Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells-derived hepatic stem cells.

Pluripotent stem cells have been investigated as a renewable source of therapeutic hepatic cells, in order to overcome the lack of transplantable donor hepatocytes. Whereas different studies were able to correct hepatic defects in animal models, they focused on the most mature phenotype of hepatocyte-like cells (HLCs) derived from pluripotent stem cells and needed freshly prepared cells, which limits clinical applications of HLCs. Here, we report the production of hepatic stem cells (pHSCs) from human-induced pluripotent stem cells (hiPSCs) in xeno-free, feeder-free, and chemically defined conditions using as extracellular matrix a recombinant laminin instead of Matrigel, an undefined animal-derived matrix. Freshly prepared and frozen pHSCs were transplanted via splenic injection in Gunn rats, the animal model for Crigler-Najjar syndrome. Following cell transplantation and daily immunosuppression treatment, bilirubinemia was significantly decreased (around 30% decrease, P < .05) and remained stable throughout the 6-month study. The transplanted pHSCs underwent maturation in vivo to restore the deficient metabolic hepatic function (bilirubin glucuronidation by UGT1A1). In conclusion, we demonstrate for the first time the differentiation of hiPSCs into pHSCs that (a) are produced using a differentiation protocol compatible with Good Manufacturing Practices, (b) can be frozen, and (c) are sufficient to demonstrate in vivo therapeutic efficacy to significantly lower hyperbilirubinemia in a model of inherited liver disease, despite their immature phenotype. Thus, our approach provides major advances toward future clinical applications and would facilitate cell therapy manufacturing from human pluripotent stem cells.

Cholic acid for primary bile acid synthesis defects: a life-saving therapy allowing a favorable outcome in adulthood.

BACKGROUND: Oral cholic acid (CA) replacement has been shown to be an effective therapy in children with primary bile acid synthesis defects, which are rare and severe genetic liver diseases. To date there has been no report of the effects of this therapy in children reaching adulthood. The aim of the study was to evaluate the long-term effectiveness and safety of CA therapy. METHODS: Fifteen patients with either 3ß-hydroxy-Δ5-C27-steroid oxidoreductase (3ß-HSD) (n = 13) or Δ4-3-oxosteroid 5ß-reductase (Δ4-3-oxo-R) (n = 2) deficiency confirmed by mass spectrometry and gene sequencing received oral CA and were followed prospectively. RESULTS: The median age at last follow-up and the median time of follow-up with treatment were 24.3 years (range: 15.3-37.2) and 21.4 years (range: 14.6-24.1), respectively. At last evaluation, physical examination findings and blood laboratory test results were normal in all patients. Liver sonograms were normal in most patients. Mean daily CA dose was 6.9 mg/kg of body weight. Mass spectrometry analysis of urine showed that excretion of the atypical metabolites remained low or traces in amount with CA therapy. Liver fibrosis scored in liver biopsies or assessed by elastography in 14 patients, after 10 to 24 years with CA therapy, showed a marked improvement with disappearance of cirrhosis (median score < F1; range: F0-F2). CA was well tolerated in all patients, including five women having 10 uneventful pregnancies during treatment. CONCLUSIONS: Oral CA therapy is a safe and effective long-term treatment of 3ß-HSD and Δ4-3-oxo-R deficiencies and allows affected children to reach adulthood in good health condition without the need for a liver transplantation.

[A wrong move in an amateur football player reveals a light chain myeloma]. / Un faux mouvement chez un footballeur amateur révélateur d'un myélome à chaînes légères.

