Survivin expression is essential for early activation of hepatic stellate cells and fibrosis progression in chronic liver injury.

AIM: Hepatic fibrosis in injured liver is characterized by the activation of hepatic stellate cells (HSCs) from their quiescent state. Survivin (BIRC5) is one of the key genes that are upregulated during activation of HSCs but their role in HSC activation and fibrosis progression is unknown. Here, we have investigated the role of survivin protein in early fibrogenic activation of HSCs and fibrosis progression in chronic liver injury. MATERIALS & METHODS: Primary quiescent HSCs were isolated from healthy mice liver through perfusion and cultured for fibrogenic activation. Survivin expression was suppressed by its pharmacological suppressant, YM155. We developed chronic liver injury induced fibrotic mice model through administrating repeated dose of CCl4 for 2 weeks and 4 weeks. Mice were pre-treated with YM155 a week before CCl4 administration till 2nd week of dosing and then discontinued. Hepatic parameters were characterized and underlying mechanisms were investigated. KEY FINDINGS: Survivin expression gradually increased along with the expression of αSMA, collagen I activation maker in HSCs during their activation from quiescent state. Survivin suppression through YM155 downregulated αSMA, collagen I. Pre-treatment of YM155 in mice ceased the early activation of HSCs and onset of fibrosis in injured liver. However, discontinuation of YM155 initiated the activation of HSCs and fibrosis progression that shows survivin expression in HSCs is essential for their early activation and onset of liver fibrosis. SIGNIFICANCE: Survivin expression induces with activation of HSCs and drives onset of liver fibrosis in injured liver. Targeting survivin protein in activated HSCs could be a potential anti-fibrotic therapeutic approach in chronic liver injury.

Hepatocyte Nuclear Factor 4 alpha 2 Messenger RNA Reprograms Liver-Enriched Transcription Factors and Functional Proteins in End-Stage Cirrhotic Human Hepatocytes.

The only definitive therapy for end-stage liver disease is whole-organ transplantation. The success of this intervention is severely limited by the complexity of the surgery, the cost of patient care, the need for long-term immunosuppression, and the shortage of donor organs. In rodents and humans, end-stage degeneration of hepatocyte function is associated with disruption of the liver-specific transcriptional network and a nearly complete loss of promoter P1-driven hepatocyte nuclear factor 4-alpha (P1-HNF4α) activity. Re-expression of HNF4α2, the predominant P1-HNF4α, reinstates the transcriptional network, normalizes the genes important for hepatocyte function, and reverses liver failure in rodents. In this study, we tested the effectiveness of supplementary expression of human HNF4α2 messenger RNA (mRNA) in primary human hepatocytes isolated from explanted livers of patients who underwent transplant for end-stage irreversibly decompensated liver failure (Child-Pugh B, C) resulting from alcohol-mediated cirrhosis and nonalcoholic steatohepatitis. Re-expression of HNF4α2 in decompensated cirrhotic human hepatocytes corrects the disrupted transcriptional network and normalizes the expression of genes important for hepatocyte function, improving liver-specific protein expression. End-stage liver disease in humans is associated with both loss of P1-HNF4α expression and failure of its localization to the nucleus. We found that while HNF4α2 re-expression increased the amount of P1-HNF4α protein in hepatocytes, it did not alter the ability of hepatocytes to localize P1-HNF4α to their nuclei. Conclusion: Re-expression of HNF4α2 mRNA in livers of patients with end-stage disease may be an effective therapy for terminal liver failure that would circumvent the need for organ transplantation. The efficacy of this strategy may be enhanced by discovering the cause for loss of nuclear P1-HNF4α localization in end-stage cirrhosis, a process not found in rodent studies.

Pruritus is common in patients with chronic liver disease and is improved by nalfurafine hydrochloride.

