Non-Alcoholic Steatohepatitis: Clinical and Translational Research.

UNLABELLED: The present review includes translational and clinical research that characterize non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Clinical and experimental evidence led to the recognition of the key toxic role played by lipotoxicity in the pathogenesis of NAFLD. The current understanding of lipotoxicity suggests that organ injury is initiated by the generation of oxidative metabolites and the translocation of gut-derived endotoxin. These processes lead to cellular injury and stimulation of the inflammatory responses mediated through a variety of molecules. The injury progresses through impairment of tissue regeneration and extracellular matrix turnover, leading to fibrogenesis and cirrhosis. Several cell types are involved in this process, predominantly stellate cells, macrophages and parenchymal cells. In response to inflammation, cytokines activate many signaling cascades that regulate fibrogenesis. This examination brings together research focusing on the underlying mechanisms of injury. It highlights the various processes and molecules that are likely involved in inflammation, immune modulation, and fibrogenesis in the liver. We searched electronic databases (Medline, Embase) for this review. This integrative work investigates different aspects of liver damage and possible repair. We aim to (1) determine the immuno-pathology of liver damage due to steatosis, (2) suggest diagnostic markers of NASH, (3) examine the role of behaviour in the development of NASH, and (4) develop common tools to study steatosis-induced effects in clinical studies. Special accent is put on co-morbidities with renal and neuropsychological disorders. Moreover, we review the evidence in literature on the role of moderate alcohol consumption in individuals that present NAFLD/NASH. KEY WORDS: behavior, diet, imaging, non-alcoholic fatty liver, nonalcoholic steatohepatitis, laboratory markers.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Hyaluronic acid as a non-invasive biomarker of liver fibrosis.

UNLABELLED: Chronic liver diseases may cause inflammation and progressive scarring, over time leading to irreversible hepatic damage (cirrhosis). As a result, the need to assess and closely monitor individuals for risk factors of components of matrix deposition and degradation, as well as the severity of the fibrosis using biomarkers, has been increasingly recognized. AIM: Our aim is to review the use of biomarker for diagnosing and defining the severity of liver fibrosis. METHODS: A systematic literature review was done using the terms "hyaluronic acid" and "liver fibrosis" as well as the name of each biomarker or algorithm known to be employed. PubMed and Google Scholar were searched, and English language articles indexed between January 2010 and October 2014 in which HA was used as a marker of liver fibrosis were retrieved, regardless of the underlying liver disease. Each author read the publications separately and the results were analyzed and discussed. RESULTS: Biomarkers offer a potential prognostic or diagnostic indicator for disease manifestation, progression, or both. Serum biomarkers, including HA, have been used for many years. Emerging biomarkers such as metalloproteinases have been proposed as tools that provide valuable complementary information to that obtained from traditional biomarkers. Moreover, markers of extracellular matrix degradation provide powerful predictions of risk. In order for biomarkers to be clinically useful in accurately diagnosing and treating disorders, age-specific reference intervals that account for differences in gender and ethnic origin are a necessity. CONCLUSIONS: This review attempts to provide a comprehensive analysis of the emerging risk biomarkers of liver fibrosis and to describe the clinical significance and analytical considerations of each biomarker pointing out sentinel features of disease progression.

Hepatotoxicity of Pyrrolizidine Alkaloids.

PURPOSE: This article aimed 1) to review herbal medicine containing pyrrolizidine alkaloids (PA)-induced toxicities of the liver; 2) to encourage the recognition and prevention of common problems encountered when using complementary and alternative medicine and 3) to review the toxic effects of herbal remedies containing PAs. DESIGN AND METHODS: We performed a systematic literature search using the PubMed and Google Scholar engines. The search was not restricted to languages. We also provide an interpretation of the data. CONCLUSIONS: Herbal remedies containing PAs can induce liver damage, including hepato- sinusoidal obstruction syndrome or veno-occlusive disease. Preventing overdose and monitoring long-term use of such remedies may avoid glutathione depletion leading to mitochondrial injury, and therefore avoid liver cell damage. Moreover, immediately stopping the herbal medication prevents further harm to the liver. Chronic consumption of hepatotoxicants can lead to cancer formation and promotion. The role of active metabolites in PA-induced liver toxicity and their mechanism of action require further investigation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Alcoholic Liver Disease: Role of Cytokines.

