Roberto Giulio Romanelli

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.)

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.

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.

Up-regulated expression of fractalkine and its receptor CX3CR1 during liver injury in humans

BACKGROUND/AIMS Little is known about the role of fractalkine (CX3CL1) in the liver. The aim of this study was to investigate the expression patterns of fractalkine and its receptor CX3CR1 in normal human liver and in conditions of injury. METHODS Distribution and expression of fractalkine and its receptor were investigated using immunohistochemistry, in situ hybridization, flow cytometry and reverse transcriptase-polymerase chain reaction. In vitro experiments were conducted in HepG2 cells. RESULTS Both fractalkine and CX3CR1 were up-regulated during chronic injury, in areas of portal and lobular inflammation. In severe acute hepatitis, fractalkine and CX3CR1 were expressed at high levels not only in areas of inflammation but also in regenerating epithelial cells within bile duct-like structures, which showed co-expression of fractalkine and cytokeratin-7 or CX3CR1. The human hepatocarcinoma cell line HepG2 expressed fractalkine at the gene and protein level, and HepG2-conditioned medium was chemotactic for cells overexpressing CX3CR1. Transcripts for CX3CR1 were detected in HepG2, and exposure of these cells to recombinant fractalkine induced cell migration. CONCLUSIONS This study shows that the fractalkine system is up-regulated during liver damage, and suggests that fractalkine may play a role in the recruitment and adhesion of inflammatory cells and in the biology of liver epithelial cells.

Terlipressin plus albumin versus midodrine and octreotide plus albumin in the treatment of hepatorenal syndrome: A randomized trial

Hepatorenal syndrome (HRS), a serious complication of cirrhosis, is associated with high mortality without treatment. Terlipressin with albumin is effective in the reversal of HRS. Where terlipressin is not available, as in the United States, midodrine and octreotide with albumin are used as an alternative treatment of HRS. The aim was to compare the effectiveness of terlipressin plus albumin versus midodrine and octreotide plus albumin in the treatment of HRS in a randomized controlled trial. Twenty‐seven patients were randomized to receive terlipressin with albumin (TERLI group) and 22 to receive midodrine and octreotide plus albumin (MID/OCT group). The TERLI group received terlipressin by intravenous infusion, initially 3 mg/24 hours, progressively increased to 12 mg/24 hours if there was no response. The MID/OCT group received midodrine orally at an initial dose of 7.5 mg thrice daily, with the dose increased to a maximum of 12.5 mg thrice daily, together with octreotide subcutaneously: initial dose 100 μg thrice daily and up to 200 μg thrice daily. Both groups received albumin intravenously 1 g/kg of body weight on day 1 and 20‐40 g/day thereafter. There was a significantly higher rate of recovery of renal function in the TERLI group (19/27, 70.4%) compared to the MID/OCT group (6/21, 28.6%), P = 0.01. Improvement in renal function and lower baseline Model for End‐Stage Liver Disease score were associated with better survival. Conclusion: Terlipressin plus albumin is significantly more effective than midodrine and octreotide plus albumin in improving renal function in patients with HRS (Hepatology 2015;62:567–574

Effects of low-dose captopril on renal hemodynamics and function in patients with cirrhosis of the liver

BACKGROUND In cirrhotic patients with ascites, captopril has deleterious effects on renal function, which have been referred to as captopril-induced arterial hypotension. The effects of this drug on renal function in cirrhosis were evaluated using low-dose captopril, thereby avoiding any change in arterial pressure. METHODS In a randomized, double-blind, placebo controlled, cross-over trial, the effects of 12.5 mg captopril on renal plasma flow, glomerular filtration rate (measured by radioisotopic techniques), and sodium excretion in healthy controls and cirrhotic patients with and without ascites were determined. RESULTS In healthy subjects, captopril only induced a significant, 18% increase in renal plasma flow. In contrast, glomerular filtration rate significantly decreased in patients with (from 108 +/- 7 to 78 +/- 9 mL/min) and without ascites (from 102 +/- 4 to 88 +/- 3 mL/min), whereas renal plasma flow did not change. Urinary sodium excretion also significantly decreased in ascitic patients (from 43.8 +/- 4.4 to 30.6 +/- 3.8 mumol/min). CONCLUSIONS These data suggest that angiotensin II contributes to maintain renal hemodynamics in cirrhosis with and without ascites.