Light chain multiple myeloma is a hematologic malignancy characterized by an excess of tumor plasma cells in the bone marrow and a monoclonal light chain in blood. It is generally diagnosed in patients aged 60-75 years old. Hypercalcemia, anemia, kidney failure, and bone pains are the main clinical and biological signs. Here is an atypical case report about a 30 year-old man who was diagnosed a light chain multiple myeloma. This patient had been suffering from back pain for 5 months. Osteolytic lesions were discovered on X-rays prescribed by the family practitioner. Admitted to the Emergency department, all blood tests showed results within the normal range. The serum protein electrophoresis was also normal. Only the urine analysis showed proteinuria. The urine immunofixation electrophoresis showed a massive κ light chain. The bone marrow aspiration cell count confirmed the myeloma diagnosis with an infiltration of dystrophic plasma cells. The patient was transferred to the hematology ward of Necker Hospital for treatment of light chain myeloma.

Human neonatal hepatocyte transplantation induces long-term rescue of unconjugated hyperbilirubinemia in the Gunn rat.

Crigler-Najjar type 1 disease is a rare inherited metabolic disease characterized by high levels of unconjugated bilirubin due to the complete absence of hepatic uridine diphosphoglucuronate-glucuronosyltransferase activity. Hepatocyte transplantation (HT) has been proposed as an alternative treatment for Crigler-Najjar syndrome, but it is still limited by the quality and the low engraftment and repopulation ability of the cells used. Because of their attachment capability and expression of adhesion molecules as well as the higher proportion of hepatic progenitor cells, neonatal hepatocytes may have an advantage over adult cells. Adult or neonatal hepatocytes were transplanted into Gunn rats, a model for Crigler-Najjar disease. Engraftment and repopulation were studied and compared by immunofluorescence (IF). Additionally, the serum bilirubin levels, the presence of bilirubin conjugates in rat serum, and the expression of uridine diphosphate glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) in rat liver samples were also analyzed. Here we show that neonatal HT results in long-term correction in Gunn rats. In comparison with adult cells, neonatal cells showed better engraftment and repopulation capability 3 days and 6 months after transplantation, respectively. Bilirubinemia decreased in the transplanted animals during the whole experimental follow-up (6 months). Bilirubin conjugates were also present in the serum of the transplanted animals. Western blots and IF confirmed the presence and expression of UGT1A1 in the liver. This work is the first to demonstrate the advantage of using neonatal hepatocytes for the treatment of Crigler-Najjar in vivo.

Correction of hyperbilirubinemia in gunn rats by surgical delivery of low doses of helper-dependent adenoviral vectors.

Helper-dependent adenoviral (HDAd) vectors are attractive for liver-directed gene therapy because they can drive sustained high levels of transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery because of a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potential lethal consequences. We have previously shown in nonhuman primates that delivery of HDAd in surgically isolated livers resulted in a significantly higher hepatic transduction with reduced systemic vector dissemination compared with intravenous delivery and multiyear transgene expression. Encouraged by these data, we have now employed a surgical vector delivery method in the Gunn rat, an animal model for Crigler-Najjar syndrome. After vector delivery into the surgically isolated liver, we show phenotypic correction at the low and clinically relevant vector dose of 1 × 10(11) vp/kg. Correction of hyperbilirubinemia and increased glucuronidation of bilirubin in bile was achieved for up to 1 year after vector administration. Surgical delivery of the vector was well tolerated without signs of acute or chronic toxicity. This method of delivery could thereby be a safer alternative to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.

Lentiviral vectors that express UGT1A1 in liver and contain miR-142 target sequences normalize hyperbilirubinemia in Gunn rats.

