Abnormal liver chemistries as a predictor of COVID-19 severity and clinical outcomes in hospitalized patients.

BACKGROUND: Abnormal liver chemistries are common findings in patients with Coronavirus Disease 2019 (COVID-19). However, the association of these abnormalities with the severity of COVID-19 and clinical outcomes is poorly understood. AIM: We aimed to assess the prevalence of elevated liver chemistries in hospitalized patients with COVID-19 and compare the serum liver chemistries to predict the severity and in-hospital mortality. METHODS: This retrospective, observational study included 3380 patients with COVID-19 who were hospitalized in the Johns Hopkins Health System (Baltimore, MD, United States). Demographic data, clinical characteristics, laboratory findings, treatment measures, and outcome data were collected. Cox regression modeling was used to explore variables associated with abnormal liver chemistries on admission with disease severity and prognosis. RESULTS: A total of 2698 (70.4%) had abnormal alanine aminotransferase (ALT) at the time of admission. Other more prevalent abnormal liver chemistries were aspartate aminotransferase (AST) (44.4%), alkaline phosphatase (ALP) (16.1%), and total bilirubin (T-Bil) (5.9%). Factors associated with liver injury were older age, Asian ethnicity, other race, being overweight, and obesity. Higher ALT, AST, T-Bil, and ALP levels were more commonly associated with disease severity. Multivariable adjusted Cox regression analysis revealed that abnormal AST and T-Bil were associated with the highest mortality risk than other liver injury indicators during hospitalization. Abnormal AST, T-Bil, and ALP were associated with a need for vasopressor drugs, whereas higher levels of AST, T-Bil, and a decreased albumin levels were associated with mechanical ventilation. CONCLUSION: Abnormal liver chemistries are common at the time of hospital admission in COVID-19 patients and can be closely related to the patient's severity and prognosis. Elevated liver chemistries, specifically ALT, AST, ALP, and T-Bil levels, can be used to stratify risk and predict the need for advanced therapies in these patients.

Global burden of disease: acute-on-chronic liver failure, a systematic review and meta-analysis.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is characterised by acute decompensation of cirrhosis associated with organ failures. We systematically evaluated the geographical variations of ACLF across the world in terms of prevalence, mortality, aetiology of chronic liver disease (CLD), triggers and organ failures. METHODS: We searched EMBASE and PubMed from 3/1/2013 to 7/3/2020 using the ACLF-EASL-CLIF (European Association for the Study of the Liver-Chronic Liver Failure) criteria. Two investigators independently conducted the abstract selection/abstraction of the aetiology of CLD, triggers, organ failures and prevalence/mortality by presence/grade of ACLF. We grouped countries into Europe, East/South Asia and North/South America. We calculated the pooled proportions, evaluated the methodological quality using the Newcastle-Ottawa Scale and statistical heterogeneity, and performed sensitivity analyses. RESULTS: We identified 2369 studies; 30 cohort studies met our inclusion criteria (43 206 patients with ACLF and 140 835 without ACLF). The global prevalence of ACLF among patients admitted with decompensated cirrhosis was 35% (95% CI 33% to 38%), highest in South Asia at 65%. The global 90-day mortality was 58% (95% CI 51% to 64%), highest in South America at 73%. Alcohol was the most frequently reported aetiology of underlying CLD (45%, 95% CI 41 to 50). Infection was the most frequent trigger (35%) and kidney dysfunction the most common organ failure (49%). Sensitivity analyses showed regional estimates grossly unchanged for high-quality studies. Type of design, country health index, underlying CLD and triggers explained the variation in estimates. CONCLUSIONS: The global prevalence and mortality of ACLF are high. Region-specific variations could be explained by the type of triggers/aetiology of CLD or grade. Health systems will need to tailor early recognition and treatment of ACLF based on region-specific data.

Augmented Liver Uptake of the Membrane Voltage Sensor Tetraphenylphosphonium Distinguishes Early Fibrosis in a Mouse Model.