Pruritus is known to be a common complication in hepatitis patients, but the exact frequency and degree are not fully elucidated. Thus, we evaluated pruritus of 450 patients with chronic liver disease at our hospital. Pruritus was observed in 240 (53%) of the patients. Pruritus was significantly associated with males (OR = 1.51, P = 0.038) and patients with alkaline phosphatase (ALP) ≥ 200 U/L (OR = 1.56, P = 0.0495) and was significantly less in HBsAg-positive patients (OR = 0.449, P = 0.004). Seasonally, there was no difference in the frequency of pruritus between summer and winter. Of the 24 refractory pruritus patients treated with nalfurafine, 17 (71%) indicated improvement of itch, which is defined as a decrease in the visual analog scale score ≥ 30 mm. Pruritus was improved by nalfurafine both during daytime and nighttime in the Kawashima's scores evaluation. All patients who received nalfurafine exhibited improved Kawashima's scores ≥ 1 point during the daytime or nighttime. In conclusion, pruritus occurred in > 50% of patients with chronic liver disease, and predictors of pruritus were males and ALP ≥ 200 U/L. Nalfurafine may be useful for pruritus, regardless of whether daytime or nighttime.

A novel homozygous frameshift variant in the ABCC2-gene in Dubin-Johnson syndrome may predispose to chronic liver disease.

Dubin-Johnson syndrome (DJS), an autosomal recessive disorder presenting with conjugated hyperbilirubinemia, is not associated with progression to chronic liver disease (CLD). Next-generation sequencing, application of bioinformatics pipeline, and segregation analysis were performed on 8 members of a consanguineous family with DJS and CLD. A novel variant, c.4406_4407delTA (p.Leu1469fs), in the ABCC2-gene in a homozygous state was found to be associated with DJS and CLD in proband and afflicted family members. DJS may not be a benign entity and novel genetic variants may be associated with progressive liver disease.

Estrogen Signaling and Portopulmonary Hypertension: The Pulmonary Vascular Complications of Liver Disease Study (PVCLD2).

BACKGROUND AND AIMS: Portopulmonary hypertension (POPH) was previously associated with a single-nucleotide polymorphism (SNP) rs7175922 in aromatase (cytochrome P450 family 19 subfamily A member 1 [CYP19A1]). We sought to determine whether genetic variants and metabolites in the estrogen signaling pathway are associated with POPH. APPROACH AND RESULTS: We performed a multicenter case-control study. POPH patients had mean pulmonary artery pressure >25 mm Hg, pulmonary vascular resistance >240 dyn-sec/cm-5 , and pulmonary artery wedge pressure ≤15 mm Hg without another cause of pulmonary hypertension. Controls had advanced liver disease, right ventricular (RV) systolic pressure <40 mm Hg, and normal RV function by echocardiography. We genotyped three SNPs in CYP19A1 and CYP1B1 using TaqMan and imputed SNPs in estrogen receptor 1 using genome-wide markers. Estrogen metabolites were measured in blood and urine samples. There were 37 patients with POPH and 290 controls. Mean age was 57 years, and 36% were female. The risk allele A in rs7175922 (CYP19A1) was significantly associated with higher levels of estradiol (P = 0.02) and an increased risk of POPH (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.12-4.91; P = 0.02) whereas other SNPs were not. Lower urinary 2-hydroxyestrogen/16-α-hydroxyestrone (OR per 1-ln decrease = 2.04; 95% CI, 1.16-3.57; P = 0.01), lower plasma levels of dehydroepiandrosterone-sulfate (OR per 1-ln decrease = 2.38; 95% CI, 1.56-3.85; P < 0.001), and higher plasma levels of 16-α-hydroxyestradiol (OR per 1-ln increase = 2.16; 95% CI, 1.61-2.98; P < 0.001) were associated with POPH. CONCLUSIONS: Genetic variation in aromatase and changes in estrogen metabolites were associated with POPH.

[Purinergic receptors and hepatic fibrosis]. / Récepteurs purinergiques et fibrose hépatique.

TITLE: Récepteurs purinergiques et fibrose hépatique. ABSTRACT: Pour la cinquième année, dans le cadre du module d'enseignement « Physiopathologie de la signalisation ¼ proposé par l'université Paris-sud, les étudiants du Master « Biologie Santé ¼ de l'université Paris-Saclay se sont confrontés à l'écriture scientifique. Ils ont sélectionné une quinzaine d'articles scientifiques récents dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant des relations hôte-pathogène aux innovations thérapeutiques, en passant par la signalisation hépatique et le métabolisme. Après un travail préparatoire réalisé avec l'équipe pédagogique, les étudiants, organisés en binômes, ont ensuite rédigé, guidés par des chercheurs, une Nouvelle soulignant les résultats majeurs et l'originalité de l'article étudié. Ils ont beaucoup apprécié cette initiation à l'écriture d'articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme ! Trois de ces Nouvelles sont publiées dans ce numéro, les autres le seront dans des prochains numéros.

