Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity.

BACKGROUND & AIMS: In patients with non-alcoholic fatty liver disease (NAFLD), liver biopsy is the gold standard to detect non-alcoholic steatohepatitis (NASH) and stage liver fibrosis. We aimed to identify differentially expressed mRNAs and non-coding RNAs in serum samples of biopsy-diagnosed mild and severe NAFLD patients with respect to controls and to each other. METHODS: We first performed a whole transcriptome analysis through microarray (n = 12: four Control: CTRL; four mild NAFLD: NAS ≤ 4 F0; four severe NAFLD NAS ≥ 5 F3), followed by validation of selected transcripts through real-time PCRs in an independent internal cohort of 88 subjects (63 NAFLD, 25 CTRL) and in an external cohort of 50 NAFLD patients. A similar analysis was also performed on liver biopsies and HepG2 cells exposed to oleate:palmitate or only palmitate (cellular model of NAFL/NASH) at intracellular/extracellular levels. Transcript correlation with histological/clinical data was also analysed. RESULTS: We identified several differentially expressed coding/non-coding RNAs in each group of the study cohort. We validated the up-regulation of UBE2V1, BNIP3L mRNAs, RP11-128N14.5 lncRNA, TGFB2/TGFB2-OT1 coding/lncRNA in patients with NAS ≥ 5 (vs NAS ≤ 4) and the up-regulation of HBA2 mRNA, TGFB2/TGFB2-OT1 coding/lncRNA in patients with Fibrosis stages = 3-4 (vs F = 0-2). In in vitro models: UBE2V1, RP11-128N14.5 and TGFB2/TGFB2-OT1 had an increasing expression trend ranging from CTRL to oleate:palmitate or only palmitate-treated cells both at intracellular and extracellular level, while BNIP3L was up-regulated only at extracellular level. UBE2V1, RP11-128N14.5, TGFB2/TGFB2-OT1 and HBA2 up-regulation was also observed at histological level. UBE2V1, RP11-128N14.5, BNIP3L and TGFB2/TGFB2-OT1 correlated with histological/biochemical data. Combinations of TGFB2/TGFB2-OT1 + Fibrosis Index based on the four factors (FIB-4) showed an Area Under the Curve (AUC) of 0.891 (P = 3.00E-06) or TGFB2/TGFB2-OT1 + Fibroscan (AUC = 0.892, P = 2.00E-06) improved the detection of F = 3-4 with respect to F = 0-2 fibrosis stages. CONCLUSIONS: We identified specific serum coding/non-coding RNA profiles in severe and mild NAFLD patients that possibly mirror the molecular mechanisms underlying NAFLD progression towards NASH/fibrosis. TGFB2/TGFB2-OT1 detection improves FIB-4/Fibroscan diagnostic performance for advanced fibrosis discrimination.

Abnormalities of Lipoprotein Levels in Liver Cirrhosis: Clinical Relevance.

Progressive lipoprotein impairment occurs in liver cirrhosis and is associated with increased morbidity and mortality. The present review aims to summarize the current evidence regarding the prognostic value of lipoprotein abnormalities in liver cirrhosis and to address the need of a better prognostic stratification of patients, including lipoprotein profile assessment. Low levels of lipoproteins are usual in cirrhosis. Much evidence supports the prognostic role of hypolipidemia in cirrhotic patients. In particular, hypocholesterolemia represents an independent predictor of survival in cirrhosis. In cirrhotic patients, lipoprotein impairment is associated with several complications: infections, malnutrition, adrenal function, and spur cell anemia. Alterations of liver function are associated with modifications of circulating lipids. Decreased levels of lipoproteins significantly impact the survival of cirrhotic patients and play an important role in the pathogenesis of some cirrhosis-related complications.

Apolipoprotein AI and HDL are reduced in stable cirrhotic patients with adrenal insufficiency: a possible role in glucocorticoid deficiency.

