Redox-sensitive high-mobility group box-1 isoforms contribute to liver fibrosis progression and resolution in mice.

BACKGROUND & AIMS: High-mobility group box-1 (HMGB1) significantly increases and undergoes post-translational modifications (PTMs) in response to liver injury. Since oxidative stress plays a major role in liver fibrosis and induces PTMs in proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. METHODS: We used ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectrometry) to study PTMs of HMGB1 during fibrosis progression and resolution. Conditional knockout mice were used for functional analyses. RESULTS: We identified that disulfide ([O]) and sulfonated ([SO3]) HMGB1 increase during carbon tetrachloride-induced liver fibrosis progression, however, while [O] HMGB1 declines, [SO3] HMGB1 drops but remains, during fibrosis resolution. Conditional knockout of Hmgb1 revealed that production of [O] and [SO3] HMGB1 occurs mostly in hepatocytes. Co-injection of [O] HMGB1 worsens carbon tetrachloride-induced liver fibrosis more than co-injection of [H] HMGB1. Conversely, ablation of [O] Hmgb1 in hepatocytes reduces liver fibrosis. Moreover, ablation of the receptor for advanced-glycation end-products (Rage) reveals that the profibrogenic effect of [O] HMGB1 is mediated by RAGE signaling in hepatic stellate cells (HSCs). Notably, injection of [SO3] HMGB1 accelerates fibrosis resolution due to RAGE-dependent stimulation of HSC apoptosis. Importantly, gene signatures activated by redox-sensitive HMGB1 isoforms in mice, classify patients with fibrosis according to fibrosis and inflammation scores. CONCLUSION: Dynamic changes in hepatocyte-derived [O] and [SO3] HMGB1 signal through RAGE-dependent mechanisms on HSCs to drive their profibrogenic phenotype and fate, contributing to progression and resolution of liver fibrosis. IMPACT AND IMPLICATIONS: Since oxidative stress plays a major role in liver fibrosis and induces post-translational modifications of proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. This study is significant because a rise in [H] HMGB1 could flag 'patient at risk', the presence of [O] HMGB1 could suggest 'disease in progress or active scarring', while the appearance of [SO3] HMGB1 could point at 'resolution under way'. The latter could be used as a readout for response to pharmacological intervention with anti-fibrotic agents.

Macrophage-derived Osteopontin (SPP1) Protects From Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, lobular inflammation, hepatocyte ballooning degeneration, and fibrosis, all of which increase the risk of progression to end-stage liver disease. Osteopontin (OPN, SPP1) plays an important role in macrophage (MF) biology, but whether MF-derived OPN affects NASH progression is unknown. METHODS: We analyzed publicly available transcriptomic datasets from patients with NASH, and used mice with conditional overexpression or ablation of Spp1 in myeloid cells and liver MFs, and fed them a high-fat, fructose, and cholesterol diet mimicking the Western diet, to induce NASH. RESULTS: This study demonstrated that MFs with high expression of SPP1 are enriched in patients and mice with nonalcoholic fatty liver disease (NAFLD), and show metabolic but not pro-inflammatory properties. Conditional knockin of Spp1 in myeloid cells (Spp1KI Mye) or in hepatic macrophages (Spp1KI LvMF) conferred protection, whereas conditional knockout of Spp1 in myeloid cells (Spp1ΔMye) worsened NASH. The protective effect was mediated by induction of arginase-2 (ARG2), which enhanced fatty acid oxidation (FAO) in hepatocytes. Induction of ARG2 stemmed from enhanced production of oncostatin-M (OSM) in MFs from Spp1KI Mye mice. OSM activated STAT3 signaling, which upregulated ARG2. In addition to hepatic effects, Spp1KI Mye also protected through sex-specific extrahepatic mechanisms. CONCLUSION: MF-derived OPN protects from NASH, by upregulating OSM, which increases ARG2 through STAT3 signaling. Further, the ARG2-mediated increase in FAO reduces steatosis. Therefore, enhancing the OPN-OSM-ARG2 crosstalk between MFs and hepatocytes may be beneficial for patients with NASH.

Deficiency of neutrophil high-mobility group box-1 in liver transplant recipients exacerbates early allograft injury in mice.