Importance of genetic polymorphisms in liver transplantation outcomes.

Although, liver transplantation serves as the only curative treatment for patients with end-stage liver diseases, it is burdened with complications, which affect survival rates. In addition to clinical risk factors, contribution of recipient and donor genetic prognostic markers has been extensively studied in order to reduce the burden and improve the outcomes. Determination of single nucleotide polymorphisms (SNPs) is one of the most important tools in development of personalized transplant approach. To provide a better insight in recent developments, we review the studies published in the last three years that investigated an association of recipient or donor SNPs with most common issues in liver transplantation: Acute cellular rejection, development of new-onset diabetes mellitus and non-alcoholic fatty liver disease, hepatocellular carcinoma recurrence, and tacrolimus concentration variability. Reviewed studies confirmed previously established SNP prognostic factors, such as PNPLA3 rs738409 for non-alcoholic fatty liver disease development, or the role of CYP3A5 rs776746 in tacrolimus concentration variability. They also identified several novel SNPs, with a reasonably strong association, which have the potential to become useful predictors of post-transplant complications. However, as the studies were typically conducted in one center on relatively low-to-moderate number of patients, verification of the results in other centers is warranted to resolve these limitations. Furthermore, of 29 reviewed studies, 28 used gene candidate approach and only one implemented a genome wide association approach. Genome wide association multicentric studies are needed to facilitate the development of personalized transplant medicine.

A Microbial Signature Identifies Advanced Fibrosis in Patients with Chronic Liver Disease Mainly Due to NAFLD.

The presence of advanced fibrosis is an important measure of the severity of chronic liver disease. Prior works that have examined the gut microbiome as a novel biomarker for advanced fibrosis have only examined patients with nonalcoholic fatty liver disease. Therefore, our goal was to examine the gut microbiome across varying etiologies of liver disease to create a predictive model for liver fibrosis based upon a microbial signature. Stool samples were obtained from patients with chronic liver disease (n = 50) undergoing FibroScan (ultrasound elastography) at the VA Greater Los Angeles Healthcare System. Healthy control patients (n = 25) were also recruited as a reference population. Fecal samples underwent 16S ribosomal RNA sequencing. Using differentially abundant microbes, a random forest classifier model was created to distinguish advanced fibrosis from mild/moderate fibrosis. The findings were then validated in a separate cohort of chronic liver disease patients (n = 37). Etiologies for liver disease included non-alcoholic liver disease (58.0%), hepatitis C (26.0%), hepatitis B (10.0%), and alcohol (6.0%). Microbiome composition was distinct in liver patients with advanced fibrosis compared to those with minimal fibrosis and healthy controls (p = 0.003). In multivariate negative binomial modeling, 26 bacterial taxa were differentially abundant in patients with advanced fibrosis as compared to those with minimal/moderate fibrosis (q-value < 0.05). A random forests classifier based on these taxa had an AUROC of 0.90 to predict advanced fibrosis. Prevotella copri, which was enriched in patients with advanced fibrosis, was the most strongly predictive microbe in the classifier. The classifier had an AUROC of 0.82 for advanced fibrosis in the validation cohort and Prevotella copri remained the strongest predictive microbe for advanced fibrosis. There is a distinct microbial signature for patients with advanced fibrosis independent of liver disease etiology and other comorbidities. These results suggest that microbial profiles can be used as a non-invasive marker for advanced fibrosis and support the hypothesis that microbes and their metabolites contribute to hepatic fibrosis.

Living-donor liver transplantation for children with tyrosinemia type I.

