BackgroundProtective properties of P42 were studied in different models of Huntington’s disease (HD): HeLa cells, but also Drosophila and R6/2 models of HD. Our results identified that P42 prevents different phenotypes induced by the expression of the mutant protein, including aggregation process, defects in axonal trafficking and neurodegeneration. As a result P42 is able to rescue motor deficits but also anxiety and memory defects. We also showed that P42 is able to prevent but also to treat some of the phenotypes/symptoms of HD.Aims and resultsThe study of the modes of action of P42 identified that P42 acts at different levels: i) it interacts with N17 domain, therefore preventing the first steps of the aggregation process; ii) as a part of the Htt protein, we analysed its role in vesicular trafficking along the axons, and found that P42 is one of the microtubule binding domain of the Htt protein and it is acting as an enhancer of the vesicular trafficking; iii) we also found that P42 is able to enhance the level of expression of cortical BDNF, defective in the context of the disease; iv) finally we identified a role of P42 in neuronal plasticity and activity.ConclusionsThese data showed that P42 allows different entry points in the disease, explaining its impact in both pre- and post-symptomatic windows. It is able to target the disease at different levels, not only by lowering the negative effect of the mutant polyQ-Htt, but also by enhancing the physiological functions of the normal Htt protein.

One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrPC level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders.

Background The starting point of our study was the observation that the ratio between wild-type and mutant polyQ-Htt is crucial in Huntington’s disease progression. Screening on polyQ-hHtt aggregation allowed the identification of a protective 23aa peptide (P42) lying within human Htt. Aims The aim was to test the protective properties of P42 on different HD models: HeLa cells, Drosophila, but also R6/2 mice. Methods/techniques The use of P42 at therapeutic ends in a mammalian model, required that this peptide was able to cross the blood-brain-barrier, and that we find a non-invasive route for chronic administration of P42. To this end, we associated two complementary strategies: i) we fused P42 to the protein transduction domain TAT to ensure its diffusion; ii) we adapted a nanostructure-based drug delivery system for brain targeting called Aonys®, where P42-TAT was formulated in a water-in-oil micro-emulsion (NP42T) and administered through buccal and/or rectal mucosa. In order to verify the pertinence of this way of administration, we also used an original imaging analysis of P42 derivatives, using MALDI Imaging Mass Spectrometry on brain sections. Results/outcome - We first verified that the fusion P42-TAT peptide did not change the functionality of P42, and identified a dose-response effect of P42-TAT action in HeLa cells. - To investigate the therapeutic potential of NP42T, buccal and rectal administrations were performed daily in R6/2 HD mouse model. The effects of NP42T treatment were analysed on a range of behavioural associated defects (foot-clasping, rotarod or body weights), and several markers (aggregation, astrogliosis or ventricle areas) recorded on brain sections. Conclusion Our data globally identify an efficient protective effect of the NP42T, leading to a clear recovery of all the phenotypes tested. This study not only shows that we identified an efficient peptide against HD, but it also describes a powerful delivery technology.

Background In Huntington’s disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice. Results To this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections. Conclusions These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.

Introduction: During embryogenesis, liver and pancreas are generated from a common endodermic precursor.Furthermore, in vivo in humans and in rodents, and in pathological environment, adult hepatocyte clusters have been observed in pancreatic tumors.These hepatic cells could come from mature pancreatic cell transdifferentiation or from pancreatic progenitor cell differentiation towards the hepatocyte lineage.These observations raised the hypothesis that progenitors from endoderm developmental stage could persist during adult life (unidentified in humans).In this study we established a similar strategy for isolation, proliferation and differentiation of human progenitor cells from adult liver and pancreas.Results: Organ fragments were dissociated with collagenase and extracted cells were seeded in human adult hepatic progenitor cell proliferation medium.After 4-7 days, various cellular populations appeared.The proliferation medium was replaced at confluence by the hepatocyte differentiation medium.We observed an increased expression of the hepatocyte genes albumin and CYP3A4, and no detection of the pancreatic genes insulin and glucagon.At day 21, clusters of positive cells for albumin and cytokeratins 7 and 8/18 were observed.In a second set of experiments, we isolated a population negative for CD105 and CD90 antigens after 7-14 days of amplification.The populations from both liver and pancreas had an identical morphology.In the hepatocyte differentiation medium, the expression of albumin and CYP3A4 mRNAs increased whereas insulin and glucagon gene expression was not detected.At day 21 positive clusters for albumin and cytokeratins 7 and 8/18 were observed. Conclusion:In conclusion, we showed the feasibility to isolate an epithelial population containing progenitor cells with hepatocyte differentiation capacity from human adult liver and pancreas.These in vitro results strongly suggest the presence of cells with hepatocyte differentiation capacity in the pancreas, as observed in vivo under particular pathological conditions.In the future, these progenitor populations could be a model for basic research and a cell source for liver and/or pancreas biotherapy.

