Clinical, experimental and pathophysiological effects of Yaq-001: a non-absorbable, gut-restricted adsorbent in models and patients with cirrhosis.

OBJECTIVE: Targeting bacterial translocation in cirrhosis is limited to antibiotics with risk of antimicrobial resistance. This study explored the therapeutic potential of a non-absorbable, gut-restricted, engineered carbon bead adsorbent, Yaq-001 in models of cirrhosis and acute-on-chronic liver failure (ACLF) and, its safety and tolerability in a clinical trial in cirrhosis. DESIGN: Performance of Yaq-001 was evaluated in vitro. Two-rat models of cirrhosis and ACLF, (4 weeks, bile duct ligation with or without lipopolysaccharide), receiving Yaq-001 for 2 weeks; and two-mouse models of cirrhosis (6-week and 12-week carbon tetrachloride (CCl4)) receiving Yaq-001 for 6 weeks were studied. Organ and immune function, gut permeability, transcriptomics, microbiome composition and metabolomics were analysed. The effect of faecal water on gut permeability from animal models was evaluated on intestinal organoids. A multicentre, double-blind, randomised, placebo-controlled clinical trial in 28 patients with cirrhosis, administered 4 gr/day Yaq-001 for 3 months was performed. RESULTS: Yaq-001 exhibited rapid adsorption kinetics for endotoxin. In vivo, Yaq-001 reduced liver injury, progression of fibrosis, portal hypertension, renal dysfunction and mortality of ACLF animals significantly. Significant impact on severity of endotoxaemia, hyperammonaemia, liver cell death, systemic inflammation and organ transcriptomics with variable modulation of inflammation, cell death and senescence in the liver, kidneys, brain and colon was observed. Yaq-001 reduced gut permeability in the organoids and impacted positively on the microbiome composition and metabolism. Yaq-001 regulated as a device met its primary endpoint of safety and tolerability in the clinical trial. CONCLUSIONS: This study provides strong preclinical rationale and safety in patients with cirrhosis to allow clinical translation. TRIAL REGISTRATION NUMBER: NCT03202498.

Sarcopenia in liver cirrhosis: Prevalence, pathophysiology and therapeutic strategies.

Sarcopenia, characterized by a loss of muscle strength, quantity/quality, and physical performance is associated with increased mortality and poor clinical outcomes in concomitant presentation with liver cirrhosis (LC). A number of mechanisms are involved in sarcopenia development in LC, many of which are secondary to liver dysfunction and/or iatrogenic involvement in treating LC. Sarcopenia severity in this population appears to be affected by patient gender, as well as the primary aetiology of LC (alcohol, non-alcoholic fatty liver disease etc.) with patient demographics shifting in recent years. Clinical detection of sarcopenia in this population may involve a combination of assessment tools, in addition to measuring muscle mass and strength separately. Muscle mass may be assessed using radiography, bioelectric impedance, ultrasound, or anthropometrics. Hand-grip strength, on the other hand, may be a useful tool for evaluating muscle strength. The role of malnutrition in sarcopenia is also a relevant factor, and screening tools such as MELD and SARC-F may be clinically useful tools for more complete diagnosis of sarcopenia in these patients. Myostatin and titin-N may represent potential diagnostic biomarkers. Lastly, physical activity and nutrition remain key elements of treatment. Further research is being conducted regarding the role of resistance vs aerobic exercise as well as the function of complementary nutrition. Continued study into the role of nutrition, physical activity and other complementary therapies will be important future endeavours in the treatment of sarcopenia in LC.

The microbiota in cirrhosis and its role in hepatic decompensation.

