Combined Analysis of Metabolomes, Proteomes, and Transcriptomes of Hepatitis C Virus-Infected Cells and Liver to Identify Pathways Associated With Disease Development.

BACKGROUND & AIMS: The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes. METHODS: Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA sequencing, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID (urokinase-type plasminogen activator/severe combined immunodeficiency) mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA sequencing and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsy specimens from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival, and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function. RESULTS: We quantified 21,950 messenger RNAs (mRNAs) and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of the mRNAs, but not proteins, that regulate the innate immune response; we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate ß-oxidation of very long-chain fatty acids; we found intracellular accumulation of very long-chain fatty acids in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of the mRNAs and proteins associated with peroxisome function, indicating perturbation of peroxisomes. We found that defects in peroxisome function were associated with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients. CONCLUSIONS: We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in the expression levels of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.

A compound heterozygote case of isolated sulfite oxidase deficiency.

We report an isolated sulfite oxidase deficiency in the first child boy of a non-consanguineous Caucasian family. He's a compound heterozygote for the sulfite oxidase gene, presenting low cystine, undetectable homocysteine and normal uric acid blood concentrations and undetectable sulfite oxidase activity in his cultured fibroblasts. Both mutations are not reported yet. The clinical presentation was typical and severe, with generalized status epilepticus and premature death.

Mitomycin C Pharmacokinetics as Predictor of Severe Neutropenia in Hyperthermic Intraperitoneal Therapy.

BACKGROUND: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is an approach to overcome peritoneal carcinomatosis from colorectal adenocarcinoma. Mitomycin C (MMC) is frequently used but not devoid of toxicity, of which the most common and feared is neutropenia. Our study explores the clinical and surgical risk factors of neutropenia and a possible link between MMC pharmacokinetics and neutropenia as HIPEC's supervention. METHODS: A total of 45 patients undergoing CRS-HIPEC for peritoneal carcinomatosis of colorectal origin between 2004 and 2010 were followed. For each patient, MMC was measured in plasma at different times during HIPEC and the area under the MMC concentration-time curve (MMC-AUC) was calculated. RESULTS: The incidence of neutropenia was 40 %. No demographic, clinical, or surgical factors increased the risk of neutropenia. However, we found that the occurrence of neutropenia and its gravity increased in direct correlation with an increase in MMC plasma concentration 30 min (T30) and 45 min (T45) after the start of HIPEC. The same correlation was observed between the MMC-AUC and the risk of neutropenia. CONCLUSIONS: Neutropenia is a frequent complication associated with MMC-HIPEC. The results of our study indicate the feasibility and the potential benefit of a protocol including the MMC dosage at T30 after the start of HIPEC. A threshold of 572 µg/L gives a predictive sensitivity of 86 % and a specificity of 80 %. These results must be considered in the management of patients undergoing MMC-HIPEC in order to place high-risk patients under neutropenic monitoring while the other patients can undergo simple hematological monitoring.

Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20.

Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca(2+) concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways.

Xanthurenic acid distribution, transport, accumulation and release in the rat brain.

Tryptophan metabolism through the kynurenine pathway leads to several neuroactive compounds, including kynurenic and picolinic acids. Xanthurenic acid (Xa) has been generally considered as a substance with no physiological role but possessing toxic and apoptotic properties. In the present work, we present several findings which support a physiological role for endogenous Xa in synaptic signalling in brain. This substance is present in micromolar amounts in most regions of the rat brain with a heterogeneous distribution. An active vesicular synaptic process inhibited by bafilomycin and nigericin accumulates xanthurenate into pre-synaptic terminals. A neuronal transport, partially dependant on adenosine 5'-triphosphate (ATP), sodium and chloride ions exists in NCB-20 neurons which could participate in the clearance of extracellular xanthurenate. Both transports (neuronal and vesicular) are greatly enhanced by the presence of micromolar amounts of zinc ions. Finally, electrical in vivo stimulation of A10-induced Xa release in the extracellular spaces of the rat prefrontal cortex. This phenomenon is reproduced by veratrine, K+ ions and blocked by EGTA and tetrodotoxin. These results strongly argue for a role for Xa in neurotransmission/neuromodulation in the rat brain, thus providing the existence of specific Xa receptors.