Age-dependent glycosylation of the sodium taurocholate co-transporte polypeptide: from neonates to adult human primary hepatocytes

Background and Aims: The sodium taurocholate cotransporter polypeptide (NTCP) is the major hepatobiliary transporter responsible for bile acids portal vein re-uptake in hepatocytes. In humans, NTCP ontogeny has not been sufficiently investigated because of limited access to human tissue. Based on rodent models, NTCP glycosylation requires four weeks to be completed (Hardikar et al., 1995; Gao et al., 2004). Therefore, the aim of this study was to examine in humans the maturational profile of NTCP glycosylation levels from birth up to adulthood. Methods: Adult and neonatal primary human hepatocytes (PHH) were obtained from UCL-St Luc biobank with ethical consent for research purposes. PHH were isolated by a one stage collagenase perfusion and cryopreserved in liquid nitrogen (Ref). Donors were selected and approved by the medical ethical committee of our institution. Two groups of PHH were established: PHH donors younger than 1 year old were considered as early infants, whereas, PHH donors older than 1 years were considered as mature hepatocytes. The two groups were tested for NTCP transcriptional, translational and glycosylation age-dependent modulation levels by RT-qPCR and SDS-PAGE analysis. Characterization of NTCP glycosylation bands was conducted with PNGase F de-glycosylation treatment. Results: As a result, we show that NTCP mature complex-glycosylated form (55 kDa) was significantly increased in an age-dependent manners as compared to non-glycosylated NTCP (38 kDa) approximately at one year of age, whereas, NTCP mRNA and native protein levels were not significantly regulated, but had a slight tendency to increase with age. The complete deglycosylation of NTCP allowed us to then quantify the amount of de-glycosylated NTCP that shift at 38 kDa band. Here we show that NTCP 38 kDa band in adults was significantly increased by PNGase F treatment as compared to non treated, whereas in infants, no significant difference was shown. Conclusions: In conclusion, our data suggests that in human neonates, NTCP complex-glycosylation maturation process requires several months to up to one year to be completed,whereas, trascriptional and translational profiles were less influenced by age

High liver expression of SPINK-1 is associated with progenitor cell and hepatocyte proliferation and determines MELD score improvement in decompensated alcoholic liver disease

Background and aims: The prognostic significance of liver progenitor cell (LPC) and macrophage expansion in the regeneration of decompensated alcoholic liver disease (ALD) remain ill defined. In a well-characterized population of patients with acutely decompensated ALD (Hepatology 2011, A62), we analysed macrophage infiltration, proliferative LPC and differential expression of hepatic genes at baseline in relation to outcome at 3 months follow up. Methods: Fifty-eight patients (MELD 20) were included. Liver biopsy at inclusion was used for (1) immunohistological analysis of proliferative LPC (MIB1+/CK7+), proliferative hepatocytes (MIB1+/CK7- parenchymal cells), morphometric analysis of macrophage infiltration (CD68) and LPC expansion (CK7), and (2) transcriptome profiling using Affymetrix GeneChip Human arrays. Serum levels of HGF were determined by immunoassay. A ≥ 3 points decrease in MELD at 3 months as compared to baseline defined the improvers. Fifteen abstinent cirrhotics served as controls. CD68 and SPINK3 mRNA expression was determined in various mice models of liver injury. Results: At baseline, patients with decompensated ALD presented a significant expansion of CD68+ macrophages and CK7+ cells compared to abstinent cirrhotics. Patients who will improve (n=34) were characterized at baseline by a higher number of CK7+/MIB1+ cells (1.9 ± 1.5 versus 0.9 ± 0.9 cells/field, p<0.01), MIB1+ hepatocytes (4.1 ± 3.6 versus 1.8 ± 1.4 cells/field, p<0.01), an increased expansion of liver macrophages (4.4% versus 3.3% of surface area, p<0.05) and a higher level of serum HGF (p<0.05), compared to those who will not (n=24). Transcriptome analysis revealed that the first pathways upregulated in improvers were related to cell cycle and a 7-fold increase of liver serine peptidase inhibitor Kazal type I (SPINK1) compared with non-improvers (p=0.005). SPINK1 liver expression positively correlated with CD68 (r=0.46) and cyclinE1 (r=0.6). In mice, a 20-fold increase in liver SPINK3 expression, the homolog of human SPINK1, was evidenced following partial hepatectomy, concurrent with hepatocyte proliferation. Conclusions: Baseline markers of liver macrophages and liver cell proliferation predict the clinical outcome in decompensated ALD. This study reveals an unexpected implication of SPINK1, an acute phase reactant, in liver regeneration and human ALD

Impact of Kupffer cells on high fat induced insulin resistance and liver fetuin-A expression.