The present review spans a broad spectrum of topics dealing with alcoholic liver disease (ALD), including clinical and translational research. It focuses on the role of the immune system and the signaling pathways of cytokines in the pathogenesis of ALD. An additional factor that contributes to the pathogenesis of ALD is lipopolysaccharide (LPS), which plays a central role in the induction of steatosis, inflammation, and fibrosis in the liver. LPS derived from the intestinal microbiota enters the portal circulation, and is recognized by macrophages (Kupffer cells) and hepatocytes. In individuals with ALD, excessive levels of LPS in the liver affect immune, parenchymal, and non-immune cells, which in turn release various inflammatory cytokines and recruit neutrophils and other inflammatory cells. In this review, we elucidate the mechanisms by which alcohol contributes to the activation of Kupffer cells and the inflammatory cascade. The role of the stellate cells in fibrogenesis is also discussed.

Alcoholic liver disease: Clinical and translational research.

The present review spans a broad spectrum of topics dealing with alcoholic liver disease (ALD), including clinical research, translational research, pathogenesis and therapies. A special accent is placed on alcohol misuse, as alcohol is a legally commercialized and taxable product. Drinking alcohol, particularly from a young age, is a major health problem. Alcoholism is known to contribute to morbidity and mortality. A systematic literature search was performed in order to obtain updated data (2008-2015). The review is focused on genetic polymorphisms of alcohol metabolizing enzymes and the role of cytochrome p450 2E1 and iron in ALD. Alcohol-mediated hepatocarcinogenesis is also discussed in the presence or absence of co-morbidities such as viral hepatitis C as well as therapeutic the role of innate immunity in ALD-HCV. Moreover, emphasis was placed on alcohol and drug interactions, as well as liver transplantation for end-stage ALD. Finally, the time came to eradicate alcohol-induced liver and intestinal damage by using betaine.

Biomolecules and Biomarkers Used in Diagnosis of Alcohol Drinking and in Monitoring Therapeutic Interventions.

BACKGROUND: The quantitative, measurable detection of drinking is important for the successful treatment of alcohol misuse in transplantation of patients with alcohol disorders, people living with human immunodeficiency virus that need to adhere to medication, and special occupational hazard offenders, many of whom continually deny drinking. Their initial misconduct usually leads to medical problems associated with drinking, impulsive social behavior, and drunk driving. The accurate identification of alcohol consumption via biochemical tests contributes significantly to the monitoring of drinking behavior. METHODS: A systematic review of the current methods used to measure biomarkers of alcohol consumption was conducted using PubMed and Google Scholar databases (2010-2015). The names of the tests have been identified. The methods and publications that correlate between the social instruments and the biochemical tests were further investigated. There is a clear need for assays standardization to ensure the use of these biochemical tests as routine biomarkers. FINDINGS: Alcohol ingestion can be measured using a breath test. Because alcohol is rapidly eliminated from the circulation, the time for detection by this analysis is in the range of hours. Alcohol consumption can alternatively be detected by direct measurement of ethanol concentration in blood or urine. Several markers have been proposed to extend the interval and sensitivities of detection, including ethyl glucuronide and ethyl sulfate in urine, phosphatidylethanol in blood, and ethyl glucuronide and fatty acid ethyl esters in hair, among others. Moreover, there is a need to correlate the indirect biomarker carbohydrate deficient transferrin, which reflects longer lasting consumption of higher amounts of alcohol, with serum γ-glutamyl transpeptidase, another long term indirect biomarker that is routinely used and standardized in laboratory medicine.

Nonmedicinal interventions in nonalcoholic fatty liver disease.

