Immune regulation and therapeutic application of T regulatory cells in liver diseases.

CD4+ CD25+ FOXP3+ T regulatory cells (Tregs) are a subset of the immunomodulatory cell population that can inhibit both innate and adaptive immunity by various regulatory mechanisms. In hepatic microenvironment, proliferation, plasticity, migration, and function of Tregs are interrelated to the remaining immune cells and their secreted cytokines and chemokines. In normal conditions, Tregs protect the liver from inflammatory and auto-immune responses, while disruption of this crosstalk between Tregs and other immune cells may result in the progression of chronic liver diseases and the development of hepatic malignancy. In this review, we analyze the deviance of this protective nature of Tregs in response to chronic inflammation and its involvement in inducing liver fibrosis, cirrhosis, and hepatocellular carcinoma. We will also provide a detailed emphasis on the relevance of Tregs as an effective immunotherapeutic option for autoimmune diseases, liver transplantation, and chronic liver diseases including liver cancer.

Senescence and senotherapies in biliary atresia and biliary cirrhosis.

BACKGROUND: Premature senescence occurs in adult hepatobiliary diseases and worsens the prognosis through deleterious liver remodeling and hepatic dysfunction. Senescence might also arises in biliary atresia (BA), the first cause of pediatric liver transplantation. Since alternatives to transplantation are needed, our aim was to investigate premature senescence in BA and to assess senotherapies in a preclinical model of biliary cirrhosis. METHODS: BA liver tissues were prospectively obtained at hepatoportoenterostomy (n=5) and liver transplantation (n=30) and compared to controls (n=10). Senescence was investigated through spatial whole transcriptome analysis, SA-ß-gal activity, p16 and p21 expression, γ-H2AX and senescence-associated secretory phenotype (SASP). Human allogenic liver-derived progenitor cells (HALPC) or dasatinib and quercetin (D+Q) were administrated to two-month-old Wistar rats after bile duct ligation (BDL). RESULTS: Advanced premature senescence was evidenced in BA livers from early stage and continued to progress until liver transplantation. Senescence and SASP were predominant in cholangiocytes, but also present in surrounding hepatocytes. HALPC but not D+Q reduced the early marker of senescence p21 in BDL rats and improved biliary injury (serum γGT and Sox9 expression) and hepatocytes mass loss (Hnf4a). CONCLUSIONS: BA livers displayed advanced cellular senescence at diagnosis that continued to progress until liver transplantation. HALPC reduced early senescence and improved liver disease in a preclinical model of BA, providing encouraging preliminary results regarding the use of senotherapies in pediatric biliary cirrhosis.

Premature senescence of the liver in Alagille patients.

INTRODUCTION: Alagille syndrome (ALGS) is an autosomal dominant disease characterized by a multisystem involvement including bile duct paucity and cholestasis, caused by JAG1 or NOTCH2 mutations in most of the cases. Jagged1-Notch2 interactions are known to be crucial for intrahepatic biliary tract development, but the Notch signaling pathway is also involved in the juxtacrine transmission of senescence and in the induction and modulation of the senescence-associated secretory phenotype (SASP). AIM: Our aim was to investigate premature senescence and SASP in ALGS livers. METHODS: Liver tissue from ALGS patients was prospectively obtained at the time of liver transplantation (n = 5) and compared to control livers (n = 5). RESULTS: We evidenced advanced premature senescence in the livers of five JAG1 mutated ALGS pediatric patients through increased senescence-associated beta-galactosidase activity (p<0.05), increased p16 and p21 gene expression (p<0.01), and increased p16 and γH2AX protein expression (p<0.01). Senescence was located in hepatocytes of the whole liver parenchyma as well as in remaining bile ducts. The classical SASP markers TGF-ß1, IL-6, and IL-8 were not overexpressed in the livers of our patients. CONCLUSIONS: We demonstrate for the first time that ALGS livers display important premature senescence despite Jagged1 mutation, underlying the complexity of senescence and SASP development pathways.

