Sukriti

Hyperoxidized albumin modulates neutrophils to induce oxidative stress and inflammation in severe alcoholic hepatitis

Albumin is a potent scavenger of reactive oxygen species (ROS). However, modifications in albumin structure may reduce its antioxidant properties and modulate its immune‐regulatory functions. We examined alterations in circulating albumin in severe alcoholic hepatitis (SAH) patients and their contribution to neutrophil activation, intracellular stress, and alteration in associated molecular pathways. Albumin modifications and plasma oxidative stress were assessed in SAH patients (n = 90), alcoholic cirrhosis patients (n = 60), and healthy controls (n = 30) using liquid chromatography/mass spectrometry and spectrophotometry. Activation and intracellular ROS were measured in healthy neutrophils after treatment with purified albumin from the study groups. Gene expression of SAH neutrophils was analyzed and compared to gene expression from healthy neutrophils after stimulation with purified albumin from SAH patient plasma. SAH‐albumin showed the highest albumin oxidative state (P < 0.05) and prominent alteration as human nonmercaptalbumin 2 (P < 0.05). Plasma oxidative stress (advanced oxidative protein product) was higher in SAH versus alcoholic cirrhosis patients and healthy controls (P < 0.05). Neutrophil gelatinase‐associated lipocalin, myeloperoxidase, and intracellular ROS levels were highest in SAH‐albumin‐treated neutrophils (P < 0.05). Genes associated with neutrophil activation, ROS production, intracellular antioxidation, and leukocyte migration plus genes for proinflammatory cytokines and various toll‐like receptors were overexpressed in SAH neutrophils compared to healthy neutrophils (P < 0.05). Expression of the above‐mentioned genes in SAH‐albumin‐stimulated healthy neutrophils was comparable with SAH patient neutrophils, except for genes associated with apoptosis, endoplasmic reticulum stress, and autophagy (P < 0.05). Conclusions: In patients with SAH, there is a significant increase in albumin oxidation, and albumin acts as a pro‐oxidant; this promotes oxidative stress and inflammation in SAH patients through activation of neutrophils. (Hepatology 2017;65:631‐646).

Impaired Antigen Processing and Presentation Machinery is Associated with Immunotolerant State in Chronic Hepatitis B Virus Infection

Background and AimsThe mechanism of hepatitis B virus (HBV)-specific T cell hyporesponsiveness in hepatitis Be antigen (HBeAg)-positive subjects is not well understood. Inefficient antigen processing and transport to major histocompatibility complex class I molecules, namely due to low molecular weight protein (LMP) 2 and 7 and transporter associated with antigen processing (TAP) 1 and 2 genes could be playing a role.Patients and MethodsForty patients with chronic hepatitis B (CHB) infection, hepatitis B surface antigen, and HBeAg positive; 26 with raised (Gr. I) and 14 with persistently normal ALT levels (Gr. II) and 11 healthy controls (Gr. III) were studied. Total RNA was isolated from peripheral blood mononuclear cells and mRNA expression of TAP1, TAP2, LMP2, and LMP7 genes was analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction method. Gamma interferon (IFN-γ) and tumor necrosis factor-alpha (TNF-α) levels were quantified by enzyme-linked immunosorbent assay (ELISA) using log-log and linear graphs, respectively.ResultsGroup II CHB patients had significantly lower mRNA expression for TAP1 (p = 0.003) and LMP2 (p = 0.002) genes as compared to Gr. I patients. The mRNA expression of TAP2 and LMP7 genes was comparable between the groups. However, expression of TAP1 (p = 0.02), TAP2 (p = 0.035), and LMP2 (p = 0.041) was found to be significantly higher in Gr. III subjects compared to Gr. I and Gr. II patients. In Gr. I and II, the IFN-γ {s54.2{9.4-165} pg/ml), (59.5{28.5-110} pg/ml)}, and TNF-α {12.0 (8.0-23.2)},{10.8(6.2-20.8)} pg/ml levels were comparable but were significantly (p = 0.00,0.004, respectively) higher than Gr. III subjects.ConclusionsLow expression of TAP1 and LMP2 suggests an important role of these genes in defective viral antigen processing in immune tolerant state of CHB patients. Higher IFN-γ and TNF-α production in CHB are probably enough to potentiate liver injury but not enough to clear the chronic HBV infection. These novel observations could pave way for new therapeutic strategies for immune restoration in CHB infected patients.