Cirrhosis - the common end-stage of chronic liver disease - is associated with a cascade of events, of which intestinal bacterial overgrowth and dysbiosis are central. Bacterial toxins entering the portal or systemic circulation can directly cause hepatocyte death, while dysbiosis also affects gut barrier function and increases bacterial translocation, leading to infections, systemic inflammation and vasodilation, which contribute to acute decompensation and organ failure. Acute decompensation and its severe forms, pre-acute-on-chronic liver failure (ACLF) and ACLF, are characterised by sudden organ dysfunction (and failure) and high short-term mortality. Patients with pre-ACLF and ACLF present with high-grade systemic inflammation, usually precipitated by proven bacterial infection and/or severe alcoholic hepatitis. However, no precipitant is identified in 30% of these patients, in whom bacterial translocation from the gut microbiota is assumed to be responsible for systemic inflammation and decompensation. Different microbiota profiles may influence the rate of decompensation and thereby outcome in these patients. Thus, targeting the microbiota is a promising strategy for the prevention and treatment of acute decompensation, pre-ACLF and ACLF. Approaches include the use of antibiotics such as rifaximin, faecal microbial transplantation and enterosorbents (e.g. Yaq-001), which bind microbial factors without exerting a direct effect on bacterial growth kinetics. This review focuses on the role of microbiota in decompensation and strategies targeting microbiota to prevent acute decompensation.

Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.

BACKGROUND & AIMS: Toll-like receptor 4 (TLR4) plays an essential role in mediating organ injury in acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). Herein, we assess whether inhibiting TLR4 signaling can ameliorate liver failure and serve as a potential treatment. METHODS: Circulating TLR4 ligands and hepatic TLR4 expression were measured in plasma samples and liver biopsies from patients with cirrhosis. TAK-242 (TLR4 inhibitor) was tested in vivo (10 mg/kg intraperitoneally) in rodent models of ACLF (bile duct ligation + lipopolysaccharide [LPS]; carbon tetrachloride + LPS) and ALF (galactosamine + LPS) and in vitro on immortalized human monocytes (THP-1) and hepatocytes (HHL5). The in vivo therapeutic effect was assessed by coma-free survival, organ injury and cytokine release and in vitro by measuring IL-6, IL-1ß or cell injury (TUNEL), respectively. RESULTS: In patients with cirrhosis, hepatic TLR4 expression was upregulated and circulating TLR4 ligands were increased (p <0.001). ACLF in rodents was associated with a switch from apoptotic cell death in ALF to non-apoptotic forms of cell death. TAK-242 reduced LPS-induced cytokine secretion and cell death (p = 0.002) in hepatocytes and monocytes in vitro. In rodent models of ACLF, TAK-242 administration improved coma-free survival, reduced the degree of hepatocyte cell death in the liver (p <0.001) and kidneys (p = 0.048) and reduced circulating cytokine levels (IL-1ß, p <0.001). In a rodent model of ALF, TAK-242 prevented organ injury (p <0.001) and systemic inflammation (IL-1ß, p <0.001). CONCLUSION: This study shows that TLR4 signaling is a key factor in the development of both ACLF and ALF; its inhibition reduces the severity of organ injury and improves outcome. TAK-242 may be of therapeutic relevance in patients with liver failure. LAY SUMMARY: Toll-like receptor 4 (or TLR4) mediates endotoxin-induced tissue injury in liver failure and cirrhosis. This receptor sensitizes cells to endotoxins, which are produced by gram-negative bacteria. Thus, inhibiting TLR4 signaling with an inhibitor (TAK-242) ameliorates organ injury and systemic inflammation in rodent models of acute and acute-on-chronic liver failure.

Treatment with non-selective beta blockers is associated with reduced severity of systemic inflammation and improved survival of patients with acute-on-chronic liver failure.

BACKGROUND & AIMS: Non-selective beta blockers (NSBBs) have been shown to have deleterious outcomes in patients with refractory ascites, alcoholic hepatitis and spontaneous bacterial peritonitis leading many physicians to stop the drug in these cases. Acute-on-chronic liver failure (ACLF) is characterized by systemic inflammation and high mortality. As NSBBs may have beneficial effects on gut motility and permeability and, systemic inflammation, the aims of this prospective, observational study were to determine whether ongoing use of NSBBs reduced 28-day mortality in ACLF patients. METHODS: The study was performed in 349 patients with ACLF included in the CANONIC study, which is a prospective observational investigation in hospitalized cirrhotic patients with acute deterioration. The data about the use of NSBBs, its type and dosage was specifically recorded. Patient characteristics at enrollment significantly associated with treatment and mortality were taken into account as potential confounders to adjust for treatment effect. A logistic regression model was fitted. RESULTS: 164 (47%) ACLF patients received NSBBs whereas 185 patients did not. Although the CLIF-C ACLF scores were similar at presentation, more patients in the NSBB treated group had lower grades of ACLF (p=0.047) at presentation and significantly more patients improved. Forty patients (24.4%) died in NSBB treated group compared with 63 patients (34.1%) (p=0.048) [estimated risk-reduction 0.596 (95%CI: 0.361-0.985; p=0.0436)]. This improvement in survival was associated with a significantly lower white cell count (NSBB: 8.5 (5.8); no NSBB: 10.8 (6.6); p=0.002). No long-term improvement in survival was observed. CONCLUSIONS: This study shows for the first time that ongoing treatment with NSBBs in cirrhosis is safe and reduces the mortality if they develop ACLF. Careful thought should be given before stopping NSBBs in cirrhotic patients.