BACKGROUND AND AIMS: Early allograft dysfunction (EAD) is a severe event leading to graft failure after liver transplant (LT). Extracellular high-mobility group box-1 (HMGB1) is a damage-associated molecular pattern that contributes to hepatic ischemia-reperfusion injury (IRI). However, the contribution of intracellular HMGB1 to LT graft injury remains elusive. We hypothesized that intracellular neutrophil-derived HMGB1 from recipients protects from post-LT EAD. APPROACH AND RESULTS: We generated mice with conditional ablation or overexpression of Hmgb1 in hepatocytes, myeloid cells, or both. We performed LTs and injected lipopolysaccharide (LPS) to evaluate the effect of intracellular HMGB1 in EAD. Ablation of Hmgb1 in hepatocytes and myeloid cells of donors and recipients exacerbated early allograft injury after LT. Ablation of Hmgb1 from liver grafts did not affect graft injury; however, lack of Hmgb1 from recipient myeloid cells increased reactive oxygen species (ROS) and inflammation in liver grafts and exacerbated injury. Neutrophils lacking HMGB1 were more activated, showed enhanced pro-oxidant and pro-inflammatory signatures, and reduced biosynthesis and metabolism of inositol polyphosphates (InsPs). On LT reperfusion or LPS treatment, there was significant neutrophil mobilization and infiltration into the liver and enhanced production of ROS and pro-inflammatory cytokines when intracellular Hmgb1 was absent. Depletion of neutrophils using anti-Ly6G antibody attenuated graft injury in recipients with myeloid cell Hmgb1 ablation. CONCLUSIONS: Neutrophil HMGB1 derived from recipients is central to regulate their activation, limits the production of ROS and pro-inflammatory cytokines, and protects from early liver allograft injury.

Hepatocellular carcinomas, exhibiting intratumor fibrosis, express cancer-specific extracellular matrix remodeling and WNT/TGFB signatures, associated with poor outcome.

BACKGROUND AND AIMS: HCC, the third leading cause of cancer-related death, arises in the context of liver fibrosis. Although HCC is generally poorly fibrogenic, some tumors harbor focal intratumor extracellular matrix (ECM) deposits called "fibrous nests." To date, the molecular composition and clinical relevance of these ECM deposits have not been fully defined. APPROACH AND RESULTS: We performed quantitative matrisome analysis by tandem mass tags mass spectrometry in 20 human cancer specific matrisome (HCCs) with high or low-grade intratumor fibrosis and matched nontumor tissues, as well as in 12 livers from mice treated with vehicle, carbon tetrachloride, or diethylnitrosamine. We found 94 ECM proteins differentially abundant between high and low-grade fibrous nests, including interstitial and basement membrane components, such as several collagens, glycoproteins, proteoglycans, enzymes involved in ECM stabilization and degradation, and growth factors. Pathway analysis revealed a metabolic switch in high-grade fibrosis, with enhanced glycolysis and decreased oxidative phosphorylation. Integrating the quantitative proteomics with transcriptomics from HCCs and nontumor livers (n = 2,285 samples), we identified a subgroup of fibrous nest HCCs, characterized by cancer-specific ECM remodeling, expression of the WNT/TGFB (S1) subclass signature, and poor patient outcome. Fibrous nest HCCs abundantly expressed an 11-fibrous-nest - protein signature, associated with poor patient outcome, by multivariate Cox analysis, and validated by multiplex immunohistochemistry. CONCLUSIONS: Matrisome analysis highlighted cancer-specific ECM deposits, typical of the WNT/TGFB HCC subclass, associated with poor patient outcomes. Hence, histologic reporting of intratumor fibrosis in HCC is of clinical relevance.

Matrisome gene-based subclassification of patients with liver fibrosis identifies clinical and molecular heterogeneities.

BACKGROUND AIMS: Excessive deposition and crosslinking of extracellular matrix increases liver density and stiffness, promotes fibrogenesis, and increases resistance to fibrinolysis. An emerging therapeutic opportunity in liver fibrosis is to target the composition of the extracellular matrix or block pathogenic communication with surrounding cells. However, the type and extent of extracellular changes triggering liver fibrosis depend on the underlying etiology. Our aim was to unveil matrisome genes not dependent on etiology, which are clinically relevant to liver fibrosis. APPROACH RESULTS: We used transcriptomic profiles from liver fibrosis cases of different etiologies to identify and validate liver fibrosis-specific matrisome genes (LFMGs) and their clinical and biological relevance. Dysregulation patterns and cellular landscapes of LFMGs were further explored in mouse models of liver fibrosis progression and regression by bulk and single-cell RNA sequencing. We identified 35 LFMGs, independent of etiology, representing an LFMG signature defining liver fibrosis. Expression of the LFMG signature depended on histological severity and was reduced in regressive livers. Patients with liver fibrosis, even with identical pathological scores, could be subclassified into LFMG Low and LFMG High , with distinguishable clinical, cellular, and molecular features. Single-cell RNA sequencing revealed that microfibrillar-associated protein 4 + activated HSC increased in LFMG High patients and were primarily responsible for the LFMG signature expression and dysregulation. CONCLUSIONS: The microfibrillar-associated protein 4 + -activated HSC-derived LFMG signature classifies patients with liver fibrosis with distinct clinical and biological characteristics. Our findings unveil hidden information from liver biopsies undetectable using traditional histologic assessments.