Unhealthy diet and lack of physical exercise are responsible for fat accumulation in the liver, which may lead to liver disease. Histologically, the severity of the disease has two stages: nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). NAFLD is defined by the presence of steatosis with no evidence of cellular injury such as hepatocyte ballooning. NASH is a distinct entity from NAFLD, and is characterized by the presence of inflammation with hepatocytes damage, with or without fibrosis. While several therapeutic strategies have been proposed to improve this condition, the present review aims to discuss nonmedicinal interventions used to reduce liver involvement or to prevent the disease altogether. The authors investigated dietary patterns and vitamin deficiencies associated with NAFLD, and their role in enhancing disease severity. Additionally, they reviewed the role of exercise and the use of interventions, such as as intragastric balloon and bariatric surgery, for improving disease progression. The authors propose monitoring disease progression or repair by following changes in cytoadipokine levels.

Quinolones-induced hypersensitivity reactions.

UNLABELLED: Quinolones are broad spectrum antibiotics that are intensively used and can induce immediate- and delayed-type hypersensitivity reactions, either IgE or T-cell mediated, in about 2-3% of treated patients. OBJECTIVE: To better understand how T cells interact with quinolones to produce the hypersensitivity and to describe the possible prevention of the reactions. METHODS: We search in PubMed for quinolones and adverse reactions naming each one of the therapeutics in use and the possible hypersensitivity reactions e.g., anaphylaxis, drug-induced delayed reactions, and hypersensitivity syndrome reactions. We also performed a search on organ-specific hypersensitivity reactions including cutaneous reactions, hepatic reactions, and renal reactions. RESULTS: Our data show that T cells are involved in delayed immune reactions to quinolones and that cross-reactivity among the different quinolones is frequent. The predictive tests for quinolone-induced hypersensitivity should be used in patients before they are given the medication. CONCLUSIONS: Early identification of the mechanism of toxicity, quantitative assessment using laboratory tests, analysis of risk factors for patient susceptibility to the quinolones, and possible drug-drug interactions may lead to appropriate patient selection for therapy, monitoring the injury early and discontinuation of the therapeutic agent.

Hyaluronic acid and wound healing.

BACKGROUND: We developed an experimental model of ethanol-induced dermatotoxicity and hepatocytoxicity using normal human keratinocytes and normal human hepatocytes that preserve inducible cytochrome p450 activities. The original work was described in several articles. The objective of this study was to determine whether hyaluronic acid attenuates skin necrosis, and to further clarify its uses in wound repair in humans, animal models and in vitro studies. METHODS: We performed a systematic review of the literature using the terms "hyaluronic acid" and "wound healing". PubMed was searched for studies published during the period 2010-2014. RESULTS: Hyaluronic acid is used in tissue regeneration alone or in combination with herbal or Western medicine. Scaffolds made up of hyaluronic acid were used to embed basic fibroblast growth factor. CONCLUSION: Hyaluronic acid extracts are safe and efficacious products to be used in skin repair.

Non-invasive biomarkers in non-alcoholic steatohepatitis-induced hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) is by far the most common form of chronic liver disease worldwide, affecting adults as well as children. Under the term of NAFLD there is a wide spectrum of diseases ranging from simple steatosis to the non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma (HCC). Several mechanisms have been described to influence the progression of the disease from the benign NAFL to the aggressive NASH. The imbalance between pro- and anti-oxidant mechanisms and between pro- and anti-inflammatory cytokines is thought to play a pivotal role in the pathogenesis of NAFLD and disease progression toward NASH and fibrosis. The present review intends to look at some of the mechanistic biomarkers to be employed in establishing an early diagnosis in HCC derived from NASH.

Alcoholic and non-alcoholic steatohepatitis.

