IL-1 Signal Inhibition in Alcohol-Related Hepatitis: A Randomized, Double-Blind, Placebo-Controlled Trial of Canakinumab.

BACKGROUND AND AIMS: Short-term mortality in alcohol-related hepatitis (AH) is high, and no current therapy results in durable benefit. A role for interleukin (IL)-1ß has been demonstrated in the pathogenesis of alcohol-induced steatohepatitis. This study explored the safety and efficacy of canakinumab (CAN), a monoclonal antibody targeting IL-1ß, in the treatment of patients with AH. METHODS: Participants with biopsy-confirmed AH and discriminant function ≥32 but Model for End-Stage Liver Disease ≤27 were randomly allocated 1:1 to receive either CAN 3 mg/kg or placebo (PBO). Liver biopsies were taken before and 28 days after treatment. The primary endpoint was the overall histological improvement in inflammation analyzed by the modified intention-to-treat principle. RESULTS: Fifty-seven participants were randomized: 29 to CAN and 28 to PBO. Two participants had histology that did not corroborate the clinical diagnosis. Of the remaining 55 participants, paired histology data were evaluable from 48 participants. In CAN-treated participants, 14 (58%) of 24 demonstrated histological improvement compared with 10 (42%) of 24 in the PBO group (P = .25). There was no improvement in prognostic scores of liver function. Four (7%) of the 55 participants died within 90 days, 2 in each group. The number of serious adverse events was similar between CAN vs PBO. In post hoc exploratory analyses after adjustment for baseline prognostic factors, CAN therapy was associated with overall histological improvement (P = .04). CONCLUSIONS: CAN therapy in severe AH participants with Model for End-Stage Liver Disease ≤27 did not alter biochemical or clinical outcomes compared with PBO. Nonsignificant histological improvements did not translate into clinical benefit. EudraCT, Number: 2017-003724-79; ClinicalTrials.gov, Number: NCT03775109.

Development and validation of the AMMON-OHE model to predict risk of overt hepatic encephalopathy occurrence in outpatients with cirrhosis.

BACKGROUND & AIMS: Neuropsychological and psychophysical tests are recommended to assess the risk of overt hepatic encephalopathy (OHE), but their accuracy is limited. Hyperammonaemia is central in the pathogenesis of OHE, but its predictive utility is unknown. In this study, we aimed to determine the role of neuropsychological or psychophysical tests and ammonia, and to develop a model (AMMON-OHE) to stratify the risk of subsequent OHE development in outpatients with cirrhosis. METHODS: This observational, prospective study included 426 outpatients without previous OHE from three liver units followed for a median of 2.5 years. Psychometric hepatic encephalopathy score (PHES) <-4 or critical flicker frequency (CFF) <39 was considered abnormal. Ammonia was normalized to upper limit of normal (AMM-ULN) at the respective reference laboratory. Multivariable frailty competing risk and random survival forest analyses were performed to predict future OHE and to develop the AMMON-OHE model. External validation was carried out using 267 and 381 patients from two independent units. RESULTS: Significant differences were found in time-to-OHE (log-rank p <0.001) according to PHES or CFF and ammonia, with the highest risk in patients with abnormal PHES plus high AMM-ULN (hazard ratio 4.4; 95% CI 2.4-8.1; p <0.001 compared with normal PHES and AMM-ULN). On multivariable analysis, AMM-ULN but not PHES or CFF was an independent predictor of the development of OHE (hazard ratio 1.4; 95% CI 1.1-1.9; p = 0.015). The AMMON-OHE model (sex, diabetes, albumin, creatinine and AMM-ULN) showed a C-index of 0.844 and 0.728 for the prediction of a first episode of OHE in two external validation cohorts. CONCLUSIONS: In this study, we developed and validated the AMMON-OHE model, comprising readily available clinical and biochemical variables that can be used to identify outpatients at the highest risk of developing a first episode of OHE. IMPACT AND IMPLICATIONS: In this study, we aimed to develop a model to predict which patients with cirrhosis are at risk of developing overt hepatic encephalopathy (OHE). Using data from three units and including 426 outpatients with cirrhosis, we developed the AMMON-OHE model - comprising sex, diabetes, albumin, creatinine and ammonia levels - which demonstrated good predictive ability. The AMMON-OHE model performs better than PHES and CFF to predict the first episode of OHE in outpatients with cirrhosis. This model was validated in 267 and 381 patients from two independent liver units. The AMMON-OHE model is available online for clinical use.

