Hepatitis C Virus (HCV) Eradication With Interferon-Free Direct-Acting Antiviral-Based Therapy Results in KLRG1+ HCV-Specific Memory Natural Killer Cells.

Direct acting antiviral therapies rapidly clear chronic hepatitis C virus (HCV) infection and restore natural killer (NK) cell function. We investigated NK-cell memory formation following HCV clearance by examining NK-cell phenotype and responses from control and chronic HCV patients before and after therapy following sustained virologic response at 12 weeks post therapy (SVR12). NK-cell phenotype at SVR12 differed significantly from paired pretreatment samples, with an increase in maturation markers CD16, CD57, and KLRG1. HCV patients possessed stronger cytotoxic responses against HCV-infected cells as compared to healthy controls; a response that further increased following SVR12. The antigen-specific response was mediated by KLRG1+ NK cells, as demonstrated by increased degranulation and proliferation in response to HCV antigen only. Our data suggest that KLRG1+ HCV-specific memory NK cells develop following viral infection, providing insight into their role in HCV clearance and relevance with regard to vaccine design.

HBV vaccination and HBV infection induces HBV-specific natural killer cell memory.

OBJECTIVE: Vaccination against hepatitis B virus (HBV) confers protection from subsequent infection through immunological memory that is traditionally considered the domain of the adaptive immune system. This view has been challenged following the identification of antigen-specific memory natural killer cells (mNKs) in mice and non-human primates. While the presence of mNKs has been suggested in humans based on the expansion of NK cells following pathogen exposure, evidence regarding antigen-specificity is lacking. Here, we demonstrate the existence of HBV-specific mNKs in humans after vaccination and in chronic HBV infection. DESIGN: NK cell responses were evaluated by flow cytometry and ELISA following challenge with HBV antigens in HBV vaccinated, non-vaccinated and chronic HBV-infected individuals. RESULTS: NK cells from vaccinated subjects demonstrated higher cytotoxic and proliferative responses against autologous hepatitis B surface antigen (HBsAg)-pulsed monocyte-derived dendritic cells (moDCs) compared with unvaccinated subjects. Moreover, NK cell lysis of HBsAg-pulsed moDCs was significantly higher than that of hepatitis B core antigen (HBcAg)-pulsed moDCs (non-vaccine antigen) or tumour necrosis factor α-activated moDCs in a NKG2D-dependent manner. The mNKs response was mediated by CD56dim NK cells coexpressing CD57, CD69 and KLRG1. Further, mNKs from chronic hepatitis B patients exhibited greater degranulation against HBcAg-pulsed moDCs compared with unvaccinated or vaccinated patients. Notably, mNK activity was negatively correlated with HBV DNA levels. CONCLUSIONS: Our data support the presence of a mature mNKs following HBV antigen exposure either through vaccination or infection. Harnessing these antigen specific, functionally active mNKs provides an opportunity to develop novel treatments targeting HBV in chronic infection.

Expansion of dysfunctional CD56-CD16+ NK cells in chronic hepatitis B patients.

BACKGROUND & AIMS: Natural killer (NK) cells are primary innate effector cells that play an important role in the control of human viral infections. During chronic viral infection, NK cells undergo significant changes in phenotype, function and subset distribution, including the appearance of CD56-CD16+ (CD56-) NK cells, previously identified in chronic human immunodeficiency virus (HIV) and hepatitis C virus infection. However, the presence of CD56- NK cells in the pathogenesis of chronic hepatitis B (CHB) remains unknown. METHODS: Phenotype and function of CD56- NK cells from patients with CHB (n = 28) were assessed using flow cytometry and in vitro stimulation with HBV antigen. RESULTS: CHB patients had a higher frequency of CD56- NK cells compared to healthy controls in peripheral blood (6.2% vs 1.4%, P < .0001). Compared to CD56+ NK cells, CD56- NK cells had increased expression of inhibitory receptors, and reduced expression of activating receptors, as measured by MFI and qPCR. CD56- NK cells were less responsive to target cell and cytokine stimulation compared to their CD56+ counterparts. In addition, CD56- NK cells demonstrated defective dendritic cells (DCs) interactions resulting in reduced DCs maturation, lower expression of NK CD69 and impaired capacity of NK cells to eliminate immature DCs in co-culture studies. Finally, frequency of CD56- NK cells was positively correlated with serum HBV DNA levels. CONCLUSION: Chronic HBV infection induces the expansion of highly dysfunctional of CD56- NK cells that likely contribute to inefficient innate and adaptive antiviral immune response in chronic HBV infection.