Backgroun and aims: Hepatokines (liver secreted proteins with possible distant action) are emerging potential players in insulin resistance in type 2 diabetic patients. Here, we explore the effect of a high fat diet on the expression of fetuin-A, one of those candidate liver proteins, and its relation with liver macrophage (Kupffer cell) activation. Methods: Male mice of 5 weeks of age were fed a normal diet (ND) or a high fat diet (HFD) for 3 days, known to initiate steatosis and insulin resistance. A preventive Kupffer cell (KC) depletion was obtained by intravenous injection of clodronate loaded liposomes and compared with PBS liposomes. The mRNA and protein expression of fetuin-A was evaluated by RT-PCR, Western-blot and immunofluorescence (IF) on different insulin-sensitive tissues (liver, adipose tissue and muscle). Results: Short term HFD induced steatosis, KC activation and insulin resistance together with a significant increased expression of liver fetuin-A mRNA (1.5 fold, p<0.01). However, liver fetuin-A protein expression remained unchanged under short term HFD. This increase in fetuin-A under high fat diet was not evidenced in the peripheral insulin sensitive tissues (skeletal muscle and adipose tissue) whether at the mRNA or at the protein level. Kupffer cell depletion in this setting did not reduce hepatic steatosis but significantly ameliorated insulin sensitivity proved by clamp studies. This amelioration in insulin sensitivity in KC-depleted mice was associated with a significant decrease in fetuin-A mRNA expression (0.7 fold, p<0.01) compared to animals with KC. On immunofluorescence, fetuin-A was mostly expressed in centrilobular hepatocytes. Interestingly, while selectively depleting liver macrophages without affecting adipose tissue macrophage infiltration, intravenous clodronate injection was associated with a significant reduction in epididymal adipose tissue expansion compared to PBS injection (1.1% of body weight versus 1.6% of body weight, p<0.001). Conclusion: This study demonstrates liver fetuin-A overexpression at the initiation of HFD feeding, concurrent with hepatic steatosis and insulin resistance. Targeting KC in this setting improved insulin sensitivity and was associated with a decreased adiposity and a reduced liver fetuin-A expression suggesting that fetuin-A acts as an hepatokine with pro-adiposity and pro-insulin resistance effects

Antibiotics induce remission in pediatric PSC-AIH overlap syndrome allowing corticosteroid-free therapy

Background and Aims: Concomitant presence of autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC) is labelled as AIH-PSC overlap syndrome or autoimmune sclerosing cholangitis (ASC). Treatment of AIH with corticosteroids and azathioprine; and of the PSC component with ursodeoxycholic acid (UDCA) is the standard practice. Antibiotics are increasingly being shown to have benefit in PSC but their role in paediatric ASC is not well evaluated. We investigated the response to oral antibiotics as initial or subsequent therapy in children with ASC. Methods: Patients diagnosed with ASC on basis of biochemical, liver biopsy and radiology findings were included. They received metronidazole or vancomycin for 14 days [10-220] either at diagnosis (i.e. initial therapy) or during their maintenance period. When antibiotics were administered as initial therapy, steroid free induction regime was adopted. In children during the maintenance phase antibiotics were administered if they had not achieved biochemical remission with their standard treatment of steroids, azathioprine and UDCA. The outcome parameters to assess the efficacy of antibiotics were achievement of biochemical remission and additionally steroid avoidance when given in the initial therapy. Results: Ten children with ASC were included, of which 6 received oral antibiotics (4 metronidazole, 2 vancomycin) at diagnosis and 4 received metronidazole during the maintenance period. All patients showed a significant decrease in their AST (-55%, p=0,005), ALT (-84%, p=0,003) and GGT (-53%, p=0,003), without significant difference across the two groups. All six children in the initial therapy group did not need corticosteroids and continued to be in remission until last follow up duration of 400 days [216-888]. Among the four children administered antibiotics in the maintenance phase, two showed biochemical remission and steroids could be tapered; while two did not show any benefit. There was transient biochemical relapse after stopping antibiotics in one responder, for which they were restarted and continued until last follow up while continuing to be in remission. Conclusions: We demonstrate the benefit of antibiotics in ASC by achieving steroid free treatment when given at diagnosis as induction regime. When given in the maintenance phase they assist in achieving long term biochemical remission in an otherwise uncontrolled ASC