BACKGROUNDS AND AIMS: Adrenal insufficiency (AI) has been reported in patients with stable cirrhosis. A lack of substrates has been suggested as a possible contributing pathogenic mechanism leading to glucocorticoid deficiency in these subjects. To better explore this hypothesis, we studied lipoproteins in cirrhotics with and without AI. METHODS: A total of 81 cirrhotic patients and 30 normal volunteers were enrolled. The severity of liver disease was graded by Child-Pugh score. Total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), and apolipoprotein AI (Apo-AI) levels were evaluated. HDL subfractions were measured by gradient gel electrophoresis. Adrenal function was assessed by the Low-Dose Short Synacthen Test. RESULTS: Cirrhotic patients showed a significant reduction of TC, HDL, LDL, TG, and Apo-AI levels compared with controls. HDL3 was significantly lower, while HDL2 was higher, in cirrhotics compared with the controls. AI was observed in 26 patients. TC, TG, HDL, and Apo-AI were significantly reduced in cirrhotics with AI compared with those with normal adrenal function. HDL2 and HDL3 did not differ between these two groups. Delta cortisol was related to TC (r = 0.30, p < 0.01), TG (r = 0.22, p = 0.05), and Apo-AI (r = 0.37, p < 0.001). Multivariate analysis revealed that Apo-AI and HDL were independently associated with AI. CONCLUSION: Our study shows that TC, TG, HDL, and Apo-AI are reduced in cirrhotics with AI. In particular, because both HDL and Apo-AI play a primary role in providing substrates for steroidogenesis to adrenal cells, this deficiency may contribute to the pathogenesis of AI in these patients.

Comparison of total cortisol, free cortisol, and surrogate markers of free cortisol in diagnosis of adrenal insufficiency in patients with stable cirrhosis.

BACKGROUND & AIMS: Measurements of serum levels of total cortisol can overestimate the prevalence of adrenal dysfunction in patients with cirrhosis because they have low concentrations of corticosteroid-binding globulin and albumin. We used measurements of serum total cortisol and serum free cortisol after the low-dose short Synacthen test (LDSST) to assess adrenal dysfunction. METHODS: We studied 79 patients with stable cirrhosis; adrenal dysfunction was defined by peak concentrations of total cortisol ≤494 mmol/L and/or peak concentrations of free cortisol ≤33 nmol/L after the LDSST. We determined free cortisol index (FCI) scores and calculated free cortisol levels by using Coolens' equation. The Cox regression model was used to assess the relationship between adrenal dysfunction and outcomes (death or liver transplant). RESULTS: On the basis of measurement of total cortisol, 34% of patients had adrenal dysfunction, and on the basis of measurement of free cortisol, 29% had adrenal dysfunction. There was agreement between total cortisol and free cortisol levels in 22% of patients; in 13%, adrenal dysfunction was diagnosed only on the basis of total cortisol and in 6% only on the basis of free cortisol (κ coefficient, 0.56; P < .01). Low concentrations of corticosteroid-binding globulin (21 vs 54 µg/mL, P < .01) led to an overestimation of adrenal dysfunction that was based on measurement of total cortisol. Measurements of calculated free cortisol constantly overestimated free cortisol concentrations, with variations as large as 87% for baseline values and up to 84% after stimulation. Adrenal insufficiency, defined by FCI scores <12, was detected in 30% of patients; among them, 23% also had subnormal peak levels of free cortisol (κ coefficient, 0.70; P < .001). Adrenal dysfunction was not significantly associated with patient outcomes, on the basis of Cox model analysis. CONCLUSIONS: Adrenal insufficiency, defined by LDSST, is frequent in patients with stable cirrhosis, on the basis of measurements of total and free cortisol. FCI scores are better than measurement of total cortisol in assessing adrenal function in patients with cirrhosis. We did not associate adrenal dysfunction with outcome, but further studies are needed.

Adrenocortical dysfunction in liver disease: a systematic review.

In patients with cirrhosis, adrenal insufficiency (AI) is reported during sepsis and septic shock and is associated with increased mortality. Consequently, the term "hepato-adrenal syndrome" was proposed. Some studies have shown that AI is frequent in stable cirrhosis as well as in cirrhosis associated with decompensation other than sepsis, such as bleeding and ascites. Moreover, other studies showed a high prevalence in liver transplant recipients immediately after, or some time after, liver transplantation. The effect of corticosteroid therapy in critically ill patients with liver disease has been evaluated in some studies, but the results remain controversial. The 250-µg adreno-cortico-tropic-hormone stimulation test to diagnose AI in critically ill adult patients is recommended by an international task force. However, in liver disease, there is no consensus on the appropriate tests and normal values to assess adrenal function; thus, standardization of normal ranges and methodology is needed. Serum total cortisol assays overestimate AI in patients with cirrhosis, so that direct free cortisol measurement or its surrogates may be useful measurements to define AI, but further studies are needed to clarify this. In addition, the mechanisms by which liver disease leads to adrenal dysfunction are not sufficiently documented. This review evaluates published data regarding adrenal function in patients with liver disease, with a particular focus on the potential limitations of these studies as well as suggestions for future studies.