OBJECTIVE: To evaluate the efficacy of living-donor liver transplantation (LDLT) in children with tyrosinemia type I. METHODS: Altogether 10 patients diagnosed with tyrosinemia type I underwent LDLT between June 2013 and April 2019. Cirrhosis was the indication for LDLT in all 10 patients, and hepatocellular carcinoma (HCC) was suspected in nine. Patients' outcomes, including liver function, restoration of metabolism, quality of life and physical development, were analyzed after LDLT. RESULTS: All recipients were alive with a normal liver function after a median follow-up period of 49 months. Pathological examinations detected HCC in one patient, dysplasia in five and cirrhosis in all. Nine patients were found to have elevated alpha-fetoprotein level, and their median alpha-fetoprotein level dropped from 2520 ng/mL to a normal level after LDLT, with no recurrence of HCC detected during the follow-up. Tyrosine metabolism was restored to its normal level with normalized plasma tyrosine and succinylacetone concentrations. Moreover, urinary succinylacetone excretion decreased significantly during the follow up. LDLT improved patients' renal tubular function, as evidenced by the normalized plasma phosphate concentration and improved glomerular filtration rate. Severe rickets symptoms, including spontaneous fractures and bone pain, were also ameliorated. Improved motor function was reported by all patients' parents during the follow-up. Dietary restriction was no longer required, which was associated with a favorable catch-up in growth and improved quality of life. Complete resolution of hypertrophic cardiomyopathy was observed one year after LDLT in one patient. CONCLUSION: LDLT is an effective treatment for patients with end-stage liver disease resulting from tyrosinemia type I.

Alloimmunity and Cholestasis After Liver Transplantation in Children With Progressive Familial Intrahepatic Cholestasis.

OBJECTIVES: Bile salt export pump (BSEP) deficiency is an important reason for chronic cholestasis leading to liver transplantation (LT) in early childhood. The underlying pathology is a dysfunction of BSEP due to various mutations in the ABCB11 gene. Cases of clinical recurrence after LT due to alloantibodies directed against BSEP (antibody-induced BSEP deficiency [AIBD]) have been reported. Most of these patients could be controlled by intensified immunosuppression. METHODS: We here report on 3 children with BSEP-deficiency and end-stage liver disease, which developed AIBD after LT refractory to extensive immunosuppressive and immunomodulatory treatments; retransplantation was necessary in all 3 patients. In 1 patient, a stem cell transplantation was performed successfully. RESULTS: AIBD seems to be induced by triggering factors such as initial impaired graft function or infections after LT. CONCLUSIONS: The underlying mutation may play a role in this process. Intensifying immunosuppression may be able to control AIBD, but some cases seem to be refractory to treatment and require retransplantation. Stem cell transplantation may provide a new therapeutic option for cases refractory to conservative treatment.

Plasma levels of soluble ST2, but not IL-33, correlate with the severity of alcoholic liver disease.

Alcoholic liver disease (ALD) is a complication that is a burden on global health and economy. Interleukin-33 (IL-33) is a newly identified member of the IL-1 cytokine family and is released as an "alarmin" during inflammation. Soluble suppression of tumourigenicity 2 (sST2), an IL-33 decoy receptor, has been reported as a new biomarker for the severity of systemic and highly inflammatory diseases. Here, we found the levels of plasma sST2, increased with the disease severity from mild to severe ALD. Importantly, the plasma sST2 levels in ALD patients not only correlated with scores for prognostic models (Maddrey's discriminant function, model for end-stage liver disease and Child-Pugh scores) and indexes for liver function (total bilirubin, international normalized ratio, albumin, and cholinesterase) but also correlated with neutrophil-associated factors as well as some proinflammatory cytokines. In vitro, lipopolysaccharide-activated monocytes down-regulated transmembrane ST2 receptor but up-regulated sST2 mRNA and protein expression and produced higher levels of tumour necrosis factor-α (TNF-α). By contrast, monocytes pretreated with recombinant sST2 showed decreased TNF-α production. In addition, although plasma IL-33 levels were comparable between healthy controls and ALD patients, we found the IL-33 expression in liver tissues from ALD patients was down-regulated at both RNA and protein levels. Immunohistochemical staining further showed that the decreased of IL-33-positive cells were mainly located in liver lobule area. These results suggested that sST2, but not IL-33, is closely related to the severity of ALD. Consequently, sST2 could be used as a potential biomarker for predicting the prognosis of ALD.

Compromised nutritional status in patients with end-stage liver disease: Role of gut microbiota.