Mitochondrial (mito-) oxidative phosphorylation (OxPhos) is a critical determinant of cellular membrane potential/voltage. Dysregulation of OxPhos is a biochemical signature of advanced liver fibrosis. However, less is known about the net voltage of the liver in fibrosis. In this study, using the radiolabeled [3H] voltage sensor, tetraphenylphosphonium (TPP), which depends on membrane potential for cellular uptake/accumulation, we determined the net voltage of the liver in a mouse model of carbon tetrachloride (CCl4)-induced hepatic fibrosis. We demonstrated that the liver uptake of 3H-TPP significantly increased at 4 weeks of CCl4-administration (6.07 ± 0.69% ID/g, p < 0.05) compared with 6 weeks (4.85 ± 1.47% ID/g) and the control (3.50 ± 0.22% ID/g). Analysis of the fibrosis, collagen synthesis, and deposition showed that the increased 3H-TPP uptake at 4 weeks corresponds to early fibrosis (F1), according to the METAVIR scoring system. Biodistribution data revealed that the 3H-TPP accumulation is significant in the fibrogenic liver but not in other tissues. Mechanistically, the augmentation of the liver uptake of 3H-TPP in early fibrosis concurred with the upregulation of mito-electron transport chain enzymes, a concomitant increase in mito-oxygen consumption, and the activation of the AMPK-signaling pathway. Collectively, our results indicate that mito-metabolic response to hepatic insult may underlie the net increase in the voltage of the liver in early fibrosis.

Human primary liver cancer organoids reveal intratumor and interpatient drug response heterogeneity.

Liver cancer is the fourth leading cause of cancer-related mortality and is distinguished by a relative paucity of chemotherapy options. It has been hypothesized that intratumor genetic heterogeneity may contribute to the high failure rate of chemotherapy. Here, we evaluated functional heterogeneity in a cohort of primary human liver cancer organoid lines. Each primary human liver cancer surgical specimen was used to generate multiple cancer organoid lines, obtained from distinct regions of the tumor. A total of 27 liver cancer lines were established and tested with 129 cancer drugs, generating 3,483 cell survival data points. We found a rich intratumor, functional (drug response) heterogeneity in our liver cancer patients. Furthermore, we established that the majority of drugs were either ineffective, or effective only in select organoid lines. In contrast, we found that a subset of drugs appeared pan-effective, displaying at least moderate activity in the majority of these cancer organoid lines. These drugs, which are FDA approved for indications other than liver cancers, deserve further consideration as either systemic or local therapeutics. Of note, molecular profiles, obtained for a reduced sample set, did not correlate with the drug response heterogeneity of liver cancer organoid lines. Taken together, these findings lay the foundation for in-depth studies of pan-effective drugs, as well as for functional personalized oncology approaches. Lastly, these functional studies demonstrate the utility of cancer organoid drug testing as part of a drug discovery pipeline.

Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin.

Activation of hepatic stellate cells (HSCs) is an integral component of the wound-healing process in liver injury/inflammation. However, uncontrolled activation of HSCs leads to constant secretion of collagen-rich extracellular matrix (ECM) proteins, resulting in liver fibrosis. The enhanced ECM synthesis/secretion demands an uninterrupted supply of intracellular energy; however, there is a paucity of data on the bioenergetics, particularly the mitochondrial (mito) metabolism of fibrogenic HSCs. Here, using human and rat HSCs in vitro, we show that the mito-respiration, mito-membrane potential (Δψm) and cellular 'bioenergetic signature' distinguish fibrogenic HSCs from normal, less-active HSCs. Ex vivo, HSCs from mouse and rat models of liver fibrosis further confirmed the altered 'bioenergetic signature' of fibrogenic HSCs. Importantly, the distinctive elevation in mito-Δψm sensitized fibrogenic HSCs for selective inhibition by mitotropic doxorubicin while normal, less-active HSCs and healthy human primary hepatocytes remained minimally affected if not, unaffected. Thus, the increased mito-Δψm may provide an opportunity to selectively target fibrogenic HSCs in liver fibrosis.

Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury.

Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.

Increased ethanol-inducible cytochrome P450-2E1 and cytochrome P450 isoforms in exosomes of alcohol-exposed rodents and patients with alcoholism through oxidative and endoplasmic reticulum stress.

This study investigated the role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in enhancing CYP2E1 and other P450 proteins in extracellular vesicles (EVs) from alcohol-exposed rodents and human patients with alcoholism and their effects on oxidative hepatocyte injury. Female Fischer rats and wild-type or Cyp2e1-null mice were exposed to three oral doses of binge ethanol or dextrose control at 12-hour intervals. Plasma EV and hepatic proteins from alcohol-exposed rodents, patients with alcoholism, and their respective controls were isolated and characterized. The number of EVs and the amounts of EV CYP2E1, CYP2A, CYP1A1/2, and CYP4B proteins were markedly elevated in both patients with alcoholism and alcohol-exposed rats and mice. The number of EVs and EV P450 proteins were significantly reduced in ethanol-exposed rats fed a diet containing polyunsaturated fatty acids. The increased number of EVs and EV CYP2E1 and other P450 isoforms in alcohol-exposed wild types were significantly reduced in the corresponding Cyp2e1-null mice. EV CYP2E1 amounts depended on increased oxidative and endoplasmic reticulum (ER) stress because their levels were decreased by cotreatment with the antioxidant N-acetylcysteine or the CYP2E1 inhibitor chlormethiazole but increased by ER stress-inducer thapsigargin, which was blocked by 4-phenylbutyric acid. Furthermore, cell death rates were elevated when primary hepatocytes or human hepatoma cells were exposed to EVs from alcohol-exposed rodents and patients with alcoholism, demonstrating that EVs from alcohol-exposed rats and patients with alcoholism are functional and can promote cell death by activating the apoptosis signaling pathway, including phospho-c-Jun N-terminal kinase, proapoptotic Bax, and activated caspase-3. Conclusion: CYP2E1 has an important role in elevating EV CYP2E1 and other P450 isoforms through increased oxidative and ER stress. Elevated EV-CYP2E1 detected after withdrawal from alcohol or exposure to the CYP2E1 inducer pyrazole can be a potential biomarker for liver injury. (Hepatology Communications 2017;1:675-690).

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

The Pathogenic Role of NLRP3 Inflammasome Activation in Inflammatory Bowel Diseases of Both Mice and Humans.

BACKGROUND AND AIMS: NLRP3 inflammasome is known to be involved in inflammatory bowel diseases. However, it is controversial whether it is pathogenic or beneficial. This study evaluated the roles of NLRP3 inflammasome in the pathogenesis of inflammatory bowel disease in IL-10-/- mice and humans. METHODS: NLRP3 inflammasome in colonic mucosa, macrophages, and colonic epithelial cells were analysed by western blotting. The NLRP3 inflammasome components were studied by sucrose density gradient fractionation, chemical cross-linking, and co-immunoprecipitation. The role of NLPR3 inflammasome in the pathogenesis of colitis was extensively evaluated in IL-10-/- mice, using a specific NLPR3 inflammasome inhibitor glyburide. RESULTS: NLRP3 inflammasome was upregulated in colonic mucosa of both IL-10-/- mice and Crohn's patients. NLRP3 inflammasome activity in IL-10-/- mice was elevated prior to colitis onset; it progressively increased as disease worsened and peaked as macroscopic disease emerged. NLRP3 inflammasome was found in both intestinal epithelial cells and colonic macrophages, as a large complex with a molecular weight of ≥ 360 kDa in size. In the absence of IL-10, NLRP3 inflammasome was spontaneously active and more robustly responsive when activated by LPS and nigericin. Glyburide markedly suppressed NLRP3 inflammasome expression/activation in IL-10-/- mice, leading to not only alleviation of ongoing colitis but also prevention/delay of disease onset. Glyburide also effectively inhibited the release of proinflammatory cytokines/chemokines by mucosal explants from Crohn's patients. CONCLUSIONS: Abnormal activation of NLRP3 inflammasome plays a major pathogenic role in the development of chronic colitis in IL-10-/- mice and humans. Glyburide, an FDA-approved drug, may have great potential in the management of inflammatory bowel diseases.