Human liver mesenchymal stem/progenitor cells inhibit hepatic stellate cell activation: in vitro and in vivo evaluation.

BACKGROUND: Progressive liver fibrosis leads to cirrhosis and end-stage liver disease. This disease is a consequence of strong interactions between matrix-producing hepatic stellate cells (HSCs) and resident and infiltrating immune cell populations. Accumulated experimental evidence supports the involvement of adult-derived human liver mesenchymal stem/progenitor cells (ADHLSCs) in liver regeneration. The aim of the present study was to evaluate the influence of ADHLSCs on HSCs, both in vitro and in vivo. METHODS: Activated human HSCs were co-cultured with ADHLSCs or ADHLSC-conditioned culture medium. The characteristics of the activated human HSCs were assessed by microscopy and biochemical assays, whereas proliferation was analyzed using flow cytometry and immunocytochemistry. The secretion profile of activated HSCs was evaluated by ELISA and Luminex. ADHLSCs were transplanted into a juvenile rat model of fibrosis established after co-administration of phenobarbital and CCl4. RESULTS: When co-cultured with ADHLSCs or conditioned medium, the proliferation of HSCs was inhibited, beginning at 24 h and for up to 7 days. The HSCs were blocked in G0/G1 phase, and showed decreased Ki-67 positivity. Pro-collagen I production was reduced, while secretion of HGF, IL-6, MMP1, and MMP2 was enhanced. Neutralization of HGF partially blocked the inhibitory effect of ADHLSCs on the proliferation and secretion profile of HSCs. Repeated intrahepatic transplantation of cryopreserved/thawed ADHLSCs without immunosuppression inhibited the expression of markers of liver fibrosis in 6 out of 11 rats, as compared to their expression in the vehicle-transplanted group. CONCLUSIONS: These data provide evidence for a direct inhibitory effect of ADHLSCs on activated HSCs, which supports their development for the treatment of liver fibrosis.

Use of mesenchymal stem cells to treat liver fibrosis: current situation and future prospects.

Progressive liver fibrosis is a major health issue for which no effective treatment is available, leading to cirrhosis and orthotopic liver transplantation. However, organ shortage is a reality. Hence, there is an urgent need to find alternative therapeutic strategies. Cell-based therapy using mesenchymal stem cells (MSCs) may represent an attractive therapeutic option, based on their immunomodulatory properties, their potential to differentiate into hepatocytes, allowing the replacement of damaged hepatocytes, their potential to promote residual hepatocytes regeneration and their capacity to inhibit hepatic stellate cell activation or induce their apoptosis, particularly via paracrine mechanisms. The current review will highlight recent findings regarding the input of MSC-based therapy for the treatment of liver fibrosis, from in vitro studies to pre-clinical and clinical trials. Several studies have shown the ability of MSCs to reduce liver fibrosis and improve liver function. However, despite these promising results, some limitations need to be considered. Future prospects will also be discussed in this review.

Treating inborn errors of liver metabolism with stem cells: current clinical development.

Advanced therapies including stem cells are currently a major biotechnological development. Adult liver stem cells can differentiate into hepatocyte like cells and be infused in the recipient's liver to bring a missing metabolic function. These cells can be produced in large quantities in vitro. Allogeneic stem cells are required to treat genetic diseases, and this approach allows to use one single source of tissue to treat different diseases and many recipients. Mesenchymal stem cells can in addition play an immunomodulatory and anti-inflammatory role and possibly prevent the accumulation of fibrous tissue in the liver. From a regulatory point of view, stem cells are considered as medicinal products, and must undergo a pharmaceutical development that goes beyond the research and proof-of-concept phases. Here, we review the track followed from the first hepatocyte transplantation in 2000 to the next generation product issued from stem cell technology, and the start of EMA approved clinical trials to evaluate the safety and potency of liver stem cells for the treatment of inborn errors of the liver metabolism.

Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells.