Gut-derived systemic inflammation as a driver of depression in chronic liver disease.

Depression and chronic liver disease (CLD) are important causes of disability, morbidity and mortality worldwide and their prevalence continues to rise. The rate of depression in CLD is high compared to that of the general population and is comparable to the increased rates observed in other medical comorbidities and chronic inflammatory conditions. Notably, a comorbid diagnosis of depression has a detrimental effect on outcomes in cirrhosis. Systemic inflammation is pivotal in cirrhosis-associated immune dysfunction - a phenomenon present in advanced CLD (cirrhosis) and implicated in the development of complications, organ failure, disease progression, increased infection rates and poor outcome. The presence of systemic inflammation is also well-documented in a cohort of patients with depression; peripheral cytokine signals can result in neuroinflammation, behavioural change and depressive symptoms via neural mechanisms, cerebral endothelial cell and circumventricular organ signalling, and peripheral immune cell-to-brain signalling. Gut dysbiosis has been observed in both patients with cirrhosis and depression. It leads to intestinal barrier dysfunction resulting in increased bacterial translocation, in turn activating circulating immune cells, leading to cytokine production and systemic inflammation. A perturbed gut-liver-brain axis may therefore explain the high rates of depression in patients with cirrhosis. The underlying mechanisms explaining the critical relationship between depression and cirrhosis remain to be fully elucidated. Several other psychosocial and biological factors are likely to be involved, and therefore the cause is probably multifactorial. However, the role of the dysfunctional gut-liver-brain axis as a driver of gut-derived systemic inflammation requires further exploration and consideration as a target for the treatment of depression in patients with cirrhosis.

Plasma ammonia levels predict hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis.

BACKGROUND & AIMS: Hyperammonaemia is central in the pathogenesis of hepatic encephalopathy. It also has pleiotropic deleterious effects on several organ systems, such as immune function, sarcopenia, energy metabolism and portal hypertension. This study was performed to test the hypothesis that severity of hyperammonaemia is a risk factor for liver-related complications in clinically stable outpatients with cirrhosis. METHODS: We studied 754 clinically stable outpatients with cirrhosis from 3 independent liver units. Baseline ammonia levels were corrected to the upper limit of normal (AMM-ULN) for the reference laboratory. The primary endpoint was hospitalisation with liver-related complications (a composite endpoint of bacterial infection, variceal bleeding, overt hepatic encephalopathy, or new onset or worsening of ascites). Multivariable competing risk frailty analyses using fast unified random forests were performed to predict complications and mortality. External validation was carried out using prospective data from 130 patients with cirrhosis in an independent tertiary liver centre. RESULTS: Overall, 260 (35%) patients were hospitalised with liver-related complications. On multivariable analysis, AMM-ULN was an independent predictor of both liver-related complications (hazard ratio 2.13; 95% CI 1.89-2.40; p <0.001) and mortality (hazard ratio 1.45; 95% CI 1.20-1.76; p <0.001). The AUROC of AMM-ULN was 77.9% for 1-year liver-related complications, which is higher than traditional severity scores. Statistical differences in survival were found between high and low levels of AMM-ULN both for complications and mortality (p <0.001) using 1.4 as the optimal cut-off from the training set. AMM-ULN remained a key variable for the prediction of complications within the random forests model in the derivation cohort and upon external validation. CONCLUSION: Ammonia is an independent predictor of hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis and performs better than traditional prognostic scores in predicting complications. LAY SUMMARY: We conducted a prospective cohort study evaluating the association of blood ammonia levels with the risk of adverse outcomes in 754 patients with stable cirrhosis across 3 independent liver units. We found that ammonia is a key determinant that helps to predict which patients will be hospitalised, develop liver-related complications and die; this was confirmed in an independent cohort of patients.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

BACKGROUND & AIMS: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE. METHODS: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days. PRIMARY OUTCOME: 50% reduction in neutrophil oxidative burst (OB) at 30 days. SECONDARY OUTCOMES: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis. RESULTS: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96). CONCLUSION: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT02019784. LAY SUMMARY: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection.