Australian consensus statements for the regulation, production and use of faecal microbiota transplantation in clinical practice.

OBJECTIVE: Faecal microbiota transplantation (FMT) has proved to be an extremely effective treatment for recurrent Clostridioides difficile infection, and there is interest in its potential application in other gastrointestinal and systemic diseases. However, the recent death and episode of septicaemia following FMT highlights the need for further appraisal and guidelines on donor evaluation, production standards, treatment facilities and acceptable clinical indications. DESIGN: For these consensus statements, a 24-member multidisciplinary working group voted online and then convened in-person, using a modified Delphi approach to formulate and refine a series of recommendations based on best evidence and expert opinion. Invitations to participate were directed to Australian experts, with an international delegate assisting the development. The following issues regarding the use of FMT in clinical practice were addressed: donor selection and screening, clinical indications, requirements of FMT centres and future directions. Evidence was rated using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system. RESULTS: Consensus was reached on 27 statements to provide guidance on best practice in FMT. These include: (1) minimum standards for donor screening with recommended clinical selection criteria, blood and stool testing; (2) accepted routes of administration; (3) clinical indications; (4) minimum standards for FMT production and requirements for treatment facilities acknowledging distinction between single-site centres (eg, hospital-based) and stool banks; and (5) recommendations on future research and product development. CONCLUSIONS: These FMT consensus statements provide comprehensive recommendations around the production and use of FMT in clinical practice with relevance to clinicians, researchers and policy makers.

KLRG1+ natural killer cells exert a novel antifibrotic function in chronic hepatitis B.

BACKGROUND & AIMS: Natural killer (NK) cells are known to exert strong antiviral activity. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is expressed by terminally differentiated NK cells and KLRG1-expressing lymphocytes are known to expand following chronic viral infections. We aimed to elucidate the previously unknown role of KLRG1 in the pathogenesis of chronic hepatitis B (CHB). METHODS: KLRG1+ NK cells were taken from the blood and liver of healthy individuals and patients with CHB. The phenotype and function of these cells was assessed using flow cytometry and in vitro stimulation. RESULTS: Patients with CHB had a higher frequency of KLRG1+ NK cells compared to healthy controls (blood 13.4 vs. 2.3%, p <0.0001 and liver 23.4 vs. 2.6%, p <0.01). KLRG1+ NK cells were less responsive to K562 and cytokine stimulation, but demonstrated enhanced cytotoxicity (9.0 vs. 4.8%, p <0.05) and IFN-γ release (8.0 vs. 1.5%, p <0.05) via antibody dependent cellular cytotoxicity compared to their KLRG1- counterparts. KLRG1+ NK cells possessed a mature phenotype, demonstrating stronger cytolytic activity and IFN-γ secretion against hepatic stellate cells (HSCs) than KLRG1- NK cells. Moreover, KLRG1+ NK cells more effectively induced primary HSC apoptosis in a TRAIL-dependent manner. Increased KLRG1+ NK cell frequency in the liver and blood was associated with lower fibrosis stage (F0/F1) in patients with CHB. Finally, the expression of CD44, degranulation and IFN-γ production were all increased in KLRG1+ NK cells following stimulation with osteopontin, the CD44 ligand, suggesting that HSC-derived osteopontin may cause KLRG1+ NK cell activation. CONCLUSIONS: KLRG1+ NK cells likely play an antifibrotic role during the natural course of CHB infection. Harnessing this antifibrotic function may provide a novel therapeutic approach to treat liver fibrosis in patients with CHB. LAY SUMMARY: Individuals that are chronically infected with hepatitis B virus (HBV) possess an increased number of immune cells, called natural killer (NK) cells expressing the surface marker KLRG1 in the blood and liver. Here, we demonstrate that these specific NK cells are able to kill activated stellate cells in the liver. Because activated stellate cells contribute to liver scarring, i.e. fibrosis, and subsequent liver dysfunction in individuals with chronic HBV infection, KLRG1+ NK cells are a novel immune cell type that can limit liver scarring.