Current Management of Alcoholic Hepatitis and Future Therapies.

Alcohol is one of the most common etiologies of liver disease, and alcoholic liver disease overall is the second most common indication for liver transplantation in the United States. It encompasses a spectrum of disease, including fatty liver disease, alcoholic hepatitis (AH), and alcoholic cirrhosis. AH can range from mild to severe disease, with severe disease being defined as: Discriminant Function (DF) ≥ 32, or Model for End-stage Liver Disease (MELD) ≥ 21, or presence of hepatic encephalopathy. Management of the mild disease consists mainly of abstinence and supportive care. Severe AH is associated with significant mortality. Currently, there is no ideal medical treatment for this condition. Besides alcohol cessation, corticosteroids have been used with conflicting results and are associated with an inherent risk of infection. Overall steroids have shown short term benefit when compared to placebo, but they have no obvious long term benefits. Pentoxifylline does not improve survival in patients with severe AH and is no longer recommended based on the results of the STOPAH (Steroid Or Pentoxifylline for Alcoholic Hepatitis) trial. Anti-tumor necrosis factor (TNF) agents are associated with increased risk of life threatening infections and death. Currently, early stage trials are underway, mainly targeting novel pathways based on disease pathogenesis, including modulation of innate immune system, inhibition of gut-liver axis and cell death pathways, and activation of transcription factor farnesyl X receptor (FXR). Future treatment may lie in human induced pluripotent stem cell (iPSC) technology, which is currently under investigation for the study of pathogenesis, drug discovery, and stem cell transplantation. Liver transplantation has been reported with good results in highly selected patients but is controversial due to limited organ supply.

Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model.

Liver fibrosis and cirrhosis result from uncontrolled secretion and accumulation of extracellular matrix (ECM) proteins by hepatic stellate cells (HSCs) that are activated by liver injury and inflammation. Despite the progress in understanding the biology liver fibrogenesis and the identification of potential targets for treating fibrosis, development of an effective therapy remains elusive. Since an uninterrupted supply of intracellular energy is critical for the activated-HSCs to maintain constant synthesis and secretion of ECM, we hypothesized that interfering with energy metabolism could affect ECM secretion. Here we report that a sublethal dose of the energy blocker, 3-bromopyruvate (3-BrPA) facilitates phenotypic alteration of activated LX-2 (a human hepatic stellate cell line), into a less-active form. This treatment-dependent reversal of activated-LX2 cells was evidenced by a reduction in α-smooth muscle actin (α-SMA) and collagen secretion, and an increase in activity of matrix metalloproteases. Mechanistically, 3-BrPA-dependent antifibrotic effects involved down-regulation of the mitochondrial metabolic enzyme, ATP5E, and up-regulation of glycolysis, as evident by elevated levels of lactate dehydrogenase, lactate production and its transporter, MCT4. Finally, the antifibrotic effects of 3-BrPA were validated in vivo in a mouse model of carbon tetrachloride-induced liver fibrosis. Results from histopathology & histochemical staining for collagen and α-SMA substantiated that 3-BrPA promotes antifibrotic effects in vivo. Taken together, our data indicate that sublethal, metronomic treatment with 3-BrPA blocks the progression of liver fibrosis suggesting its potential as a novel therapeutic for treating liver fibrosis.

Activation of liver X receptor/retinoid X receptor pathway ameliorates liver disease in Atp7B(-/-) (Wilson disease) mice.