Dynamics of dissociative electron attachment to aliphatic thiols.

Dissociative electron attachment (DEA) shows functional group-dependent site selectivity in H- ion channels. In this context, thiol functional groups have yet to be studied in great detail, although they carry importance in radiation damage studies where low-energy secondary electrons are known to induce damage through the DEA process. In this context, we report detailed measurements of absolute cross-sections and momentum images of various anion fragments formed in the DEA process in simple aliphatic thiols. We also compare the observed dynamics with that reported earlier in hydrogen sulphide, the precursor molecule for this functional group, and with that in aliphatic alcohols. Our findings show substantial resemblance in the underlying dynamics in these compounds and point to a possible generalisation of these features in the DEA to thiols. In addition, we identify various pathways that contribute to the S- and SH- channels.

Anxiety and Stress Related to COVID-19 Among the Community Dwelling Older Adults Residing in the Largest Refugee Camp of the World.

The current cross-sectional study was conducted among 864 older adults aged ≥ 60 years residing in Rohingya refugee camp through face-to-face interviews during November-December 2021. COVID-19-related anxiety was measured using the five-point Coronavirus Anxiety Scale (CAS) and perceived stress using the 10-point Perceived Stress Scale (PSS). The linear regression model identified the factors associated with COVID-19-related anxiety and perceived stress. The prevalence of COVID-19-related anxiety and perceived stress was 68% and 93%, respectively. The average COVID-19-related anxiety score expected to be significantly higher among those who were physically inactive, concerned about COVID-19, had a close friend/family member diagnosed with COVID-19, and had some difficulty in getting food and routine medical care during the COVID-19 pandemic. Meanwhile, the average perceived stress score was expected to be significantly higher among those without partners, who were feeling overwhelmed by COVID-19, and who experienced COVID-19-related anxiety during the pandemic. The findings suggest providing immediate psychosocial support to older Rohingya adults.

Osteopontin Takes Center Stage in Chronic Liver Disease.

Osteopontin (OPN) was first identified in 1986. The prefix osteo- means bone; however, OPN is expressed in other tissues, including liver. The suffix -pontin means bridge and denotes the role of OPN as a link protein within the extracellular matrix. While OPN has well-established physiological roles, multiple "omics" analyses suggest that it is also involved in chronic liver disease. In this review, we provide a summary of the OPN gene and protein structure and regulation. We outline the current knowledge on how OPN is involved in hepatic steatosis in the context of alcoholic liver disease and non-alcoholic fatty liver disease. We describe the mechanisms whereby OPN participates in inflammation and liver fibrosis and discuss current research on its role in hepatocellular carcinoma and cholangiopathies. To conclude, we highlight important points to consider when doing research on OPN and provide direction for making progress on how OPN contributes to chronic liver disease.

Molecular surveillance for operationally relevant genetic polymorphisms in Plasmodium falciparum in Southern Chad, 2016-2017.