Gastric Cancer Screening in Common Variable Immunodeficiency.

Individuals with common variable immunodeficiency (CVID) have an increased risk of gastric cancer, and gastrointestinal lymphoma, yet screening for premalignant gastric lesions is rarely offered routinely to these patients. Proposed screening protocols are not widely accepted and are based on gastric cancer risk factors that are not applicable to all CVID patients. Fifty-two CVID patients were recruited for screening gastroscopy irrespective of symptoms or blood results and were compared to 40 controls presenting for gastroscopy for other clinical indications. Overall, 34% of CVID patients had intestinal metaplasia (IM), atrophic gastritis or moderate to severe non-atrophic gastritis, which can increase the risk of gastric cancer, compared to 7.5% of controls (p < 0.01). Focal nodular lymphoid hyperplasia, a precursor lesion for gastrointestinal lymphoma, was seen in eight CVID patients (16%), one of whom was diagnosed with gastrointestinal lymphoma on the same endoscopy. High-risk gastric pathology was associated with increased time since diagnosis of CVID, smoking, Helicobacter pylori, a low-serum pepsinogen I concentration, and diarrhea, but not pepsinogen I/II ratio, iron studies, vitamin B12 levels or upper gastrointestinal symptoms. There was a lower rate of detection of IM when fewer biopsies were taken, and IM and gastric atrophy were rarely predicted by the endoscopist macroscopically, highlighting the need for standardized biopsy protocols. The prevalence of premalignant gastric lesions in patients with CVID highlights the need for routine gastric screening. We propose a novel gastric screening protocol to detect early premalignant lesions and reduce the risk of gastric cancer and gastric lymphoma in these patients.

Diverse impacts of the rs58542926 E167K variant in TM6SF2 on viral and metabolic liver disease phenotypes.

UNLABELLED: A genome-wide exome association study has identified the transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 variant encoding an E167K substitution as a genetic determinant of hepatic steatosis in nonalcoholic fatty liver disease (NAFLD). The roles of this variant across a spectrum of liver diseases and pathologies and on serum lipids comparing viral hepatitis to NAFLD and viral load in chronic viral hepatitis, as well as its intrahepatic molecular signature, have not been well characterized. We undertook detailed analyses in 3260 subjects with viral and nonviral liver diseases and in healthy controls. Serum inflammatory markers and hepatic expression of TM6SF2 and genes regulating lipid metabolism were assessed in a subset with chronic hepatitis C (CHC). The rs58542926 T allele was more prevalent in 502 NAFLD patients than controls (P = 0.02) but not different in cohorts with CHC (n = 2023) and chronic hepatitis B (n = 507). The T allele was associated with alterations in serum lipids and hepatic steatosis in all diseases and with reduced hepatic TM6SF2 and microsomal triglyceride transfer protein expression. Interestingly, the substitution was associated with reduced CHC viral load but increased hepatitis B virus DNA. The rs58542926 T allele had no effect on inflammation, impacted ≥F2 fibrosis in CHC and NAFLD assessed cross-sectionally (odds ratio = 1.39, 95% confidence interval 1.04-1.87, and odds ratio = 1.62, 95% confidence interval 1.03-2.52, respectively; P < 0.03 for both), but had no effect on fibrosis progression in 1174 patients with CHC and a known duration of infection. CONCLUSION: The TM6SF2 E167K substitution promotes steatosis and lipid abnormalities in part by altering TM6SF2 and microsomal triglyceride transfer protein expression and differentially impacts CHC and chronic hepatitis B viral load, while effects on fibrosis are marginal. (Hepatology 2016;64:34-46).

The gut microbiome.

