Defining the temporal evolution of gut dysbiosis and inflammatory responses leading to hepatocellular carcinoma in Mdr2 -/- mouse model.

BACKGROUND: Emerging evidence implicates the gut microbiome in liver inflammation and hepatocellular carcinoma (HCC) development. We aimed to characterize the temporal evolution of gut dysbiosis, in relation to the phenotype of systemic and hepatic inflammatory responses leading to HCC development. In the present study, Mdr2 -/- mice were used as a model of inflammation-based HCC. Gut microbiome composition and function, in addition to serum LPS, serum cytokines/chemokines and intrahepatic inflammatory genes were measured throughout the course of liver injury until HCC development. RESULTS: Early stages of liver injury, inflammation and cirrhosis, were characterized by dysbiosis. Microbiome functional pathways pertaining to gut barrier dysfunction were enriched during the initial phase of liver inflammation and cirrhosis, whilst those supporting lipopolysaccharide (LPS) biosynthesis increased as cirrhosis and HCC ensued. In parallel, serum LPS progressively increased during the course of liver injury, corresponding to a shift towards a systemic Th1/Th17 proinflammatory phenotype. Alongside, the intrahepatic inflammatory gene profile transitioned from a proinflammatory phenotype in the initial phases of liver injury to an immunosuppressed one in HCC. In established HCC, a switch in microbiome function from carbohydrate to amino acid metabolism occurred. CONCLUSION: In Mdr2 -/- mice, dysbiosis precedes HCC development, with temporal evolution of microbiome function to support gut barrier dysfunction, LPS biosynthesis, and redirection of energy source utilization. A corresponding shift in systemic and intrahepatic inflammatory responses occurred supporting HCC development. These findings support the notion that gut based therapeutic interventions could be beneficial early in the course of liver disease to halt HCC development.

Animal and translational models of SARS-CoV-2 infection and COVID-19.

COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

Virus Genotype-Dependent Transcriptional Alterations in Lipid Metabolism and Inflammation Pathways in the Hepatitis C Virus-infected Liver.

Despite advances in antiviral therapy, molecular drivers of Hepatitis C Virus (HCV)-related liver disease remain poorly characterised. Chronic infection with HCV genotypes (1 and 3) differ in presentation of liver steatosis and virological response to therapies, both to interferon and direct acting antivirals. To understand what drives these clinically important differences, liver expression profiles of patients with HCV Genotype 1 or 3 infection (n = 26 and 33), alcoholic liver disease (n = 8), and no liver disease (n = 10) were analysed using transcriptome-wide microarrays. In progressive liver disease, HCV genotype was the major contributor to altered liver gene expression with 2151 genes differentially expressed >1.5-fold between HCV Genotype 1 and 3. In contrast, only 6 genes were altered between the HCV genotypes in advanced liver disease. Induction of lipogenic, lipolytic, and interferon stimulated gene pathways were enriched in Genotype 1 injury whilst a broad range of immune-associated pathways were associated with Genotype 3 injury. The results are consistent with greater lipid turnover in HCV Genotype 1 patients. Moreover, the lower activity in inflammatory pathways associated with HCV genotype 1 is consistent with relative resistance to interferon-based therapy. This data provides a molecular framework to explain the clinical manifestations of HCV-associated liver disease.

Sofosbuvir and daclatasvir therapy in patients with hepatitis C-related advanced decompensated liver disease (MELD ≥ 15).

BACKGROUND: Antiviral therapy for hepatitis C has the potential to improve liver function in patients with decompensated cirrhosis. AIMS: To examine the virological response and effect of viral clearance in patients with decompensated hepatitis C cirrhosis all with MELD scores ≥15 following sofosbuvir/daclatasvir ± ribavirin. METHODS: We prospectively collected data on patients who commenced sofosbuvir/daclatasvir for 24-weeks under the Australian patient supply program (TOSCAR) and analysed outcomes including sustained viral response at 12 weeks (SVR12), death and transplant. RESULTS: 108 patients (M/F, 79/29; median age 56years; Child-Pugh 10; MELD 16; genotype 1/3, 55/47) received sofosbuvir/daclatasvir and two also received ribavirin. On intention-to-treat, the SVR12 rate was 70% (76/108). Seventy-eight patients completed 24-weeks therapy. SVR12 was achieved in 56 of these patients on per-protocol-analysis (76%). SVR12 was 80% in genotype 1 compared to 69% in genotype 3. Thirty patients failed to complete therapy. In patients achieving SVR12, median MELD and Child-Pugh fell from 16(IQR15-17) to 14(12-17) and 10(9-11) to 8(7-9), respectively (P<.001). In those who died, MELD increased from 16 to 23 at death (P=.036). Patients who required transplantation had a significantly higher baseline MELD (20) compared to those patients completing treatment (16) (P=.0010). The odds ratio for transplant in patients with baseline MELD ≥20 was 13.8(95%CI 2.78-69.04). CONCLUSIONS: SVR12 rates with sofosbuvir/daclatasvir in advanced liver disease are lower than in compensated disease. Although treatment improves MELD and Child-Pugh in most patients, a significant proportion will die or require transplantation. In those with MELD ≥20, it may be better to delay treatment until post-transplant.