This paper is based upon the "Charles Lieber Satellite Symposia" organized by Manuela G. Neuman at the Research Society on Alcoholism (RSA) Annual Meetings, 2013 and 2014. The present review includes pre-clinical, translational and clinical research that characterize alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH). In addition, a literature search in the discussed area was performed. Strong clinical and experimental evidence lead to recognition of the key toxic role of alcohol in the pathogenesis of ALD. The liver biopsy can confirm the etiology of NASH or alcoholic steatohepatitis (ASH) and assess structural alterations of cells, their organelles, as well as inflammatory activity. Three histological stages of ALD are simple steatosis, ASH, and chronic hepatitis with hepatic fibrosis or cirrhosis. These latter stages may also be associated with a number of cellular and histological changes, including the presence of Mallory's hyaline, megamitochondria, or perivenular and perisinusoidal fibrosis. Genetic polymorphisms of ethanol metabolizing enzymes such as cytochrome p450 (CYP) 2E1 activation may change the severity of ASH and NASH. Alcohol mediated hepatocarcinogenesis, immune response to alcohol in ASH, as well as the role of other risk factors such as its co-morbidities with chronic viral hepatitis in the presence or absence of human immunodeficiency virus are discussed. Dysregulation of hepatic methylation, as result of ethanol exposure, in hepatocytes transfected with hepatitis C virus (HCV), illustrates an impaired interferon signaling. The hepatotoxic effects of ethanol undermine the contribution of malnutrition to the liver injury. Dietary interventions such as micro and macronutrients, as well as changes to the microbiota are suggested. The clinical aspects of NASH, as part of metabolic syndrome in the aging population, are offered. The integrative symposia investigate different aspects of alcohol-induced liver damage and possible repair. We aim to (1) determine the immuno-pathology of alcohol-induced liver damage, (2) examine the role of genetics in the development of ASH, (3) propose diagnostic markers of ASH and NASH, (4) examine age differences, (5) develop common research tools to study alcohol-induced effects in clinical and pre-clinical studies, and (6) focus on factors that aggravate severity of organ-damage. The intention of these symposia is to advance the international profile of the biological research on alcoholism. We also wish to further our mission of leading the forum to progress the science and practice of translational research in alcoholism.

Safety of anti-tumor necrosis factor therapies in arthritis patients.

PURPOSE: Inflammatory and rheumatic arthritis remain leading causes of disability worldwide. The arthritis therapeutic area commands the largest market for the prescription of biological and non-steroidal anti-inflammatory drugs (NSAID). Yet biotechnology and pharmaceutical companies conducting research and providing therapeutics in this area frequently face challenges in patient safety. The purpose of our study was to assess safety of anti-tumor necrosis factor therapies in arthritis patients. METHODS: The present study systematically reviews adverse events of biologicals alone or in the presence of NSAIDs and other immunosuppressant therapeutics such as disease-modifying antirheumatic drugs (DMARD). We assessed the rheumatology literature that included clinical trials with anti-tumor necrosis factor (TNF) biologicals and case reports published between 2010 and 2014. RESULTS: Currently approved anti-TNF biologicals in arthritis include the monoclonal antibodies infliximab, adalimumab, certolizumab pegol and golimumab, and the fusion protein etanercept. The most frequently-reported adverse event was infection. We grouped the adverse reactions as immune-mediated, hypersensitivity syndrome reactions including cutaneous and hepatic manifestation, neurological, hematological, and malignancy. DISCUSSION: Most adverse events are due to the failure of host immunological control, which involves susceptibility to the drug itself, or de novo infection or reactivation of a latent bacterial or viral infection, often with a different expression of disease. Drug-induced liver injury associated with anti-TNF biologicals must be kept in mind when evaluating patients with increased liver enzymes. CONCLUSION: Risk assessment in individuals undergoing treatment with biologicals represents a step towards achieving a personalized medicine approach to identify those patients that will safely benefit from this therapeutic approach. Patients and physicians must be alert of anti-TNF agents as potential causes of drug-induced liver injury and monitor the therapies. Personalizing therapeutic pharmacovigilance promises to optimize benefits while minimizing side effects.

Alcoholic liver disease: a synopsis of the Charles Lieber's Memorial Symposia 2009-2012.

This paper is based upon the 'Charles Lieber Satellite Symposia' organized by Manuela G. Neuman at each of the 2009-2012 Research Society on Alcoholism (RSA) Annual Meetings. The presentations represent a broad spectrum dealing with alcoholic liver disease (ALD). In addition, a literature search (2008-2013) in the discussed area was performed in order to obtain updated data. The presentations are focused on genetic polymorphisms of ethanol metabolizing enzymes and the role of cytochrome P4502E1 (CYP2E1) in ALD. In addition, alcohol-mediated hepatocarcinogenesis, immune response to alcohol and fibrogenesis in alcoholic hepatitis as well as its co-morbidities with chronic viral hepatitis infections in the presence or absence of human deficiency virus are discussed. Finally, emphasis was led on alcohol and drug interactions as well as liver transplantation for end-stage ALD.