BACKGROUND: More than a trillion, mostly good, microbes live within our gastrointestinal tract and are responsible for vital metabolic, immune and nutritional functions. Dysbiosis, meaning a maladaptive imbalance of the microbiome, is associated with many common diseases and is a target for therapy. OBJECTIVE: This article provides an overview of the gut microbiome in health and disease, highlighting conditions such as Clostridium difficile infection, inflammatory bowel disease, irritable bowel syndrome, obesity and non-alcoholic fatty liver disease, with which dysbiosis is associated. Information about treatments that affect the gut microbiome, including probiotics and faecal microbiota transplant, are discussed. DISCUSSION: As our knowledge of the microbiome increases, we are likely to better understand the complex interactions that cause disease, and develop new and more effective treatments for many common conditions.

Adiponectin reduces hepatic stellate cell migration by promoting tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion.

Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/ß1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin.

Hepatic fat loss in advanced nonalcoholic steatohepatitis: are alterations in serum adiponectin the cause?

UNLABELLED: Advanced liver fibrosis in nonalcoholic steatohepatitis (NASH) is often accompanied by a reduction in hepatic fat to the point of complete fat loss (burnt-out NASH), but the mechanisms behind this phenomenon have not been elucidated. Adiponectin is raised in cirrhosis of any cause and has potent antisteatotic activity. In this study we examined 65 patients with advanced biopsy-proven NASH (fibrosis stage 3-4) and 54 with mild disease (fibrosis stage 0-1) to determine if disappearance of steatosis correlated with changes in serum adiponectin. All patents had fasting blood tests and anthropometric measures at the time of liver biopsy. Liver fat was accurately quantitated by morphometry. Serum adiponectin was measured by immunoassay. When compared to those with early disease, patients with advanced NASH were more insulin-resistant, viscerally obese, and older, but there was no difference in liver fat content or adiponectin levels. Adiponectin had a significant negative correlation with liver fat percentage in the whole cohort (r = -0.28, P < 0.01), driven by patients with advanced NASH (r = -0.40, P < 0.01). In advanced NASH, for each 4 µg/L increase in adiponectin there was an odds ratio OR of 2.0 (95% confidence interval [CI]: 1.3-3.0, P < 0.01) for a 5% reduction in hepatic fat. Adiponectin was highly and significantly associated with almost complete hepatic fat loss or burnt-out NASH (12.1 versus 7.4 µg/L, P = 0.001) on multivariate analysis. A relationship between adiponectin, bile acids, and adipocyte fexaramine activation was demonstrated in vivo and in vitro, suggestive of hepatocyte-adipocyte crosstalk. CONCLUSION: Serum adiponectin levels in advanced NASH are independently associated with hepatic fat loss. Adiponectin may in part be responsible for the paradox of burnt-out NASH. (HEPATOLOGY 2012).

Using a human colonoid-derived monolayer to study bacteriophage translocation.

Bacteriophages (phages) are estimated to be the most abundant microorganisms on Earth. Their presence in human blood suggests that they can translocate from non-sterile sites such as the gastrointestinal tract where they are concentrated. To examine phage translocation ex vivo, we adapted a primary colonoid monolayer model possessing cell diversity and architecture, and a thick layer of mucus akin to the colonic environment in vivo. We show that the colonoid monolayer is superior to the Caco-2 cell-line model, possessing intact and organized tight junctions and generating a physiologically relevant mucus layer. We showed, using two different phages, that translocation across the colonoid monolayer was largely absent in differentiated monolayers that express mucus, unlike Caco-2 cultures that expressed little to no mucus. By stimulating mucus production or removing mucus, we further demonstrated the importance of colonic mucus in preventing phage translocation. Finally, we used etiological drivers of gut permeability (alcohol, fat, and inflammatory cytokines) to measure their effects on phage translocation, demonstrating that all three stimuli have the capacity to amplify phage translocation. These findings suggest that phage translocation does occur in vivo but may be largely dependent on colonic mucus, an important insight to consider in future phage applications.

Clostridioides difficile Infection: Clinical Practice and Health Outcomes in 6 Large Tertiary Hospitals in Eastern Australia.