Gene Expression Profiles of T Cells from Hepatitis E Virus Infected Patients in Acute and Resolving Phase

Background and AimsApproximately 50% of acute viral hepatitis in young adults and in pregnant women is due to hepatitis E virus (HEV) infection in developing countries. T cell-mediated immune injury probably plays a key role in the pathogenesis of acute hepatitis illness. However, there is a paucity of data on the global gene expression programs activated on T cells, which are subsequently responsible for T cell recruitment to the liver and triggering of immune injury.Patients and MethodsWe performed a flow cytometric analysis of T cells in individuals with acute hepatitis E (AVH-E; n = 10), resolving phase of HEV (n = 9), and ten healthy controls (HC). Further transcriptional profiling analysis was performed using Affymetrix GeneChip DNA microarrays to identify the genes that were differentially expressed in AVH-E and HC.ResultsPatients with AVH-E showed higher frequencies of CD8+ (27 ± 4%; P = 0.02) and activated CD38+ CD69+ T cells (25% ± 3%; P = 0.04) than in resolving phase patients (20 ± 2% and 9.1 ± 4%, respectively), who in turn exhibited higher CCR9 expression than cells from patients in active phase. The naïve T cell population (CD3+ CD45RA+) was decreased upon HEV infection (29 ± 4% in AVH-E vs. 53.1 ± 3.2% in HC; P = 0.05); however, the CD11a high subpopulation within CD4+ CD45RA+ cells was increased in both AVH-E (6.1%) and resolving phase (7.7%) patients. Gene ontology analysis suggested that during AVH-E infection, there is in CD4+ T cells an activation of genes involved in pro-inflammatory responses. Additional RT-PCR analysis confirmed that in cells from AVH-E patients, there is an increased expression of CCR5, CCR9, CXCR3, CXCR4, STAT1, IRF-9, IFN-α, and TNF-α, together with a down-regulation of IL-2, SOCS3, and IL-10, with respect to cells from resolving phase patients.ConclusionsOur findings suggest the involvement of a circulating CD45RA+ CD11a high population with CCR5 expression in the pathogenesis processes of AVH-E. The obtained results help to understand the underlying inflammatory process occurring in HEV infection, which can lead to either resolution or immunopathology.

Interleukin-28b CC genotype predicts early treatment response and CT/TT genotypes predicts non-response in patients infected with HCV genotype 3.

Response to antiviral therapy for hepatitis C virus (HCV) depends upon the genotype and host immune response. IL28b gene mutations have been shown to modulate host antiviral immune response against genotype 1. However, the predictive value of IL28b polymorphism in genotype 3 HCV patients is largely unknown. The association of IL28b polymorphism with virological response was studied in 356 patients with genotype 3 chronic HCV undergoing treatment with peg‐interferon and ribavirin and was compared with matched controls. IL28b genotyping followed by DNA sequencing was performed to identify the CC, CT, or TT genotypes. Two log reduction of HCV RNA at Day 7 (Quick Viral Response, QVR) and HCV RNA negativity at Day 28 (Rapid Viral Response, RVR) were analyzed with CC and non‐CC genotypes in addition to other predictors of response. The associations of alleles with the response patterns were predicted. Sustained viral response was seen in 250 (70.2%) patients and the IL28b genotype CC/CT/TT distribution was 61.1%; 30.5%; and 8.4%, respectively. The non‐CC genotypes were significantly higher in non‐responders when compared to responders (67.6% vs. 38.9%, P < 0.001). Interestingly, the rapid viral response in responders was observed in 72.7% with the CC genotype and in 27.2% with the non‐CC genotype (P < 0.001). Multivariate analysis showed CC genotype as an independent factor predicting the sustained viral response in patients infected with HCV genotype 3. In conclusion, the IL28b CT/TT genotype strongly correlates with treatment non‐response in patients infected with HCV genotype 3 and CC genotype of IL28b is associated with higher quick viral response. J. Med. Virol. 86:707–712, 2014.

