Francesco Vizzutti

Acute viral hepatitis increases liver stiffness values measured by transient elastography

Liver tissue alterations other than fibrosis may have an impact on liver stiffness measurement. In this study we evaluated 18 patients without a previous clinical history of liver disease, consecutively admitted for acute viral hepatitis. In each patient, aminotransferase determination and liver stiffness measurement were performed on the same study day, at 3 different points: (1) peak increase in aminotransferase; (2) aminotransferase 50% or less of the peak; (3) aminotransferase levels ≤2× the upper limit of normal. In all patients, the degree of liver stiffness at the time of the peak increase in aminotransferases exceeded the cutoff values proposed for the prediction of significant fibrosis or cirrhosis. A progressive significant reduction in liver stiffness values was observed (P < 0.0001) in the follow‐up period in parallel with the reduction of aminotransferase levels (P < 0.0001). Moreover, a statistically significant, positive correlation between aminotransferases and liver stiffness measurement (LSM) at the onset of acute viral hepatitis was found (r = 0.53, P = 0.02 and r = 0.51, P = 0.03 for alanine aminotransferase and aspartate aminotransferase, respectively). In conclusion, the extent of necroinflammatory activity needs to be carefully considered in future studies aimed at further validating transient elastography, particularly in patients with absent or low‐stage liver fibrosis (in other words, F0‐F2 METAVIR). LSM does not represent a reliable instrument to detect the presence of advanced fibrosis and cirrhosis in patients presenting with a clinical picture of acute hepatitis. (HEPATOLOGY 2007.)

Liver stiffness measurement predicts severe portal hypertension in patients with HCV‐related cirrhosis

Measurement of hepatic venous pressure gradient (HVPG) is a standard method for the assessment of portal pressure and correlates with the occurrence of its complications. Liver stiffness measurement (LSM) has been proposed as a noninvasive technique for the prediction of the complications of cirrhosis. In this study, we evaluated the ability of LSM to predict severe portal hypertension compared with that of HVPG in 61 consecutive patients with HCV‐related chronic liver disease. A strong relationship between LSM and HVPG measurements was found in the overall population (r = 0.81, P < 0.0001). However, although the correlation was excellent for HVPG values less than 10 or 12 mm Hg (r = 0.81, P = 0.0003 and r = 0.91, P < 0.0001, respectively), linear regression analysis was not optimal for HVPG values ≥10 mm Hg (r2 = 0.35, P < 0.0001) or ≥12 mm Hg (r2 = 0.17, P = 0.02). The AUROC for the prediction of HVPG ≥10 and ≥12 mm Hg were 0.99 and 0.92, respectively and at LSM cutoff values of 13.6 kPa and 17.6 kPa, sensitivity was 97% and 94%, respectively. In patients with cirrhosis, LSM positively correlated with the presence of esophageal varices (P = 0.002), although no correlation between LSM and esophageal varices size was detected. The area under the ROC for the prediction of EV was 0.76 and at a LSM cutoff value of 17.6 kPa sensitivity was 90%. Conclusion: LSM represents a non‐invasive tool for the identification of chronic liver disease patients with clinically significant or severe portal hypertension and could be employed for screening patients to be subjected to standard investigations including upper GI endoscopy and hemodynamic studies. (HEPATOLOGY 2007;45:1290–1297.)

Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells

Leptin upregulates collagen expression in hepatic stellate cells (HSCs), but the possible modulation of other actions has not been elucidated. The aim of this study was to investigate the expression and function of leptin receptors (ObR) in human HSCs and the biological actions regulated by leptin. Exposure of HSCs to leptin resulted in upregulation of monocyte chemoattractant protein 1 (MCP‐1) expression. Leptin also increased gene expression of the proangiogenic cytokines vascular endothelial growth factor (VEGF) and angiopoietin‐1, and VEGF was also upregulated at the protein level. Activated HSCs express ObRb and possibly other ObR isoforms. Exposure to leptin increased the tyrosine kinase activity of ObR immunoprecipitates and resulted in activation of signal transducer and activator of transcription 3. Several signaling pathways were activated by leptin in HSCs, including extracellular‐signal–regulated kinase, Akt, and nuclear factor κB, the latter being relevant for chemokine expression. Leptin also increased the abundance of hypoxia‐inducible factor 1α, which regulates angiogenic gene expression, in an extracellular‐signal–regulated kinase– and phoshatidylinositol 3‐kinase–dependent fashion. In vivo, leptin administration induced higher MCP‐1 expression and more severe inflammation in mice after acute liver injury. Conversely, in leptin‐deficient mice, the increase in MCP‐1 messenger RNA and mononuclear infiltration was less marked than in wild‐type littermates. Finally, ObR expression colocalized with VEGF and α‐smooth muscle actin after induction of fibrosis in rats. In conclusion, ObR activation in HSCs leads to increased expression of proinflammatory and proangiogenic cytokines, indicating a complex role for leptin in the regulation of the liver wound‐healing response.(HEPATOLOGY 2005;42:1339–1348.)

Accuracy and reproducibility of transient elastography for the diagnosis of fibrosis in pediatric nonalcoholic steatohepatitis

Transient elastography (TE) has received increasing attention as a means to evaluate disease progression in chronic liver disease patients. In this study, we assessed the value of TE for the prediction of fibrosis stage in a cohort of pediatric patients with nonalcoholic steatohepatitis. Furthermore, TE interobserver agreement was evaluated. TE was performed in 52 consecutive biopsy‐proven nonalcoholic steatohepatitis patients (32 males, 20 females, age 13.6 ± 2.44 years). The area under the receiver operating characteristic curves for the prediction of “any” (≥1), significant (≥2), or advanced fibrosis (≥3) were 0.977, 0.992, and 1, respectively. Calculation of multilevel likelihood ratios showed that TE values <5, <7, and <9 kPa, suggest the presence of “any” fibrosis, significant fibrosis, and advanced fibrosis, respectively. TE values between 5 and 7 kPa predict a fibrosis stage of 1, but with some degree of uncertainty. TE values between 7 and 9 kPa predict fibrosis stages 1 or 2, but cannot discriminate between these two stages. TE values of at least 9 kPa are associated with the presence of advanced fibrosis. The intraclass correlation coefficient for absolute agreement was 0.961. Conclusion: TE is an accurate and reproducible methodology to identify pediatric subjects without fibrosis or significant fibrosis, or with advanced fibrosis. In patients in which likelihood ratios are not optimal to provide a reliable indication of the disease stage, liver biopsy should be considered when clinically indicated. (HEPATOLOGY 2008.)

Reliability of transient elastography for the diagnosis of advanced fibrosis in chronic hepatitis C

Background: Transient elastography (TE) has received increasing attention as a means to evaluate disease progression in patients with chronic liver disease. Aim: To assess the value of TE for predicting the stage of fibrosis. Methods: Liver biopsy and TE were performed in 150 consecutive patients with chronic hepatitis C-related hepatitis (92 men and 58 women, age 50.6 (SD 12.5) years on the same day. Necro-inflammatory activity and the degree of steatosis at biopsy were also evaluated. Results: The areas under the curve for the prediction of significant fibrosis (⩾F2), advanced fibrosis (⩾F3) or cirrhosis were 0.91, 0.99 and 0.98, respectively. Calculation of multilevel likelihood ratios showed that values of TE <6 or ⩾12, <9 or ⩾12, and <12 or ⩾18, clearly indicated the absence or presence of significant fibrosis, advanced fibrosis, and cirrhosis, respectively. Intermediate values could not be reliably associated with the absence or presence of the target condition. The presence of inflammation significantly affected TE measurements in patients who did not have cirrhosis (p<0.0001), even after adjusting for the stage of fibrosis. Importantly, TE measurements were not influenced by the degree of steatosis. Conclusions: TE is more suitable for the identification of patients with advanced fibrosis than of those with cirrhosis or significant fibrosis. In patients in whom likelihood ratios are not optimal and do not provide a reliable indication of the disease stage, liver biopsy should be considered when clinically indicated. Necro-inflammatory activity, but not steatosis, strongly and independently influences TE measurement in patients who do not have cirrhosis.