UNLABELLED: Wilson disease (WD) is a hepatoneurological disorder caused by mutations in the copper-transporter, ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurological symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified dysregulation of nuclear receptors (NRs), especially the liver X receptor (LXR)/retinoid X receptor heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist, T0901317, ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized, and liver function and histology were improved. CONCLUSIONS: The results demonstrate the major role of an altered NR function in the pathogenesis of WD and suggest that modulation of NR activity should be explored as a supplementary approach to improving liver function in WD. (Hepatology 2016;63:1828-1841).

MicroRNA-224 Induces G1/S Checkpoint Release in Liver Cancer.

Profound changes in microRNA (miR) expression levels are frequently found in liver cancers compared to the normal liver. In this study, we evaluate the expression of miR-224 in human HCC and CCA, as well as its downstream targets and affected pathways. We show that miR-224 is upregulated in a large cohort of human CCA, similar to its upregulation in human HCC. For the purpose of studying the roles of miR-224 in HCC and CCA, we enforced miR-224 expression in cells. mRNA arrays followed by Ingenuity Pathway Analysis (IPA)-identified putative molecules and pathways downstream of miR-224. Phenotypically, we report that enforced expression of miR-224 increases the growth rate of normal cholangiocytes, CCA cell lines, and HCC cell lines. In addition, we identified, in an unbiased fashion, that one of the major biologic processes affected by miR-224 is Gap1 (G1) to Synthesis (S) transition checkpoint release. We next identified p21, p15, and CCNE1 as downstream targets of miR-224 and confirmed the coordinated downregulation results in the increased phosphorylation of Retinoblastoma (Rb) with resulting G1/S checkpoint release. Our data suggest that miR-224 is a master regulator of cell cycle progression, and that its overexpression results in G1/S checkpoint release followed by accelerated cell growth.

Pseudogene INTS6P1 regulates its cognate gene INTS6 through competitive binding of miR-17-5p in hepatocellular carcinoma.

The complex regulation of tumor suppressive gene and its pseudogenes play key roles in the pathogenesis of hepatocellular cancer (HCC). However, the roles played by pseudogenes in the pathogenesis of HCC are still incompletely elucidated. This study identifies the putative tumor suppressor INTS6 and its pseudogene INTS6P1 in HCC through the whole genome microarray expression. Furthermore, the functional studies - include growth curves, cell death, migration assays and in vivo studies - verify the tumor suppressive roles of INTS6 and INTS6P1 in HCC. Finally, the mechanistic experiments indicate that INTS6 and INTS6P1 are reciprocally regulated through competition for oncomiR-17-5p. Taken together, these findings demonstrate INTS6P1 and INTS6 exert the tumor suppressive roles through competing for oncomiR-17-5p. Our investigation of this regulatory circuit reveals novel insights into the underlying mechanisms of hepatocarcinogenesis.

Assessing liver fibrosis: comparison of arterial enhancement fraction and diffusion-weighted imaging.

PURPOSE: Noninvasive markers have been developed to reduce the need for liver biopsy. The aim of this study was to compare the strength of association of the arterial enhancement fraction (AEF), apparent diffusion coefficient (ADC), and serum biomarkers for staging hepatic fibrosis. MATERIALS AND METHODS: Eighty-five patients with chronic liver disease underwent triple-phase contrast-enhanced MRI, used to calculate AEF, and diffusion-weighted MRI (b = 0,750 s/mm(2) ), used to calculate ADC. Hepatic fibrosis was staged according METAVIR criteria. The overall association of the four biomarkers (AEF, ADC, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, and aspartate aminotransferase to platelet ratio index [APRI]) was compared using nonparametric tests and receiver operating characteristic (ROC) curve, using histopathologic analysis as the reference standard. RESULTS: AEF and ADC values differed significantly between histopathologic fibrosis stages. AEF values correlated with fibrosis stage, ADC values correlated negatively with fibrosis stage. Compared with ADC, AEF showed a trend toward an improved capability of discriminating fibrosis stages. A weighted composite score of AEF and ADC had significantly better diagnostic accuracy than ADC alone (P ≤ 0.023). Imaging parameters had a significantly better diagnostic accuracy than AST/ALT ratio or APRI. CONCLUSION: AEF may be able to detect the presence of mild, moderate, and advanced liver fibrosis, and its value is increased with concomitant use of ADC.

Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis.

UNLABELLED: Cholangiocarcinoma (CCA) presents significant diagnostic challenges, resulting in late patient diagnosis and poor survival rates. Primary sclerosing cholangitis (PSC) patients pose a particularly difficult clinical dilemma because they harbor chronic biliary strictures that are difficult to distinguish from CCA. MicroRNAs (miRs) have recently emerged as a valuable class of diagnostic markers; however, thus far, neither extracellular vesicles (EVs) nor miRs within EVs have been investigated in human bile. We aimed to comprehensively characterize human biliary EVs, including their miR content. We have established the presence of extracellular vesicles in human bile. In addition, we have demonstrated that human biliary EVs contain abundant miR species, which are stable and therefore amenable to the development of disease marker panels. Furthermore, we have characterized the protein content, size, numbers, and size distribution of human biliary EVs. Utilizing multivariate organization of combinatorial alterations (MOCA), we defined a novel biliary vesicle miR-based panel for CCA diagnosis that demonstrated a sensitivity of 67% and specificity of 96%. Importantly, our control group contained 13 PSC patients, 16 with biliary obstruction of varying etiologies (including benign biliary stricture, papillary stenosis, choledocholithiasis, extrinsic compression from pancreatic cysts, and cholangitis), and 3 with bile leak syndromes. Clinically, these types of patients present with a biliary obstructive clinical picture that could be confused with CCA. CONCLUSION: These findings establish the importance of using extracellular vesicles, rather than whole bile, for developing miR-based disease markers in bile. Finally, we report on the development of a novel bile-based CCA diagnostic panel that is stable, reproducible, and has potential clinical utility.

Effects of vitamin D3 stimulation of thioredoxin-interacting protein in hepatocellular carcinoma.

AIM: Thioredoxin-interacting protein (TXNIP) promotes oxidative stress by inactivating thioredoxin (TXN). This protein is involved in diverse disease processes, including insulin resistance, atherosclerosis and carcinogenesis. The aim of the present study was to measure the expression and function of TXNIP in in vitro models of liver disease, as well as in primary human hepatocellular carcinoma (HCC) tissue specimens. In addition, we wanted to determine the effects of vitamin D3-induced TXNIP stimulation in HCC-derived cell lines. METHODS: TXNIP expression was measured by quantitative reverse transcription polymerase chain reaction and western blots. TXNIP expression was stimulated by vitamin D exposure and by transfection. Cell proliferation, apoptosis and reactive oxygen species were determined by standard assays. RESULTS: TXNIP expression levels were low in HCC cell lines, and vitamin D3 stimulated TXNIP expression in vitro. In HCC cells transfected with a TXNIP expression vector or treated with exogenous vitamin D3, there was a reduction in cell proliferation and an increase in apoptosis. Cells expressing TXNIP were markedly susceptible to oxidative injury induced by cobalt chloride or bacterial lipopolysaccharide. TXNIP expression was reduced or absent in a majority of primary human HCC specimens relative to matching, non-cancerous liver tissue. CONCLUSION: TXNIP expression is low or absent in human HCC specimens and HCC-derived cell lines. Vitamin D3 stimulates TXNIP expression, resulting in diminished proliferation and enhanced apoptosis. Liver cells expressing TXNIP are primed for oxidative injury. These findings suggest that stimulation of TXNIP expression, by factors such as vitamin D3, may attenuate carcinogenesis in patients with chronic liver disease.