Expression and Function of Gonadotropin-releasing Hormone (GnRH) Receptor in Human Olfactory GnRH-secreting Neurons AN AUTOCRINE GnRH LOOP UNDERLIES NEURONAL MIGRATION

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmanns syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3–4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

Activation of p38MAPK mediates the angiostatic effect of the chemokine receptor CXCR3-B

Chemokines binding the CXCR3 receptor have been shown to inhibit angiogenesis via the CXCR3-B isoform, but the underlying molecular mechanisms are unknown. Aim of this study was to elucidate the effects of CXCR3-B on activation of members of the mitogen-activated protein kinase family, and to explore the relevance of defined signaling pathways to the angiostatic effects of CXCR3-B ligands. Human embryonic kidney (HEK) 293 cells were transfected with expression vectors encoding for CXCR3-A or CXCR3-B. In cells expressing CXCR3-A, CXCL10 (IP-10) at nanomolar concentrations induced activation of ERK, Akt, and Src, as previously described in human vascular pericytes. In HEK-293 cells expressing CXCR3-B, exposure to CXCL10 in the micromolar concentration range led to activation of the p38(MAPK) pathway, as indicated by phosphorylation of p38(MAPK) itself, and of MKK3/6 and MAPKAPK-2, that lie upstream and downstream of p38(MAPK), respectively. Similar results were obtained in cells stimulated with CXCL4 (PF4), a specific ligand of CXCR3-B. In contrast, CXCL4 was unable to activate p38(MAPK) in mock-transfected HEK-293 cells. Only a modest induction of ERK or JNK was observed upon CXCR3-B activation. In human microvascular endothelial cells, which selectively express CXCR3-B, in a cell cycle-dependent fashion, CXCL10 and CXCL4 increased the enzymatic activity of p38(MAPK). Pharmacologic inhibition of p38(MAPK) by SB302580 resulted in a significant increase in DNA synthesis and in reversal of the inhibitory action of CXCL10. In conclusion, the p38(MAPK) pathway is a downstream effector of CXCR3-B implicated in the angiostatic action of this chemokine receptor.

The Ras antagonist, farnesylthiosalicylic acid (FTS), inhibits experimentally-induced liver cirrhosis in rats

BACKGROUND/AIMS Protooncogenes may play an important role, not only in carcinogenesis, but also in the regulation of normal cellular proliferation and differentiation. Several studies have indicated increased expression of the Ras protooncogenes in the liver in animal models and in patients with liver cirrhosis. The aim of the present study was to examine whether a synthetic Ras antagonist, S-farnesylthiosalicylic acid (FTS), which specifically dislodges Ras from the membrane of Ras-transformed fibroblasts (EJ cells), can prevent experimentally-induced liver cirrhosis in rats. METHODS Cirrhosis was induced in male Wistar rats by intraperitoneal administration of thioacetamide (200 mg/kg twice weekly for 12 weeks). The Ras antagonist, farnesylthiosalicylic acid (FTS, 5 mg/kg), was administered during the study period 3 times a week. Ras expression in the liver was determined by Western blot analysis with pan anti-Ras antibodies and by immunohistochemistry. RESULTS Rats treated with thioacetamide and the Ras antagonist, farnesylthiosalicylic acid (FTS), for 12 weeks had lower histopathologic scores of fibrosis and inflammation (p-values of 0.003 and 0.008, respectively) than those treated with thioacetamide only. There were no differences between the histopathologic scores in vehicle (control) and in Ras-antagonist (FTS) only treatments. Analysis of hepatic hydroxyproline levels from the two thioacetamide-treated groups and controls confirmed the histopathologic scores (7.7+/-0.9 mg/g protein in the TAA-treated vs. 3.8+/-0.5 mg/g protein in the TAA+FTS treated group, p = 0.007). Ras levels, determined by Western blot analysis, were markedly increased in the livers treated with TAA (17-fold over control) and significantly decreased (by about 70%) in the livers of rats treated with TAA and FTS. Studies in isolated human hepatic stellate cells demonstrated that FTS inhibited both DNA synthesis and migration of those cells (p<0.05). CONCLUSION These results indicate that inhibition of Ras expression in the liver during fibrogenesis, prevents the development of experimentally-induced hepatic cirrhosis.

Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor‐β1

1 Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver. 2 Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor‐β1 (TGF‐β1) for different time periods in the presence or absence of PTF. TGF‐β1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3–6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9–12 h after the addition of TGF‐β1. Co‐incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell‐associated expression of the corresponding protein. Conversely, PTF dose‐dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells. 3 Pulse‐chase experiments employing L‐[3H]‐proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP‐1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP‐1) mRNA. Accordingly incubation with PTF increased the levels of ‘activated MMP‐1’ in cell supernatants in both basal and stimulated conditions. 4 These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.