BACKGROUND & AIMS: Crigler-Najjar type 1 (CN-I) is an inherited liver disease caused by an absence of bilirubin-uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1) activity. It results in life-threatening levels of unconjugated bilirubin, and therapeutic options are limited. We used adult Gunn rats (an animal model of the disease) to evaluate the efficiency of lentiviral-based gene therapy to express UGT1A1 in liver. METHODS: Gunn rats were given intraportal injections of VSVG-pseudotyped lentiviral vectors that encode UGT1A1 under the control of a liver-specific transthyretin promoter (mTTR.hUGT1A1); this vector does not contain target sequences for miR-142, a microRNA that is expressed specifically in hematopoietic cells. Rats were also injected with the vector mTTR.hUGT1A1.142T, which contains 4 copies of the miR-142 target sequences; its messenger RNA should be degraded in antigen-presenting cells. Bilirubinemia was monitored, and the presence of transduced hepatocytes was analyzed by quantitative polymerase chain reaction. Vector expression was tested in vitro in rat hematopoietic cells. RESULTS: In Gunn rats, bilirubin levels normalized 2 weeks after administration of mTTR.hUGT1A1. However, hyperbilirubinemia resumed 8 weeks after vector administration, concomitant with the induction of an immune response. In contrast, in rats injected with mTTR-UGT1A1.142T, bilirubin levels normalized for up to 6 months and transduced cells were not eliminated. CONCLUSIONS: Lentiviral vectors that express UGT1A1 reduce hyperbilirubinemia in immunocompetent Gunn rats for at least 6 months. The immune response against virally expressed UGT1A1 can be circumvented by inclusion of miR-142 target sequences, which reduce vector expression in antigen-presenting cells. This lentiviral-based gene therapy approach might be developed to treat patients with CN-I.

Transient increase in intrahepatic pressure mediates successful treatment of the Gunn rat with reduced doses of lentiviral vector.

Lentiviral vectors can stably transduce hepatocytes and are promising tools for gene therapy of hepatic diseases. Although hepatocytes are accessible to blood-borne viral vectors through fenestrations of the hepatic endothelium, improved liver transduction after delivery of vectors to the blood stream is needed. As the normal endothelial fenestration and lentiviral vectors are similar in size (150 nm), we hypothesized that a transient increase in hepatic blood pressure may enhance in vivo gene transfer to hepatocytes. We designed a simple surgical procedure, by which the liver is temporarily excluded from blood flow. Lentiviral vectors were injected in a large volume to increase intrahepatic pressure. We demonstrated that in the Gunn rat, a model of Crigler-Najjar disease, the administration of low vector doses (corresponding to a multiplicity of infection of 0.2) by this procedure resulted in therapeutic correction of hyperbilirubinemia, without toxicity. The correction was sustained for 10 months (end of study). The same vector amounts yielded only partial correction after intraportal delivery. We believe that this new and clinically applicable strategy may broaden the range of genetic liver diseases accessible to gene therapy.

Ex vivo lentivirus transduction and immediate transplantation of uncultured hepatocytes for treating hyperbilirubinemic Gunn rat.

BACKGROUND: Ex vivo liver gene therapy provides an attractive alternative to orthotopic liver transplantation for the treatment of liver diseases. We previously reported a protocol in which human primary hepatocytes are highly transduced in Suspension with Lentiviral vectors and Immediately Transplanted (SLIT). Here, we evaluated the SLIT approach in Gunn rats, the animal model for Crigler-Najjar syndrome type 1, a defect in bilirubin UDP-glucuronosyltransferase (BUGT). METHODS: We constructed lentiviral vectors coding for BUGT under control of an ubiquitous promoter. Control vectors contained Green Fluorescent Protein (GFP) under control of the same promoter. Hepatocytes were isolated from jaundiced Gunn rats and transduced in suspension for four hr. After washing, 2x10 hepatocytes were immediately transplanted into syngeneic rats. Bilirubinemia and bile pigments were regularly assessed after cell transplantation. The percentage and presence of transduced hepatocytes was analyzed by immunohistochemistry in GFP-transplanted animals. RESULTS: In rats receiving BUGT-transduced hepatocytes, bilirubinemia decreased by about 30%. The level of correction remained stable for up to 240 days. Bilirubin glucuronides were present in the bile of treated animals, indicating the metabolic activity of engrafted hepatocytes. In contrast, bilirubinemia in GFP-transplanted rats did not decline but rather increased. GFP-positive hepatocytes amounted to 0.5-1% of the liver, which is in agreement with the number of transplanted and genetically-modified hepatocytes (6x10). CONCLUSIONS: This work reports the first demonstration of long-term metabolic benefit after rapid transplantation of ex vivo lentivirally tranduced hepatocytes. Therefore, this study demonstrates the therapeutic proof-of-principle and potential of the SLIT approach for treating inherited metabolic liver diseases.

Taurine status and response to intravenous taurine supplementation in adults with short-bowel syndrome undergoing long-term parenteral nutrition: a pilot study.

Taurine deficiency in patients on long-term parenteral nutrition may be involved in cholestasis. We aimed to assess plasma taurine and tauro-conjugated bile acids in adults with short-bowel syndrome and their response to intravenous taurine. Thirty-two adult patients, who had been on taurine-free parenteral nutrition for a mean of 59 (SE 14) months for short-bowel syndrome, were studied retrospectively. In a second study, a subgroup of ten patients with chronic cholestasis received taurine-enriched (6.0 (SE 0.6) mg/kg per d) parenteral nutrition for 55 (SE 13) months. Post-absorptive plasma taurine and bile acid concentrations were measured and liver function tests routinely sampled. At baseline, plasma taurine was lower in patients with a jejunal length of less than 35 cm (group A, n 16) than in those with a jejunal length of 35 cm or more (group B, n 16): 43 (SE 3) v. 58 (SE 4) micromol/l (P=0.01). The groups were no different in terms of chronic cholestasis (12/16 v.13/16 patients), total bile acids (26 (SE 13) v.14 (SE 5) micromol/l) or the ratio of tauro-conjugated:glyco-conjugated bile acids (5 (SE 2) v.8(SE 4)%, usual range 30-60%). After supplementation, there was an increase in plasma taurine level (63 (SE 8) v. 43 (SE 4), P=0.007) but was no change in either total bile acids or the ratio of tauro-conjugated: glyco-conjugated bile acids. There was a significant decrease in aspartate aminotransferase level. Long-term parenteral nutrition for short-bowel syndrome is associated with an impaired tauro-conjugation of bile acids (enterohepatic pool), irrespective of plasma taurine level (systemic pool) and despite long-term taurine intravenous supplementation.

Hyperhomocysteinaemia, methylenetetrahydrofolate reductase polymorphism and risk of coronary artery disease.

BACKGROUND: Hyperhomocysteinaemia is an independent, graded risk factor for coronary artery disease (CAD). The methylenetetrahydrofolate reductase (MTHFR) polymorphism is associated with hyperhomcysteinaemia and may therefore influence individual susceptibility to CAD. We have investigated this risk factor in a Tunisian Arab population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the C677T and A1298C variants of the MTHFR gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed as the number of affected vessels. Plasma total homocysteine (tHcy) concentration was determined using a direct chemiluminescence assay. RESULTS: MTHFR CC, CT and TT genotype frequencies in the CAD group were significantly different from those observed in the control group (49%, 35% and 16% versus 48.3%, 45.8% and 5.8%, respectively; P = 0.031). However, MTHFR AA, AC and CC genotypes frequencies in the CAD group were not significantly different from the control group ( P = 0.568). Patients with CAD showed higher plasma tHcy concentrations than patients without CAD (15.86 +/- 8.63 micromol/L versus 11.90 +/- 3.25 micromol/L, P < 0.001). There was no association between the MTHFR polymorphisms and the number of stenosed vessels. Patients with the MTHFR TT genotype had higher plasma tHcy, serum creatinine, cholesterol and triglyceride concentrations than patients with the MTHFR CC genotype. CONCLUSIONS: The C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinaemia, lipid dysregulation and the presence of CAD in this Tunisian Arab population.

Hyperhomocysteinemia, endothelial nitric oxide synthase polymorphism, and risk of coronary artery disease.

