Non-alcoholic fatty liver disease.

Together with that of obesity, the prevalence of non-alcoholic fatty liver disease is increasing. NAFLD is associated with insulin resistance and participate to the metabolic syndrome. A proportion of NAFLD patient will present a progressive disease characterized by, in addition to steatosis, hepatocellualr damage, chronic inflammation and progressive fibrosis and termed non-alcoholic steatohepatitis (NASH). NAHS is recognized as the hepatic complication of the metabolic syndrome. Its severity is related to fibrosis progression. Pathophysiological mechanisms underlying NASH remain ill-defined, the tools to identify NAFLD patients at risk for NASH progression and validate therapy are lacking. In this review, we concentrate on 2 aspects of NAFLD/NASH pathogenesis: the mechanisms of hepatic insulin resistance and the determinants of fibrosis in the context of metabolic syndrome.

[The virophages: new predators for viruses?] / Les virophages: de nouveaux prédateurs pour les virus?

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CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) and alcoholic liver disease have similar pathological features. Because CYP2E1 plays a key role in alcoholic liver disease with its ability to stimulate lipid peroxidation, we tested the proposal that CYP2E1 could also be a factor in the development of NASH. In a dietary model - mice fed a methionine- and choline-deficient (MCD) diet - liver injury was associated with both induction of CYP2E1 and a 100-fold increase in hepatic content of lipid peroxides. Microsomal NADPH-dependent lipid oxidases contributed to the formation of these lipid peroxides, and in vitro inhibition studies demonstrated that CYP2E1 was the major catalyst. To further define the role of CYP2E1 as an initiator of oxidative stress in NASH, Cyp2e1(-/-)mice were administered the MCD diet. CYP2E1 deficiency neither prevented the development of NASH nor abrogated the increased microsomal NADPH-dependent lipid peroxidation, indicating the operation of a non-CYP2E1 peroxidase pathway. In Cyp2e1(-/-) mice with NASH (but not in wild-type mice), CYP4A10 and CYP4A14 were upregulated. Furthermore, hepatic microsomal lipid peroxidation was substantially inhibited by anti-mouse CYP4A10 antibody in vitro. These results show that experimental NASH is strongly associated with hepatic microsomal lipid peroxidation. CYP2E1, the main enzyme associated with that process in wild-type mice, is not unique among P450 proteins in catalyzing peroxidation of endogenous lipids. We have now identified CYP4A enzymes as alternative initiators of oxidative stress in the liver.

The PPARgamma agonist pioglitazone inhibits early neoplastic occurrence in the rat liver.

Hepatocellular carcinoma (HCC) is increasing worldwide and is the fifth main cause of cancer-related death. HCC develops on a preneoplastic organ, the cirrhotic liver. Therefore, chemoprevention could play a role in the therapy of HCC. We evaluated the preventive effects of pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on the induction of early carcinogenic events. We monitored pre-neoplastic foci induced by a two-stage initiation/promotion model of hepatocarcinogenesis in rats, using diethylnitrosamine and acetylaminofluorene. Pioglitazone treatment was initiated the day after the first diethylnitrosamine injection. By quantitative morphometry and Western blot, we showed that pioglitazone significantly decreases the size of pre-neoplastic foci. Analysis of proliferation and apoptosis, assessed by immunohistochemistry, demonstrated decreased proliferation but no effect on cell death in rats treated with pioglitazone. These events were associated with an increased expression of the cyclin-dependent kinase inhibitor p27(kip1), compared to the non treated group. In conclusion, pioglitazone inhibits early carcinogenic transformation in a two-step rat model. As pioglitazone has a low toxicity profile, we believe it would be interesting to evaluate its effect in chemoprevention of HCC in humans in a clinical setting.

Somatic mutation in human T-cell leukemia virus type 1 provirus and flanking cellular sequences during clonal expansion in vivo.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma, shows intrapatient genetic variability. Although HTLV-1 can replicate via the reverse transcription of virion RNA to a double-stranded DNA provirus (the conventional manner for retroviruses), its predominant mode of replication is via the clonal expansion (mitosis) of the infected cell. This expansion is achieved by the viral oncoprotein Tax, which keeps the infected CD4 T lymphocyte cycling. Because Tax also interferes with cellular DNA repair pathways, we investigated whether somatic mutations of the provirus that occur during the division of infected cells could account for HTLV-1 genetic variability. METHODS: An inverse polymerase chain reaction strategy was designed to distinguish somatic mutations from reverse transcription-associated substitutions. This strategy allows the proviral sequences to be isolated together with flanking cellular sequences. Using this method, we sequenced 208 HTLV-1 provirus 3' segments, together with their integration sites, belonging to 29 distinct circulating cellular clones from infected individuals. RESULTS: For 60% of the clones, 8%-80% of infected cells harbored a mutated HTLV-1 provirus, without evidence of reverse transcription-associated mutations. Mutations within flanking cellular sequences were also identified at a frequency of 2.8 x 10(-4) substitution per base pair. Some of these clones carried multiple discrete substitutions or deletions, indicating progressive accumulation of mutations during clonal expansion. The overall frequency of somatic mutations increased with the degree of proliferation of infected T cells. CONCLUSIONS: These data indicate that, in vivo, HTLV-1 variation results mainly from postintegration events that consist of somatic mutations of the proviral sequence occurring during clonal expansion. The finding of substitutions in flanking sequences suggests that somatic mutations occurring after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which adult T-cell leukemia/lymphoma is the end point.