Efficacy and safety of prasugrel in acute coronary syndrome patients.

Ischemic heart disease is the primary cause of death worldwide. The pathophysiological process of cardiovascular diseases is linked to atheromatous plaque formation, while plaque rupture releases thrombogenic elements, which lead to activation of platelets, blood clotting and formation of thrombi. Platelet inhibitors are used to prevent thrombosis. The present systematic review discusses the efficacy of prasugrel in terms of platelet inhibition potential and clinical prevention of cardiovascular outcomes. The balance between reduction of ischemic events as a measure of drug efficacy and the risk of bleeding is reviewed. Other adverse events observed in patients treated with this platelet inhibitor are discussed, including hematological complications, and cutaneous and hepatic idiosyncratic reactions. The complex relationship between prasugrel use and cancer promotion is also described.

Biologic therapies in inflammatory bowel disease.

Inflammatory bowel disease, including its 2 entities ulcerative colitis and Crohn's disease, is a chronic medical condition characterized by the destructive inflammation of the intestinal tract. Biologics represent a class of therapeutics with immune intervention potential. These agents block the proinflammatory cascade that triggers the activation and proliferation of T lymphocytes at the level of the intestine, therefore reestablishing the balance between the pro- and anti-inflammatory messages. All 7 biologics showing clinical benefits in inflammatory bowel disease are monoclonal antibodies. The following systematic review discusses the pharmacokinetics and efficacy of the tumor necrosis factor blockers infliximab, adalimumab, certolizumab pegol, and golimumab. In addition, we describe the α4 integrin inhibitors natalizumab and vedolizumab, which are directed against cell adhesion molecules, as well as the interleukin 12/23 blocker ustekinumab.

Biomarkers in nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver condition characterized by insulin resistance, type 2 diabetes and fat accumulation in the liver that may cause hepatic inflammation and progressive scarring leading to nonalcoholic steatohepatitis (NASH) and irreversible liver damage (cirrhosis). As a result, there has been increased recognition of the need to assess and closely monitor individuals for risk factors of components of NAFLD and NASH, as well as the severity of these conditions using biomarkers. AIM: To review the biomarkers used to diagnose and define the severity of NAFLD and NASH. METHODS: A comprehensive PubMed and Google Scholar literature search was performed using the terms "non-alcoholic fatty liver disease", "non-alcoholic steatohepatitis", as well as the name of each biomarker known to be used. Articles indexed between 2004 and 2014 were used. Each author read the publications separately and the results were discussed. RESULTS: Biomarkers offer a potential prognostic or diagnostic indicator for disease manifestation, progression or both. Serum biomarkers, including total cholesterol, triglycerides, insulin resistance and C-peptide, have been used for many years. Emerging biomarkers, such as apolipoprotein A1, apolipoprotein B, leptin, adiponectin, free fatty acids, ghrelin and tumour necrosis factor-alpha, have been proposed as tools that could provide valuable complementary information to that obtained from traditional biomarkers. Moreover, markers of cell death and mitochondrial dysfunction (cytokeratins) represent powerful predictors of risk. For biomarkers to be clinically useful in accurately diagnosing and treating disorders, age-specific reference intervals that account for differences in sex and ethnic origin are a necessity. CONCLUSIONS: The present review attempts to provide a comprehensive analysis of the emerging risk biomarkers of NAFLD and NASH, and to use the clinical significance and analytical considerations of each biomarker pointing out sentinel features of disease progression.

Risk of infections of biological therapies with accent on inflammatory bowel disease.

