Giacomo Laffi

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.)

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.

Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells.

BACKGROUND & AIMS Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI 3-K) activation in mediating the biological effects of PDGF on cultured HSCs and its involvement in vivo. METHODS HSCs were isolated from normal human livers. PI 3-K was assayed on phosphotyrosine or PDGF-receptor immunoprecipitates by in vitro kinase assay. RESULTS Incubation of HSCs with PDGF caused a time-dependent increase in PI 3-K activity. Immunoprecipitation of PDGF-alpha and -beta receptors showed that both subunits associate with active PI 3-K in PDGF-stimulated HSCs. Wortmannin, a specific PI 3-K inhibitor, dose-dependently blocked PI 3-K activity induced by PDGF and inhibited DNA synthesis. PDGF (homodimer)-BB also stimulated HSC chemotaxis, which was inhibited by pretreatment with wortmannin. To explore the potential role of PI 3-K in vivo, liver homogenates from rats treated with CCl4 and from control rats were immunoprecipitated with anti-PDGF-beta-receptor antibodies. Liver injury was associated with increased PDGF-beta-receptor autophosphorylation, and greater PI 3-K activity associated with the receptor itself. CONCLUSIONS This study shows that in cultured HSCs, PI 3-K activation is necessary for both mitogenesis and chemotaxis induced by PDGF and that this pathway is up-regulated during liver injury in vivo.

Prevalence of bcl-2 Rearrangement in Patients with Hepatitis C Virus–Related Mixed Cryoglobulinemia with or without B-Cell Lymphomas