Persistent oligoclonal expansion of human T-cell leukemia virus type 1-infected circulating cells in patients with Tropical spastic paraparesis/HTLV-1 associated myelopathy.

The pattern of HTLV-1 replication was assessed through PCR amplification of the 3' proviral integration sites in patients with TSP/HAM at different times. Integration sites were sequenced and oligonucleotides specific for the flanking sequences were synthesized. Together with HTLV-1 LTR specific primers, clonotypic nested PCR was performed on peripheral blood from two patients. The frequencies of five clones studied ranged from 1/300 to 1/1500 PBMCs while four clones persisted for more than 1-5 years. It would seem that Tax driven expansion of T cells may persist for considerable periods of time in TSP/HAM despite strong cellular immunity. This may provide a background for the accumulation of subsequent mutations leading to malignancy.

High circulating proviral load with oligoclonal expansion of HTLV-1 bearing T cells in HTLV-1 carriers with strongyloidiasis.

Adult T cell leukemia (ATLL) develops in 3 - 5% of HTLV-1 carriers after a long period of latency during which a persistent polyclonal expansion of HTLV-1 infected lymphocytes is observed in all individuals. This incubation period is significantly shortened in HTLV-1 carrier with Strongyloides stercoralis (Ss) infection, suggesting that Ss could be a cofactor of ATLL. As an increased T cell proliferation at the asymptomatic stage of HTLV-1 infection could increase the risk of malignant transformation, the effect of Ss infection on infected T lymphocytes was assessed in vivo in HTLV-1 asymptomatic carriers. After real-time quantitative PCR, the mean circulating HTLV-1 proviral load was more than five times higher in HTLV-1 carriers with strongyloidiasis than in HTLV-1+ individuals without Ss infection (P<0.009). This increased proviral load was found to result from the extensive proliferation of a restricted number of infected clones, i.e. from oligoclonal expansion, as evidenced by the semiquantitative amplification of HTLV-1 flanking sequences. The positive effect of Ss on clonal expansion was reversible under effective treatment of strongyloidiasis in one patient with parasitological cure whereas no significant modification of the HTLV-1 replication pattern was observed in an additional case with strongyloidiasis treatment failure. Therefore, Ss stimulates the oligoclonal proliferation of HTLV-1 infected cells in HTLV-1 asymptomatic carriers in vivo. This is thought to account for the shortened period of latency observed in ATLL patients with strongyloidiasis. Oncogene (2000) 19, 4954 - 4960

Hepatic regeneration in a rat model is impaired by chemotherapy agents used in metastatic colorectal cancer.

PURPOSE: Administering Oxaliplatin prior to resection of colorectal liver metastases often induces a Sinusoidal Obstruction Syndrome (SOS), which can affect postoperative patient outcome. Bevacizumab (Anti-VEGF-A) can decrease the severity of SOS and the associated risk of postoperative liver failure. We investigated the impact of both Oxaliplatin (Oxali) and Bevacizumab on liver regeneration in a rat model. MATERIAL AND METHODS: Male Wistar rats underwent a 70% partial hepatectomy (PH) 3 days after a 2 ml intraperitoneal injection of either saline (controls, n = 17), or Oxaliplatin 10, 20 or 50 mg/kg, 5-Fluorouracil 100 mg/kg (5-FU) and Bevacizumab 5 or 10 mg/kg in various combinations (total 98 rats, 11 groups, n = 5-18/group). Liver regeneration was assessed by remnant liver weight recovery and cell proliferation by immunodetection of BrDU incorporation (days 1, 2, 3, 7). Hepatic mRNA expression levels of VEGF-A and of its 2 receptors (Flt-1 and KDR) were quantified by PCR technique. RESULTS: Liver regeneration was impaired for 3 days post PH by Oxali 20 alone and Oxali 10 + 5-FU, without any rescue effect by neither Bevacizumab 5 nor 10 mg/kg. Unlike in humans, there were no sinusoidal changes. VEGF-A mRNA expression and receptor 2 (KDR) expressions decreased 24 h post PH in a similar fashion in controls, Oxali 20 and Oxali 10 + 5-FU groups. All groups had recovered over 60% of their liver weight by day 7. CONCLUSION: Oxaliplatin causes early hepatocyte proliferation impairment post PH, unaffected by Bevacizumab and unexplained by changes in VEGF-A signalling in a Wistar rat model.