Background Huntington9s disease (HD) pathogenesis results from a dominant effect of polyQ-hHtt and a loss of function of Htt. Indeed, addition of wild-type Htt improves polyQ-hHtt-induced defects. Aims Screening on polyQ-hHtt aggregation in HeLa cells, we identified an inhibitory 23aa peptide (Pep42) lying within human Htt. Interestingly Pep42 acts specifically on HD model through a direct interaction with N17 domain, suggesting a role of Pep42 on nucleation and aggregation processes. The protective properties of Pep42 were confirmed on different polyQ-hHtt-induced phenotypes in HD Drosophila model. Therefore Pep42 presents a clear therapeutical potential that we further tested in R6(2) mice. Methods/techniques In order to attempt to cross the brain-blood barrier, Pep42 was fused to TAT-HIV. Then, we compared its protective effect after intraventricular injections, intravenous injections, vs buccal and rectal mucosa administrations in a water-in-oil microemulsion-based delivery vector (called Aonys, MedesisPharma). Results/outcome Pharmacokinetics analysed on brain sections at 6hrs and 24hrs after Pep42 treatment showed a nice spread of the peptide in the brain, still visible at 24 h. Finally, administration of Pep42/Aonys or empty vector was performed daily in 2 wks–11 wks old R6(2) mice for pre-symptomatic treatment and in 9–11 wks old R6(2) mice for post-symptomatic treatment. Several polyQ-hHtt behavioural associated defects were analysed (rotarod, foot-clasping or body weights), and several markers were followed on brain sections. These data globally show the protective effect of Pep42-TAT when delivered through Aonys vector, in both pre- and post-symptomatic treatments. Conclusions Altogether these data highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.

patients (2.9±1.5 fold). No change is recorded for IRS1, PTEN4 and AKT. The expression of IRS1, AKT, PTP, PTEN1, PTEN4, PI3K, MTOR and MAP3K – belonging to the insulin signaling pathway-, did not change when the cells are infected with JFH1, although protein levels did change: IRS1 protein is decreased when the cells were infected and insulin was present in the medium. Two IRS1 inhibitory phosphorylation (S612 and S307) were also decreased in virus+insulin. MTOR protein is increased in the presence of viral particles. PTEN is down-regulated in infected cells and this effect is more evident when insulin is present in the medium. Conclusions: Hepatitis C virus modifies both gene and protein expression in a genotype-dependent manner: in patients infected by HCV genotype 1 PTP and MTOR increased their expression in NR patients, while PTEN1 is down-regulated. However, JFH1 inhibits the amount of PTEN protein and increased IRS1 and mTOR.

La fraction de cellules non parenchymateuses épithéliales (NPE) isolée à partir de pièces d'hépatectomie contient des progéniteurs intrahépatiques capables de se différencier in vitro en cellules proches de l'hépatocyte. Nous avons testé leur capacité à se greffer et se différencier dans le foie de souris NOD/SCID en régénération hépatique. Afin d'évaluer la greffe de façon non invasive, les NPE non différenciées ont été préalablement transduites par un vecteur lentiviral exprimant la luciférase sous promoteur constitutif. La bioluminescence est suivie grâce à des caméras après injection de luciférine et anesthésie de l'animal. Un avantage prolifératif a été apporté aux cellules transplantées par l'utilisation de la rétrorsine, un agent alkylant qui bloque la réplication des hépatocytes endogènes, suivie d'une hépatectomie partielle au 2/3 pour stimuler la régénération. Un million de cellules NPE-luc a été injecté directement dans le parenchyme hépatique, dans deux sites différents. Chaque semaine, les souris sont observées à la caméra, et un prélèvement sanguin est réalisé. Lors du sacrifice des animaux, 4 semaines après transplantation, le sang, le foie et la rate ont été recueillis. La présence et la fonctionnalité des cellules humaines sont analysées par des techniques d'immunohistochimie à l'aide d'anticorps dirigés contre les noyaux humains et l'albumine humaine, de techniques de PCR, et de dosage ELISA de protéines plasmatiques circulantes humaines. Nous avons observé la présence des NPE-Luc, signée par l'activité luminescente, dans le foie des souris transplantées, avec un signal fort à j3, plus faible à j10, puis absent à j21. Les analyses immunohistochimiques réalisées à l'aide de l'anticorps anti-albumine humaine sur des coupes du foie et de la rate révèlent la présence des cellules NPE dans ces tissus. Dans le parenchyme hépatique, elles sont généralement localisées près des espaces portes, le plus souvent isolées, et rarement en clusters. Elles sont abondantes dans la rate, également situées près des vaisseaux sanguins. L'expression de l'albumine humaine est confirmée dans ces tissus par la détection de l'ARNm par RT-PCR, et par la présence d'albumine humaine circulante dans le sang périphérique dès la première semaine post-transplantation, avec un pic de sécrétion à 3 semaines puis une diminution à 4 semaines. Les cellules NPE non différenciées peuvent s'implanter dans un modèle animal de régénération hépatique et acquérir in situ la fonction de sécrétion d'albumine humaine. Le degré de différenciation des NPE vers l'hépatocyte est en cours d'évaluation. Ces progéniteurs intrahépatiques, de part leur capacité à proliférer in vitro, pourraient constituer une source cellulaire alternative aux hépatocytes dans une stratégie de biothérapie cellulaire des insuffisances hépatiques.

Recent findings show that colchicine (COL) in submicromolar concentrations downregulates the expression of major drug-metabolizing P450 enzymes in human hepatocytes. Concomitantly, the expression of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) was diminished by COL, whereas expression of glucocorticoid receptor (GR) remained unaltered. A tentative mechanism is perturbation of the GR-PXR/CAR-CYP2/3 signaling cascade, resulting in restricted transcriptional activity of GR receptor by colchicine. In this work we focused on 10-demethylcolchicine (colchiceine; EIN), a structural analogue and a putative metabolite of COL that lacks tubulin-binding activity. We investigated the effects of EIN on the expression of PXR, CAR, and GR receptors in primary cultures of human hepatocytes. In contrast with the effects of COL, EIN does not alter the expression of PXR, CAR, and/or GR receptors mRNAs. In addition, EIN had no effects on transcriptional activities of PXR, CAR, and GR receptors in reporter gene assays using transfected cell lines. Considering that COL and EIN are structurally very close and differ only in their tubulin-binding activity, the data presented imply that the deleterious effects of COL on the GR-PXR/CAR-CYP2/3 cascade are primarily due to perturbation of the microtubule network. Our data support the idea of replacing COL by EIN, which is less toxic and does not interact with xenoreceptors.

Primary culture of human hepatocytes is an in vitro model widely used to investigate numerous aspects of liver physiology and pathology. The technique used to isolate human hepatocytes is based on two-step collagenase perfusion. Originally performed in situ for obtaining hepatocytes from the adult rat, this technique has been adapted to the ex vivo treatment of human liver from organ donors or from lobectomy resection for medical purposes. This chapter describes experimental protocols for the isolation of hepatocytes from human liver tissue and for the preparation of short- and long-term cultures in which cells retain a differentiated phenotype for at least 1 mo. The various aspects emphasized here include the conditions for obtaining tissue, quality control of tissue for efficient perfusion, collagenase perfusion parameters, solutions for perfusion and culture media, cell substrate, cell plating, specific equipment, and safety conditions.