Implications and Management of Cirrhosis-Associated Immune Dysfunction Before and After Liver Transplantation.

Cirrhosis-associated immune dysfunction (CAID) describes a panacea of innate and adaptive deficits that result from the sequelae of cirrhotic portal hypertension that is similar in its manifestations regardless of etiology of chronic liver injury. CAID is associated with synchronous observations of dysregulated priming of innate immune effector cells that demonstrate a proinflammatory phenotype but are functionally impaired and unable to adequately prevent invading pathogens. CAID is mainly driven by gut-barrier dysfunction and is associated with deficits of microbial compartmentalization and homeostasis that lead to tonic activation, systemic inflammation, and exhaustion of innate-immune cells. CAID leads to a high frequency of bacterial and fungal infections in patients with cirrhosis that are often associated with acute decompensation of chronic liver disease and acute-on-chronic liver failure and carry a high mortality rate. Understanding the deficits of mucosal and systemic immunity in the context of chronic liver disease is essential to improving care for patients with cirrhosis, preventing precipitants of acute decompensation of cirrhosis, and improving morbidity and survival. In this review, we summarize the detailed dynamic immunological perturbations associated with advanced chronic liver disease and highlight the importance of recognizing immune dysregulation as a sequela of cirrhosis. Furthermore, we address the role of screening, prevention, and early treatment of infections in cirrhosis in improving patient outcomes in transplant and nontransplant settings.

Targeting the gut-liver-immune axis to treat cirrhosis.

Cirrhotic portal hypertension is characterised by development of the decompensating events of ascites, encephalopathy, portal hypertensive bleeding and hepatorenal syndrome, which arise in a setting of cirrhosis-associated immune dysfunction (CAID) and define morbidity and prognosis. CAID describes the dichotomous observations that systemic immune cells are primed and display an inflammatory phenotype, while failing to mount robust responses to pathogen challenge. Bacterial infections including spontaneous bacterial peritonitis are common complications of advanced chronic liver disease and can precipitate variceal haemorrhage, hepatorenal syndrome and acute-on-chronic liver failure; they frequently arise from gut-derived organisms and are closely linked with dysbiosis of the commensal intestinal microbiota in advanced chronic liver disease.Here, we review the links between cirrhotic dysbiosis, intestinal barrier dysfunction and deficits of host-microbiome compartmentalisation and mucosal immune homoeostasis that occur in settings of advanced chronic liver disease. We discuss established and emerging therapeutic strategies targeted at restoring intestinal eubiosis, augmenting gut barrier function and ameliorating the mucosal and systemic immune deficits that characterise and define the course of decompensated cirrhosis.

Dysfunctional neutrophil effector organelle mobilization and microbicidal protein release in alcohol-related cirrhosis.