Molecular Ellipticity of Circulating Albumin-Bilirubin Complex Associates With Mortality in Patients With Severe Alcoholic Hepatitis

BACKGROUND & AIMS Hyperbilirubinemia and hypoalbuminemia are features of hepatic dysfunction that associate with disease severity. This is because hepatic insufficiency causes hypoalbuminemia, which indirectly increases the circulating levels of free bilirubin. Circular dichroism (CD) spectroscopy can be used to quantify the molecular ellipticity (ME) of the albumin–bilirubin complex, and might associate with the severity or outcome of severe alcoholic hepatitis (SAH). METHODS We performed a cross‐sectional study of 265 patients with SAH admitted in the Department of Hepatology, Institute of Liver and Biliary Sciences in New Delhi, India from January 2014 through January 2016. Blood samples were collected and patients were followed for 12 months or death. The molar ratios of bilirubin: albumin and albumin–bilirubin complexes were determined for a discovery cohort (30 patients who survived the study period and 60 patients who did not survive) and compared with those of 60 patients with alcoholic cirrhosis and 30 healthy individuals (controls). Optical activities of albumin–bilirubin complexes in blood samples were determined by CD spectroscopy and compared among groups. Findings were validated in a separate cohort of 150 patients with SAH from the same institute. We studied the correlation between ME and albumin binding capacity (ABiC). RESULTS The molar ratio of bilirubin: albumin was higher in patients with SAH than with alcoholic cirrhosis or controls (P < .05). Patients with SAH had different CD spectra and higher ME than the other groups (P < .01); ME correlated with model for end‐stage liver disease score (with and without Na) and discriminant function (r2 > .3; P < .01). ME values above a cut off of 1.84 mdeg predicted 3‐month mortality in patients with SAH with an area under receiver operating characteristic curve of 0.87 (95% CI, 0.79–0.95), a 77% positive predictive value, and a 90% negative predictive value. The hazard ratio and concordance index of ME values for 3‐month mortality in patients with SAH was 10% higher than the hazard ratio and concordance index of model for end‐stage liver disease score. In patients with SAH, there was an inverse correlation between ME and ABiC (r2 > 0.7; P < .01). We observed a significant reduction in ABiC with increasing levels of bilirubin in vitro prepared albumin–bilirubin complex. CONCLUSION In a cross‐sectional study of patients with SAH, we associated ME of the albumin–bilirubin complex, measured by CD spectroscopy, with outcomes of patients with SAH. Increased loading of bilirubin on albumin could explain reduced albumin function. Bilirubin removal by albumin dialysis might benefit patients with SAH.

Iron-Overload triggers ADAM-17 mediated inflammation in Severe Alcoholic Hepatitis

Severe alcoholic hepatitis (SAH) is associated with iron accumulation in hepatocytes/macrophages. This possibly correlates with inflammation and stress but the exact mechanism still remains obscure. To understand the role of iron and the mechanisms of systemic iron-overload, a transcriptomic study of liver and Peripheral Blood -Mononuclear-Cells (PBMCs) was undertaken in SAH patients, with and without hepatic iron-overload. Our results show that iron-overload in hepatocytes/macrophages is due to an increased expression of iron-loading receptors and CD163 signaling cascade. Increase in labile iron pool induces expression of iron-loading, oxidative-stress and inflammatory genes along with expression of CD163 and ADAM17. Increased liver iron correlated with circulatory iron, TNF-α, macrophage activation (sCD163) and peroxide-stress in CD163+macrophages in patients who were iron-overloaded and died. Circulatory TNF-α and sCD163 levels were associated with poor outcome. Temporal iron/Fenton stress induced in healthy monocyte-derived-macrophage (MDM)/Tohoku-Hospital-Pediatrics-1(THP1) cells showed higher expression of iron-regulatory, inflammatory and oxidative-stress genes. These genes could be suppressed by iron-chelation. These results suggest that iron mediates inflammation through ADAM17 induction, resulting in macrophage activation and increased shedding of TNF-α and sCD163. These events could be inhibited with iron chelation or with ADAM17-blockade, postulating a therapeutic strategy for SAH patients with iron overload.