BACKGROUND: Patients with end-stage liver disease (ESLD) have a compromised nutritional status because of the liver crucial role in regulating metabolic homeostasis and energy balance. DATA SOURCES: A systematic review of literature based on extensive relevant articles published from 2001 to 2017 in English in PubMed database was performed by searching keywords such as liver disease, non-alcoholic liver disease, alcoholic liver disease, malnutrition, epigenetics, gut microbiota, and probiotics. RESULTS: Liver transplantation would be one eligible therapy for ESLD patients, even if, the clinical outcome is negatively influenced by malnutrition and/or infections. The malnutrition is a condition of nutrient imbalance with a high incidence in ESLD patients. An accurate evaluation of nutritional status could be fundamental for reducing complications and prolonging the survival of ESLD patients including those undergoing liver transplantation. In addition, the interaction among nutrients, diet and genes via epigenetics has emerged as a potential target to reduce the morbidity and mortality in ESLD patients. The malnutrition induces changes in gut microbiota causing dysbiosis with a probable translocation of bacteria and/or pathogen-derived factors from the intestine to the liver. Gut microbiota contribute to the progression of chronic liver diseases as well as hepatocellular carcinoma. The administration of probiotics modulating gut microbiota could improve all chronic liver diseases. CONCLUSIONS: This review provides an update on malnutrition status linked to epigenetics and the potential benefit of some probiotics on the management of ESLD patients. In support of this view and to reveal the constant and growing interest in this field, some clinical trials are reported.

Identification of IL-28B Genotype Modification in Hepatocytes after Living Donor Liver Transplantation by Laser Capture Microdissection and Pyrosequencing Analysis.

The aim of this study is to elucidate the biogenetic modification of donor and recipient interleukin-28B (IL-28B) genotypes in liver graft biopsies after living donor liver transplantation (LDLT) for chronic hepatitis C virus- (HCV-) related, end-stage liver disease. Fifty liver graft biopsies were collected from recipients during LDLT treatment for HCV-related, end-stage liver disease. DNA was extracted from all 50 liver tissues, and the IL-28B single-nucleotide polymorphisms (SNPs) rs8099917 and rs12979860 were studied for allelic discrimination by real-time PCR analysis. Blood samples were obtained from donors and recipients on postoperative day 0 (POD0), POD7, and POD30. We randomly selected five liver biopsies and isolated the hepatocytes by laser capture microdissection (LCM) to evaluate genotype modifications resulting from LDLT. After LDLT, the IL-28B SNP rs8099917 was identified not only in the liver graft biopsies and donors' sera (TT = 41 : 43; GT = 9 : 5; GG = 0 : 2), but also in liver graft biopsies and recipients' sera on POD0 (TT = 41 : 44; GT = 9 : 4; GG = 0 : 2), POD7 (TT = 41 : 30; GT = 9 : 18; GG = 0 : 2), and POD30 (TT = 41 : 29; GT = 9 : 19; GG = 0 : 2). A significant difference was observed between the rs8099917 allele frequencies of liver graft biopsies and recipients' sera on POD30 (p = 0.039). In addition, a significant difference was also noted between the rs12979860 allele frequencies of liver graft biopsies and donors' sera (CT = 49 : 39; TT = 1 : 10) (p = 0.012) and of liver graft biopsies and recipients' sera on POD0 (CT = 49 : 39; TT = 1 : 11) (p = 0.002), POD7 (CT = 49 : 42; TT = 1 : 8) (p = 0.016), and POD30 (CT = 49 : 41; TT = 1 : 9) (p = 0.008). This phenomenon was confirmed by pyrosequencing of hepatocytes isolated by LCM. Following LDLT, the TT-to-GT IL-28B genotype modification predominated in rs8099917, and the CC-to-CT modification predominated in rs12979860. In conclusion, these modified phenomena suggested that the selected donor with a predictable and favourable IL-28B genotype will not confer a benefit on the recipient in the living donor liver transplantation setting.

Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer.