Background: Clostridioides difficile infection (CDI) is associated with significant morbidity and mortality in both healthcare and community settings. We aimed to define the predisposing factors, risks for severe disease, and mortality determinants of CDI in eastern Australia over a 1-year period. Methods: This is an observational retrospective study of CDI in hospitalized patients aged ≥18 years in 6 tertiary institutions from 1 January 2016 to 31 December 2016. Patients were identified through laboratory databases and medical records of participating institutions. Clinical, imaging, and laboratory data were input into an electronic database hosted at a central site. Results: A total of 578 patients (578 CDI episodes) were included. Median age was 65 (range, 18-99) years and 48.2% were male. Hospital-onset CDI occurred in 64.0%. Recent antimicrobial use (41.9%) and proton pump inhibitor use (35.8%) were common. Significant risk factors for severe CDI were age <65 years (P < .001), malignancy within the last 5 years (P < .001), and surgery within the previous 30 days (P < .001). Significant risk factors for first recurrence included severe CDI (P = .03) and inflammatory bowel disease (P = .04). Metronidazole was the most common regimen for first episodes of CDI with 65.2% being concordant with Australian treatment guidelines overall. Determinants for death at 60 days included age ≥65 years (P = .01), severe CDI (P < .001), and antibiotic use within the prior 30 days (P = .02). Of those who received metronidazole as first-line therapy, 10.1% died in the 60-day follow-up period, compared to 9.8% of those who received vancomycin (P = .86). Conclusions: Patients who experience CDI are vulnerable and require early diagnosis, clinical surveillance, and effective therapy to prevent complications and improve outcomes.

Visceral adiposity index is not a predictor of liver histology in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Visceral adiposity is associated with hepatic steatosis, inflammation, and fibrosis in non-alcoholic fatty liver disease (NAFLD). The visceral adiposity index (VAI), a novel marker of visceral fat distribution and dysfunction, has been correlated with histology in hepatitis C. We assessed the ability of VAI to predict disease severity in NAFLD and hence its role as a non-invasive marker of liver damage. METHODS: We examined 190 adults with biopsy-proven NAFLD and 129 controls. All had anthropometric and metabolic profiling. VAI was calculated using waist circumference (WC), body mass index, triglycerides, and HDL-cholesterol. Abdominal fat was quantified by magnetic resonance imaging (MRI) in 38 patients. RESULTS: On multivariate analysis, NAFLD diagnosis and fasting glucose were independently associated with VAI (p <0.05). VAI increased across control, steatosis, and NASH groups (1.5, 2.3, and 3.2, respectively; p=0.000), however, this association was no stronger than the increase in WC across groups (r=0.452 vs. 0.540 respectively, p <0.001). VAI was not associated with steatosis, lobular inflammation or fibrosis, but WC was associated with fibrosis (p=0.01). VAI and WC correlated with an increasing number of metabolic syndrome components (r=0.623 vs. 0.614, p <0.001) and with metabolic syndrome diagnosis (r=0.559 vs. 0.509, p <0.001). VAI only modestly correlated with visceral fat on MRI (r=0.39, p <0.05) compared to WC (r=0.52, p <0.01). CONCLUSIONS: In NAFLD, VAI is not associated with steatosis, inflammation or fibrosis. VAI is no more powerful than WC in discriminating steatosis from steatohepatitis, reflecting limitations of the formula with what is known about the pathogenesis of NAFLD.

The natural history of nonalcoholic fatty liver disease with advanced fibrosis or cirrhosis: an international collaborative study.