Assessment of adrenocortical reserve in stable patients with cirrhosis.

BACKGROUND & AIMS: Adrenal insufficiency (AI) is reported in critically ill patients with cirrhosis and is associated with increased mortality. It is unclear if AI is an underlying condition or triggered by critical events (e.g. sepsis). We investigated AI in cirrhosis without infection or hemodynamic instability. METHODS: A total of 101 consecutive patients with cirrhosis were studied. AI was defined by a total serum cortisol (TC) <18 µg/dl at 20 or 30 min after injection of 1 µg of tetracosactrin. Transcortin, calculated free cortisol (cFC), and free cortisol index (FCI) were assessed in a subgroup of 41 patients, with FCI>12 representing normal adrenal function. RESULTS: AI was present in 38 patients (38%). Child score (median, 10 vs 7, p<0.0001), MELD score (median, 17 vs 12, p<0.0001), ascites (68% vs 37%, p<0.01), basal TC (median,7.6 vs 14.9 µg/dl, p<0.001), albumin (28 ± 0.8 vs 33 ± 0.7 g/L, p<0.0001), INR (median, 1.6 vs 1.2, p<0.0001), total bilirubin (median, 51 vs 31 µmol/L, p<0.05), total cholesterol (median, 120 vs 142, p<0.05), and LDL (median, 76 vs 81, p<0.05) were significantly different between those with and without AI. ROC curves showed a basal TC ≤ 12.8 µg/dl to be a cut-off value closely associated with AI. The cFC was significantly related to TC for baseline values (R=0.94, p<0.0001), peak values (R=0.90, p<0.0001), and delta values (R=0.95, p<0.0001), in patients with and without AI. However, no patient had a FCI<12. CONCLUSIONS: AI defined by an abnormal response to 1 µg tetracosactrin is frequent in stable patients with cirrhosis, in the absence of infections or hemodynamic instability and is related to the severity of liver disease. However, evaluation of the true incidence of AI should comprise direct assays of free cortisol. Clinical consequences of AI need to be explored.

Direct oral anticoagulant treatment of deep vein thrombosis reduces IL-6 expression in peripheral mono-nuclear blood cells.

Multiple factors play a pathophysiologic role for the venous thromboembolism (VTE) as a multi-factorial disease. Inflammation might play a peculiar role in shifting towards a pro-thrombotic state. Anticoagulant drugs are the first cure line for VTE. The low-molecular-weight heparins (LMWH) show anti-coagulant capability as well as reducing levels of inflammatory factors, including interleukin (IL)-6. The direct oral anticoagulants (DOACs) have shown efficacy in threating VTE, additionally to the anti-activated factor X these drugs seem able to reduce the abnormal release of pro-inflammatory agents. The present study evaluated the capability of DOACs in reducing plasma level of IL-6 in patients suffered from deep vein thrombosis (DVT) of the lower limbs. Our results showed reduced IL-6 expression levels in the peripheral lymphocytes of DVT compared to controls (fold-change, 2.8; P<0.05). We postulate that lowered IL-6 expression in the lymphocytes of DVT patients may mediate the anti-inflammatory action of DOACs. The present study is the first evidence concerning the anti-inflammatory properties of DOACs in specific setting of VTE patients such as DVT.

Guidelines on the management of atrial fibrillation in the emergency department: a critical appraisal.

Several guidelines often exist on the same topic, sometimes offering divergent recommendations. For the clinician, it can be difficult to understand the reasons for this divergence and how to select the right recommendations. The aim of this study is to compare different guidelines on the management of atrial fibrillation (AF), and provide practical and affordable advice on its management in the acute setting. A PubMed search was performed in May 2014 to identify the three most recent and cited published guidelines on AF. During the 1-week school of the European School of Internal Medicine, the attending residents were divided in five working groups. The three selected guidelines were compared with five specific questions. The guidelines identified were: the European Society of Cardiology guidelines on AF, the Canadian guidelines on emergency department management of AF, and the American Heart Association guidelines on AF. Twenty-one relevant sub-questions were identified. For five of these, there was no agreement between guidelines; for three, there was partial agreement; for three data were not available (issue not covered by one of the guidelines), while for ten, there was complete agreement. Evidence on the management of AF in the acute setting is largely based on expert opinion rather than clinical trials. While there is broad agreement on the management of the haemodynamically unstable patient and the use of drugs for rate-control strategy, there is less agreement on drug therapy for rhythm control and no agreement on several other topics.