Immunomodulatory and antioxidant function of albumin stabilises the endothelium and improves survival in a rodent model of chronic liver failure.

BACKGROUND & AIMS: Liver failure is characterized by endothelial dysfunction, which results in hemodynamic disturbances leading to renal failure. Albumin infusion improves hemodynamics and prevents renal dysfunction in advance liver failure. These effects are only partly explained by the oncotic properties of albumin. This study was designed to test the hypothesis that albumin exerts its beneficial effects by stabilising endothelial function. METHODS: In vivo: systemic hemodynamics, renal function, markers of endothelial dysfunction (ADMA) and inflammation were studied in analbuminaemic and Sprague-Dawley rats, 6-weeks after sham/bile duct ligation surgery. In vitro: human umbilical vein endothelial cells were stimulated with LPS with or without albumin. We studied protein expression and gene expression of adhesion molecules, intracellular reactive oxygen species, and cell stress markers. RESULTS: Compared to controls, analbuminaemic rats had significantly greater hemodynamic deterioration after bile duct ligation, resulting in worse renal function and shorter survival. This was associated with significantly greater plasma renin activity, worse endothelial function, and disturbed inflammatory response. In vitro studies showed that albumin was actively taken up by endothelial cells. Incubation of albumin pre-treated endothelial cells with LPS was associated with significantly less activation compared with untreated cells, decreased intracellular reactive oxygen species, and markers of cell stress. CONCLUSIONS: These results show, for the first time, that absence of albumin is characterised by worse systemic hemodynamics, renal function and higher mortality in a rodent model of chronic liver failure and illustrates the important non-oncotic properties of albumin in protecting against endothelial dysfunction.

Clinical and pathophysiological consequences of alterations in the microbiome in cirrhosis.

Cirrhosis is a major cause of mortality worldwide. Exponential rises in prevalence have been observed secondary to increases in obesity and alcohol consumption. Multiple lines of evidence implicate gut-derived bacteria and bacterial ligands as a central driver of pathogenesis. Recent developments in culture-independent techniques have facilitated a more accurate description of microbiome composition in cirrhosis and led to the description of measures of dysbiosis shown to be associated with disease. More importantly, metagenomic studies are adding to an understanding of the functional contribution of the microbiota and may prove to be a more clinically relevant biomarker than phylogenetic studies. Much like other dysbiotic states such as inflammatory bowel disease, the microbiota in cirrhosis is characterized by a low microbial and genetic diversity. Therapeutic strategies to diminish this process are currently limited to selective intestinal decontamination with antibiotics. This review summarizes the available data and develops a framework for the use of current and future treatment strategies to diminish the consequences of dysbiosis in cirrhosis. Interventional strategies to bind bacterial products in the gut lumen and blood, and modulate the magnitude of host sensing mechanisms remain an unmet clinical need. A greater understanding of the host-microbiota interaction in cirrhosis is of key importance to inform future interventional strategies to diminish the currently escalating burden of the disease.

Increased renal expression and urinary excretion of TLR4 in acute kidney injury associated with cirrhosis.