Liver diseases are a growing epidemic worldwide. If unresolved, liver fibrosis develops and can lead to cirrhosis and clinical decompensation. Around 5% of cirrhotic liver diseased patients develop hepatocellular carcinoma (HCC), which in its advanced stages has limited therapeutic options and negative survival outcomes. CEPBA is a master regulator of hepatic function where its expression is known to be suppressed in many forms of liver disease including HCC. Injection of MTL-CEBPA, a small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposomal nanoparticles (NOV340- SMARTICLES) upregulates hepatic CEBPA expression. Here we show how MTL-CEBPA therapy promotes disease reversal in rodent models of cirrhosis, fibrosis, hepatosteatosis, and significantly reduces tumor burden in cirrhotic HCC. Restoration of liver function markers were observed in a carbon-tetrachloride-induced rat model of fibrosis following 2 weeks of MTL-CEBPA therapy. At 14 weeks, animals showed reduction in ascites and enhanced survival rates. MTL-CEBPA reversed changes associated with hepatosteatosis in non-alcoholic methionine and cholic-deficient diet-induced steaotic liver disease. In diethylnitrosamine induced cirrhotic HCC rats, MTL-CEBPA treatment led to a significant reduction in tumor burden. The data included here and the rapid adoption of MTL-CEBPA into a Phase 1 study may lead to new therapeutic oligonucleotides for undruggable diseases.

Intrahepatic MicroRNA Profile of Liver Transplant Recipients with Hepatitis C Virus Co-Infected with Human Immunodeficiency Virus.

BACKGROUND In patients with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) co-infection, HIV can modulate HCV replication and immune response as well as accelerate liver fibrosis. The role of miRNA in HIV/HCV co-infection is not fully elucidated. The aim of this study was to examine the differential expression of miRNAs in the liver. MATERIAL AND METHODS Thirteen patients who had undergone a liver transplant (7 HCV-infected and 6 HIV/HCV-co-infected patients) were examined using a miRNA array containing 1347 human miRNAs. To confirm the microarray results, data for 20 patients (10 HCV-infected and 10 HIV/HCV-co-infected) were validated using real-time polymerase chain reaction probing for miR101b, miR149, and miR200c. This miRNA was selected based on microarray results and its biological significance in liver fibrosis. RESULTS Microarray analysis revealed 22 miRNAs that were differentially expressed in the HIV/HCV-co-infected group compared to the HCV-infected group (p<0.05). The expression of miR-101b and miR149 was significantly decreased in the HIV/HCV-co-infected group compared to that in the HCV-infected group (miR101b, 0.103±0.09 vs. 0.0157±0.0093, p=0.007; miR149, 0.152±0.159 vs. 0.0192±0.015, p=0.025). CONCLUSIONS HIV/HCV co-infection may promote liver fibrosis by modulating miRNA expression.

Differential carbonylation of proteins in end-stage human fatty and nonfatty NASH.

OBJECTIVE: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease is oxidative stress leading to the accumulation of highly reactive electrophilic α/ß unsaturated aldehydes. The objective of this study was to determine if significant differences were evident when evaluating carbonylation in human end-stage fatty nonalcoholic steatohepatitis (fNASH) compared to end-stage nonfatty NASH (nfNASH). METHODS: Using hepatic tissue obtained from healthy humans and patients diagnosed with end stage nfNASH or fNASH, overall carbonylation was assessed by immunohistochemistry (IHC) and LC-MS/MS followed by bioinformatics. RESULTS: Picrosirius red staining revealed extensive fibrosis in both fNASH and nfNASH which corresponded with increased reactive aldehyde staining. Although significantly elevated when compared to normal hepatic tissue, no significant differences in overall carbonylation and fibrosis were evident when comparing fNASH with nfNASH. Examining proteins that are critical for anti-oxidant defense revealed elevated expression of thioredoxin, thioredoxin interacting protein, glutathione S-transferase p1 and mitochondrial superoxide dismutase in human NASH. As important, using immunohistochemistry, significant colocalization of the aforementioned proteins occurred in cytokeratin 7 positive cells indicating that they are part of the ductular reaction. Expression of catalase and Hsp70 decreased in both groups when compared to normal human liver. Mass spectrometric analysis revealed a total of 778 carbonylated proteins. Of these, 194 were common to all groups, 124 unique to tissue prepared from healthy individuals, 357 proteins exclusive to NASH, 124 proteins distinct to samples from patients with fNASH and 178 unique to nfNASH. Using functional enrichment analysis of hepatic carbonylated proteins revealed a propensity for increased carbonylation of proteins regulating cholesterol and Huntington's disease related pathways occurred in nfNASH. Examining fNASH, increased carbonylation was evident in proteins regulating Rho cytoskeletal pathways, nicotinic acetylcholine receptor signaling and chemokine/cytokine inflammatory pathways. Using LC-MS/MS analysis and trypsin digests, sites of carbonylation were identified on peptides isolated from vimentin, endoplasmin and serum albumin in nfNASH and fNASH respectively. CONCLUSIONS: These results indicate that cellular factors regulating mechanisms of protein carbonylation may be different depending on pathological diagnosis of NASH. Furthermore these studies are the first to use LC-MS/MS analysis of carbonylated proteins in human NAFLD and explore possible mechanistic links with end stage cirrhosis due to fatty liver disease and the generation of reactive aldehydes.