BACKGROUND & AIMS: Umbilical cord matrix stem cells (UCMSCs) are able to differentiate into mesodermal and ectodermal lineages. The present study investigates the differentiation potential of human UCMSCs into hepatic lineage. METHODS: We isolated human UCMSCs and characterized them in vitro by measuring their expansion potential, by assessing expression of mesenchymal stem cell (MSC) markers, and by evaluating their ability to differentiate into adipocytes and osteocytes. UCMSCs were thereafter subjected to a hepatogenic differentiation protocol. Expression of hepatic and MSC markers in differentiated cells was analyzed by reverse-transcription polymerase chain reaction, flow cytometry, and immunocytochemical assays and compared with undifferentiated UCMSCs and freshly isolated liver cells. UCMSCs were transplanted into livers of hepatectomized-SCID mice, and engraftment capacity was investigated by detection of human nucleus and mitochondria and human hepatic-specific proteins. RESULTS: In vitro expanded UCMSCs constitutively expressed markers of hepatic lineage, including albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. In vitro-differentiated UCMSCs exhibited hepatocyte-like morphology, up-regulated several hepatic markers, stored glycogen, produced urea, and exhibited an inducible CYP 3A4 activity. However, absence of some hepatic markers in differentiated UCMSCs, as HepPar1 or hepatocyte nuclear factor 4 (HNF-4), implied that their differentiation did not reach the level of mature hepatocytes. We also noticed that differentiated UCMSCs partially preserved MSC markers. Engraftment capacity of UCMSCs was observed, and expression of human albumin and alpha-fetoprotein was detected 2, 4, and 6 weeks after transplantation in mice livers, while cytokeratin 19 was completely down-regulated. CONCLUSIONS: We conclude that UCMSCs, with a newly demonstrated endodermic differentiation potential, might be an alternative source for liver-directed cell therapies.

Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes?

It is currently accepted that adult tissues may develop and maintain their own stem cell pools. Because of their higher safety profile, adult stem cells may represent an ideal candidate cell source to be used for liver cell therapies. We therefore evaluated the differentiation potential of mesenchymal-like cells isolated from adult human livers. Mesenchymal-like cells were isolated from enzymatically digested adult human liver and expanded in vitro. Cell characterization was performed using flow cytometry, RT-PCR, and immunofluorescence, whereas the differentiation potential was evaluated both in vitro after incubation with specific media and in vivo after intrasplenic transplantation of uPA(+/+)-SCID and SCID mice. Adult-derived human liver mesenchymal-like cells expressed both hepatic and mesenchymal markers among which albumin, CYP3A4, vimentin, and alpha-smooth muscle actin. In vitro differentiation studies demonstrated that these mesenchymal-like cells are preferentially determined to differentiate into hepatocyte-like cells. Ten weeks following intrasplenic transplantation into uPA(+/+)-SCID mice, recipient livers showed the presence of human hepatocytic cell nodules positive for human albumin, prealbumin, and alpha-fetoprotein. In SCID transplanted liver mice, human hepatocyte-like cells were mostly found near vascular structures 56 days posttransplantation. In conclusion, the ability of isolated adult-derived liver mesenchymal stem-like cells to proliferate and differentiate into hepatocyte-like cells both in vitro and in vivo leads to propose them as an attractive expandable cell source for stem cell therapy in human liver diseases.

Risk of hepatocellular carcinoma in liver mitochondrial respiratory chain disorders.

Mitochondrial respiratory chain disorders (MRCD) are a large group of disorders that can affect any organ besides muscles or the central nervous system. We report two children who presented with neonatal cholestasis and progressive cirrhosis, who subsequently developed hepatocellular carcinoma (HCC). This suggests a particular risk of degeneration in these patients and the importance of a regular screening for secondary liver cancer. Suggestion of HCC should lead to early liver transplantation, which was successful without tumor recurrence in the two patients.

Chronic hepatitis B infection: long term comparison of children receiving interferon alpha and untreated controls.