BACKGROUND: Biological therapies using anti-tumor necrosis factor (TNF)-α agents have an important impact in the treatment of inflammatory bowel disease, rheumatoid arthritis, psoriasis, and other inflammatory conditions. However, a significant number of patients lose their response to these medications over time. Clinical trials have demonstrated that antibodies against anti-TNF agents may impact treatment response and increase the risk of infusion reactions. Of concern is also the possibility of developing adverse events induced by anti-TNF agents. The purpose of the present systematic review is to describe the current knowledge on the risk of infections associated with anti-TNF agents antagonists, as well as integrin antagonists. We also intend to describe case reports of these adverse events in inflammatory bowel disease patients. METHODS: Currently approved anti-TNF biologicals in IBD include the monoclonal antibodies infliximab, adalimumab, certolizumab pegol and golimumab. Integrin antagonists include natalizumab, etrolizumab and vedolizumab. RESULTS: The most frequently-reported adverse events of these biologicals were infections, and these are described in detail in this study. DISCUSSION: Most adverse events are due to the failure of host immunological control, which involves de novo infection, or reactivation of latent bacterial or viral infection, often with a different expression of disease. CONCLUSION: Risk assessment in individuals undergoing treatment with biologicals represents a step towards achieving treatment personalization to identify those patients that will safely benefit from this therapeutic approach. Patients and physicians must be alert for anti-TNF agents and anti-integrin medication as potential causes of drug-induced infections and monitor the therapies. Personalizing therapeutic vigilance promises to optimize benefits while minimizing infections.

Changes in the pathogenesis of alcohol-induced liver disease -- preclinical studies.

Excessive alcohol consumption presents considerable health risks in humans. A variety of morphologic and functional changes contribute to hepatic injury produced by heavy drinking. The present review summarizes the current knowledge of alcohol-induced liver disease and describes preclinical experimental approaches used to understand alcoholic liver disease (ALD), with a particular emphasis on impaired protein and lipid trafficking, disruption of proteolysis and autophagy, alterations in methionine metabolism and perturbations in metabolic signaling that cause dysfunctional gene expression and the eventual formation of aggresomal Mallory-Denk bodies (MDB) in liver cells. These changes eventually lead to some of the more severe hepatic impairments, including alcoholic hepatitis and fibrosis. Moreover the misuse of alcohol contributes to immune dysfunction and inadequate immune response to viral infections.

Valproic acid derivatives signal for apoptosis and repair in vitro.

PURPOSE: To determine the cytotoxicity of valproic acid (VPA) and its derivatives in human hepatoblastoma (HepG2) cells, and to study the possible toxicity of these compounds in human lymphocytes from patients with known hypersensitivity syndrome reactions (HSRs) to other medication. METHODS: Cells were exposed to physiological doses of VPA, valnoctamide (VCD) and its one carbon homologue sec-Butyl-propyl-acetamide (SPD) for 2h and for 24h. Cell viability was measured using succinate dehydrogenase activity for hepatocytes and lymphocyte toxicity assay (LTA) for lymphocytes. Cytokines and apoptosis [cytokeratine 18 (cCK18-M30)] markers were quantitated by ELISA. RESULTS: VCD and SPD presented lower cytotoxicity compared to VPA in cultured HepG2 cells. SPD led to cytotoxicity in lymphocytes. VPA and its derivatives increased the release of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in media, but had no influence on the release of either interleukin (IL)-1 or IL-6. Significant increases in the release of IFN-γ and TNF-α were observed in lymphocytes exposed to high doses of VPA, and this increased further with exposure time. SIGNIFICANCE: HepG2 cells exposed to VCD and SPD experienced lower direct cytotoxicity than those treated with VPA. Lymphocytes from patients that experienced HSR to other medication have shown cytotoxicity to VPA and its VPA derivatives-induced. High levels of pro-inflammatory cytokines were released in the cell culture media, suggesting that inflammation plays a key role in VPA-derivatives induced lymphocyte toxicity.

Adverse drug reactions induced by valproic acid.

Valproic acid is a widely-used first-generation antiepileptic drug, prescribed predominantly in epilepsy and psychiatric disorders. VPA has good efficacy and pharmacoeconomic profiles, as well as a relatively favorable safety profile. However, adverse drug reactions have been reported in relation with valproic acid use, either as monotherapy or polytherapy with other antiepileptic drugs or antipsychotic drugs. This systematic review discusses valproic acid adverse drug reactions, in terms of hepatotoxicity, mitochondrial toxicity, hyperammonemic encephalopathy, hypersensitivity syndrome reactions, neurological toxicity, metabolic and endocrine adverse events, and teratogenicity.