Patients with alcohol-related cirrhosis (ALD) are prone to infection. Circulating neutrophils in ALD are dysfunctional and predict development of sepsis, organ dysfunction, and survival. Neutrophil granules are important effector organelles containing a toxic array of microbicidal proteins, whose controlled release is required to kill microorganisms while minimizing inflammation and damage to host tissue. We investigated the role of these granular responses in contributing to immune disarray in ALD. Neutrophil granular content and mobilization were measured by flow cytometric quantitation of cell-surface/intracellular markers, [secretory vesicles (CD11b), secondary granules (CD66b), and primary granules (CD63; myeloperoxidase)] before and after bacterial stimulation in 29 patients with ALD cirrhosis (15 abstinent; 14 actively drinking) compared with healthy controls (HC). ImageStream Flow Cytometry characterized localization of granule subsets within the intracellular and cell-surface compartments. The plasma cytokine environment was analyzed using ELISA/cytokine bead array. Circulating neutrophils were primed in the resting state with upregulated surface expression of CD11b (P = 0.0001) in a cytokine milieu rich in IL-8 (P < 0.001) and lactoferrin (P = 0.035). Neutrophils showed exaggerated mobilization to the cell surface of primary granules at baseline (P = 0.001) and in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (P = 0.009) and Escherichia coli (P = 0.0003) in ALD. There was no deficit in granule content or mobilization to the cell membrane in any granule subset observed. Paradoxically, active alcohol consumption abrogated the hyperresponsive neutrophil granular responses compared with their abstinent counterparts. Neutrophils are preprimed at baseline with augmented effector organelle mobilization in response to bacterial stimulation; neutrophil degranulation is not a mechanism leading to innate immunoparesis in ALD.NEW & NOTEWORTHY Neutrophil granule release is dysregulated in patients with alcohol-related cirrhosis (ALD) with augmented effector organelle mobilization and microbiocidal protein release. Neutrophil granules are upregulated in ALD at baseline and demonstrate augmented responses to bacterial challenge. The granular responses in ALD did not contribute to the observed functional deficit in innate immunity but rather were dysregulated and hyperresponsive, which may induce bystander damage to host tissue. Paradoxically, active alcohol consumption abrogated the excessive neutrophil granular responses to bacterial stimulus compared with their abstinent counterparts.

Neutrophil Toll-Like Receptor 9 Expression and the Systemic Inflammatory Response in Acetaminophen-Induced Acute Liver Failure.

OBJECTIVES: There is a marked propensity for patients with acetaminophen-induced acute liver failure to develop sepsis, which may culminate in multiple organ failure and death. Toll-like receptors sense pathogens and induce inflammatory responses, but whether this is protective or detrimental in acetaminophen-induced acute liver failure remains unknown. DESIGN, SETTING, AND PATIENTS: We assessed Toll-like receptor expression on circulating neutrophils and their function in 24 patients with acetaminophen-induced acute liver failure and compared with 10 healthy controls. INTERVENTIONS: Neutrophil Toll-like receptor 2, -4, and -9 expression and cytokine production and function were studied ex vivo at baseline and following stimulation with lipopolysaccharide, oligodeoxynucleotides, ammonium chloride, and interleukin-8. To examine the influence of acetaminophen-induced acute liver failure plasma and endogenous DNA on Toll-like receptors-9 expression, healthy neutrophils were incubated with acetaminophen-induced acute liver failure plasma with and without deoxyribonuclease-I. MEASUREMENTS AND MAIN RESULTS: Circulating neutrophil Toll-like receptor 9 expression was increased in acetaminophen-induced acute liver failure on day 1 compared with healthy controls (p = 0.0002), whereas Toll-like receptor 4 expression was decreased compared with healthy controls (p < 0.0001). Toll-like receptor 2 expression was unchanged. Neutrophil phagocytic activity was decreased, and spontaneous oxidative burst increased in all patients with acetaminophen-induced acute liver failure compared with healthy controls (p < 0.0001). Neutrophil Toll-like receptor 9 expression correlated with plasma interleukin-8 and peak ammonia concentration (r = 0.6; p < 0.05) and increased with severity of hepatic encephalopathy (grade 0-2 vs 3/4) and systemic inflammatory response syndrome score (0-1 vs 2-4) (p < 0.05). Those patients with advanced hepatic encephalopathy (grade 3/4) or high systemic inflammatory response syndrome score (2-4) on day 1 had higher neutrophil Toll-like receptor 9 expression, arterial ammonia concentration, and plasma interleukin-8 associated with neutrophil exhaustion. Healthy neutrophil Toll-like receptor 9 expression increased upon stimulation with acetaminophen-induced acute liver failure plasma, which was abrogated by preincubation with deoxyribonuclease-I. Intracellular Toll-like receptor 9 was induced by costimulation with interleukin-8 and ammonia. CONCLUSION: These data point to neutrophil Toll-like receptor 9 expression in acetaminophen-induced acute liver failure being mediated both by circulating endogenous DNA as well as ammonia and interleukin-8 in a synergistic manner inducing systemic inflammation, neutrophil exhaustion, and exacerbating hepatic encephalopathy.