OBJECTIVES: To investigate the virological outcome of chronic hepatitis B (CH-B) in children who received interferon alpha (IFN) compared with no treatment. METHODS: Seventy-four children with CH-B (median age, 6.1 years; 44 boys) selected from a cohort of 158 cases were included and divided into two groups: IFN-treated (n = 37) and control (n = 37). The controls were matched with the treated children by baseline alanine aminotransferase (ALT) levels, sex and age. The Kaplan-Meier method was performed to estimate the time to clearance of hepatitis B e antigen (HbeAg) and hepatitis B surface antigen (HbsAg). RESULTS: Mean duration of follow-up was comparable in two groups (5.2 +/- 3.8 years in treatment group versus 5.2 +/- 3.7 years in control group, NS). HBeAg and HBsAg loss occurred in 20 (54.1%) and three treated children versus 13 (35.1%) and one untreated children (NS), respectively. The 7-year cumulative HBeAg and HBsAg clearance rates were 47.5% and 8.9% after the first visit in the treatment group versus 33.5% and 4.0% in untreated children (NS), respectively. Elevated baseline ALT (two times upper limit of normal) had a significant effect on the long-term cumulative rate of HBeAg seroconversion in treated patients (P = 0.01) but not in the untreated group. CONCLUSIONS: These findings show that the overall long-term virological outcome does not differ significantly between IFN-treated and untreated children but that a significant benefit of treatment on the long term rate of HBeAg seroconversion is obtained in children with higher baseline ALT levels.

Primary duodenogastric reflux in children and adolescents.

UNLABELLED: Primary duodenogastric reflux is a rare disorder in adults which has not yet been documented in children. Six young patients, aged 4.5 to 16.5 years (median 13.5 years) presented with atypical reflux symptoms persisting from 1 to 84 months (median 8 months) and unresponsive to classical antacid therapy. In all six patients, 24 h gastric bilimetry showed excessive bile exposures for absorbances ranging from 0.25 to 0.60. The fraction of time (supine period) above the 0.25 absorbance threshold ranged from 30% to 75% while the 95th percentile value for healthy adults is 31%. In all patients tested, hepato-iminodiacetic acid scintigraphy revealed the occurrence of a massive duodenogastric reflux and four out of five patients had an alkaline shift (fraction of time pH >8 on 24 h lower oesophageal pH monitoring) ranging from 4.2% to 20% (control values 0.0% to 2.9%). Endoscopic findings included abundant bilious gastric leak (6/6) and chronic prepyloric Helicobacter pylorinegative gastritis (2/6). Daily administration of cisapride, sucralfate with or without omeprazole resulted in an improvement of symptoms in five patients within 15 days. This treatment was ineffective in one patient who became symptom-free only after a surgical duodenal switch with fundoplication was performed. CONCLUSION: primary duodenogastric reflux is a rare foregut disorder of unknown origin occurring in late childhood. If suspected, 24 h intragastric bilimetry appears to be a useful investigation to confirm the diagnosis.

Steroid withdrawal after pediatric liver transplantation: a long-term follow-up study in 109 recipients.

BACKGROUND: Steroids remain an important component of maintenance immunosuppression in liver transplantation, but when administered for a long period they may be associated with multiple severe side effects, particularly growth suppression in children. This study was conducted to clarify the balance of potential benefits and risks of steroid withdrawal (SW) in pediatric liver transplantation. METHODS: Between April 1984 and July 2000, 109 pediatric recipients with SW and at least 12 months of follow-up after SW were retrospectively reviewed and divided into three groups according to the type of anticalcineurin at SW: group I (cyclosporine, n=25), group II (cyclosporine microemulsion, n=25), and group III (tacrolimus, n=59). Steroids were withdrawn after a three-step reduction of steroid dosage (taper down to the substitution dose of 0.25 mg/kg/day, switch to alternate-day therapy, progressive SW). Patients were regularly followed up for clinical and biochemical monitoring. RESULTS: Median follow-up was 8.1 (range, 1.6-16.8) years. After SW, neither chronic rejection nor graft nor patient loss occurred. A trend toward lower anticalcineurin trough levels was observed in all groups. Glomerular filtration rate and fasting cholesterol were significantly better in group III (P<0.05). Median height z-score in all patients was -1.1 SD on alternate-day steroids versus -0.2 SD at the time of SW. Height z-score was slightly better in group III (NS). Early SW within 2 years after transplantation allowed a slightly better gain in growth. CONCLUSIONS: SW in pediatric liver transplantation is safe and may be beneficial to height outcome. Tacrolimus seems to offer several advantages in the long-term outcome.