An unusual cause of anemia in cirrhosis: spur cell anemia, a case report with review of literature.

Chronic anemia is common in liver cirrhosis. In this setting, the pathogenesis of anemia is complex and multifactorial. Spur cell anemia is a serious disorder in cirrhotic patients and is associated with poor prognosis. Liver transplantation constitutes the only therapeutic tool. We report a case with severe spur cell anemia in alcoholic liver cirrhosis. In the attempt to investigate the origin of the disorder, we have evaluated the lipoprotein profile and found a significant reduction of apolipoprotein AI and HDL3 subclass as a possible cause of the disease.

Emerging hepatic syndromes: pathophysiology, diagnosis and treatment.

Liver cirrhosis is a major cause of morbidity and mortality worldwide, mainly due to complications of portal hypertension. In this article, we review the current understanding on the pathophysiology, the diagnostic criteria and the available therapeutic options for patients with emerging hepatic syndromes in cirrhosis, namely the hepatorenal, hepato-adrenal and hepatopulmonary syndrome. The hepatorenal syndrome is a well-recognized complication of advanced cirrhosis and is usually associated with an accelerated course to death unless liver transplantation is performed. The hepatopulmonary syndrome is often missed in the evaluation of patients with cirrhosis; however, early recognition is essential for the efficient management of individual patients. The hepato-adrenal syndrome, although not fully characterized, offers an exciting field for research and potential therapeutic interventions.

Hepatic insulin resistance in NAFLD: relationship with markers of atherosclerosis and metabolic syndrome components.

AIMS: Fat accumulation in the liver and in the muscle results in hepatic and muscle insulin resistance and has been associated with increased cardiovascular risk. It is unclear whether the individual role of hepatic and muscle insulin resistance in the onset of dyslipidaemia is observed in nonalcoholic fatty liver disease (NAFLD) patients and whether this association is mediated through traditional risk factors. The aim of this study was to assess hepatic and muscle insulin resistance in NAFLD and its relationship with carotid artery intima-media thickness (IMT) and the apoB/apoAI ratio as markers of atherosclerosis. METHODS: We studied 132 patients with a non-invasive diagnosis of NAFLD stratified into two groups according to the severity of steatosis at ultrasound scan. In all subjects, we measured hepatic insulin resistance (H-IR) and muscle insulin sensitivity index (MISI) by oral glucose tolerance test as proposed by DeFronzo, IMT, apoB/apoAI and the components of the metabolic syndrome (MS) as defined by ATP III. RESULTS: H-IR was significantly higher in moderate/severe steatosis than in the mild steatosis group (p < 0.0001). By contrast, MISI did not differ between the two groups. There was a significant correlation between H-IR, MISI and all of the components of MS. H-IR was significantly correlated with carotid IMT (r = 0.35; p < 0.0001) and the apoB/apoAI ratio (r = 0.43; p < 0.0001). Otherwise, a significant correlation was observed only between MISI and apoB/apoAI ratio. Multivariate analysis revealed that H-IR is related to early markers of atherosclerosis independent of MS components. CONCLUSIONS: In our study population, NAFLD was positively associated with carotid IMT, and this association is independent of MS components, but strictly related to H-IR that might contribute to the development of atherosclerosis through an impairment of the lipid profile in terms of the apoB/apoAI ratio. By contrast, no significant relation was observed between MISI and carotid IMT.

Cardiovascular dysfunction in patients with liver cirrhosis.

Hyperdynamic syndrome is a well-known clinical condition found in patients with cirrhosis and portal hypertension, characterized by increased heart rate and cardiac output, and reduced systemic vascular resistance and arterial blood pressure. The leading cause of hyperdynamic circulation in cirrhotic patients is peripheral and splanchnic vasodilatation, due to an increased production/activity of vasodilator factors and decreased vascular reactivity to vasoconstrictors. The term "cirrhotic cardiomyopathy" describes impaired contractile responsiveness to stress, diastolic dysfunction and electrophysiological abnormalities in patients with cirrhosis without known cardiac disease. Underlying circulatory and cardiac dysfunctions are the main determinant in the development of hepatorenal syndrome in advanced cirrhosis. Moreover, the clinical consequences of cirrhosis-related cardiovascular dysfunction are evident during and after liver transplantation, and after transjugular intrahepatic portosystemic shunt insertion. Cardiovascular complications following these procedures are common, with pulmonary edema being the most common complication. Other complications include overt heart failure, arrhythmia, pulmonary hypertension, pericardial effusion, and cardiac thrombus formation. This review discusses the circulatory and cardiovascular dysfunctions in cirrhosis, examining the pathophysiologic and clinical implications in light of the most recent published literature.