BACKGROUND: Resistance to anti-malarials is a serious threat to the efforts to control and eliminate malaria. Surveillance based on simple field protocols with centralized testing to detect molecular markers associated with anti-malarial drug resistance can be used to identify locations where further investigations are needed. METHODS: Dried blood spots were collected from 398 patients (age range 5-59 years, 99% male) with Plasmodium falciparum infections detected using rapid diagnostic tests over two rounds of sample collection conducted in 2016 and 2017 in Komé, South-West Chad. Specimens were genotyped using amplicon sequencing or qPCR for validated markers of anti-malarial resistance including partner drugs used in artemisinin-based combination therapy (ACT). RESULTS: No mutations in the pfk13 gene known to be associated with artemisinin resistance were found but a high proportion of parasites carried other mutations, specifically K189T (190/349, 54.4%, 95%CI 49.0-59.8%). Of 331 specimens successfully genotyped for pfmdr1 and pfcrt, 52% (95%CI 46.4-57.5%) carried the NFD-K haplotype, known to be associated with reduced susceptibility to lumefantrine. Only 20 of 336 (6.0%, 95%CI 3.7-9.0%) had parasites with the pfmdr1-N86Y polymorphism associated with increased treatment failures with amodiaquine. Nearly all parasites carried at least one mutation in pfdhfr and/or pfdhps genes but 'sextuple' mutations in pfdhfr-pfdhps including pfdhps -A581G were rare (8/336 overall, 2.4%, 95%CI 1.2-4.6%). Only one specimen containing parasites with pfmdr1 gene amplification was detected. CONCLUSIONS: These results provide information on the likely high efficacy of artemisinin-based combinations commonly used in Chad, but suggest decreasing levels of sensitivity to lumefantrine and high levels of resistance to sulfadoxine-pyrimethamine used for seasonal malaria chemoprevention and intermittent preventive therapy in pregnancy. A majority of parasites had mutations in the pfk13 gene, none of which are known to be associated with artemisinin resistance. A therapeutic efficacy study needs to be conducted to confirm the efficacy of artemether-lumefantrine.

COVID-19 related anxiety and its associated factors: a cross-sectional study on older adults in Bangladesh.

BACKGROUND: The COVID-19 pandemic has resulted in serious mental health conditions, particularly among older adults. This research explored the prevalence of COVID-19-related anxiety and its associated factors among older adults residing in Bangladesh. METHODS: This cross-sectional study was conducted among 1,045 older Bangladeshi adults aged ≥ 60 years through telephone interviews in September 2021. A semi-structured interview schedule was used to collect data on participants' characteristics and COVID-19-related anxiety. The anxiety level was measured using the Bengali version of the five-point Coronavirus Anxiety Scale (CAS). A linear regression model explored the factors associated with COVID-19-related anxiety. RESULTS: Overall, the prevalence of COVID-19-related anxiety was 23.2%. The regression analysis revealed that the average COVID-19-related anxiety score was significantly higher among females (ß: 0.43, 95% CI: 0.05 to 0.81), and among those who faced difficulty getting medicine (ß: 0.57, 95% CI: 0.16 to 0.97), felt isolated (ß: 0.60, 95% CI: 0.24 to 0.95), and felt requiring additional care during the pandemic (ß: 0.53, 95% CI: 0.16 to 0.91). Alternatively, the average COVID-19-related anxiety score was significantly lower among those who were widowed (ß: -0.46, 95% CI: -0.87 to -0.04) and living distant from the health centre (ß: -0.48, 95% CI: -0.79 to -0.17). CONCLUSION: The findings of the present study suggest providing immediate psychosocial support package to the older adults, particularly females and those who are vulnerable to receive health and social care support during the COVID-19 pandemic in Bangladesh.

The pace of secular changes of body measurements of children and adolescents from Kolkata (India) in the context of socioeconomic inequalities between the sexes.

OBJECTIVES: To examine the pace of secular changes of selected body measurements and proportions of children and adolescents from Kolkata (India), between 1952-1966 and 1999-2011 in the context of differences between the sexes. METHODS: The study group consisted of 7753 children, adolescents and young adults (7-21 years of age) included in two series of studies (1952-1966 and 2005-2011). The measurements included: body height, sitting height, biacromial and biiliocristal diameters, as well as body mass. Additionally, subishial leg length was derived. The pace of the observed intergenerational trends was estimated on the basis of the differences of the mean values of the analyzed characteristics between both cohorts and expressed as the change of a given parameter for a decade. Information regarding the educational and professional status of the parents of participants was obtained using a questionnaire. RESULTS: In the majority of the analyzed characteristics, the pace of intergenerational changes was significantly higher among males, in comparison to females. It was visible especially during adolescence-between 11 and 19 years of age. The positive trends, especially, for characteristics such as body height or limb lengths occurred significantly quicker in males, in comparison to females. CONCLUSIONS: There were significant differences between the sexes in the pace of secular changes regarding the growth of the examined population. Considering the relatively homogenous economic situation of the families of the participants, it was more likely that those discrepancies effected from the social and domestic division of sexes, and the resulting differences in growth and development conditions.