Effect of viral suppression on hepatic venous pressure gradient in hepatitis C with cirrhosis and portal hypertension.

Portal hypertension is a predictor of liver-related clinical events and mortality in patients with hepatitis C and cirrhosis. The effect of interferon-free hepatitis C treatment on portal pressure is unknown. Fifty patients with Child-Pugh-Turcotte (CPT) A and B cirrhosis and portal hypertension (hepatic venous pressure gradient [HVPG] >6 mm Hg) were randomized to receive 48 weeks of open-label sofosbuvir plus ribavirin at Day 1 or after a 24-week observation period. The primary endpoint was sustained virologic response 12 weeks after therapy (SVR12) in patients who received ≥1 dose of treatment. Secondary endpoints included changes in HVPG, laboratory parameters, and MELD and CPT scores. A subset of patients was followed 48 weeks posttreatment to determine late changes in HVPG. SVR12 occurred in 72% of patients (33/46). In the 37 patients with paired HVPG measurements at baseline and the end of treatment, mean HVPG decreased by -1.0 (SD 3.97) mm Hg. Nine patients (24%) had ≥20% decreases in HVPG during treatment. Among 39 patients with pretreatment HVPG ≥12 mm Hg, 27 (69%) achieved SVR12. Four of the 33 (12%) patients with baseline HVPG ≥12 mm Hg had HVPG <12 mm Hg at the end of treatment. Of nine patients with pretreatment HVPG ≥12 mm Hg who achieved SVR12 and completed 48 weeks of follow-up, eight (89%) had a ≥20% reduction in HVPG, and three reduced their pressure to <12 mm Hg. Patients with chronic HCV and compensated or decompensated cirrhosis who achieve SVR can have clinically meaningful reductions in HVPG at long-term follow-up. (EudraCT 2012-002457-29).

Have we significantly underestimated the capacity in the Australian health system to treat chronic hepatitis C infection in an interferon-free era?

BACKGROUND: New antiviral therapies for hepatitis C infection may lead to an increase in capacity to treat because of their simplicity and safety. AIMS: To determine the likely current capacity of accredited treatment centres in treating hepatitis C (HCV) patients with interferon (IFN)-free direct acting antiviral (DAA) agents in Australia. METHOD: Data were collected from 22 centres before the introduction of DAA therapy - 11 sites from the Study 1 survey and an additional 11 sites from the Study 2 survey. The sites were selected based on consensus by viral hepatitis experts, in consultation with the NSW Agency for Clinical Innovation. The services were selected as they were experienced in the delivery of IFN-based treatments and/or had knowledge about IFN-free regimens. The sites selected offered a mix of metropolitan and regional clinics, opioid substitution clinics, Aboriginal services, general practitioner-initiated, criminal justice and nurse-led services. Following the survey, the first 3 months of actual treatment uptake, since listing in March and May 2016, became available for a comparison and were subsequently analysed. RESULTS: The survey indicated that an average of 27 h would be required to treat patients with IFN-based regimens. This was reduced to an average of 9 h if IFN-free regimens were used. The average number of patients on IFN-based treatment regimens per site was 87 per year compared to 493 per site if IFN-free therapy was freely available. There is capacity in the current health system to treat five times the numbers of patients with chronic HCV in Australia. When applying a weighted ratio; the results for all centres show a 3.6-fold increase in the capacity to treat with IFN-free regimens. However, these numbers may be an underestimate based on the first 3 months of actual treatment uptake since Pharmaceutical Benefits Scheme listing in March and May 2016. CONCLUSION: Although current sites have a significant capacity to increase rapidly HCV treatment numbers, expansion of treatment settings and new models of care and training may be required to deliver HCV treatment to all HCV-infected individuals.