Inflammation and hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome associated with both acute and chronic liver dysfunction, spanning a spectrum that ranges from mild neuropsychological disturbances to coma. The central role of ammonia in the pathogenesis of HE remains incontrovertible however, there is a robust evidence base indicating the important role of inflammation in exacerbating the neurological effects of HE. Inflammation can arise directly within the brain itself as a result of deranged nitrogen and energy homeostasis, with resultant neuronal, astrocyte and microglial dysfunction. Inflammation may also originate in the peripheral circulation and exert effects on the brain indirectly, via the release of pro-inflammatory mediators which directly signal to the brain via the vagus nerve. This review summarises the data that demonstrate the synergistic relationship of inflammation and ammonia that culminates in the manifestation of HE. Sterile inflammation arising from the inflamed or necrotic liver, circulating endotoxin arising from the gut (bacterial translocation) inducing immune dysfunction, and superimposed sepsis will be comprehensively discussed. Finally, this review will provide an overview of the existing and novel treatments on the horizon which can target the inflammatory response, and how they might translate into clinical practise as therapies in the prophylaxis and treatment of HE.

Acute liver failure and the brain: a look through the crystal ball.

Over the past 35 years, the outlook for a patient presenting with acute liver failure (ALF) has changed beyond all recognition. A patient presenting in 1984 had an 80 % likelihood of succumbing to intracranial hypertension. Today due to dramatic improvements in intensive care in dedicated liver transplant units, this has been reduced to just 20 %. Prompt fluid resuscitation, empirical treatment for sepsis and standardised management protocols that include early intubation and high flow hemofiltration for ammonia removal, limit the numbers of patients who die from the sequelae of cerebral edema and ALF. With the evolution and development of bedside prognostic markers that will include personalised genomic, metabonomic and immune profiling, rationalisation of grafts to those who are not predicted to survive is likely to further minimise the number of grafts utilised. Furthermore, in those patients with a dismal prognosis, the use of plasmapheresis, immunomodulatory therapies, biological liver support systems and hepatocyte transplantation offer a potential bridge until the injured liver can begin to regenerate avoiding transplantation and life-long immunosuppressant therapy.

Systemic inflammation and ammonia in hepatic encephalopathy.

Infection and inflammation have been associated with the development of delirium for many centuries and there is a rapidly growing evidence base supporting the role of inflammation in exacerbating the neurological manifestations of both acute and chronic liver failure. Inflammation in the context of hepatic encephalopathy (HE) can arise directly within the brain itself resulting in astrocytic, microglial and neuronal dysfunction, impacting on the development of 'brain failure'. Inflammation may also develop systemically and indirectly influence brain function. Systemic inflammation develops following liver injury, resulting in hyperammonemia and a 'cytotoxic soup' of pro-inflammatory mediators which are released into the circulation and modulate the impact of ammonia on the brain. The aim of this review is to summarise the current evidence base supporting the synergistic role of systemic inflammation and hyperammonemia in the pathogenesis of hepatic encephalopathy. Systemic inflammation and ammonia induce neutrophil degranulation and release reactive oxygen species into the peripheral circulation that may ultimately cross the blood brain barrier. Circulating endotoxin arising from the gut (bacterial translocation), superimposed sepsis, and hyperammonemia upregulate the expression of microbial pattern recognition receptors such as Toll-like receptors. The early recognition and management of systemic inflammation may not only facilitate improved outcomes in HE but supports the development of novel therapeutic strategies that reduce circulating endotoxemia and immune cell dysfunction.