Cholestasis After Pediatric Liver Transplantation-Recurrence of a Progressive Familial Intrahepatic Cholestasis Phenotype as a Rare Differential Diagnosis: A Case Report.

INTRODUCTION: Nonobstructive cholestasis after pediatric liver transplantation is a common diagnostic and therapeutic dilemma. We describe a girl with neonatal cholestasis because of progressive familial intrahepatic cholestasis 2 (PFIC-2) and presence of a homozygous splice site mutation in the ABCB11 gene. Liver transplantation was performed because of end-stage liver disease at the age of 6. Cholestasis with normal gamma-glutamyl transferase (GGT) developed 8 years after liver transplantation. A liver biopsy showed canalicular cholestasis and giant cell hepatitis without evidence of rejection, mimicking PFIC-2. Immunofluorescence staining of normal human liver sections with patient's serum revealed reactivity toward a canalicular epitope, which could be identified as bile salt export pump (BSEP) using BSEP-yellow fluorescent protein (YFP) transfected cells. Our patient developed a recurrence of a PFIC-2 phenotype due to production of antibodies against BSEP (alloimmune BSEP disease [AIBD]). Intensification of immunosuppressive therapy as well as antibody treatment with plasmapheresis and Rituximab were initiated, leading to stabilization of the clinical condition and depletion of anti-BSEP antibodies in serum. However, after 1 year liver transplantation was necessary again because of end-stage liver insufficiency. Afterward, immunomodulatory treatment consisted of tacrolimus, mycophenolate mofetil, prednisone, immunoadsorption, and high-dose immunoglobulin therapy (1 g/kg/d). CONCLUSION: Cholestasis after liver transplantation may indicate an AIBD with a PFIC-2 phenotype. Besides enhancement of immunosuppressive therapy, an antibody depletion with plasmapheresis, immunoadsorption, immunoglobulins, and B-cell depletion represents a therapeutic option.

Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia.

BACKGROUND: Hepatitis B virus (HBV) occult infection (OBI) is a risk factor to be taken into account in transfusion, hemodialysis and organ transplantation. The aim of this study was to identify and characterize at the molecular level OBI cases in patients with end-stage liver disease. METHODS: Sixty-six liver samples were obtained from patients with diagnosis of end-stage liver disease submitted to liver transplantation in Medellin (North West, Colombia). Samples obtained from patients who were negative for the surface antigen of HBV (n = 50) were tested for viral DNA detection by nested PCR for ORFs S, C, and X and confirmed by Southern-Blot. OBI cases were analyzed by sequencing the viral genome to determine the genotype and mutations; additionally, viral genome integration events were examined by the Alu-PCR technique. RESULTS: In five cases out of 50 patients (10%) the criteria for OBI was confirmed. HBV genotype F (subgenotypes F1 and F3), genotype A and genotype D were characterized in liver samples. Three integration events in chromosomes 5q14.1, 16p13 and 20q12 affecting Receptor-type tyrosine-protein phosphatase T, Ras Protein Specific Guanine Nucleotide Releasing Factor 2, and the zinc finger 263 genes were identified in two OBI cases. Sequence analysis of the viral genome of the 5 OBI cases showed several punctual missense and nonsense mutations affecting ORFs S, P, Core and X. CONCLUSIONS: This is the first characterization of OBI in patients with end-stage liver disease in Colombia. The OBI cases were identified in patients with HCV infection or cryptogenic cirrhosis. The integration events (5q14.1, 16p13 and 20q12) described in this study have not been previously reported. Further studies are required to validate the role of mutations and integration events in OBI pathogenesis.