Assessment of adult patients with chronic liver failure for liver transplantation in 2015: who and when?

In 2015, there are a few absolute contraindications to liver transplantation. In adult patients, survival post-liver transplant is excellent, with 1-year survival rate >90% and 5-year survival rates >80% and predicted median allograft survival beyond 20 years. Patients with a Child-Turcotte Pugh score ≥9 or a model for end-stage liver disease (MELD) score >15 should be referred for liver transplantation, with patients who have a MELD score >17 showing a 1-year survival benefit with liver transplantation. A careful selection of hepatocellular cancer patients results in excellent outcomes, while consideration of extra-hepatic disease (reversible vs irreversible) and social support structures are crucial to patient assessment. Alcoholic liver disease remains a challenge, and the potential to cure hepatitis C virus infection together with the emerging issue of non-alcoholic fatty liver disease-associated chronic liver failure will change the landscape of the who in the years ahead. The when will continue to be determined largely by the severity of liver disease based on the MELD score for the foreseeable future.

Suicidal emperipolesis: a process leading to cell-in-cell structures, T cell clearance and immune homeostasis.

"Suicidal emperipolesis" is one of the most recently reported processes leading to cell-in-cell structures that promote cell death. This process was discovered in studies investigating the fate of autoreactive CD8 T cells activated within the liver. Recently, we reported that activated T cells invaded hepatocytes, formed transient cell-in-cell structures, and were rapidly degraded within endosomal/lysosomal compartments by a non-apoptotic pathway. Importantly, pharmacological inhibition of this process caused intrahepatic accumulation of tissue-reactive T cells and breach of immune tolerance. The characterization of the molecular mechanisms of suicidal emperipolesis is still in its infancy, but initial studies suggest this phenomenon is distinct from other reported cell-in-cell structures. As opposed to the formation of other cell-in-cell structures, suicidal emperipolesis takes place in a non-malignant environment, and without obvious pathology. It is therefore the first cell-in-cell structure described to have a role in maintaining homeostasis in normal physiology in higher organisms. T cell emperipolesis within hepatocytes has also been observed by pathologists in a range of chronic human liver pathologies. As T cell-in-hepatocyte structures resulting from suicidal emperipolesis are very transiently observed in normal physiology, their accumulation during liver disease would suggest that severe tissue injury is promoted by, or associated with, defective T cell clearance. In this review, we compare "suicidal emperipolesis" to other processes leading to cell-in-cell structures, and consider its potential biological roles in maintaining immune homeostasis and tolerance in the context of the hepatic environment.

Percutaneous Retroperitoneal Splenorenal Shunt for Symptomatic Portal Vein Thrombosis After Liver Transplantation.

Acute or recurrent bleeding from ectopic varices is a potentially life-threatening condition in rare patients with extrahepatic complete portal vein thrombosis (PVT) after liver transplantation (LT). In this setting, the role of interventional radiology is very limited and surgical shunts, in particular splenorenal shunts are usually used, despite the high associated mortality. We present the first reports of the clinical use of a new minimally invasive technique, percutaneous retroperitoneal splenorenal shunt (PRESS), in two LT recipients with life-threatening variceal hemorrhage secondary to PVT. Both patients had a successful PRESS using a transplenic approach with resolution of bleeding, avoiding the need for a potentially complicated laparotomy. The PRESS procedure is a useful addition to the interventional armamentarium that can be used in cases unsuitable for surgical shunt, and refractory to endoscopic management. In the future, this technique may be an alternative to surgical shunts as the standard procedure in patients with extra-hepatic PVT, just as the transjugular intrahepatic portosystemic shunt (TIPS) procedure has become for the management of portal hypertension in the absence of PVT. Longer-term follow-up will be needed to establish the long-term success of this procedure.

Lower serum fibroblast activation protein shows promise in the exclusion of clinically significant liver fibrosis due to non-alcoholic fatty liver disease in diabetes and obesity.

UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) is common in diabetes and obesity but few have clinically significant liver fibrosis. Improved risk-assessment is needed as the commonly used clinical-risk algorithm, the NAFLD fibrosis score (NFS), is often inconclusive. AIMS: To determine whether circulating fibroblast activation protein (cFAP), which is elevated in cirrhosis, has value in excluding significant fibrosis, particularly combined with NFS. METHODS: cFAP was measured in 106 with type 2 diabetes who had transient elastography (Cohort 1) and 146 with morbid obesity who had liver biopsy (Cohort 2). RESULTS: In Cohort 1, cFAP (per SD) independently associated with median liver stiffness (LSM) ≥ 10.3 kPa with OR of 2.0 (95% CI 1.2-3.4), p=0.006. There was 0.12 OR (95% CI 0.03-0.61) of LSM ≥ 10.3 kPa for those in the lowest compared with the highest FAP tertile (p=0.010). FAP levels below 730 pmol AMC/min/mL had 95% NPV for LSM ≥ 10.3 kPa and reclassified 41% of 64 subjects from NFS 'indeterminate-risk' to 'low-risk'. In Cohort 2, cFAP (per SD), associated with 1.7 fold (95% CI 1.1-2.8) increased odds of significant fibrosis (F ≥ 2), p=0.021, and low cFAP reclassified 49% of 73 subjects from 'indeterminate-risk' to 'low-risk'. CONCLUSIONS: Lower cFAP, when combined with NFS, may have clinical utility in excluding significant fibrosis in diabetes and obesity.

Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection.

Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus-cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver.

De novo cancer-related death in Australian liver and cardiothoracic transplant recipients.

Evidence is sparse on the relative mortality risk posed by de novo cancers in liver and cardiothoracic transplant recipients. A retrospective cohort study was conducted in Australia using population-based liver (n = 1926) and cardiothoracic (n = 2718) registries (1984-2006). Standardized mortality ratios (SMRs) were computed by cancer type, transplanted organ, recipient age and sex. During a median 5-year follow-up, de novo cancer-related mortality risk in liver and cardiothoracic recipients was significantly elevated compared to the matched general population (n = 171; SMR = 2.83; 95% confidence interval [95%CI], 2.43-3.27). Excess risk was observed regardless of transplanted organ, recipient age group or sex. Non-Hodgkin lymphoma was the most common cancer-related death (n = 38; SMR = 16.6; 95%CI, 11.87-22.8). The highest relative risk was for nonmelanocytic skin cancer (n = 23; SMR = 49.6, 95%CI, 31.5-74.5), predominantly in males and in recipients of heart and lung transplants. Risk of death from de novo cancer was high in pediatric recipients (n = 5; SMR = 41.3; 95%CI, 13.4-96.5), four of the five deaths were non-Hodgkin lymphoma. De novo cancer was a leading cause of late death, particularly in heart and liver transplantation. These findings support tailored cancer prevention strategies, surveillance to promote early detection, and guidelines for managing immunosuppression once cancer occurs.

Comparison of de novo cancer incidence in Australian liver, heart and lung transplant recipients.

Population-based evidence on the relative risk of de novo cancer in liver and cardiothoracic transplant recipients is limited. A cohort study was conducted in Australia using population-based liver (n = 1926) and cardiothoracic (n = 2718) registries (1984-2006). Standardized incidence ratios (SIRs) were computed by cancer type, transplanted organ and recipient age. Cox regression models were used to compare cancer incidence by transplanted organ. During a median 5-year follow-up, the risk of any cancer in liver and cardiothoracic recipients was significantly elevated compared to the general population (n = 499; SIR = 2.62, 95%CI 2.40-2.86). An excess risk was observed for 16 cancer types, predominantly cancers with a viral etiology. The pattern of risk by cancer type was broadly similar for heart, lung and liver recipients, except for Merkel cell carcinoma (cardiothoracic only). Seventeen cancers (10 non-Hodgkin lymphomas), were observed in 415 pediatric recipients (SIR = 23.8, 95%CI 13.8-38.0). The adjusted hazard ratio for any cancer in all recipients was higher in heart compared to liver (1.29, 95%CI 1.03-1.63) and lung compared to liver (1.65, 95%CI 1.26-2.16). Understanding the factors responsible for the higher cancer incidence in cardiothoracic compared to liver recipients has the potential to lead to targeted cancer prevention strategies in this high-risk population.

Terlipressin therapy for moderate-to-severe hyponatraemia in patients with liver failure.