UNLABELLED: Information on the long-term prognosis of nonalcoholic fatty liver disease (NAFLD) is limited. We sought to describe the long-term morbidity and mortality of patients with NAFLD with advanced fibrosis or cirrhosis by prospectively studying 247 such patients from four international centers (in Australia, USA, UK and Italy). Their natural history was then compared with 264 patients with HCV infection who were either naïve or non-responders to treatment. Both cohorts were Child-Pugh class A and had advanced fibrosis (stage 3) or cirrhosis (stage 4) confirmed by liver biopsy at enrollment. In the NAFLD cohort, followed up for a mean of 85.6 months (range, 6-297), there were 48 (19.4%) liver-related complications and 33 (13.4%) deaths or liver transplants. In the HCV cohort, followed up for 74.9 months (mean; range, 6-238), there were 47 (16.7%) liver-related complications and 25 (9.4%) deaths or liver transplants. When adjusting for baseline differences in age and gender, the cumulative incidence of liver-related complications was lower in the NAFLD than the HCV cohort (P = 0.03), including incident hepatocellular cancer (6 versus 18; P = 0.03), but that of cardiovascular events (P = 0.17) and overall mortality (P = 0.6) were similar in both groups. In the NAFLD cohort, platelet count, stage 4 fibrosis, lowered platelet count, and lowered serum cholesterol and alanine aminotransferase (ALT) levels were associated with liver-related complications; an aspartate aminotransferase/ALT ratio >1 and older age were associated with overall mortality, and higher serum bilirubin levels and stage 4 fibrosis were associated with liver-related mortality. CONCLUSIONS: Patients with NAFLD with advanced fibrosis or cirrhosis have lower rates of liver-related complications and hepatocellular cancer than corresponding patients with HCV infection, but similar overall mortality. Some clinical and laboratory features predict liver-related complications and other outcomes in patients with NAFLD.

Chronic hepatitis C is associated with peripheral rather than hepatic insulin resistance.

BACKGROUND & AIMS: Chronic hepatitis C (CHC) is associated with insulin resistance (IR), liver steatosis (genotype 3), and increased diabetes risk. The site and mechanisms of IR are unclear. METHODS: We compared cross-sectionally 29 nonobese, normoglycemic males with CHC (genotypes 1 and 3) to 15 adiposity and age-matched controls using a 2-step hyperinsulinemic-euglycemic clamp with [6,6-(2)H(2)] glucose to assess insulin sensitivity in liver and peripheral tissues and (1)H-magnetic resonance spectroscopy to evaluate liver and intramyocellular lipid. Insulin secretion was assessed after intravenous glucose. RESULTS: Insulin secretion was not impaired in CHC. Peripheral insulin sensitivity was 35% higher in controls vs CHC (P < .001) during high-dose (264.3 +/- 25 [standard error] mU/L) insulin (P < .001); this was negatively associated with viral load (R(2) = .12; P = .05) and subcutaneous fat (R(2) = .41; P < .001). IR was similar in both genotypes despite 3-fold increased hepatic fat in genotype 3 (P < .001). Hepatic glucose production (P = .25) and nonesterified free fatty acid (P = .84) suppression with insulin were not different between CHC and controls inferring no adipocyte IR, and suggesting IR is mainly in muscle. In CHC, intramyocellular lipid was nonsignificantly increased but levels of glucagon (73.8 +/- 3.6 vs 52.8 +/- 3.1 ng/mL; P < .001), soluble tumor necrosis factor receptor 2 (3.1 +/- 0.1 vs 2.3 +/- 0.1 ng/mL; P < .001), and Lipocalin-2 (36.4 +/- 2.9 vs 19.6 +/- 1.6 ng/mL; P < .001) were elevated. CONCLUSIONS: CHC represents a unique infective/inflammatory model of IR, which is predominantly in muscle, correlates with subcutaneous, not visceral, adiposity, and is independent of liver fat.

Complex non-invasive fibrosis models are more accurate than simple models in non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Significant hepatic fibrosis is prognostic of liver morbidity and mortality in non-alcoholic fatty liver disease (NAFLD); however, it remains unclear whether non-invasive fibrosis models can determine this end-point. We therefore compared the accuracy of simple bedside versus complex fibrosis models across a range of fibrosis in a multi-centre NAFLD cohort. METHODS: Simple (APRI, BARD) and complex (Hepascore, Fibrotest, FIB4) fibrosis models were calculated in 242 NAFLD subjects undergoing liver biopsy. Significant (F2-4) and advanced fibrosis (F3,4) were defined using Kleiner criteria. Models were compared using area under the receiver operator characteristic curves (AUC). Cut-offs were determined by Youden Index or 90% predictive values. RESULTS: For significant fibrosis, non-invasive fibrosis models had modest accuracy (AUC 0.707-0.743) with BARD being least accurate (AUC 0.609, P < 0.05 vs others). Using single cut-offs, sensitivities and predictive values were < 80%; using two cut-offs, > 75% of subjects fell within indeterminate ranges. Simple models had significantly more subjects within indeterminate ranges than complex models (99.1-100% vs 82.1-84.4% respectively, P < 0.05 for all). For advanced fibrosis, complex models were more accurate than BARD (AUC 0.802-0.858 vs 0.701, P < 0.05). Using two cut-offs, complex models had fewer individuals within indeterminate ranges than BARD (11.1-32.3% vs 70.7%, P < 0.01 for all). For cirrhosis, complex models had higher AUC values than simple models. CONCLUSIONS: In NAFLD subjects, non-invasive models have modest accuracy for determining significant fibrosis and have predictive values less than 90% in the majority of subjects. Complex models are more accurate than simple bedside models across a range of fibrosis.