Diagnosis and management of ectopic varices in portal hypertension.

Ectopic variceal bleeding is a rare cause of gastrointestinal bleeding that can occur in settings of cirrhotic and non-cirrhotic portal hypertension and is characterised by its development at locations remote from the oesophagus and stomach. Ectopic varices can be difficult to identify and access, and, although a relatively uncommon cause of portal hypertensive bleeding, can represent a difficult diagnostic and therapeutic challenge associated with considerable mortality. Low incidence and variance in variceal anatomy preclude large randomised controlled trials, and clinical practice is based on experience from case reports, case series, and specialist centre expertise. Optimisation of survival outcomes relies on understanding a patient's portal venous anatomy and functional hepatic reserve to guide timely and targeted endoscopic and endovascular interventions to facilitate the rapid control of ectopic variceal bleeding.

An 18-year-old girl with retrosternal burning discomfort: a bitter pill to swallow.

Caustic injury secondary to impaction of ingested batteries is a potentially severe cause of oesophageal injury with an increasing incidence that reflects consumer trends and the utilisation of compact electronic devices. Delays to recognition and management are associated with increased risk of complications, morbidity and mortality. In this manuscript, we describe a case presentation and literature review of a patient presenting with upper oesophageal odynophagia after the deliberate ingestion of multiple foreign bodies.

Modulation of collagen type I, fibronectin and dermal fibroblast function and activity, in systemic sclerosis by the antioxidant epigallocatechin-3-gallate.

OBJECTIVES: SSc is characterized by the overproduction of extracellular matrix (ECM) proteins, such as collagen and fibronectin, by activated fibroblasts, as well as oxidative stress. This study investigates the anti-fibrotic potential of the antioxidant epigallocatechin-3-gallate (EGCG) on activated dermal fibroblasts from SSc patients. METHODS: Dermal fibroblasts from a cell line (AG), healthy individuals (CON) and SSc patients were treated with EGCG, TGF-ß, PDGF-BB or other antioxidants [antioxidants superoxide dismutase (SOD), catalase, N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI)]. Collagen type I, fibronectin, connective tissue growth factor (CTGF), α-smooth muscle actin and mitogen-activated protein (MAP) kinases were measured by ELISA and western blot. Fibroblast contractile forces were measured by collagen gel contraction. Reactive oxygen species (ROS) were assessed by dichlorofluorescein assay and nuclear factor κ beta (NF-κB) activity by DNA binding assay. RESULTS: EGCG (1-100 µM) dose-dependently decreased collagen type I secretion in culture medium after 24 h in AG fibroblasts. Collagen type I protein expression in cell lysates was also significantly reduced by 40% in EGCG-treated cells (40 µM). Furthermore, EGCG also down-regulated TGF-ß-induced collagen type I, fibronectin and CTGF. Similarly, in CON fibroblasts EGCG decreased basal and stimulated collagen type I, fibronectin and CTGF after 24 h, while in SSc the effects of the antioxidant were apparent after 48 h. Fibroblast-mediated contraction of collagen gels was inhibited by EGCG as early as 1 h in AG fibroblasts, and in the CON and SSc fibroblasts. Additionally, EGCG also inhibited TGF-ß-stimulated gel contraction similar to other antioxidants DPI and NAC, but not SOD or catalase. EGCG suppressed TGF-ß-induced ROS production in all fibroblasts. Furthermore, EGCG inhibited TGF-ß or PDGF-BB-induced phospho-extracellular signal-regulated kinase (ERK)1/2 MAP kinase and NF-κB activity in SSc fibroblasts. CONCLUSION: The results suggest that the antioxidant, EGCG, can reduce ECM production, the fibrotic marker CTGF and inhibit contraction of dermal fibroblasts from SSc patients. Furthermore, EGCG was able to suppress intracellular ROS, ERK1/2 kinase signalling and NF-κB activity. Taken together, EGCG may be a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc.