Véronique Barbu

Epimutation of the telomeric imprinting center region on chromosome 11p15 in Silver-Russell syndrome

Silver-Russell syndrome (SRS, OMIM 180860) is a congenital disorder characterized by severe intrauterine and postnatal growth retardation, dysmorphic facial features and body asymmetry. SRS is genetically heterogenous with maternal uniparental disomy with respect to chromosome 7 occurring in ∼10% of affected individuals. Given the crucial role of the 11p15 imprinted region in the control of fetal growth, we hypothesized that dysregulation of genes at 11p15 might be involved in syndromic intrauterine growth retardation. We identified an epimutation (demethylation) in the telomeric imprinting center region ICR1 of the 11p15 region in several individuals with clinically typical SRS. This epigenetic defect is associated with, and probably responsible for, relaxation of imprinting and biallelic expression of H19 and downregulation of IGF2. These findings provide new insight into the pathogenesis of SRS and strongly suggest that the 11p15 imprinted region, in addition to those of 7p11.2-p13 and 7q31-qter, is involved in SRS.

Epidermal growth factor receptor and HER-3 restrict cell response to sorafenib in hepatocellular carcinoma cells

BACKGROUND & AIMS Sorafenib is the standard of care for the treatment of advanced hepatocellular carcinoma (HCC). However, primary and acquired resistance is observed in patients. We examined whether gefitinib, which inhibits both epidermal growth factor receptor (EGFR) and HER-3 phosphorylation, could improve HCC cell response to sorafenib. METHODS Sorafenib and gefitinib were tested in HCC tumor xenografts and in sorafenib-sensitive and sorafenib-resistant HCC cell lines. Biomarkers relevant to the HER system were analyzed by Western blotting and ELISA. RNA interference was used to downregulate the HER system. Amphiregulin concentrations were measured by ELISA in sera from patients under sorafenib treatment. RESULTS Sorafenib combined with gefitinib significantly inhibited tumor growth in mice and reduced cell viability in vitro compared to single agents. In cell lines cultured in 10% serum or treated with EGF, sorafenib alone inhibited phospho-STAT3 while it maintained or even increased phospho-ERK and/or phospho-AKT. The paradoxical effects of sorafenib were prevented by gefitinib or by downregulation of EGFR and HER-3 expression. In cells with acquired resistance to sorafenib, aberrant activation of EGFR/HER-3 receptors as well as overexpression of several EGFR ligands were observed. These enhanced autocrine/paracrine loops led to the constitutive activation of ERK and AKT and conferred increased sensitivity to gefitinib. Increased serum concentrations of amphiregulin were observed in 10 out of 14 patients under sorafenib treatment compared to baselines. CONCLUSIONS Signaling pathways controlled by EGFR and HER-3 restrict sorafenib effects both in naive and sorafenib-resistant HCC cells. Consequently, gefitinib cooperates with sorafenib to increase antiproliferative response and to prevent resistance.

Fetal microchimerism in primary biliary cirrhosis

BACKGROUND/AIMS Recent studies have suggested a role of fetal microchimerism in the pathogenesis of scleroderma. The present study investigated the potential role of fetal microchimerism in primary biliary cirrhosis (PBC), a closely related disease. METHODS A quantitative nested polymerase chain reaction was used to detect Y-chromosome sequences in the peripheral blood or the liver of PBC women and controls having male children and no transfusion or miscarriage history. RESULTS Male microchimerism was found in the peripheral blood from 45% (9 of 20) of PBC women and 25% (5 of 20) of healthy controls matched for the number of male children and age of the youngest son (p=0.28), and in the liver-biopsy specimens from 33% (5 of 15) of PBC women and 32% (8 of 25) of controls. The level of chimerism did not differ between patients and controls either in blood or in liver. Microchimerism was not related to the severity of the disease but was more frequent in PBC patients with anticentromere antibodies (p=0.049). CONCLUSIONS Fetal microchimerism does not seem to play a major role in most cases of PBC. However, the association with anticentromere antibodies suggests a possible role in the subgroup of patients with CREST syndrome or scleroderma.

Frequency of cytochrome P450 2C9 allelic variants in the Chinese and French populations.

Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of different drugs with low therapeutic index such as oral anticoagulants. CYP2C9*2 and CYP2C9*3 are two single nucleotide polymorphic allelic variants. The frequency of these alleles in different ethnic populations is extremely variable. In this study, we compared the frequencies of CYP2C9 allelic variants among 394 Chinese living in Shanghai to 151 French Caucasians living in Paris. The allelic frequencies of CYP2C9 variants of the Chinese and the French subjects were 0.963, 0.001, 0.036 and 0.77, 0.15, 0.08 for CYP2C9*1, CYP2C9*2, CYP2C9*3, respectively. Chinese CYP2C9*3 allelic frequency was twice as lower as the French subjects, but three times higher than Korean (0.036 vs. 0.011). The CYP2C9*2 allele could be detected in only one Chinese subject, whereas it represented the major allelic variant in French Caucasians. The low frequency of the CYP2C9*2 and CYP2C9*3 allelic variants in Chinese subjects does not justify their detection in clinical practice, unlike French Caucasians.

Rapamycin inhibits cdk4 activation, p 21WAF1/CIP1 expression and G1‐phase progression in transformed mouse fibroblasts

Rapamycin, a bacterial macrolide antibiotic, is a potent immunosuppressant agent that blocks cell proliferation by inhibiting the G1/S transition in several cell types. In sensitive cells, rapamycin inhibits the phosphorylation of p70 S6K and of Rb; however, the precise mechanisms involved have not been elucidated. In the mouse BP‐A31 fibroblasts, synchronised in G0/G1 phase by serum starvation and induced to reinitiate the G1‐phase progression, rapamycin inhibited the entry into S phase. The effect of rapamycin was situated in early G1 phase. The assembly of the cyclin D1/cdk4 complexes that phosphorylate Rb early in the G1 phase was not modified by the drug. Nevertheless, an inhibition of the activation of cyclin D1/cdk4 and cyclin E/cdk2 as well as of Rb phosphorylation accompanied the cell cycle arrest. Remarkably, rapamycin reduced the level of total p21WAF1/CIP1 as well as that of p21WAF1/CIP1 associated with the cyclin D1/cdk4 complexes. Besides its inhibitory activity toward cdk, p21WAF1/CIP1 has been recently found to participate in the formation/stabilisation/nuclear translocation of cyclin D1/cdk4 complexes. We propose that the inhibition of the expression of p21WAF1/CIP1 is a mechanism by which rapamycin inhibits the triggering of the cdk cascade in the BP‐A31 cells.

Genotype‐phenotype relationships in the low‐phospholipid‐associated cholelithiasis syndrome: A study of 156 consecutive patients

The low‐phospholipid‐associated cholelithiasis syndrome (LPAC; OMIM 171060) is a peculiar form of intrahepatic cholelithiasis occurring in young adults, associated with ABCB4/MDR3 gene sequence variations. Our aim was to determine the genotype‐phenotype relationships in 156 consecutive patients with the criteria of LPAC syndrome. A variant was detected in 79 (61 missense and 18 truncating sequence variants), 63 being monoallelic. The clinical features (age at onset, high prevalence in women, frequency and severity of acute and chronic complications, intrahepatic cholestasis of pregnancy [ICP]) were similar in the patients with or without ABCB4 gene sequence variation. Truncating variations were associated with an earlier onset of symptoms both in women and men. Acute and chronic biliary complications were variant‐independent. Half of the women who had pregnancy developed ICP. The frequency of ICP and fetal complications were similar in patients with missense and truncating variants. Conclusion: The LPAC syndrome is more frequent in women and highly associated with ICP. Half of the patients harbored missense or truncating variants of the ABCB4 gene. The characteristics of the patients without detectable variant are similar to those with variant, indicating that yet unexplored regions of the ABCB4 and other genes may be involved. (Hepatology 2013;53:1105–1110)

Defects in Gallbladder Emptying and Bile Acid Homeostasis in Mice With Cystic Fibrosis Transmembrane Conductance Regulator Deficiencies

BACKGROUND & AIMS Patients with cystic fibrosis (CF) have poorly defined defects in biliary function. We evaluated the effects of cystic fibrosis transmembrane conductance regulator (CFTR) deficiency on the enterohepatic disposition of bile acids (BAs). METHODS Bile secretion and BA homeostasis were investigated in Cftr(tm1Unc) (Cftr-/-) and CftrΔF508 (ΔF508) mice. RESULTS Cftr-/- and ΔF508 mice did not grow to normal size, but did not have liver abnormalities. The gallbladders of Cftr-/- mice were enlarged and had defects in emptying, based on (99m)technetium-mebrofenin scintigraphy or post-prandial variations in gallbladder volume; gallbladder contraction in response to cholecystokinin-8 was normal. Cftr-/- mice had abnormal gallbladder bile and duodenal acidity, and overexpressed the vasoactive intestinal peptide-a myorelaxant factor for the gallbladder. The BA pool was larger in Cftr-/- than wild-type mice, although there were no differences in fecal loss of BAs. Amounts of secondary BAs in portal blood, liver, and bile of Cftr-/- mice were much lower than normal. Expression of genes that are induced by BAs, including fibroblast growth factor-15 and BA transporters, was lower in the ileum but higher in the gallbladders of Cftr-/- mice, compared with wild-type mice, whereas enzymes that synthesize BA were down-regulated in livers of Cftr-/- mice. This indicates that BAs underwent a cholecystohepatic shunt, which was confirmed using cholyl-(Ne-NBD)-lysine as a tracer. In Cftr-/- mice, cholecystectomy reversed most changes in gene expression and partially restored circulating levels of secondary BAs. The ΔF508 mice overexpressed vasoactive intestinal peptide and had defects in gallbladder emptying and in levels of secondary BAs, but these features were less severe than in Cftr-/- mice. CONCLUSIONS Cftr-/- and CftrΔF508 mice have defects in gallbladder emptying that disrupt enterohepatic circulation of BAs. These defects create a shunt pathway that restricts the amount of toxic secondary BAs that enter the liver.

Lymphocyte P‐glycoprotein expression and activity before and after rifampicin in man

Abstract— It has recently been shown that P‐glycoprotein (P‐gp) is inducible by rifampicin in the human gut as shown in intestinal biopsies. The present study was performed in order to test the hypothesis that human peripheral lymphocytes can be used to assess such an inducibility. We also assessed inter‐ and intra‐individual variability of P‐gp expression and activity in peripheral lymphocytes. Blood samples from 13 healthy volunteers were collected 1.7, 14 and 19 days after inclusion. Rifampicin treatment (600 mg/day) was administered from clay 15 to day 18. Lymphocyte P‐gp expression was measured at the messenger RNA level by semi‐quantitative RT‐PCR and at the protein level by immunostaining flow cytometry. P‐gp activity was determined by flow cytometry with rhodamine 123 efflux. Cytochrome P4503A4 (CYP3A4) inducibility was measured by comparing the urinary metabolic ratio of 6β‐hydroxycortisol/cortisol on day 14 and 19. Lymphocyte P‐gp expression and activity was not induced by rifampicin, while it increased CYP3A4 activity from 5.0 ± 4.0 to 22.9 ± 16.6 (P < 0.001). There was a 3–4‐fold inter‐individual variability and a 3–44% intra‐individual variability of lymphocyte P‐gp expression and activity. Peripheral lymphocytes are not an appropriate material to assess P‐gp inducibility in humans. P‐gp shows significant inter‐ and intra‐individual variability in human lymphocytes.

Survival and differentiation of porcine hepatocytes encapsulated by semiautomatic device and allotransplanted in large number without immunosuppression

BACKGROUND/AIMS The aim of this study was to evaluate the survival and functions of porcine hepatocytes transplanted in large quantities in the peritoneal cavity of allogeneic animals following semiautomatic encapsulation. METHODS Isolated porcine hepatocytes and a polymer solution composed of AN69 were coextruded through a double lumen spinneret. Minitubes containing hepatocytes were transplanted in the peritoneal cavity of 12 pigs (4 x 10(9) cells/animal) in the absence of immunosuppressive therapy. Seven, 15, and 21 days after transplantation, minitubes was collected and processed for analyses. The morphology was examined under light and electron microscopy. Albumin synthesis was assessed by semi-quantitative reverse transcription-polymerase chain reaction. Cytochrome P450 3A (CYP3A) gene expression was analyzed by Western blot and by testosterone 6-beta-hydroxylation assay. RESULTS The device allowed to encapsulate 55 x 10(6) hepatocytes/min. Hepatocytes exhibited normal structural and ultrastructural features up to day 21. Albumin gene expression decreased progressively between days 0 and 21. The amount of CYP3A protein and 6-beta-hydroxylase activity were approximately 2-fold lower at days 7 and 15 than in freshly encapsulated hepatocytes, and further decreased thereafter. CONCLUSIONS The preservation of hepatocyte functions during 1-2 weeks is encouraging for potential short-term use of such bioartificial liver in future clinical application.

Low levels of lymphocyte MDR1 gene expression during early renal transplantation in patients treated with tacrolimus.

Abstract— P‐glycoprotein (P‐gp) is a membrane efflux pump increasing the transport of drugs such as tacrolimus out of the cells. The aim of the study was to determine the kinetics of lymphocyte P‐gp expression in patients treated with tacrolimus during the first 3 months following renal transplantation. Lymphocyte MDR1 gene expression was measured by semi‐quantitative RT‐PCR a few hours before transplantation, 3 weeks and 3 months after the graft. Lymphocyte MDR1 gene expression was low in all the 10 patients compared to 10 healthy volunteers: 0.30 ± 0.07 arbitrary units (patients) vs. 1.74 ± 0. 55 (healthy volunteers) (P = 0.0002). MDR1 gene expression decreased among the patients during the study: 0.28 ± 0.12 (3 weeks later) and 0.12 ± 0.09 (3 months later) (P = 0.006). We can conclude that lymphocyte MDR1 gene expression among patients before renal transplantation is low and remains low during the first 3 months following the graft.