Utility of Fibroscan XL to assess the severity of non-alcoholic fatty liver disease in patients undergoing bariatric surgery.

Significant weight loss can modify the progression of Nonalcoholic fatty liver disease (NAFLD) with the most convincing evidence coming from bariatric surgery cohorts. Effective ways to non-invasively characterise NAFLD in these patients has been lacking, with high Fibroscan failure rates reported. We prospectively evaluated the utility of Fibroscan using XL-probe over a two-year period. 190 consecutive patients undergoing bariatric surgery were followed as part of their routine care. All patients had Fibroscan performed on the day of surgery and at follow-up a mean of 13 months (± 6.3) later. The majority of patients were female (82%) with mean age of 42. Fibroscan was successful in 167 (88%) at baseline and 100% at follow up. Patients with a failed Fibroscan had higher body mass index (BMI) and alanine transaminase (ALT), but no difference in FIB-4/NAFLD score. Mean baseline Liver stiffness measurement was 5.1 kPa, with 87% of patients classified as no fibrosis and 4% as advanced fibrosis. Mean baseline controlled attenuation parameter was 291, with 78% having significant steatosis, 56% of which was moderate-severe. Significant fibrosis was associated with higher BMI and HbA1c. Significant steatosis was associated with higher BMI, ALT, triglycerides and insulin resistance. Mean follow up time was 12 months with weight loss of 25.7% and BMI reduction of 10.4 kg/m2. Seventy patients had repeat fibroscan with reductions in steatosis seen in 90% and fibrosis in 67%. Sixty-four percent had complete resolution of steatosis. Fibroscan can be performed reliably in bariatric cohorts and is useful at baseline and follow-up. Significant steatosis, but not fibrosis was seen in this cohort with substantial improvements post-surgery.

Duodenal plasma cells correspond to serum IgA in common variable immunodeficiency.

Common variable immunodeficiency (CVID) can be associated with a range of serum IgA concentrations, from absent, to variably reduced, and in some patients classified as 'possible CVID', even normal. The aim of this study was to assess the proportion of duodenal plasma cells in patients with CVID and determine whether there was an association with serum IgA concentration. Duodenal biopsies obtained at upper endoscopy from 35 patients with CVID were assessed for the presence of plasma cells and compared with serum IgA concentrations. A reduction or absence of duodenal plasma cells in 60% of patients with CVID and an association between the proportions of duodenal plasma cells and serum IgA concentrations was demonstrated. The presence of duodenal plasma cells associated with numbers of isotype switched memory B cells in the peripheral blood. A reduction in serum IgA over time was observed in 19% of CVID patients. The gastrointestinal tract provides a window into the immune system in CVID, and these results reinforce the association between gastrointestinal plasma cells and serum IgA concentrations. Preservation of gastrointestinal plasma cells and serum IgA in some patients with CVID, and the sequential decline of both in others, highlight the heterogeneity of this disorder.

Adipocyte fatty acid binding protein levels relate to inflammation and fibrosis in nonalcoholic fatty liver disease.