Unresectable hepatocellular carcinoma: MR imaging after intraarterial therapy. Part II. Response stratification using volumetric functional criteria after intraarterial therapy.

PURPOSE: To assess whether volumetric functional magnetic resonance (MR) results 3-4 weeks after initial intraarterial therapy can aid accurate distinction between responders and nonresponders, to determine whether overall survival (OS) is improved, and to compare volumetric functional MR response with anatomic response criteria (Response Evaluation Criteria in Solid Tumors [RECIST], modified RECIST [mRECIST], European Association for the Study of the Liver [EASL]), as well as α1-fetoprotein [AFP] level. MATERIALS AND METHODS: In this single-institution HIPAA-compliant retrospective, institutional review board-approved study, informed consent was waived; 143 patients with hepatocellular carcinoma underwent intraarterial therapy between October 2005 and February 2011. Volumetric functional MR response (25% or more increase in apparent diffusion coefficient, 65% or more decrease in enhancement) was stratified as follows: Dual-parameter responders fulfilled both criteria, single-parameter responders fulfilled one criterion, and those with stable disease (SD) fulfilled neither. RECIST, mRECIST, EASL, and AFP response criteria were determined. Kaplan-Meier technique, log-rank tests, and the Cox proportional hazards model were used to test whether OS was different per response. RESULTS: OS differed significantly between single-parameter responders and dual-parameter responders (P = .01) and between single-parameter responders and those with SD (P = .001). Dual-parameter responders' response improved OS compared with single-parameter responders; risk of death decreased (hazard ratio [HR] = 0.28, P = .01). In those with SD compared with single-parameter responders, risk of death increased (HR = 2.09, P = .001). RECIST, mRECIST, and EASL stratification was short of significant; most lesions were classified as SD. Baseline AFP level increased in 55 patients; AFP responders versus AFP nonresponders had decreased risk of death (HR = 0.36, P = .002). Agreement between anatomic response criteria and volumetric functional MR findings (κ = 0.06-0.12) and between AFP response and imaging criteria (κ = -0.04 to 0.14) was low. CONCLUSION: Volumetric functional MR response 3-4 weeks after initial intraarterial therapy showed improved OS. Volumetric functional MR was superior to current imaging (RECIST, mRECIST, and EASL) and biochemical (AFP level) response criteria.

1,25-dihydroxyvitamin D3 and its nuclear receptor repress human α1 (I) collagen expression and type I collagen formation.

BACKGROUND: Vitamin D deficiency is common in chronic liver disease particularly in those with severe liver fibrosis. AIMS: To determine the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ) on the human α(1) (I) collagen promoter and collagen formation by human stellate LX-2 cells and the mechanism of the effect of the vitamin D receptor (VDR) on the promoter. METHODS: Type I collagen was assessed by measurements of collagen mRNA and collagen protein and by transfection experiments. Binding of VDR to the α(1) (I) collagen promoter was determined by EMSA and ChIP assays. RESULTS: 1,25-(OH)2 D3 decreased human α(1) (I) collagen mRNA and protein and the secretion of type I collagen by stellate cells after exposure to TGFß1. Furthermore, 1,25-(OH)2 D3 inhibited TGFß1-induced activation of the α(1) (I) collagen promoter in transfected LX-2 cells. The effect of 1,25-(OH)2 D3 is mediated by the VDR, which binds at a proximal Sp1 site and also at a newly identified distal site on the collagen promoter. A VDR expression vector reduced the activities of the collagen promoter in transfected LX-2 cells. CONCLUSIONS: 1,25-(OH)2 D3 inhibits type I collagen formation in human stellate cells. The effect of 1,25-(OH)2 D3 is mediated by its receptor which binds at a proximal Sp1.1 site and at a newly identified distal site on the collagen promoter. Correction of vitamin D deficiency in patients with chronic liver disease is a potential therapy to inhibit progression of fibrosis.