectively. According to FT and LS cutoffs, 123 (63.4%) and 124 (63.9%) patients were correctly classified consistent with histological fibrosis (F1, F2, F3, and F4), respectively. Overall concordance between each fibrosis stage estimated by FT and LS was observed in 111 patients, where 88 were correctly classified with histological results. A combination formula adding LS to FT (LS+FT) showed similar AUROC levels (0.885, 0.905, and 0.915), while another multiplying LS by FT (LS×FT) showed the best AUROCs (0.941, 0.931, and 0.929 for F≥2, F≥3, and F4, respectively).

Conclusions

FT provides good fibrosis prediction, with comparable outcomes to LS in Asian CHB patients. FT substantially reduces need for liver biopsy, especially when used in combination with LS.     

Emergence of minor drug-resistant HIV-1 variants after triple antiretroviral prophylaxis for prevention of vertical HIV-1 transmission

Background

WHO-guidelines for prevention of mother-to-child transmission of HIV-1 in resource-limited settings recommend complex maternal antiretroviral prophylaxis comprising antenatal zidovudine (AZT), nevirapine single-dose (NVP-SD) at labor onset and AZT/lamivudine (3TC) during labor and one week postpartum. Data on resistance development selected by this regimen is not available. We therefore analyzed the emergence of minor drug-resistant HIV-1 variants in Tanzanian women following complex prophylaxis.

Method

1395 pregnant women were tested for HIV-1 at Kyela District Hospital, Tanzania. 87/202 HIV-positive women started complex prophylaxis. Blood samples were collected before start of prophylaxis, at birth and 1–2, 4–6 and 12–16 weeks postpartum. Allele-specific real-time PCR assays specific for HIV-1 subtypes A, C and D were developed and applied on samples of mothers and their vertically infected infants to quantify key resistance mutations of AZT (K70R/T215Y/T215F), NVP (K103N/Y181C) and 3TC (M184V) at detection limits of <1%.

Results

50/87 HIV-infected women having started complex prophylaxis were eligible for the study. All women took AZT with a median duration of 53 days (IQR 39–64); all women ingested NVP-SD, 86% took 3TC. HIV-1 resistance mutations were detected in 20/50 (40%) women, of which 70% displayed minority species. Variants with AZT-resistance mutations were found in 11/50 (22%), NVP-resistant variants in 9/50 (18%) and 3TC-resistant variants in 4/50 women (8%). Three women harbored resistant HIV-1 against more than one drug. 49/50 infants, including the seven vertically HIV-infected were breastfed, 3/7 infants exhibited drug-resistant virus.

Conclusion

Complex prophylaxis resulted in lower levels of NVP-selected resistance as compared to NVP-SD, but AZT-resistant HIV-1 emerged in a substantial proportion of women. Starting AZT in pregnancy week 14 instead of 28 as recommended by the current WHO-guidelines may further increase the frequency of AZT-resistance mutations. Given its impact on HIV-transmission rate and drug-resistance development, HAART for all HIV-positive pregnant women should be considered.     

Total and high molecular weight adiponectin and hepatocellular carcinoma with HCV infection

Background

Adiponectin is shown to be inversely associated with development and progression of various cancers. We evaluated whether adiponectin level was associated with the prevalence and histological grade of hepatocellular carcinoma (HCC), and liver fibrosis in patients with hepatitis C virus (HCV) infection.

Methods

A case-control study was conducted on 97 HCC patients (cases) and 97 patients (controls) matched for sex, Child-Pugh grade and platelet count in patients with HCV infection. The serum total and high molecular weight (HMW) adiponectin levels were measured by enzyme-linked immunosorbent assays and examined in their association with the prevalence of HCC. In addition, the relationship between these adiponectin levels and body mass index (BMI), progression of liver fibrosis, and histological grade of HCC was also evaluated. Liver fibrosis was assessed using the aspartate aminotransferase to platelet ratio index (APRI).

Results

There were no significant differences in the serum total and HMW adiponectin levels between cases and controls. Moreover, there were no inverse associations between serum total and HMW adiponectin levels and BMI in both cases and controls. On the other hand, serum total and HMW adiponectin levels are positively correlated with APRI in both cases (r = 0.491, P<0.001 and r = 0.485, P<0.001, respectively) and controls (r = 0.482, P<0.001 and r = 0.476, P<0.001, respectively). Interestingly, lower serum total (OR 11.76, 95% CI: 2.97–46.66 [P<0.001]) and HMW (OR 10.24, CI: 2.80–37.40 [P<0.001] adiponectin levels were independent risk factors of worse histological grade of HCC.

Conclusions

Our results suggested that serum total and HMW adiponectin levels were predictors of liver fibrosis, but not prevalence of HCC in patients with HCV infection. Moreover, low these adiponectin levels were significantly associated with worse histological grades.     

Hepatitis C viral evolution in genotype 1 treatment-naïve and treatment-experienced patients receiving telaprevir-based therapy in clinical trials

Background

In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR.

Methods

Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment.

Results

Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients.

Conclusions

A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment.     