UNLABELLED: Several circulating cytokines are increased with obesity and may combine with the influence of visceral fat to generate insulin resistance, inflammation, and fibrosis in nonalcoholic fatty liver disease (NAFLD). Little information exists in NAFLD about three recently recognized tissue-derived cytokines that are all lipid-binding and involved in inflammation, namely adipocyte fatty acid-binding protein (AFABP), lipocalin-2, and retinol-binding protein 4 (RBP4). We examined the association of these three peptides with hepatic steatosis, inflammation, and fibrosis plus indices of adiposity, insulin resistance, and dyslipidaemia in 100 subjects with NAFLD and 129 matched controls. Levels of AFABP and lipocalin-2, but not RBP4, were significantly elevated in NAFLD versus control (AFABP, 33.5 +/- 14.4 versus 23.1 +/- 12.1 ng/mL [P < 0.001]; lipocalin-2, 63.2 +/- 26 versus 48.6 +/- 20 ng/mL [P < 0.001]) and correlated with indices of adiposity. AFABP correlated with indices of subcutaneous rather than visceral fat. AFABP alone distinguished steatohepatitis from simple steatosis (P= 0.02). Elevated AFABP independently predicted increasing inflammation and fibrosis, even when insulin resistance and visceral fat were considered; this applied to lobular inflammation and ballooning (odds ratio 1.4, confidence interval 1.0-1.8) and fibrosis stage (odds ratio 1.3, confidence interval 1.0-1.7) (P < or = 0.05 for all). None of the cytokines correlated with steatosis grade. AFABP levels correlated with insulin resistance (homeostasis model assessment of insulin resistance) in controls and NAFLD, whereas lipocalin-2 and RBP4 only correlated positively with insulin resistance in controls. CONCLUSION: Circulating AFABP, produced by adipocytes and macrophages, and lipocalin-2, produced by multiple tissues, are elevated and may contribute to the metabolic syndrome in NAFLD. AFABP levels, which correlate with subcutaneous, but not visceral fat, independently predict inflammation and fibrosis in NAFLD and may have a direct pathogenic link to disease progression.

Cross-Linked Multimeric Pro-Peptides of Type III Collagen (PC3X) in Hepatocellular Carcinoma - A Biomarker That Provides Additional Prognostic Value in AFP Positive Patients.

PURPOSE: Non-invasive biomarkers for diagnosing and prognosing hepatocellular carcinoma (HCC) are urgently needed. Cirrhosis is present in 80-90% of HCC patients. Cirrhosis is characterized by deposition and cross-linking of collagens that have crucial roles in HCC initiation and progression. We evaluated circulating cross-linked pro-peptides of type III collagen (PC3X) as a diagnostic and prognostic biomarker for HCC. PATIENTS AND METHODS: PC3X was measured by ELISA in plasma from patients with HCC (n=79), cirrhosis (n=86), non-cirrhotic hepatitis-B infection (n=74) and from healthy controls (n=44). PC3X was compared to the liver fibrosis marker PRO-C3 and the HCC tumor-cell derived marker alpha-fetoprotein (AFP). Diagnostic and prognostic potential was evaluated by AUROC and by calculating hazard ratios (HR) for progression-free survival (PFS) and overall survival (OS). RESULTS: PC3X, PRO-C3 and AFP were significantly elevated in patients with HCC compared to other liver diseases and healthy controls (p=0.0002, p<0.0001). In patients with normal AFP (<20 IU/mL), PC3X and PRO-C3 separated HCC from cirrhosis with an AUROC of 0.72 and 0.68, respectively. High PC3X and AFP predicted for poor PFS (HRPC3X=1.80, p=0.032; HRAFP=1.70, p=0.031) and OS (HRPC3X=2.12, p=0.024; HRAFP=2.55; p=0.003), whereas PRO-C3 did not (PFS: HR=1.19, p=0.059 and OS: HR=1.12, p=0.324). PC3X was independent of AFP (PFS: HR=1.74, p=0.045 and OS: HR=2.21, p=0.018) and combining the two improved prognostic value (PFS: HR=2.66, p=0.004 and OS: HR=5.86, p<0.0001). CONCLUSION: PC3X is associated with HCC independent of AFP and provides diagnostic and prognostic value for HCC patients. If validated, this suggests that PC3X has biomarker potential for HCC.