Ductular reaction on protocol biopsy post liver transplant corresponds to concurrent fibrosis and does not predict fibrogenesis

Background and Aims: Ductular reaction (DR) is identified by using CK7 staining and represents a ductular phenotype, possibly arising from proliferation of cholangiocytes and progenitor cells. DR has been linked to hepatic fibrosis progression in HCV and hemochromatosis. It is frequently seen on protocol biopsies (PB) post-liver transplant (LT) and given its origin it’s unclear if its linked to fibrogenesis or hepatocyte regeneration after an insult. This was analyzed in the current study using paired PB’s to test if CK7 quantification on the baseline PB could predict the course of fibrogenesis in the follow up PB. Methods: Children who underwent LT from 2012 to 2014 and had ≥2 PB at an interval of 1-2 years between each other were included. The first PB was labeled as “baseline biopsy” and the successive biopsy as “follow-up biopsy.” The change of fibrosis severity between the “baseline” and “follow-up” biopsy was termed as “prospective change in fibrosis.” Each biopsy was evaluated for inflammation and fibrosis using the Metavir and LAFSc system. The baseline biopsy was stained with CK7 and digitally evaluated to obtain “CK7 – positive area percentage” i.e. CK7-stained area expressed as percentage of the total biopsy area. The association between CK7-positive area percentage on baseline biopsy and “prospective change in fibrosis” scores, ductular reaction, lobular inflammation, and portal tract inflammation was assessed using ordinal logistic regression models. Results: Our study included 64 pared PB from 32 children. The baseline PB stained for CK7, were taken at a mean of 2.89 years post-LT. The time interval between the two paired PB’s was 1.41 years. CK7-positive area percentage was significantly associated with baseline fibrosis, extent of ductular reaction (p = 0.006) and portal tract inflammation (p = 0.01). There was a no significant association between the CK7-positive area percentage of the baseline biopsy and the “prospective change in fibrosis” as assessed by the Metavir score (p = 0.36) or cumulative LAFSc (p = 0.25) or with recipient or donor age, donor type, HLA antibodies, or recipient or donor gender. Conclusions: Ductular reaction, CK7 expression is significantly associated with extent of portal tract inflammation and concurrent severity of fibrosis but does not predict the future course of fibrogenesis

Sustained biochemical response to oral antibiotics in pediatric PSC and ASC are correlated to changes in gut microbiota during therapy

Background and aim Concomitant presence of autoimmune hepatitis and primary sclerosing cholangitis (PSC) is labelled as autoimmune sclerosing cholangitis (ASC) in children. Based upon the possible implication of microbiota in the pathogenesis of PSC, oral antibiotics are increasingly being used as a novel therapeutic approach and shown to have benefit in PSC but their role in pediatric ASC is not well evaluated. We prospectively analysed the gut microflora before and after antibiotic therapy in children with ASC or PSC alone, and evaluated whether changes in gut microflora correlated with response to treatment. Methods Patients diagnosed with ASC or PSC on basis of biochemical, liver biopsy and radiology findings were included. They prospectively received metronidazole (MTZ) for 14 days as induction or rescue therapy. Antibiotics were administrated in addition to the standard treatment of UDCA for PSC patients and azathioprine, UDCA and/or steroids for ASC patients. Stool samples were collected before and after antibiotic therapy. DNA isolation, amplification and Illumina sequencing to profile the microbiota composition were performed using the bacterial 16s rRNA. The beta-diversity measured the dissimilarity between each paired stool samples. The outcome parameters to assess the efficacy of antibiotics were reduction liver enzymes and subsequently achievement of sustained biochemical remission. Results Seven children (4 ASC, 3 PSC) were included, of which 5 have a concomitant ulcerative colitis (UC). All patients showed a significant decrease in their AST (-44%, p<0.025), ALT (-56%, p<0.025) and GGT (-41%, p<0.025) under MTZ. Three children relapsed after stopping MTZ while the four others children showed a sustained biochemical remission (liver enzymes below 1.5 times upper limit of normal) after a median follow-up of 375 days. Among these four patients, three exhibited a wide different microbial composition before and after MTZ as expressed by the beta-diversity variation. On the contrary, the microbiota of patients who relapsed remained unchanged pre- and post-MTZ. Conclusions Our study suggests that oral antibiotic could be an effective treatment of ASC and PSC, especially those with a concomitant UC, and that intestinal microflora play a major role in these diseases as sustained biochemical remission is associated with wide changes in gut microbiota communities after taking antibiotics