BACKGROUND: Hyperhomocysteinemia is an independent, graded risk factor for coronary artery disease (CAD). The G894T variant of endothelial nitric oxide synthase (eNOS) was postulated to be associated with hyperhomocysteinemia and could influence individual susceptibility to CAD. The aims of this study were to investigate (a) the relationship of the eNOS G894T polymorphism with the presence and the severity of CAD and (b) the possible relationship between hyperhomocysteinemia and the eNOS G894T variant for the risk of CAD severity in a Tunisian population. METHODS: We used PCR with restriction fragment length polymorphism analysis to detect the G894T variant of the eNOS gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed by the number of affected vessels. Total plasma homocysteine concentrations were determined by direct chemiluminescence assay. RESULTS: The frequencies of the eNOS GG, GT, and TT genotypes in the CAD group were significantly different from those in the control group (45%, 44%, and 11% vs 60%, 35.8% and 4.2%, respectively; P = 0.035). There was no association between the eNOS G894T genotype frequencies and the number of stenosed vessels (P = 0.149). In the CAD group, the coexistence of the 894 GT or TT genotypes and hyperhomocysteinemia led to an increased risk of CAD severity. CONCLUSION: The G894T polymorphism of the eNOS gene is associated with the presence of CAD, and in conjunction with hyperhomocysteinemia, increased the risk of CAD severity in a Tunisian population.

Therapeutic lentivirus-mediated neonatal in vivo gene therapy in hyperbilirubinemic Gunn rats.

Crigler-Najjar type 1 disease (CN-1) is a genetic disorder characterized by high levels of unconjugated bilirubin due to the absence of hepatic UDPglucuronosyltransferase (UGT1) activity. Here we show that in vivo neonatal hepatocyte transduction with a lentiviral vector expressing the defective enzyme resulted in long-term correction in Gunn rats, a model of CN-1. Lentiviral vectors harboring the human UGT1 cDNA (approved symbol UGT1A1) under the control of a liver-specific transthyretin promoter were produced. Two-day-old Gunn rats were injected with 50 microl of vector. Bilirubinemia was monitored at 6 weeks and monthly thereafter. At 6 weeks, bilirubinemia was completely normalized in treated animals, whereas it remained around 100 microM in control rats. The level of correction remained stable for up to 42 weeks. Large amounts of bilirubin conjugates were present in the bile of corrected animals. PCR and Western blots confirmed the presence and expression of UGT1 in liver. The estimated proportion of transduced hepatocytes was 40% and transduced cells were not detected in extrahepatic tissues except bone marrow in some animals. This work represents the first demonstration of a complete and permanent correction of hyperbilirubinemia in Gunn rats using lentiviral vectors.

Successful gene therapy of the Gunn rat by in vivo neonatal hepatic gene transfer using murine oncoretroviral vectors.

Crigler-Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-glucuronosyl transferase (UGT1), resulting in high levels of unconjugated bilirubin. CN1 is an attractive candidate disease for gene therapy. Here we show that in vivo neonatal hepatocyte transduction using recombinant oncoretroviral vectors results in long-term and complete phenotype correction in Gunn rats, a model for CN1. Two-day-old newborn Gunn rats were injected via the temporal vein with 200 microL UGT1 or control beta-galactosidase retroviral vectors. In UGT1-injected animals, bilirubinemia was normal at 6 weeks (3 micromol/L) and remained in the normal range (i.e., <10 micromol/L) for more than 34 weeks. In contrast, in beta-galactosidase-injected animals as well as in noninjected controls, bilirubinemia remained at a high level (i.e., >100 micromol/L) during the whole experimental follow-up. Large amounts of bilirubin monoglucuronides and diglucuronides were present in the bile of treated animals. Finally, polymerase chain reaction and reverse transcription polymerase chain reaction analysis as well as Western blot confirmed the presence and expression of UGT1 almost exclusively in the liver. The estimated proportion of transduced hepatocytes was in the range of 5% to 10%. In conclusion, complete and permanent correction of hyperbilirubinemia in newborn Gunn rats using retroviral vectors can be obtained, paving the way for future gene therapy for CN1.