Elastography, spleen size, and platelet count identify portal hypertension in patients with compensated cirrhosis.

BACKGROUND & AIMS Noninvasive methods are needed to identify clinically significant portal hypertension (CSPH) and esophageal varices (EVs) in patients with compensated cirrhosis. We looked for markers of the presence of CSPH and EVs in patients with cirrhosis. METHODS We performed a cross-sectional study that included a training set of 117 patients with compensated cirrhosis, confirmed by histology, from a tertiary referral center. Spleen diameter was measured by ultrasound, and liver stiffness (LS) was measured by transient elastography; endoscopy was used as the standard for detection of EVs, and measurements of hepatic venous pressure gradient were used as the standard for identifying CSPH. We assessed the ability of platelet count, spleen diameter, LS, and combinations of these factors (ie, ratio of platelet count to spleen size, and LS × spleen size/platelet count [LSPS]) to identify patients with CSPH and EV. The analysis included 2 new statistical models: the PH risk score and the varices risk score. Results were validated using an independent series of 56 patients with compensated patients from another center. RESULTS LS was the best single noninvasive variable for identifying patients with CSPH (area under the receiver operating characteristic, 0.883; 95% confidence interval [CI], 0.824-0.943; P < .0001). The area under the receiver operating characteristic value increased when LS was combined with platelet count and spleen size, either as LSPS (0.918; 95% CI, 0.872-0.965; P < .0001) or PH risk score (0.935; 95% CI, 0.893-0.977; P < .0001). More than 80% of patients were accurately classified using LSPS and PH risk score. Analyses of the varices risk score and LSPS were superior to all other noninvasive tests for identifying patients with EVs (area under the receiver operating characteristic, 0.909; 95% CI, 0.841-0.954 and 0.882; 95% CI, 0.810-0.935, respectively); they correctly classified 85% of patients in the training set and 75% in the validation set. CONCLUSIONS Combined data on LS, spleen diameter, and platelet count can be used to identify patients with compensated cirrhosis most likely to have CSPH and EV.

Silybin, a component of sylimarin, exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells

BACKGROUND/AIMS Hepatic fibrogenesis, a consequence of chronic liver tissue damage, is characterized by activation of the hepatic stellate cells (HSC). Silybin has been shown to exert anti-fibrogenic effects in animal models. However, scant information is available on the fine cellular and molecular events responsible for this effect. The aim of this study was to assess the mechanisms regulating the anti-fibrogenic and anti-inflammatory activity of Silybin. METHODS Experiments were performed on HSC isolated from human liver and activated by culture on plastic. RESULTS Silybin was able to inhibit dose-dependently (25-50 microM) growth factor-induced pro-fibrogenic actions of activated human HSC, including cell proliferation (P < 0.001), cell motility (P < 0.001), and de novo synthesis of extracellular matrix components (P < 0.05). Silybin (25-50 microM), inhibited the IL-1-induced synthesis of MCP-1 (P < 0.01) and IL-8 (P < 0.01) showing a potent anti-inflammatory activity. Silybin exerts its effects by directly inhibiting the ERK, MEK and Raf phosphorylation, reducing the activation of NHE1 (Na+/H+ exchanger, P < 0.05) and the IkBalpha phosphorylation. In addition, Silybin was confirmed to act as a potent anti-oxidant agent. CONCLUSION The results of the study provide molecular insights into the potential therapeutic action of Silybin in chronic liver disease. This action seems to be mostly related to a marked inhibition of the production of pro-inflammatory cytokines, a clear anti-oxidant effect and a reduction of the direct and indirect pro-fibrogenic potential of HSC.

Adenosine monophosphate–activated protein kinase modulates the activated phenotype of hepatic stellate cells

Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate–activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time‐dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5‐aminoimidazole‐4‐carboxamide‐1‐beta‐4‐ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet‐derived growth factor (PDGF)‐stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein‐1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF‐dependent activation of extracellular signal‐regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short‐term phosphorylation of ribosomal S6 kinase (p70S6K) and 4E binding protein‐1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin‐a (IL‐1) to activate nuclear factor kappa B (NF‐κB) was also reduced by AICAR. Conclusion: Activation of AMPK negatively modulates the activated phenotype of HSCs. (HEPATOLOGY 2007.)