BACKGROUND: Patients with cirrhosis frequently develop renal dysfunction, a proportion of who do not fulfill criteria for hepatorenal syndrome (HRS). We hypothesized that the kidneys in these patients would exhibit histological and biomarker evidence of kidney injury. We looked specifically for TLR expression as they may mediate kidney injury. METHODS: Sixty seven subjects (6); alcoholic cirrhosis: compensated (9), acute deterioration of alcoholic cirrhosis (52)] were included. Renal dysfunction was defined as a creatinine of >133 µmol/L and/or according to the AKI network criteria. Urinary biomarkers, KIM-1, πGST, αGST and a novel biomarker, urinary TLR4 were measured. Renal biopsies were also available from eight other alcoholic cirrhosis patients (three non-HRS renal dysfunction; five HRS) that were stained for TLR4 and caspase-3. RESULTS: Fourteen patients developed renal dysfunction, amongst these three had type 2 HRS. KIM-1, πGST and αGST were higher in patients with acute deterioration of cirrhosis compared with patients with compensated cirrhosis, but did not differ between those with and without renal dysfunction. Urinary TLR4 was significantly higher in patients with renal dysfunction associated with infection/inflammation. Kidney biopsies from non-HRS renal dysfunction patients showed tubular damage with evidence of increased tubular expression of TLR4, and caspase-3. Minor changes were observed in HRS patients. CONCLUSIONS: The data provide proof of concept that renal dysfunction in patients with cirrhosis with superimposed inflammation is associated with significant tubular injury and apoptosis and with increased renal expression and urinary excretion of the TLR4, suggesting a potential role of TLR4 as mediator of renal injury.

α-Synuclein expression in response to bacterial ligands and metabolites in gut enteroendocrine cells: an in vitro proof of concept study.

Caudo-rostral migration of pathological forms of α-synuclein from the gut to the brain is proposed as an early feature in Parkinson's disease pathogenesis, but the underlying mechanisms remain unknown. Intestinal epithelial enteroendocrine cells sense and respond to numerous luminal signals, including bacterial factors, and transmit this information to the brain via the enteric nervous system and vagus nerve. There is evidence that gut bacteria composition and their metabolites change in Parkinson's disease patients, and these alterations can trigger α-synuclein pathology in animal models of the disorder. Here, we investigated the effect of toll-like receptor and free fatty acid receptor agonists on the intracellular level of α-synuclein and its release using mouse secretin tumour cell line 1 enteroendocrine cells. Secretin tumour cell line 1 enteroendocrine cells were treated for 24 or 48 h with toll-like receptor agonists (toll-like receptor 4 selective lipopolysaccharide; toll-like receptor 2 selective Pam3CysSerLys4) and the free fatty acid receptor 2/3 agonists butyrate, propionate and acetate. The effect of selective receptor antagonists on the agonists' effects after 24 hours was also investigated. The level of α-synuclein protein was measured in cell lysates and cell culture media by western blot and enzyme-linked immunosorbent assay. The level of α-synuclein and tumour necrosis factor messenger RNA was measured by quantitative reverse transcription real-time polymerase chain reaction. Stimulation of secretin tumour cell line 1 enteroendocrine cells for 24 and 48 hours with toll-like receptor and free fatty acid receptor agonists significantly increased the amount of intracellular α-synuclein and the release of α-synuclein from the cells into the culture medium. Both effects were significantly reduced by antagonists selective for each receptor. Toll-like receptor and free fatty acid receptor agonists also significantly increased tumour necrosis factor transcription, and this was effectively inhibited by corresponding antagonists. Elevated intracellular α-synuclein increases the likelihood of aggregation and conversion to toxic forms. Factors derived from bacteria induce α-synuclein accumulation in secretin tumour cell line 1 enteroendocrine cells. Here, we provide support for a mechanism by which exposure of enteroendocrine cells to specific bacterial factors found in Parkinson's disease gut dysbiosis might facilitate accumulation of α-synuclein pathology in the gut.

Evaluation of an Adapted Semi-Automated DNA Extraction for Human Salivary Shotgun Metagenomics.

Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.

Prevention of acute kidney injury in a rodent model of cirrhosis following selective gut decontamination is associated with reduced renal TLR4 expression.