Inhibition of chlorzoxazone metabolism, a clinical probe for CYP2E1, by a single ingestion of watercress.

To investigate the effect of watercress on the metabolism of chlorzoxazone, an in vivo probe for CYP2E1, the oral pharmacokinetics of chlorzoxazone was studied in 10 healthy volunteers before and after a single ingestion of a watercress homogenate (50 gm). A third chlorzoxazone pharmacokinetic study was performed after a 1-week treatment with isoniazid (300 mg/day), a well-known CYP2E1 inhibitor. Ingestion of watercress or isoniazid did not affect the oral absorption of chlorzoxazone. The area under the chlorzoxazone plasma concentration-time curve was significantly increased by 56% (p < 0.05) after watercress ingestion and by 135% (p < 0.001) with isoniazid treatment. Similarly, chlorzoxazone elimination half-life was prolonged after watercress (53%; p < 0.05) and isoniazid (104%; p < 0.01) administration. These results show that a single ingestion of watercress inhibits the hydroxylation of chlorzoxazone, an in vivo probe for CYP2E1.

Basis of HTLV type 1 target site selection.

Sequencing integration sites from >/=200 proviruses isolated from infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. However, about 6% of HTLV-1 proviruses were found to be integrated into transcription units, suggesting that in some cells, HTLV-1 integration may alter gene expression in vivo. Therefore, the target choice in vivo seems to be determined by local features rather than by the accessibility of specific regions. This led us subsequently to analyze the role of the DNA structure in HTLV-1 integration in vitro. Double-strand HTLV-1 or HIV-1 3' LTR extremities were used as substrates for in vitro strand transfer reactions using highly purified HTLV-1 and HIV-1 integrases (INs) expressed in Escherichia coli, and two synthetic naked 50-bp double-strand DNA molecules harboring different structures were used as targets. A fluorometric quantitative analysis of integration products was designed to assess the reaction efficiency for both target sequences. As suggested for HTLV-1 in vivo (present results), and, as previously described for other retroviruses in vitro, the structure of the target was found to greatly influence the site and the efficiency of integration. Both HIV-1 and HTLV-1 INs underwent the same target structural constraint, i.e., a strong preference for curved DNA. Altogether these results indicate that if most or all the regions of the genome appear to be accessible to HTLV-1 integration, local DNA curvature seems to confer a kinetic advantage for both in vitro and in vivo HTLV-1 integration.

PCR detection of aminoglycoside resistance genes: a rapid molecular typing method for Acinetobacter baumannii.

Aminoglycoside resistance is common among strains of Acinetobacter baumannii responsible for nosocomial infections, and inactivation of these antibiotics by enzymatic modification is the main mechanism. Different types of aminoglycoside acetyltransferases (AAC), nucleotidyltransferases (ANT), and phosphotransferases (APH) are synthesized by clinical isolates, and several enzymes can be produced by a single strain. Using a multiplex PCR procedure carried out on bacterial thermolysates, we analyzed the aminoglycoside resistance gene content of strains belonging to eight clusters identified by pulsed-field gel electrophoresis. In a single reaction were combined three primer pairs in order to amplify the genes coding for AAC(6')-Ih, AAC(3)-I, and AAC(3)-II, three primer pairs for the genes coding for ANT(2'')-I, APH(3')-VI, and rRNA 16S as internal control, and finally two primer pairs for the genes coding for AAC(6')-Ib and APH(3')-I. According to the aminoglycoside resistance gene patterns, the strains of the eight clusters were distributed into seven classes. This simple and rapid (< 8 h) fingerprinting technique could be a useful tool for the epidemiological investigation of A. baumannii nosocomial infections.

Estimation of chlorzoxazone hydroxylase activity in liver microsomes and of the plasma pharmacokinetics of chlorzoxazone by the same high-performance liquid chromatographic method.

We have developed a HPLC method which allows the determination of chlorzoxazone and its hydroxy metabolite in rat liver microsomes and in human plasma. We found that dehalogenated chlorzoxazone or 2-benzoxazolinone was a convenient and stable internal standard. Proteins were precipitated with diluted perchloric acid and the supernatant was extracted with ethyl acetate. Complete resolution of the peaks was achieved within 20 min with a Spherisorb ODS-1 column. The inter-day R.S.D.s were 6.5% at 0.5 microgram/ml of hydroxychlorzoxazone and 5.8% at 1 microgram/ml of chlorzoxazone in human plasma. The reproducibility of the method has been demonstrated for a large number of samples over a long period.

Midazolam and cortisol metabolism before and after CYP3A induction in humans.