Hypothalamus-pituitary dysfunction is common in patients with stable cirrhosis and abnormal low dose synacthen test.

BACKGROUND: Adrenal insufficiency is often present in cirrhosis. We hypothesize that a prolonged adrenocorticotropic hormone (ACTH) stimulus can restore cellular capacity of adrenal glands to secrete cortisol. Aim of our study was to assess adrenal responsiveness to prolonged ACTH stimulation in cirrhotics. METHODS: Prospective observational study in 121 consecutively admitted cirrhotic patients undergoing a low dose short synacthen test and plasma ACTH measurement using a chemiluminescence immunoassay. Long synacthen test was performed if the low dose was abnormal. RESULTS: 46 patients had abnormal low dose short test (38%), and 29 underwent the long test: 41% showed normal response (Group 1), 55% showed delayed response (Group 2) and 1 had abnormal response (4%). Baseline ACTH levels did not significantly differ between the two groups. Median basal cortisol was higher in Group 1 (296 vs. 198 nmol/L; p=0.02). Using ROC curve basal cortisol <254 nmol/L was associated with a delayed long synacthen test response (AUC 0.78, p=0.001) with good accuracy (sensitivity 67%, specificity 81%). CONCLUSION: A delayed cortisol response after a prolonged ACTH stimulation is found in over fifty percent of cirrhotics with abnormal low dose short synacthen test, confirming that the mechanism of hypoadrenalism in these patients could be related both to adrenal cellular dysfunction and hypothalamus-pituitary adrenal axis impairment.

Diabetes increases renovascular impedance in patients with liver cirrhosis.

Renal failure is a common complication of cirrhosis and is associated with poor prognosis. Several reports have demonstrated the clinical utility of renal resistive indices in the assessment of renal function in cirrhosis patients. It is unknown whether the occurrence of diabetes affects renal haemodynamic indices in patients with cirrhosis. Therefore, the aim of our study was to compare renal Doppler indices in cirrhotic patients with and without type 2 diabetes mellitus (T2DM) and in diabetics without cirrhosis, and to relate the Doppler parameters to albuminuria. We evaluated 89 consecutive patients with normal renal functioning, including 37 with cirrhosis and T2DM (CD-Group), 41 with cirrhosis without diabetes (C-Group) and 11 with diabetes without cirrhosis (D-Group). The kidney pulsatility index (PI) and resistance index (RI) were measured by Doppler ultrasound. Renal function was expressed as the estimated glomerular filtration rate (eGFR) using the modification of diet in renal disease (MDRD) formula. Microalbuminuria (µAlb) was also evaluated. No significant differences were observed with respect to age, the Child-Pugh class or the serum creatinine level. The eGFR was mildly reduced in the CD-Group compared with the C-Group and D-Group, and µAlb was present in 24.4 % of the patients in the CD-Group and in 9 % of those in the D-Group. The PI and RI were significantly increased in the CD-Group and D-Group compared with the C-Group. Both the PI and RI were significantly associated with µAlb independent of age and Child-Pugh class. The novel finding of this study was that T2DM potentially impairs renal haemodynamics in patients with cirrhosis.

Corticosteroid replacement therapy in hepatoadrenal syndrome: case report with a review of the literature.

In liver cirrhosis, hepatoadrenal syndrome has been described recently as a progressive impairment in the adrenocortical reserve, with deficient production or action of glucocorticoids resulting in adrenal insufficiency. Data on the treatment of this syndrome are scarce. We report a case of a 60-year-old male patient referred to our hospital because of rectal bleeding and bilateral leg swelling. He complained of marked weakness, bilateral leg swelling, and shortness of breath with exertion for the last 2 months. Biochemistry and imaging indicated liver cirrhosis. Because of the weakness and persistent hypotension, we performed a low-dose synacthen test, which indicated adrenal insufficiency (baseline cortisol level 1.8 µg/dl, increasing to 3.5 and 3.7 µg/dl at 20 and 30 min, respectively). The patient's general condition improved promptly after corticosteroid supplementation.