BACKGROUND & AIMS: Superimposed infection and/or inflammation precipitate renal failure in cirrhosis. This study aimed at testing the hypothesis that increased gut bacterial translocation in cirrhosis primes the kidney to the effect of superimposed inflammation by upregulating expression of Toll-like receptor 4 (TLR4), NFκB, and cytokines. A well-characterized bile-duct ligated (BDL) model of cirrhosis, which develops renal failure following superimposed inflammatory insult with lipopolysaccharide (LPS), was used and selective gut decontamination was performed using norfloxacin. METHODS: Sprague-Dawley rats were studied: Sham, Sham+LPS; BDL, BDL+LPS; an additional BDL and BDL+LPS groups were selectively decontaminated with norfloxacin. Plasma biochemistry, plasma renin activity (PRA) and cytokines and, protein expression of TLR4, NFκB, and cytokines were measured in the kidney homogenate. The kidneys were stained for TLR4, TLR2, and caspase-3. Endotoxemia was measured using neutrophil burst and Limulus amoebocyte lysate (LAL) assays. RESULTS: The groups treated with norfloxacin showed significant attenuation of the increase in plasma creatinine, plasma and renal TNF-α and renal tubular injury on histology. The increased renal protein expression of TLR4, NFκB, and caspase-3 in the untreated animals was significantly attenuated in the norfloxacin treated animals. PRA was reduced in the treated animals and severity of endotoxemia was also reduced. CONCLUSIONS: The results show for the first time that kidneys in cirrhosis show an increased expression of TLR4, NFκB, and the pro-inflammatory cytokine TNF-α, which makes them susceptible to a further inflammatory insult. This increased susceptibility to LPS can be prevented with selective decontamination, providing novel insights into the pathophysiology of renal failure in cirrhosis.

Bile acids and neurological disease.

This review will focus on how bile acids are being used in clinical trials to treat neurological diseases due to their central involvement with the gut-liver-brain axis and their physiological and pathophysiological roles in both normal brain function and multiple neurological diseases. The synthesis of primary and secondary bile acids species and how the regulation of the bile acid pool may differ between the gut and brain is discussed. The expression of several bile acid receptors in brain and their currently known functions along with the tools available to manipulate them pharmacologically are examined, together with discussion of the interaction of bile acids with the gut microbiome and their lesser-known effects upon brain glucose and lipid metabolism. How dysregulation of the gut microbiome, aging and sex differences may lead to disruption of bile acid signalling and possible causal roles in a number of neurological disorders are also considered. Finally, we discuss how pharmacological treatments targeting bile acid receptors are currently being tested in an array of clinical trials for several different neurodegenerative diseases.

An evidence-based surveillance tool to identify and report catheter/cannula bloodstream infection in patients receiving parenteral nutrition.

OBJECTIVE: Catheter/cannula-bloodstream infection (CBI) has been proposed as a marker of the quality of care provided to patients receiving parenteral nutrition (PN). However, surveillance criteria for CBI are variable, inconsistent, and sometimes confusing and impractical. Surveillance criteria were developed to simply and accurately demonstrate the presence or absence of CBI. The aim of this study was to establish a simple and valid surveillance tool, with consideration of changes in vital signs, to identify CBI in patients receiving PN. METHODS: Adult (≥18 y) inpatients prescribed PN at a single large teaching hospital were recruited between October 11, 2017 and November 16, 2018. Common clinical and laboratory criteria, including blood culture, associated with 100 consecutive PN episodes associated with suspected CBI were examined for potential predictive markers of CBI. Using binary logistic regression, criteria were incorporated into an instrument that was validated against a reference classification of CBI established by an expert multidisciplinary group. RESULTS: The reference classification comprised 12 PN episodes with CBI and 88 without. Abnormal vital signs did not significantly predict CBI, but resolution of fever (≥38°C) and low systolic blood pressure (<100 mm Hg) in response to a specific treatment for CBI (line removal/antibiotics) did. Two other criteria were also significant predictors: positive blood culture; and absence of an alternative source that could explain the septic episode other than the catheter/cannula supplying PN. These two criteria together with a positive response to treatment (temperature and/or blood pressure, incorporated into a single binary variable), resulted in 100% correct CBI classification (100% sensitivity, 100% specificity, and 1.000 area under the curve in receiver operating characteristic analysis). All criteria could be retrospectively extracted from the medical records of all PN episodes. CONCLUSION: A CBI tool shows promise as a surveillance instrument for benchmarking and interinstitutional comparisons of the care received by hospitalized patients given PN.