INTRODUCTION: CYP3A is responsible for the metabolism of numerous endogenous and exogenous compounds. Several substrates of CYP3A have been investigated to assess the CYP3A-metabolizing capacity of an individual in an attempt to predict the rate of metabolism of other CYP3A substrates. Two such tests of CYP3A activity are the midazolam plasma clearance after its intravenous administration and the 6beta-OH cortisol urinary ratio. Possible correlations between these 2 tests were investigated before and after treatment with rifampin in a group of healthy volunteers. METHODS: Pharmacokinetic parameters of midazolam and 6beta-OH cortisol urinary ratio were evaluated in 8 volunteers before and after 6 days treatment with rifampin, a potent inducer of CYP3A, and after cessation of rifampin treatment. RESULTS: Midazolam systemic clearance and the 6beta-OH cortisol urinary ratio were significantly higher at Days 7 and 10 than at Day 0. There was a strong positive correlation between these 2 parameters (r = 0.70, p < 0.001). In contrast, no correlation was observed between the ratio of the AUCs of 1'-OH midazolam vs. midazolam (AUC0-1(1'-OH)/AUC0-t(MDZ)) or the ratio of plasma concentration of 1'-OH midazolam vs. midazolam (C30 min(1'-OH)/C30 min(MDZ)) and the 6beta-OH cortisol urinary ratio (r = 0.05, p = 0.82; r = 0.04, p = 0.88, respectively). Considering only data obtained before or after treatment with rifampin, however, no correlation was observed between midazolam systemic clearance and the 6beta-OH cortisol urinary ratio. CONCLUSIONS: These data demonstrate that there is a strong positive correlation between systemic midazolam clearance and 6beta-OH cortisol urinary ratio before and after induction. This suggests that the 6beta-OH cortisol urinary ratio test is a non-invasive alternative to the use of systemic midazolam clearance for monitoring the time-course of CYP3A induction.

[Adenoid cystic carcinoma. Apropos of a case localized in the lacrimal apparatus]. / Carcinome adénoide kystique. A propos d'une localisation au sac lacrymal.

The authors report one case of adenoid cystic carcinoma of the medical canthus of the orbit. This carcinoma is typified by slow but aggressive growth with multiple local recurrences. Surgery is necessary but radical excision with total exenteration is a difficult decision. Radiotherapy associated to surgery might be usefull.

[Prevention of infectious complications of major upper respiratory and digestive tract surgery by the combination of pefloxacin-amoxicillin-clavulanic acid]. / Prévention des complications infectieuses de la chirurgie majeure des cancers des voies aéro-digestives supérieures par l'association: pefloxacine-amoxicilline acide clavulanique.

The authors report the results of a therapeutic trial aimed at assessing the efficacy and costs of a twin-antibiotic therapy associating Pefloxacin and Amoxicillin-clavulanic acid for the prevention of infectious complications in major surgery for cancer of the upper respiratory and digestive tracts. The results obtained show 10% of complications, including 6.5% of local complications, with 4.5 to 9.5 day treatments. In our opinion, the poor results noted in the literature in studies on a simple intraoperative antibiotic prophylaxis confirm the need to use a real, so-called "curative" antibiotic therapy over a sufficient period for this type of surgery. This antibiotic therapy must be well tolerated, active for the contaminating organisms, produce no selection of resistant bacteria, and raise hopes to reduce postoperative infectious morbidity, therefore the costs of treatment.

Association of endothelial lipase Thr111Ile polymorphism with proliferative retinopathy in type 2 diabetes patients.

AIM: Our previous study demonstrated that the endothelial lipase (EL) C.584C>T polymorphism (rs2000813, p.Thr111Ile) was significantly associated with diabetic retinopathy (DR). The present work was conducted to see if this specific variant of the EL gene was more specifically linked to the severity of DR. METHODS: This retrospective cohort study was based on a review of the institutional charts of 287 type 2 diabetes patients (mean age = 59.7 years; mean BMI = 29.0 kg/m(2); mean HbA1c=8.4%) genotyped for the EL C.584C>T polymorphism (rs2000813, p.Thr111Ile). The stage of DR was also determined for each genotype (CC, CT, TT). RESULTS: On univariate analysis, the minor allele homozygote TT variant was significantly associated with severe DR (OR: 4.3; 95% CI: 1.4, 13.1) compared with the major CC homozygote. No significant result was found for the CT heterozygote. Multivariate analysis revealed an increased risk for TT homozygotes to present with severe non-proliferative DR (OR: 8.09; 95% CI: 1.23, 53.1) or proliferative DR. Other associations were not significant. CONCLUSION: Minor allele homozygosity for this EL variant (c.584C>T) could be a significant risk factor for developing severe, sight-threatening disease due to proliferative DR. Further prospective studies of this EL polymorphism in a larger population sample are needed to confirm these results.

Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms.

Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.