BACKGROUND: Hyponatraemia in liver failure is associated with increased morbidity and mortality. Improving serum sodium in liver failure has been observed in patients receiving terlipressin. METHODS: We assessed the response of hyponatraemia in patients with liver failure to terlipressin using comparative retrospective analysis. RESULTS: Twenty-three patients received terlipressin for hyponatraemia after failed conservative management (median age 52 years (27-67), model for end-stage liver disease score 28 (16-38)). The median therapy was 7 days (1-27), with an average total dose of 25 mg (4-90) and a mean follow up of 51 days (5-1248). These patients were compared with 11 hyponatraemic patients managed conservatively during the same period with comparable age, baseline serum sodium and follow up. After 1 week of terlipressin therapy, serum sodium increased from a median of 120 (115-128) to 129 mmol/L (121-144) (P < 0.001), and at the end of terlipressin therapy, the serum sodium had increased significantly to 131 mmol/L (120-148) (P < 0.001). In comparison, in the conservatively managed group, the serum sodium did not increase significantly from the baseline of 123 (117-127) mmol/L. Adverse events occurred in 26% of patients receiving terlipressin, which predominantly pulmonary oedema. Importantly, more hyponatraemic patients treated with terlipressin (48%) were alive compared with the conservative group (18%), despite the latter having a significantly lower baseline median MELD score of 21 (16-30) (P = 0.008). Moreover, the transplant-free survival was higher in the terlipressin (30%) compared with the conservative group (0%). CONCLUSIONS: Terlipressin is effective in treating hyponatraemia in liver failure. Importantly, terlipressin use results in better transplant-free survival but also more adverse events.

Soluble CD26 / dipeptidyl peptidase IV enhances human lymphocyte proliferation in vitro independent of dipeptidyl peptidase enzyme activity and adenosine deaminase binding.

Human CD26 has dipeptidyl peptidase-4 (DPP IV) enzyme activity and binds to adenosine deaminase (ADA). CD26 is costimulatory for lymphocytes and has a circulating soluble form (sCD26). DPP IV enzyme inhibition is a new successful type 2 diabetes therapy. We examined whether the ADA binding and catalytic functions of sCD26 contribute to its effects on T-cell proliferation. Wildtype soluble recombinant human CD26 (srhCD26), an enzyme inactive mutant (srhCD26E-) and an ADA non-binding mutant (srhCD26A-) were co-incubated in in vitro T-cell proliferation assays with peripheral blood mononuclear cells (PBMC) stimulated with phytohaemagglutinin (PHA), muromonab-CD3 or Herpes simplex virus antigen (HSV Ag). Both srhCD26 and srhCD26E- enhanced PHA-induced T-cell proliferation dose-dependently in all six subjects tested. srhCD26 and srhCD26A- had no overall effect on anti-CD3-stimulated PBMC proliferation in four of five subjects. srhCD26, srhCD26E- and srhCD26A- enhanced HSV Ag induced PBMC proliferation in low responders to HSV Ag, but had no effect or inhibited proliferation in HSV-high responders. Thus, effects of soluble human CD26 on human T-cell proliferation are mechanistically independent of both the enzyme activity and the ADA-binding capability of sCD26.

Increasing liver transplantation waiting list mortality: a report from the Australian National Liver Transplantation Unit, Sydney.

BACKGROUND: We aimed to describe the demand for liver transplantation (LTx) and patient outcomes on the waiting list at the Australian National Liver Transplantation Unit, Sydney over the last 20 years. METHODS: We performed a retrospective analysis with the data divided into three eras: 1985-1993, 1994-2000 and 2001-2008. RESULTS: The number of patients accepted for LTx increased from 320 to 372 and 548 (P < 0.001) with the number of LTx being performed increasing from 262 to 312 and 452 respectively (P < 0.001). The median adult recipient age increased from 45 to 48 and 52 years (P < 0.001) while it decreased in children from 4 to 2 and 1 years respectively (P = 0.001). In parallel, the deceased donor offers decreased from 1003 to 720 and 717 (P < 0.001). Methods to improve access to donor livers have been used with the use of split livers, extended criteria and non-heart beating donors, resulting in increased acceptance of deceased donor offers by 65% and 115% in the second and third eras when compared with the first era (P < 0.001). However, the adult median waiting time has increased from 23 to 41 and 120 days respectively (P < 0.001). This was associated with increased adult mortality on the waiting list from 23 to 40 and 122 respectively (P < 0.001). CONCLUSIONS: Despite the increasing proportion of donor offers being used, the waiting list mortality is increasing. A solution to this problem is an increase in organ donation to keep pace with the escalating demand for LTx.