Ontogeny of MAP kinases in rat small intestine: premature stimulation by insulin of BBM hydrolases is regulated by ERKs but not by p-38 MAP kinase.

Although mitogen-activating protein (MAP) kinases are crucial signal transduction molecules regulating cellular proliferation, differentiation, and morphology, their ontogenic changes in the small intestine have not been analyzed. Also, it remains unknown which pathway of activated MAP kinases regulates the expression of brush border membrane hydrolases during growth. Therefore, we have analyzed the mucosal distribution, ontogeny, and responses to insulin and to inhibitors of p44, p42, and p38 MAP kinases in immature and mature enterocytes using Western blot analysis and autoradiography after immunoprecipitation, immunohistochemistry, and in vitro phosphorylation assays. Between d 10 and 40 postpartum, diphosphorylated active p44/p42 extracellular regulated protein kinases (ERKs) increased in abundance compared with total immunoprecipitated ERKs, and were highly responsive to exogenous insulin. In concordance, ERK total activity increased by 4-fold during the same period of growth and was further enhanced 2-fold by exogenous insulin. In weaning rats, ERKs were mainly located in membranes of villus cells and with less intensity in crypt cells. By contrast, p38 MAP kinase was unresponsive to insulin and was confined to nuclei. Administration to sucklings of PD 098059, a specific inhibitor of ERKs, not only inhibited the premature stimulation of sucrase, lactase, and maltase total activities in response to exogenous insulin, but also depressed the natural expression of these brush border membrane enzymes in the absence of insulin stimulation. In concordance, administration of SB 203580, a specific inhibitor of p38 MAP kinase, failed to inhibit both the response of brush border membrane hydrolases to insulin and their natural expression in the absence of insulin stimulation. We conclude that the ontogenic expression of brush border membrane hydrolases and their premature stimulation by insulin are regulated at least in part by the activation of p44/p42 ERKs but not by p38 MAP kinase.

Acute tonsillitis as the first manifestation of post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication that may follow orthotopic liver transplantation (OLT) in children. The first symptoms are often in the ear, nose, or throat (ENT) area. This abnormal proliferation of lymphoid cells is related to Epstein-Barr virus (EBV) infection in immunocompromised children. The incidence of PTLD, EBV status before OLT and at the diagnosis of PTLD, delay between OLT and PTLD, localization, pathological investigations, and the treatment and evolution of PTLD were prospectively evaluated in 77 pediatric liver transplant recipients. Eight patients (10%) developed PTLD, all with an ENT presentation. Seven had acute nonbacterial tonsillitis (with a negative throat swab), and 1 had a pharyngolaryngeal localization at the time of the diagnosis. Four patients had associated involvement outside the ENT area. All patients were EBV-seronegative at the time of OLT; 6 underwent seroconversion at the time of diagnosis, and 2 within 9 and 20 months of diagnosis. All patients presented with low-grade PTLD. All patients with acute tonsillitis associated with EBV seroconversion underwent immediate tonsillectomy, and immunosuppression was decreased as much as tolerated. This therapeutic protocol led to complete recovery in all patients. After OLT in children, nonbacterial tonsillar inflammation or hypertrophy associated with an EBV infection is often the first manifestation of PTLD. Tonsillectomy combined with tapering of immunosuppression offers the best chance for a complete recovery.