Curcumin limits the fibrogenic evolution of experimental steatohepatitis

Nonalcoholic steatohepatitis is characterized by the association of steatosis with hepatic cell injury, lobular inflammation and fibrosis. Curcumin is known for its antioxidant, anti-inflammatory and antifibrotic properties. The aim of this study was to test whether the administration of curcumin limits fibrogenic evolution in a murine model of nonalcoholic steatohepatitis. Male C57BL/6 mice were divided into four groups and fed a diet deficient in methionine and choline (MCD) or the same diet supplemented with methionine and choline for as long as 10 weeks. Curcumin (25 μg per mouse) or its vehicle (DMSO) was administered intraperitoneally every other day. Fibrosis was assessed by Sirius red staining and histomorphometry. Intrahepatic gene expression was measured by quantitative PCR. Hepatic oxidative stress was evaluated by staining for 8-OH deoxyguanosine. Myofibroblastic hepatic stellate cells (HSCs) were isolated from normal human liver tissue. The increase in serum ALT caused by the MCD diet was significantly reduced by curcumin after 4 weeks. Administration of the MCD diet was associated with histological steatosis and necro-inflammation, and this latter was significantly reduced in mice receiving curcumin. Curcumin also inhibited the generation of hepatic oxidative stress. Fibrosis was evident after 8 or 10 weeks of MCD diet and was also significantly reduced by curcumin. Curcumin decreased the intrahepatic gene expression of monocyte chemoattractant protein-1, CD11b, procollagen type I and tissue inhibitor of metalloprotease (TIMP)-1, together with protein levels of α-smooth muscle-actin, a marker of fibrogenic cells. In addition, curcumin reduced the generation of reactive oxygen species in cultured HSCs and inhibited the secretion of TIMP-1 both in basal conditions and after the induction of oxidative stress. In conclusion, curcumin administration effectively limits the development and progression of fibrosis in mice with experimental steatohepatitis, and reduces TIMP-1 secretion and oxidative stress in cultured stellate cells.

Elastography for the non-invasive assessment of liver disease: limitations and future developments

Hepatology is still a relatively young discipline in medicine. The birth of hepatology in the 1950s was largely dependent on the introduction of liver biopsy performed with the Menghini’s needle1 and its modifications. Ever since, diagnosis and staging of chronic liver diseases (CLDs) has been based on the morphological evaluation of liver tissue associated with grading and staging according to established scoring systems.2 3 The introduction of ultrasound scanning, practically the first easily available imaging tool, at the end of the 1970s, provided further diagnostic advantages for the hepatologist, and made liver biopsy a safer procedure. Nowadays, liver biopsy remains an essential tool for the diagnosis of the majority of liver diseases, while its clinical relevance is more limited to the staging of the disease in settings where the diagnosis is based on other established parameters, ie, viral markers in chronic viral hepatitis. Even though the prevalence of biopsy-related complications is very limited in tertiary centres, this procedure still carries a burden of potentially severe events.4 Moreover, sampling error and intra- and inter-observer variability represent major drawbacks.5 6 Overall, liver biopsy should be considered the “best possible reference standard” and does not allow discrimination of adjacent stages of fibrosis (ie, F2 vs F1 or F3 vs F2)6 7 with adequate diagnostic accuracy. Along this trend, hepatologists have started to develop non-invasive systems able to provide a surrogate estimate of disease staging, ie, semi-quantitative assessment of fibrosis. Overall, these attempts are aimed at fulfilling the dream of developing an accurate, easy and cost/effective non-invasive test that allows (1) fast screening, (2) longitudinal follow-up, (3) efficacy of therapeutic treatments; and (4) prognostic evaluation. This assumes a particular relevance in the paediatric setting, where the use of liver biopsy is perceived as bearing higher risks and is less …