Expression of pro-inflammatory and hepatoprotective factors at early stages of alcoholic liver disease in humans and the impact of short term abstinence.

Background and Aims: Animal models imperfectly mimic the spectrum of alcoholic liver disease (ALD) seen in humans. Some studies have investigated late stages and severe forms of human ALD, but little is known about the pathophysiological mechanisms occurring in the human liver at early stages of the disease. Here,we investigated inflammatory mechanisms in alcohol dependent patients undergoing a standardized inpatient alcohol withdrawal program. Methods: Patients with suspicion of significant ALD (ALT, AST increase, fibroscan >7.8 kPa) were randomly assigned to undergo liver biopsy either in the active drinking phase or after 2 weeks of abstinence. Patients without significant fibrosis on histology were included in the study (n = 40). Liver tissue (n = 6) from size-reduced liver grafts was used as normal controls. Expression and cellular localization of various factors was assessed by Western-blotting, qPCR and immunohistochemistry and immunofluorescence. Results: the active drinking phase, a strong activation of the Kupffer cell (KC) compartment was found with KCs forming clusters adjacent to ballooned, steatotic hepatocytes. KC showed a pro-inflammatory M1 phenotype with activation of NFkB and increased expression of TNF, IL-1 and iNOS. After 2 weeks of abstinence, the staining pattern of KC returned to normal and NFkB, IL-1 and iNOS levels but not TNF decreased to almost control levels. In addition, abstinence induced a partial shift to a M2 phenotype with increased production of the anti-inflammatory cytokine IL-10. Interestingly,we did not find activation of TLR4 since TLR4, CD14, and LBP levels remained at control values in active drinkers. By contrast, we found a strong and persistent upregulation of the intracellular TLR3 and TLR7 which correlated with high production of interferon beta and gamma principally located to hepatocytes and bile ducts. Moreover, the hepatoprotective factors IL-6, IL-22, MCP-1 and Stat3 DNA-binding were strongly down-regulated in active drinkers and did not recover after short term abstinence. Hepatocyte Ki67 proliferation index was low in active drinkers and increased modestly but significantly after 2 weeks of abstinence. Conclusions: At early stages of ALD, a strong pro-inflammatory, KC-dependent response is observed which rapidly reversed upon abstinence. By contrast, down-regulation of hepatoprotective factors is more long lasting and might significantly impair liver repair mechanisms in sustained drinkers

Intraportal infusion of human liver-mesenchymal stem cells in rats lead to transient interruption of the hepatic blood flow: intravital microscopy and anapathological analysis