CAR (Constitutive Androstane Receptor, NR1I3) and PXR (Pregnane X Receptor, NR1I2) are nuclear receptors that control both endogenous and exogenous toxic compounds metabolism and elimination. In contrasts to PXR, CAR is expressed only in adult liver and not in fetal liver. Factors regulating human CAR (hCAR) gene expression remain largely unexplored. We focused our attention on the effect of Hepatocyte Nuclear Factor (HNF)-4a isoforms onto hCAR gene promoter. HNF-4a gene possesses two promoters, proximal P1 and distal P2, whose use results in HNF-4a1 and HNF4a7 transcripts, respectively. While HNF-4a7 is mainly expressed in the embryonic liver, HNF-4a1 is almost exclusively in the adult liver. Transient transfections showed that HNF-4a1, but not HNF-4a7, enhances hCAR promoter activity. 5′-Deletion and mutation analysis of hCAR promoter identified a proximal DR1 regulatory element, termed CAR HNF-4a-RE (−114ccAGGCCTtTGCCCTga). HNF-4a1 increased the activity of a heterologous promoter driven by two copies of the CAR HNF-4a-RE. We demonstrate that in vitro synthetized HNF-4a binds to this element in gel shift assays, while endogenous HNF4-a interacts with hCAR promoter in chromatin immunoprecipitation studies performed with human hepatocytes. In addition, we observed that glucocorticoid receptor and HNF-4a1 (but not HNF4-a7) cooperate to maximally transactivate CAR gene expression, while HNF4-a7 repressed this cooperation. Finally, we observed a strong correlation between CAR and HNF-4a1 mRNA expression level in human liver tissu samples, and an inverse correlation between CAR and HNF-4a7 in human hepatoma suggesting that, while HNF-4a1 positively regulates CAR expression in adult, the fetal HNF-4a7 isoform represses CAR gene expression.

Cytochrome P450 (CYP) 1A1 attracts attention mainly because of its role in production of carcinogenic reactive metabolites from polycyclic aromatic hydrocarbons such as benzo[a]pyrene, but recent developments indicate its apparent role in cell cycle progression. Expression of the enzyme is subject to regulation by aryl hydrocarbon receptor (AhR). It has been shown that induction of CYP 1A1 in HepG2 cells and primary rat hepatocytes by tetrachloro-p-dibenzodioxin (TCDD) is diminished by colchicine and nocodazole. Both compounds decrease CYP1A1 mRNA, protein, and activity levels in HepG2 cells and mRNA level in primary rat hepatocytes. Neither compound significantly affected [(3)H]-TCDD binding to AhR, thus their effect on AhR transcriptional activity proceeds via indirect means. For colchicine and nocodazole are well-known microtubule interfering agents, we also assessed their effect on microtubule integrity in both cell types under investigation. Both compounds disrupt cytoskeleton integrity with differential potency depending on cell type. The observed suppression of AhR transcriptional activity by colchicine and nocodazole can be associated with G2/M cell cycle arrest in HepG2 cells, as demonstrated by Myt1 protein hyperphosphorylation and FACS analysis. However, in primary rat hepatocytes, cytoskeleton disruption is independent of cell cycle while displaying the same influence on AhR-dependent gene transcription. In our view, this is evidence in favor of modulatory role of cytoskeleton in AhR-dependent expression.

In the last few years, several studies have provided a causal link between constitutive activation of nuclear factor kappa-B (NF-kappaB) and the initiation and development of cancer. More recently, it appears that a cancer-induced inflammatory response may be an important factor in the inter-individual variability of the response to and toxic effects of cancer chemotherapy, as well as in the alteration of drug metabolism enzyme expression in patients. The relationships between chronic inflammation (or infection), cancer and drug metabolism are many: chronic infections lead to inflammation, inflammation may lead to cancer, cancer usually leads to an inflammatory syndrome, and inflammation alters the expression of drug metabolising enzymes and thus of the efficiency of cancer chemotherapy. This review focuses on the functional consequences of NF-kappaB activation during oncogenesis and on the expression of the major cytochrome P450s (CYP) involved in anticancer therapies. Finally, the potential role of NF-kappaB as the missing link between inflammation, cancer and alteration in hepatic drug metabolism in patients with cancer is discussed.