Time trends in mid-upper-arm anthropometry from 1982 to 2011 in male children and adolescents from Kolkata, India.

The aim of this study was to investigate inter-generational changes in selected mid-upper-arm measurements of boys from Kolkata, India. The analysis was based on the anthropometric measurements of two cohorts of Bengali boys aged 7-16 from middle-class families, in 1982-83 and 2005-11. The two cohorts were compared in terms of their mid-upper-arm circumference (MUAC) and mid-upper-arm area (MUAA), mid-upper-arm muscle area (MUAMA), mid-upper-arm fat area (MUAFA) and Arm Fat Index (AFI). The significances of the differences were determined using two-way ANOVA. All features differed significantly between the examined cohorts and all showed a general positive secular trend. In most cases, the biggest differences were noted for 14- and 16-year olds and the smallest for the youngest boys. The contemporary boys seemed to have more favourable overall developmental conditions, probably related to socioeconomic progress in India over recent decades.

Danger signals in liver injury and restoration of homeostasis.

Damage-associated molecular patterns are signalling molecules involved in inflammatory responses and restoration of homeostasis. Chronic release of these molecules can also promote inflammation in the context of liver disease. Herein, we provide a comprehensive summary of the role of damage-associated molecular patterns as danger signals in liver injury. We consider the role of reactive oxygen species and reactive nitrogen species as inducers of damage-associated molecular patterns, as well as how specific damage-associated molecular patterns participate in the pathogenesis of chronic liver diseases such as alcohol-related liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cancer. In addition, we discuss the role of damage-associated molecular patterns in ischaemia reperfusion injury and liver transplantation and highlight current studies in which blockade of specific damage-associated molecular patterns has proven beneficial in humans and mice.

Secular changes in limb lengths and proportions from 1952 to 2011 in children, adolescents, and young adults from Kolkata (India).

OBJECTIVES: The aim of the study was to investigate the intergenerational changes of upper and lower limb lengths as well as the values of the upper, lower limb, and intermembral indicators of children, adolescents, and young adults from Kolkata (India) between 1952 to 1966 and 2005 to 2011. METHODS: The analysis was based on the results of anthropometric measurements of 7488 Bengali children, adolescents, and young adults. They were included in three cross-sectional surveys, carried out in 1955 to 1966, 1982 to 1983 (only males), and 2005 to 2011. The upper and lower limb lengths were obtained and the upper and lower limb indicators, as well as an intermembral index, were calculated. The differences between the cohorts were assessed using two-way ANOVA. RESULTS: Positive, statistically significant, secular trends regarding the length of the lower and upper limbs as well as the value of the lower limb index were observed. Negative intergenerational changes were noted for the values of the upper limb indicator and intermembral index, suggesting that the secular increase of the length of the upper limbs was less pronounced than those of the body height and lower limbs length. CONCLUSIONS: The secular increase regarding the lower limbs length was associated with the socioeconomic progress of the country, but the length of the upper limbs was less sensitive for those factors. It is also important to mention that there is still very little information on those characteristics in general, which further proves the need for similar studies.

Modification Patterns of Urinary Albumin Correlates With Serum Albumin and Outcome in Severe Alcoholic Hepatitis.

BACKGROUND AND AIMS: Albumin modifications and deranged functions are well documented in serum of severe alcoholic hepatitis (SAH). We investigated whether urinary albumin (u-Alb) can serve as surrogate marker of circulatory albumin phenotype, functionality, and could predict outcome in SAH patients. PATIENTS AND METHODS: Baseline serum and urine samples from 100 SAH, 20 alcoholic cirrhosis, and 20 healthy controls were subjected to u-Alb, ischemia modified albumin (IMA), IMA to albumin ratio (IMAr), advanced oxidation protein products, advanced glycation end-products, albumin-binding capacity determination. In addition, SAH urinary samples were also analyzed at day 4 and day 7 to predict nonresponse to corticosteroid therapy. RESULTS: Urine and serum levels of IMA, advanced oxidation protein products and advanced glycation end-products were higher (P<0.05) in SAH versus alcoholic cirrhosis and healthy controls. IMAr was low in urine but high in serum of SAH (P<0.05). Albumin-binding capacity was lower (P<0.05) in both urinary and serum albumin of SAH. Urinary and serum albumin parameters showed direct correlation, whereas IMAr showed inverse correlation (cc>0.2, P<0.05). Baseline u-Alb level was significantly higher in SAH, and was correlated directly with corticosteroid treatment outcome and 12-month mortality in SAH. Baseline u-Alb showed an area under the receivers operating curve analysis of 0.7 and a hazard ratio of 1.23 for prediction of 12-month mortality in SAH. Baseline u-Alb level >9.0 mg/dL was associated with reduced 12-month survival in SAH (log rank <0.01). CONCLUSIONS: u-Alb modifications are reflective of serum albumin modifications. Further baseline u-Alb levels could be exploited to predict steroid response and mortality in SAH patients.