Previous studies showed that liver Mesenchymal Stem Cells (MSC) express a procoagulant activity (PCA) that can be controlled in vitro by an anticoagulant cocktail, combining an antithrombin activator (Heparin) and a thrombin inhibitor (Bivalirudin). First we would like to confirm in vivo the PCA of human liver MSCs in a rat model, then study it’s inhibition by a specific anticoagulant cocktail and finally assess the effect of this cocktail on cell implantation. The main aim of this study is to control the PCA induced by infused liver MSCs without reducing the implantation of cells. Methods: Wistar rat were transplanted with 50x106/kg fluorescent (cell tracker red) human liver MSCs by intraportal infusion with (n=12) or without (n=13) anticoagulant drugs. Using an intra-femoral catheter we injected FITC-dextran and Hoechst to visualise liver vasculature and cell nuclei. By intravital microscopy (IVM) we analysed the left liver lobe at different time points, 1h-24h-48h and 7days post-infusion to study liver MSCs localisation and their effect on liver microvasculature. By pathological examination, cell localisation, cell implantation, vascular and tissue alterations were studied at the same time points. Serial slides were performed for a standard haematoxylin eosin staining and a human cell staining (human B-1-integrin). Results: By IVM we observed that the transplanted cells in the first hour formed aggregates in the larger liver vessels, then cells migrate to the sinusoids after 24h, to be quickly cleared after 7days. After 24h, large defect of perfusion were observed in both groups, but normal hepatic vascularisation was restored after 48h. These observations were confirmed by pathological examination. Large necrotic zones surrounding the infused cells were observed after 24h, with respectively 14.7% and 19.5% of the liver tissue in the non anticoagulated and anticoagulated group. This necrosis decreased rapidly after 48h to 0.5% and 1.9%. The anticoagulant drugs didn’t prevent this necrosis, no difference has been observed between the 2 groups (p>0.05).We confirmed that the infused cells are rapidly cleared after 24h from the liver over time, with respectively after 1h, 24h, 48h and 7days a decrease from 1.3%, 0.3%, 0.07% till 0.03% of infused cells. No difference in cell implantation has been observed between the groups with or without anticoagulation (p>0.05). Conclusion: Intraportal infusion of human liver MSCs to Wistar rats induces a transient alteration in liver vascular flow after 24h. This could be explained by the temporary localisation of liver MSCs in large portal veins and sinusoids up to 1hour after the infusion, in addition to a possible xenotransplantation acute reaction. After 24h more than 70% of cells are cleared and cells are surrounded by transient necrotico-hemorragic regions that regress almost completely after 48h. After 7days no necrosis and very few cells are seen. We observed no difference between the two groups, with or without anticoagulation

Results of Liver Transplantation in Adult Polycystic Liver Disease: Report of a Single Center Experience

Introduction: Polycystic liver disease (PCLD) occurs either in an isolated form (Autosomal Dominant Polycystic Liver Disease [ADPLD]) or in association with Autosomal Dominant Polycystic Kidney Disease (ADPKD). It remains an uncommon and controversial indication for liver transplantation (LT). Objectives: 1. to assess the results of LT in patients with massive PCLD; 2. to determine whether previous hepatic surgery is associated with a higher rate of complications following LT; 3. to examine the evolution of renal function after LT in order to determine whether pre-emptive renal transplantation is justified in case of ADPKD without (pre-)terminal renal failure. Methods: We retrospectively reviewed the medical charts of 19 patients (15 females) who underwent LT for PCLD between 1999 and 2008. Fifteen patients had ADPKD (12 females). Three received a combined liver and kidney transplantation (LKT) for associated terminal (n = 1) or pre-terminal renal failure. Two patients had previous kidney transplantation (KT) and 7 had undergone hepatic surgery. Results: Median duration of follow-up is 30 months [5-112]. All patients are alive and relieved of their symptoms. Their median Karnofsky score is 90% [80-100]. Intervention tended to be longer (10:30h vs. 7:20h, p=0.098) in the group who had previous surgery. There was no significant difference in intra-operative blood transfusion, severity of postoperative complications, length of ICU or hospital stay. Median pre-LT GFR of ADPLD and ADPKD patients (excluding those who underwent LKT or previous KT) is 89.5ml/min/1.73m2 [69-114] and 69ml/min/1.73m2 [33-120] respectively (p=0.129). Median GFR 1 year post-LT is 68ml/min/1.73m2 [57-95] and 51ml/min/1.73m2 [29-85] respectively (p=0.089). Median decrease in GFR at one year post-LT is 17.5 ml/min/1.73m2 and 18 ml/min/1.73m2 respectively (p=0.694). Conclusions: LT is an effective treatment for selected patients with massive PCLD. Prior conservative surgery tends to increase the length of the transplant operation. Renal function decreases at an identical rate after LT in ADPLD and ADPKD patients with pre-LT GFR >30 ml/min/1.73m2. Thus, pre-emptive renal transplantation would not have been justified in the latter