Body fat composition determines outcomes before and after liver transplantation in patients with cirrhosis.

Cachexia occurs in late stages of liver cirrhosis, and a low-fat mass is potentially associated with poor outcome. This study compared different computed tomography (CT)-derived fat parameters with respect to its prognostic impact on the development of complications and death before and after liver transplantation. Between 2001 and 2014, 612 patients with liver cirrhosis without hepatocellular carcinoma listed for liver transplantation met the inclusion criteria, including abdominal CT scan (±200 days to listing). A total of 109 patients without cirrhosis served as controls. The subcutaneous fat index (SCFI), the paraspinal muscle fat index, and the visceral fat index were assessed at L3/L4 level and normalized to the height (cm2 /m2 ). Data were collected and analyzed retrospectively. Low SCFI was associated with a higher rate of ascites and increased C-reactive protein levels (p < 0.001). In addition, multivariate Cox regression analysis adjusting for sex, age, body mass index (BMI), and Model for End-Stage Liver Disease showed that decreasing SCFI was also associated with an increased risk of cirrhosis-related complications (p = 0.003) and death on the transplant wait list (p = 0.013). Increased paraspinal and visceral fat were not only positively correlated with creatinine levels (p < 0.001), BMI, and metabolic comorbidities (all p < 0.001) before transplantation, but also predictive for 1-year mortality after transplantation. Conclusion: The distribution of body fat is a major determinant for complications and outcome in cirrhosis before and after liver transplantation.

The gut-brain axis and Parkinson disease: clinical and pathogenetic relevance.

Gastrointestinal disorders are one of the most significant non-motor problems affecting people with Parkinson disease (PD). Pathogenetically, the gastrointestinal tract has been proposed to be the initial site of pathological changes in PD. Intestinal inflammation and alterations in the gut microbiota may contribute to initiation and progression of pathology in PD. However, the mechanisms underlying this "gut-brain" axis in PD remain unclear. PD patients can display a large variety of gastrointestinal symptoms, leading to reduced quality of life and psychological distress. Gastrointestinal disorders can also limit patients' response to medications, and consequently negatively impact on neurological outcomes. Despite an increasing research focus, gastrointestinal disorders in PD remain poorly understood and their clinical management often suboptimal. This review summarises our understanding of the relevance of the "gut-brain" axis to the pathogenesis of PD, discusses the impact of gastrointestinal disorders in patients with PD, and provides clinicians with practical guidance to their management.

The Unique Impact of COVID-19 on Human Gut Microbiome Research.

The coronavirus (COVID-19) pandemic has disrupted clinical trials globally, with unique implications for research into the human gut microbiome. In this mini-review, we explore the direct and indirect influences of the pandemic on the gut microbiome and how these can affect research and clinical trials. We explore the direct bidirectional relationships between the COVID-19 virus and the gut and lung microbiomes. We then consider the significant indirect effects of the pandemic, such as repeated lockdowns, increased hand hygiene, and changes to mood and diet, that could all lead to longstanding changes to the gut microbiome at an individual and a population level. Together, these changes may affect long term microbiome research, both in observational as well as in population studies, requiring urgent attention. Finally, we explore the unique implications for clinical trials using faecal microbiota transplants (FMT), which are increasingly investigated as potential treatments for a range of diseases. The pandemic introduces new barriers to participation in trials, while the direct and indirect effects laid out above can present a confounding factor. This affects recruitment and sample size, as well as study design and statistical analyses. Therefore, the potential impact of the pandemic on gut microbiome research is significant and needs to be specifically addressed by the research community and funders.

The role of RIPK1 mediated cell death in acute on chronic liver failure.