Context Rearrangement of bcl-2 has an antiapoptotic effect and has been implicated as a potential cause of benign lymphoproliferation (causing mixed cryoglobulinemia) and B-cell lymphoma. Mixed cryoglobulinemia is strongly associated with hepatitis C virus (HCV) infection. Contribution In patients with HCV-associated chronic liver disease, bcl-2 rearrangement occurred significantly more often in patients with chronic HCV infection and mixed cryoglobulinemia than in HCV-infected patients without mixed cryoglobulinemia; it also occurred in three of four patients with B-cell lymphoma. Transient suppression of HCV in two patients was associated with remission of clinical manifestations of mixed cryoglobulinemia. Implications Viral induction of gene sequence translocations may help explain some benign and malignant lymphoproliferative disorders. The Editors Mixed cryoglobulinemia is a distinct syndrome clinically characterized by purpura; weakness; arthralgia; and such conditions as membranoproliferative glomerulonephritis, peripheral neuropathy, skin ulcers, and diffuse vasculitis (1, 2). Cryoprecipitable immune complexes, specifically mixed (IgG-IgM) cryoglobulins, are the serologic hallmark of the disease. Immunoglobulin Gs are the autoantigens, and IgMs with rheumatoid factor activity are the autoantibodies. Mixed cryoglobulinemia is classified as type II or type III according to the presence of polyclonal or monoclonal IgMs (3, 4). Because expansion of rheumatoid factorproducing B cells is the underlying disorder of mixed cryoglobulinemia, this condition is considered a benign B-cell lymphoproliferative disease. Type II and III mixed cryoglobulinemia are similar in terms of organ involvement and clinical course, except that type II disease may evolve into cancer. Type II mixed cryoglobulinemia is often observed in conjunction with bone marrow findings consistent with indolent B-cell lymphoma (5-9) and evolves to frank B-cell malignancy in about 10% of cases (10). A strong association between mixed cryoglobulinemia and infection with hepatitis C virus (HCV), a hepatotropic and lymphotropic virus (10, 11), has been shown. A pathogenetic role of chronic infection with HCV in mixed cryoglobulinemia has been suggested. The mechanisms involved in benign lymphoproliferation of mixed cryoglobulinemia and its evolution to lymphoma remain unknown. However, rearrangement of the antiapoptotic B-cell lymphoma/leukemia 2 (bcl-2) genethe t(14;18) translocationis suggested to play a role in the pathogenesis of HCV-associated mixed cryoglobulinemia (12, 13). The t(14;18) translocation, the most frequent genetic aberration in human lymphoma (14, 15), may be favored by sustained, strong antigenic stimulation (16-18). As a result of bcl-2 rearrangement, the bcl-2 gene on chromosome 18q21 is coupled with the immunoglobulin heavy chain gene (IgH) on chromosome 14q32 by a process frequently involving IgH joining segments (JH) (Figure 1, top). At the junction of the two genes, insertions of variable lengths (N segments) due to random addition of nongermline nucleotides result in a DNA pattern that is clone specific (19, 20). As a consequence of this rearrangement, bcl-2 is activated and B cells bearing the t(14;18) translocation express inappropriately elevated levels of the Bcl-2 protein. Figure 1. Schematic representation of the t(14; 18) translocation and its effects on B cells. Top. bcl-2 Bottom. Bcl-2 is a member of a larger family. Family members can interact with each other in a complex manner; some act to promote and others to inhibit apoptosis (14). The Bcl-2 protein protects cells from apoptosis, whereas its homologue, Bax, kills cells (21). Thus, the ratio of Bcl-2 to Bax is a determinant of susceptibility to apoptosis (14) (Figure 1, bottom). Strong expression of Bcl-2 protein has been observed in lymphoid infiltrates in liver and bone marrow specimens of patients with mixed cryoglobulinemia (22). In a previous study, the prevalence of bcl-2 rearrangement in peripheral blood mononuclear cells was significantly higher in patients with chronic HCV infection than in healthy persons or those without HCV infection but with chronic liver diseases or systemic autoimmune disorders (13). Of note, the prevalence of bcl-2 rearrangement was particularly high in patients with HCV-associated type II mixed cryoglobulinemia. We sought to evaluate the prevalence of bcl-2 rearrangement in peripheral blood cells of patients with mixed cryoglobulinemia, to confirm that results are patient specific by sequencing studies, to analyze Bcl-2 expression and the ratio of Bcl-2 to Bax in these patients, and to observe the effect of antiviral therapy. Methods Patients We enrolled 37 patients (12 men and 25 women; mean age SD, 64 9 years) with HCV infection and mixed cryoglobulinemia who were consecutively referred to the outpatient clinic of the Department of Internal Medicine, University of Florence School of Medicine, a tertiary hepatology center, and the rheumatologic section of the Department of Internal Medicine, University of Pisa School of Medicine, from January 1999 to May 2000. These patients were compared with 101 consecutively recruited patients (62 men and 39 women; mean age, 51 11 years) who had HCV-related chronic liver diseases but not mixed cryoglobulinemia or another lymphoproliferative disease. Hepatitis C virus infection was established by detection of circulating anti-HCV antibodies (EIA-2 and RIBA-2, Ortho Diagnostic Systems, Raritan, New Jersey) and HCV RNA (nested polymerase chain reaction [PCR] for HCV) (10, 13, 23). Essential mixed cryoglobulinemia was diagnosed according to published criteria (10, 13). Serum cryoglobulins, complement fraction levels, rheumatoid factor, and autoantibodies were routinely measured and characterized in all patients as described elsewhere (10, 13, 23). Diagnosis of liver disease was based on results of liver biopsy. Lymphomas were diagnosed by an independent pathologist and classified according to the revised European-American classification of lymphoid neoplasms (24). No patient tested positive for hepatitis B surface antigen, IgM anti-HBc, hepatitis B virus DNA, IgM anti-delta, antiEpsteinBarr virus, anti-cytomegalovirus, antiherpes simplex virus, or anti-HIV. No patient had a history of alcohol abuse or previous antiviral or immunosuppressive treatment. All patients gave informed consent to participate in the study, which was performed in accordance with the principles of the Declaration of Helsinki, and the study was approved by the local ethics committee. Detection of the t(14; 18) Translocation The t(14; 18) translocation in peripheral blood mononuclear cells was detected on total DNA by using nested PCR (major breakpoint region), as described elsewhere (13). Nested PCR is a variant of PCR; after an initial series of amplification cycles, templates are again amplified by using a second set of primers internal to the first ones. The resulting reaction is very specific and sensitive owing to specific binding to the target sequences of four instead of two specific primers. The limit of sensitivity was one rearranged cell in 105 to 106 normal cells. Amplification products were analyzed by both ethidium bromide staining and hybridization with a specific digoxigenin-labeled probe (Southern blot analysis). Each sample was analyzed at least twice, and all samples that tested negative on PCR were analyzed at least four times. Different cell samples that were obtained at the same time (synchronous) or at different times (metachronous) were also analyzed when possible. Approximately 2.5 105 mononuclear cells were tested in each reaction, corresponding to about 1 g of DNA. Positive and negative control samples were included in each experiment (13). To avoid false-positive results caused by carryover of PCR product, precautions were taken, as described elsewhere (10, 13). To ensure DNA amplificability, PCR was also performed by using primers for the human HLA gene (exon 2 of HLA-DRB gene), as previously reported (13). Finally, bcl-2/JH junction sequence was determined in part by cycle sequencing and solid-phase sequencing techniques (13, 25) and in part by automated sequencing (Abi Prism, Perkin Elmer, Norwalk, Connecticut). Measurement of Bcl-2 and Bax Proteins Bcl-2 and Bax proteins were measured as described elsewhere (13) on freshly isolated peripheral blood mononuclear cells and, when possible (9 patients), in separated cell subgroups (T cells, B cells, and monocytes and macrophages). Bcl-2 was detected by using monoclonal mouse anti-human Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, California), and Bax was detected by using polyclonal rabbit anti-human Bax (Upstate Biotechnology, Inc., Lake Placid, New York). The CD2+ T cells, CD19+ B cells, and CD14+monocytes and macrophages from peripheral blood were separated by immunomagnetic isolation using Dynabeads M450 Pan-T, M-450 Pan-B, and M-450 CD14+, respectively (Dynal A.S., Oslo, Norway), according to the manufacturers instructions. Statistical Analysis Data are expressed as the mean SD. Data were analyzed by performing the Fisher exact test, using True Epistat 4.0 statistical software (Epistat Service, Richardson, Texas). A P value less than 0.05 was considered significant. Role of the Funding Sources The funding sources had no role in the analysis, reporting, or interpretation of the data or in the decision to submit the report for publication. Results The Table shows the clinical, epidemiologic, and pathologic characteristics of patients with HCV-related mixed cryoglobulinemia. The mean duration of mixed cryoglobulinemia syndrome was 9.2 5.2 years. Most of these patients (91%) had chronic liver diseases. Liver biopsy showed chronic hepatitis in 27 patients (72.9%) and cirrhosis in 7 patients (18.9%); of the latter patients, 1 also had superimposed hepatocellular carcinoma. Liver biopsy was not performed in the remaining 3 patients becaus

Albumin improves the response to diuretics in patients with cirrhosis and ascites: results of a randomized, controlled trial.

BACKGROUND/AIMS Diuretic treatment of ascites could result in intravascular volume depletion, electrolyte imbalance and renal impairment. We investigated whether intravascular volume expansion with albumin exert beneficial effects in cirrhosis with ascites. METHODS In protocol 1, 126 cirrhotic inpatients in whom ascites was not relieved following bed rest and a low-sodium diet, were randomly assigned to receive diuretics (group A) or diuretics plus albumin, 12.5 g/day (group B). In protocol 2, group A patients continued to receive diuretics and group B diuretics plus albumin (25 g/week) as outpatients and were followed up for 3 years. End points were: disappearance of ascites, duration of hospital stay (protocol 1), recurrence of ascites, hospital readmission and survival (protocol 2). RESULTS The cumulative rate of response to diuretic treatment of ascites was higher (p < 0.05) and hospital stay was shorter (20 +/- 1 versus 24 +/- 2 days, p < 0.05) in group B than in group A patients. After discharge, group B patients had a lower cumulative probability of developing ascites (19%, 56%, 69% versus 30%, 79% and 82% at 12, 24 and 36 months, p < 0.02) and a lower probability of readmission to the hospital (15%, 56%, 69% versus 27%, 74% and 79%, respectively, p < 0.02). Survival was similar in the two groups. CONCLUSIONS Albumin is effective in improving the rate of response and preventing recurrence of ascites in cirrhotic patients with ascites receiving diuretics. However, the cost/benefit ratio was favorable to albumin in protocol 1 but not in protocol 2.

Liver stiffness is influenced by a standardized meal in patients with chronic hepatitis C virus at different stages of fibrotic evolution

Transient elastography (TE) is increasingly employed in clinical practice for the noninvasive detection of tissue fibrosis in patients with chronic liver disease (CLD), and particularly chronic hepatitis C virus (HCV)‐related hepatitis. The present study was designed to provide a definitive characterization of the “confounding” increase in liver stiffness (LS) following a standardized meal in a consecutive population of 125 patients with chronic HCV infection at different stages of fibrotic evolution. LS values were obtained after overnight fasting and 15, 30, 45, 60, and 120 minutes following the onset of a standardized liquid meal (400 mL, 600 Kcal, 16.7% protein, 53.8% carbohydrates, 29.5% fat). An evident increase in LS values was observed 15 to 45 minutes after the onset of the meal with return to baseline premeal levels within 120 minutes in all patients. The peak postmeal delta increase in LS was progressively more marked with increasing stages of fibrosis (P < 0.001), becoming maximal in patients with cirrhosis. However, the probability of identifying the Metavir stage of fibrosis, the Child‐Pugh class, or the presence/absence of esophageal varices with the postmeal delta increase in LS was inferior to that obtained with baseline LS values. Conclusion: The results of the present study provide definitive evidence of the confounding effect of a meal on the accuracy of LS measurements for the prediction of fibrosis stage in patients with chronic HCV hepatitis and suggest that a fasting period of 120 minutes should be observed before the performance of TE. (HEPATOLOGY 2013;)

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.)

Evidence for a Storage Pool Defect in Platelets From Cirrhotic Patients With Defective Aggregation

The mechanisms underlying the defective platelet function in cirrhotic patients were investigated. Eleven cirrhotic patients with mild disease (group 1), 20 patients with severe cirrhosis (group 2), and 31 controls were studied. Platelet aggregation was significantly reduced in cirrhotics compared with controls. Compared with controls, cirrhotic patients in group 2 showed a significant reduction in the total content of adenosine triphosphate (57.8 +/- 7.8 vs. 26.1 +/- 6.3 mumol/10(11) platelets; P less than 0.05), 5-hydroxytryptamine (285 +/- 26 vs. 104 +/- 38 nmol/10(11) platelets; P less than 0.05), beta-thromboglobulin (2129 +/- 120 vs. 1223 +/- 161 ng/10(8) platelets; P less than 0.01), and platelet factor 4 (1389 +/- 108 vs. 805 +/- 176 ng/10(8) platelets; P less than 0.05). In patients with severe disease, an increase in plasma beta-thromboglobulin-platelet factor 4 ratio, an index of in vivo platelet activation, was observed (controls, 3.50 +/- 0.50; group 1, 4.02 +/- 0.80; and group 2, 6.59 +/- 1.15). Our data indicate the existence of a platelet storage pool defect, which may favor the bleeding tendency of cirrhotic patients.