A secreted form of the asialoglycoprotein receptor, sH2a, as a novel potential noninvasive marker for liver fibrosis

Background and Aim

The human asialoglycoprotein receptor is a membrane heterooligomer expressed exclusively in hepatocytes. A soluble secreted form, sH2a, arises, not by shedding at the cell surface, but by intracellular cleavage of its membrane-bound precursor, which is encoded by an alternatively spliced form of the receptor H2 subunit. Here we determined and report that sH2a, present at constant levels in serum from healthy individuals is altered upon liver fibrosis, reflecting the status of hepatocyte function.

Methods

We measured sH2a levels in serum using a monoclonal antibody and an ELISA assay that we developed, comparing with routine liver function markers. We compared blindly pretreatment serum samples from a cohort of 44 hepatitis C patients, which had METAVIR-scored biopsies, with 28 healthy individuals.

Results

sH2a levels varied minimally for the healthy individuals (150±21 ng/ml), whereas the levels deviated from this normal range increasingly in correlation with fibrosis stage. A simple algorithm combining sH2a levels with those of alanine aminotransferase allowed prediction of fibrosis stage, with a very high area under the ROC curve of 0.86.

Conclusions

sH2a has the potential to be a uniquely sensitive and specific novel marker for liver fibrosis and function.     

Genomic characterization of mutant laboratory mouse strains by exome sequencing and annotation lift-over

Background

Exome sequencing has become a popular method to evaluate undirected mutagenesis experiments in mice. However, the most suitable mouse strain for the biological model may be relatively distant from the standard mouse reference genome. For pinpointing causative variants, a matching reference with gene annotations is essential, but not always readily available.

Results

We present an approach that allows to use murine Ensembl annotations on alternative mouse strain assemblies. We resolved ENU-induced mutation screening for 8 phenotypic mutant lines generated on C3HeB/FeJ background aligning the sequences against the closely related, but not annotated reference of C3H/HeJ. Variants occurring in all strains were filtered out as specific for the C3HeB/FeJ strain but unrelated to mutagenesis. Variants occurring exclusively in all individuals of one mutant line and matching the inheritance model were selected as mutagenesis-related. These variants were annotated with gene and exon names lifted over from the standard murine reference mm9 to C3H/HeJ using megablast. For each mutant line, we could restrict the results to exonic variants in between 1 and 23 genes.

Conclusions

The presented method of exonic annotation lift-over proved to be a valuable tool in the search for mutagenesis-derived coding genomic variants and the assessment of genotype-phenotype relationships.     

Hedgehog signaling antagonist promotes regression of both liver fibrosis and hepatocellular carcinoma in a murine model of primary liver cancer

Objective

Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2^−/− mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC.

Methods

Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2^−/− mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging.

Results

Unlike controls, Mdr2^−/− mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality.

Conclusions

Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.     

Exercise related ventricular arrhythmias are related to cardiac fibrosis in hypertrophic cardiomyopathy mutation carriers

Aims

Hypertrophic cardiomyopathy (HCM) is a frequent cause of sudden cardiac death (SCD) due to exercise-related ventricular arrhythmias (ERVA); however the pathological substrate is uncertain. The aim was to determine the prevalence of ERVA and their relation with fibrosis as determined by cardiac magnetic resonance imaging (CMR) in carriers of an HCM causing mutation.

Methods

We studied the prevalence and origin of ERVA and related these with fibrosis on CMR in a population of 31 HCM mutation carriers.

Results

ERVA occurred in seven patients (23%) who all showed evidence of fibrosis (100% ERVA(+) vs. 58% ERVA(-), p = 0.04). No ventricular tachycardia or ventricular fibrillation occurred. In patients with ERVA, the extent of fibrosis was significantly larger (8 ± 4% vs. 3 ± 4%, p = 0.02). ERVA originated from areas with a high extent of fibrosis or regions directly adjacent to these areas.

Conclusions

ERVA in HCM mutation carriers arose from the area of fibrosis detected by CMR; ERVA seems closely related to cardiac fibrosis. Fibrosis as detected by CMR should be evaluated as an additional risk factor to further delineate risk of SCD in carriers of an HCM causing mutation.     

Serum markers of hepatocyte death and apoptosis are non invasive biomarkers of severe fibrosis in patients with alcoholic liver disease

Background

Quantification of hepatotocyte death is useful to evaluate the progression of alcoholic liver diseases. Our aims were to quantify and correlate the circulating levels of Cytokeratin 18 (CK18) and its caspases-generated fragment to disease severity in heavy alcoholics.

Methodology/Principal Findings

CK18 and CK18-fragment were evaluated in the serum of 143 heavy alcoholics. Serum levels of markers of hepatocyte death (CK18), apoptosis (CK18 fragment) and necrosis (CK18 -CK18 fragment) increased in patients with severe fibrosis compared to patients with mild fibrosis. These markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, fibrosis and with hepatic TNFα and TGFβ assessed in the liver of 24 patients. Elevated levels of serum hepatocyte death and apoptotic markers were independent risk factors in predicting severe fibrosis in a model combining alkaline phosphatase, bilirubin, prothrombin index, hyaluronate, hepatocyte death and apoptotic markers. The level of markers of hepatocyte death and apoptosis had an area under the receiving operator curve that predicted severe fibrosis of 0.84 and 0.76, respectively.

Conclusion/Significance

Death of hepatocytes can be easily evaluated with serum markers and correlated with severe fibrosis in heavy alcohol drinkers. These biomarkers could be useful to rapidly evaluate liver injuries and the efficacy of therapies.