Acute-on-chronic liver failure (ACLF) is characterized predominantly by non-apoptotic forms of hepatocyte cell death. Necroptosis is a form of programmed lytic cell death in which receptor interacting protein kinase (RIPK) 1, RIPK3 and phosphorylated mixed lineage kinase domain-like (pMLKL) are key components. This study was performed to determine the role of RIPK1 mediated cell death in ACLF. RIPK3 plasma levels and hepatic expression of RIPK1, RIPK3, and pMLKL were measured in healthy volunteers, stable patients with cirrhosis, and in hospitalized cirrhotic patients with acutely decompensated cirrhosis, with and without ACLF (AD). The role of necroptosis in ACLF was studied in two animal models of ACLF using inhibitors of RIPK1, necrostatin-1 (NEC-1) and SML2100 (RIPA56). Plasma RIPK3 levels predicted the risk of 28- and 90-day mortality (AUROC, 0.653 (95%CI 0.530-0.776), 0.696 (95%CI 0.593-0.799)] and also the progression of patients from no ACLF to ACLF [0.744 (95%CI 0.593-0.895)] and the results were validated in a 2nd patient cohort. This pattern was replicated in a rodent model of ACLF that was induced by administration of lipopolysaccharide (LPS) to bile-duct ligated rats and carbon tetrachloride-induced fibrosis mice administered galactosamine (CCL4/GalN). Suppression of caspase-8 activity in ACLF rodent model was observed suggesting a switch from caspase-dependent cell death to necroptosis. NEC-1 treatment prior to administration of LPS significantly reduced the severity of ACLF manifested by reduced liver, kidney, and brain injury mirrored by reduced hepatic and renal cell death. Similar hepato-protective effects were observed with RIPA56 in a murine model of ACLF induced by CCL4/GalN. These data demonstrate for the first time the importance of RIPK1 mediated cell death in human and rodent ACLF. Inhibition of RIPK1 is a potential novel therapeutic approach to prevent progression of susceptible patients from no ACLF to ACLF.

Biomarkers of extracellular matrix formation are associated with acute-on-chronic liver failure.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by organ failure(s), high short-term mortality, and, pathophysiologically, deranged inflammatory responses. The extracellular matrix (ECM) is critically involved in regulating the inflammatory response. This study aimed to determine alterations in biomarkers of ECM turnover in ACLF and their association with inflammation, organ failures, and mortality. METHODS: We studied 283 patients with cirrhosis admitted for acute decompensation (AD) with or without ACLF, 64 patients with stable cirrhosis, and 30 healthy controls. A validation cohort (25 ACLF, 9 healthy controls) was included. Plasma PRO-C3, PRO-C4, PRO-C5, PRO-C6, and PRO-C8 (i.e. collagen type III-VI and VIII formation) and C4M and C6M (i.e. collagen type IV and VI degradation) were measured. Immunohistochemistry of PRO-C6 was performed on liver biopsies (AD [n = 7], ACLF [n = 5]). A competing-risk regression analysis was performed to explore the prognostic value of biomarkers of ECM turnover with 28- and 90-day mortality. RESULTS: PRO-C3 and PRO-C6 were increased in ACLF compared to AD (p = 0.089 and p <0.001, respectively), whereas collagen degradation markers C4M and C6M were similar. Both PRO-C3 and PRO-C6 were strongly associated with liver function and inflammatory markers. Only PRO-C6 was associated with extrahepatic organ failures and 28- and 90-day mortality (hazard ratio [HR; on log-scale] 6.168, 95% CI 2.366-16.080, p <0.001, and 3.495, 95% CI 1.509-8.093, p = 0.003, respectively). These findings were consistent in the validation cohort. High PRO-C6 expression was observed in liver biopsies of patients with ACLF. CONCLUSIONS: This study shows, for the first time, evidence of severe net interstitial collagen deposition in ACLF and makes the novel observation of the association between PRO-C6 and (extrahepatic) organ failures and mortality. Further studies are needed to define the pathogenic significance of these observations. LAY SUMMARY: This study describes a disrupted turnover of collagen type III and VI in Acute-on-chronic liver failure (ACLF). Plasma biomarkers of these collagens (PRO-C3 and PRO-C6) are associated with the severity of liver dysfunction and inflammation. PRO-C6, also known as the hormone endotrophin, has also been found to be associated with multi-organ failure and prognosis in acute decompensation and ACLF.

Liver failure at the front door.

Patients with ALF or ACLF frequently develop multiple organ failure and should be managed by senior physicians in a high dependency or intensive care environment (Table 4).