Extracellular vesicles from hepatitis B patients serve as reservoir of hepatitis B virus DNA

Hepatitis B virus persists in all patients with infection, even those with evidence of serological recovery. Patients with inactive or resolved HBV infection are at a high risk of HBV reactivation. Extracellular vesicles have been shown to serve as vehicles for intercellular communication and transfer of genetic material. We investigated whether extracellular vesicles carry infection and serve as reservoir of HBV DNA. EVs were isolated from chronic hepatitis B (CHB) patients, Group1: qHBsAg+ve with undetectable HBV DNA post-antiviral therapy [n=9]; Group 2: CHB with detectable HBV DNA>103 [n=12], compared with healthy controls (HC) (n=20). The cellular origin of extracellular vesicles was confirmed using flow cytometer. HBV transmission and replication was assessed in Huh7, HepG2 and immortalized human hepatocytes (IHH) cell lines. In Group1, HBV DNA was detected in EVs in 5 of 9 (60%) (p=0.00) patients, whereas undetectable in plasma. The probability of detecting HBV DNA in extracellular vesicles was significant (p=0.03) with odds ratio 28.1(95% of CI -1.27 to 619.9). In Group2, the HBV DNA was detected in all the patients, but levels in extracellular vesicles were significantly lower than in plasma (p=0.00) (median: 2.2X103 IU/ml versus 6.3X 106 IU/ml). Hepatocyte extracellular vesicles detected in CHB patients transmitted HBV to naïve hepatocyte cell lines (detected at 12h, 24h and 72h). The uptake of HBV infected EVs by hepatocytes was confirmed by PKH26 labeled EVs internalization in Huh7 cells. Our results show that HBV persists in extracellular vesicles (even though undetectable in plasma) and such extracellular vesicles are infectious in hepatocytes. This article is protected by copyright. All rights reserved.

Suboptimal Level of Bone‐Forming Cells in Advanced Cirrhosis are Associated with Hepatic Osteodystrophy

Bone loss is common in advanced cirrhosis, although the precise mechanisms underlying bone loss in cirrhosis are unknown. We studied the profile and functionality of bone‐forming cells and bone‐building proteins in bone marrow (BM) of individuals with cirrhosis (n = 61) and individuals without cirrhosis as normal controls (n = 50). We also performed dual energy X‐ray absorptiometry for clinical correlation. BM mesenchymal cells (MSCs) were analyzed for colony‐forming units‐fibroblasts and their osteogenic (fibronectin‐1 [FN1], insulin‐like growth factor binding protein 3 [IGFBP3], collagen type 1 alpha 1 chain [COL1A1], runt‐related transcription factor 2 [RUNX2], and alkaline phosphatase, liver [ALPL]) and adipogenic ( adiponectin, C1Q, and collagen domain containing [ADIPOQ], peroxisome proliferator‐activated receptor gamma [PPARγ], and fatty acid binding protein 4 [FABP4]) potentials. Colony‐forming units‐fibroblasts were lower in patients with cirrhosis (P = 0.002) than in controls. Cirrhotic BM‐MSCs showed >2‐fold decrease in osteogenic markers. Compared to controls, patients with cirrhosis showed fewer osteocytes (P = 0.05), osteoblasts, chondroblasts, osteocalcin‐positive (osteocalcin+) area, clusters of differentiation (CD)169+ macrophages (P < 0.001, each), and nestin+ MSCs (P = 0.001); this was more apparent in Child‐Turcotte‐Pugh (CTP) class C than A (P < 0.001). Multivariate logistic regression showed low nestin+ MSCs (P = 0.004) as a predictor of bone loss. Bone‐resolving osteoclasts were comparable among CTP groups, but >2‐fold decreased anti‐osteoclastic and increased pro‐osteoclastic factors were noted in patients with CTP C compared to CTP A. Bone‐building proteins (osteocalcin [P = 0.008], osteonectin [P < 0.001], and bone morphogenic protein 2 [P = 0.001]) were decreased while anti‐bone repair factors (fibroblast growth factor 23 [P = 0.015] and dipeptidyl peptidase 4 [P < 0.001]) were increased in BM and peripheral blood; this was more apparent in advanced cirrhosis. The dual energy X‐ray absorptiometry scan T score significantly correlated with the population of osteoblasts, osteocytes, MSCs, and CD169+ macrophages. Conclusion: Osteoprogenitor cells are substantially reduced in patients with cirrhosis and more so in advanced disease. Additionally, increased anti‐bone repair proteins enhance the ineffective bone repair and development of osteoporosis in cirrhosis. Hepatology Communications 2018;0:0‐0)