Anthropometric variations in different BMI and adiposity levels among children, adolescents and young adults in Kolkata, India.

The objective of the study was to analyse selected anthropometric features of children, adolescents and young adults from middle-class families in Kolkata, India, by BMI and adiposity categories. Standardized anthropometric measurements of 4194 individuals (1999 male and 2195 female) aged 7-21 were carried out between the years 2005 and 2011. The results were compared by BMI and adiposity categories. Statistical significance was assessed using two-way-ANOVA and linear regression analysis was performed. The study population could be differentiated in terms of BMI and adiposity categories for all examined anthropometric characteristics (p ≤ 0.001). After taking age into consideration, differences were observed for males in the case of body height and humerus breadth in BMI and adiposity categories, and for femur breadth in the case of adiposity categories. For females, differences were noted in body height measurements in BMI and adiposity categories, a sum of skinfold thicknesses in BMI categories, and upper-arm and calf circumferences in adiposity categories. The patterns of differences in the BMI categories were found to be similar to those in adiposity categories. The linear regression analysis results showed that there was a significant relationship between BMI and body fat ratio in the examined population. Underweight individuals, and those with low adiposity, were characterized by lower extremity circumferences and skeletal breadths. These features reached highest values in overweight/obese persons, characterized by high body fat. However, the differences observed between each BMI and adiposity category, in most cases, were only present in early childhood.

BMI and adiposity based approach to obesity: the need for ethnic specificity. A reply to Kapoor et al. (2019).

The ethnicity of the studied group is one of the key characteristics that should be taken into consideration when analysing the problem of overweight and obesity. It is especially crucial in populations of countries such as India, where the proportion of the fat to lean mass and general adiposity are significantly different from those observed among Europeans. This can cause a higher risk of various metabolic-related diseases to appear at relatively lower absolute adiposity. Therefore, there is a need for further research regarding the issues of body mass and composition in Indian populations, to obtain additional information as well as to develop ethnically specific cut-off points.

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.

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).

Dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute-on-chronic liver failure.

UNLABELLED: Acute-on-chronic liver failure (ACLF) is an ailment with high incidence of multiorgan failure (MOF) and consequent mortality. Dysregulated iron homeostasis and macrophage dysfunction are linked to increased incidence of MOF. We investigated whether a panel of circulating iron-regulating proteins are associated with development of MOF and can predict 15- or 30-day mortality in ACLF patients. One hundred twenty patients with ACLF, 20 patients with compensated cirrhosis, and 20 healthy controls were studied. Relative protein expression profiling was performed in the derivative cohort and confirmed in the validation cohort. A panel of iron regulators and indices were determined. Multiparametric flow cytometry for quantitation of labile iron pool (LIP) was performed. Validation studies confirmed lower serum transferrin (Tf) and ceruloplasmin levels in ACLF and ACLF-MOF, compared to patients with cirrhosis and controls (P < 0.01). Serum iron and ferritin levels were markedly elevated (P < 0.001; P < 0.05) and hepcidin levels were lower (P < 0.001) in ACLF patients with MOF than those without and other groups (P < 0.001). Percentage Tf saturation (%SAT) was higher in ACLF-MOF (39.2%; P < 0.001) and correlated with poor outcome (hazard ratio: 6.970; P < 0.01). Intracellular LIP indices were significantly elevated in the subsets of circulating macrophages in ACLF-MOF, compared to other groups (P < 0.01). Whereas expression of iron-regulatory genes was markedly down-regulated, genes related to endoplasmic reticulum stress, apoptosis, and inflammation were up-regulated in ACLF patients, compared to patients with cirrhosis. Severe dysregulation of autophagy mechanisms was also observed in the former. CONCLUSIONS: Iron metabolism and transport are severely deranged in ACLF patients and more so in those with MOF. %SAT, circulating hepcidin, and LIP in macrophages correlate with disease severity and %SAT could be used for early prognostication in ACLF patients.