Effects of a 3-mo consumption of short-chain fructo-oligosaccharides on parameters of colorectal carcinogenesis in patients with or without small or large colorectal adenomas.

Intervention studies of colorectal adenoma recurrence have demonstrated the need for surrogate markers of the cancer risk. Short-chain fructo-oligosaccharides (sc-FOS) have protective actions on colon carcinogenesis in animal models. We investigated differences in biological markers between adenoma and adenoma-free subjects, before and after 3 mo of daily intake of 10 g sc-FOS, within a multicenter study. After a full colonoscopy, 3 groups were studied at baseline and after 3 mo: 26 subjects with small colorectal adenoma(s), 18 with large adenoma(s), and 30 with no adenoma. At baseline, the mean fecal butyrate concentration was significantly lower in the adenoma groups than in the adenoma-free group (12.01 +/- 5.08 vs. 17.28 +/- 7.34 mmol/g dry weight) but was significantly increased in that group after 3-mo ingestion of sc-FOS (15.7 +/- 8.0 mmol/g; P = 0.02). In subjects without adenoma, sc-FOS ingestion was associated with a decrease in fecal lithocholic acid (P = 0.02) and an increase in cholic acid (P = 0.02), chenodeoxycholic acid (P = 0.04), total primary bile acids (P = 0.03), and ursodeoxycholic acid (P = 0.05). Fecal pH, blood parameters, and crypt cell proliferation were not significantly modified by sc-FOS ingestion in either group. In subjects with and without adenoma, sc-FOS affects some aspects of the colonic environment, which may be involved in prevention of colorectal neoplasia.

Fecal primary bile acids and serum cholesterol are associated with colorectal adenomas.

In order to identify biomarkers of colorectal tumors, 20 subjects with colorectal adenomas were compared with 20 controls as regards fecal parameters (pH, short-chain fatty acids, bile acids, and sterols), blood parameters (bile acids, cholesterol, triglycerides, glycemia and insulinemia), and rectal cell proliferation. Variables were compared by unconditional logistic regression, controlling for gender. There were significant and positive associations between risk of adenoma and total fecal primary bile acids and serum cholesterol, with odds ratios for the third versus first tertile = 9.4 (P for trend = 0.03) and 8.6 (P for trend = 0.04), respectively. There was a trend towards an increased triglycerides level in adenoma subjects compared with controls (P = 0.08). These three parameters correlated with cell proliferation, although cell proliferation itself was not significantly associated with adenomas. In conclusion, these results suggest that fecal primary bile acids and serum cholesterol are markers of early events of colorectal carcinogenesis.

Effects of ursodeoxycholic acid (ursodiol) treatment on chronic viral hepatitis in heart transplant patients: results of a prospective, double-blind, placebo-randomized study.

BACKGROUND: Chronic viral hepatitis averages 15% to 20% in heart transplant patients. Several studies have shown that ursodiol may improve liver biochemistry in patients with chronic hepatitis. We used a double-blind randomized controlled trial to evaluate the effect of ursodiol in heart transplant patients with chronic viral hepatitis. METHODS: Thirty heart patients with chronic viral hepatitis B, C, or non-A-G received ursodiol, 800 mg per day (group 1), and 30 received placebo (group 2) for 12 months. Endpoints were improvement in liver biochemical tests and in total Knodell score. Intent-to-treat and per-protocol analyses were performed. RESULTS: At entry, both groups were comparable for all of the studied parameters. During the study period, serum alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase variations were not different between group 1 and group 2 patients. Knodell score improved in 20% of group 1 patients and in 43% of group 2 patients (NS). Adverse events or mortality were not different in the two groups during the study period. Similar results were observed by intent-to-treat and per-protocol analyses. CONCLUSIONS: A 12-month course of ursodiol therapy had no effect on liver enzymes or liver histology in heart transplant patients with chronic hepatitis.