[Show abstract][Hide abstract] ABSTRACT: Opioids are the cornerstone of treatment for moderate to severe pain, but chronic use leads to maladaptations that include: tolerance, dependence and opioid-induced hyperalgesia (OIH). These responses limit the utility of opioids, as well as our ability to control chronic pain. Despite decades of research, we have no therapies or proven strategies to overcome this problem. However, murine haplotype based computational genetic mapping and a SNP data base generated from analysis of whole-genome sequence data (whole-genome HBCGM), provides a hypothesis-free method for discovering novel genes affecting opioid maladaptive responses.
[Show abstract][Hide abstract] ABSTRACT: Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers.
Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers.
FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants. Please see later in the article for the Editors' Summary.
PLoS Medicine 04/2014; 11(4):e1001628. · 15.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a novel method for differentiating adipocyte-derived stem cells (ASCs) into hepatocyte-like cells (iHeps). ASCs are cultured as spherical cellular aggregates, and are then induced by culture in chemically defined media for a short time period to differentiate into spherical-culture iHeps (SCi-Heps). SCi-Heps have many of the in vitro functional properties of mature hepatocytes, and they can stably reconstitute functioning human liver in vivo in a murine model system, and implantation studies demonstrate that SCi-Heps have a very low malignant potential. All human liver regenerative procedures, including ultrasound-guided direct liver implantation, are scalable and appropriate for human clinical use. These methods can be used to achieve the major promise of regenerative medicine; it may now be possible to regenerate human liver using autologous stem cells obtained from a readily accessible tissue.
[Show abstract][Hide abstract] ABSTRACT: CRPS is a painful, debilitating, and often-chronic condition characterized by various sensory, motor, and vascular disturbances. Despite many years of study, current treatments are limited by our understanding of the underlying mechanisms. Little is known on the molecular level concerning changes in gene expression supporting the nociceptive sensitization commonly observed in CRPS limbs, or how those changes might evolve over time.
We used a well-characterized mouse tibial fracture/cast immobilization model of CRPS to study molecular, vascular and nociceptive changes. We observed that the acute (3 weeks after fracture) and chronic (7 weeks after fracture) phases of CRPS-like changes in our model were accompanied by unique alterations in spinal gene expression corresponding to distinct canonical pathways. For the acute phase, top regulated pathways were: chemokine signaling, glycogen degradation, and cAMP-mediated signaling; while for the chronic phase, the associated pathways were: coagulation system, granzyme A signaling, and aryl hydrocarbon receptor signaling. We then focused on the role of CcL2, a chemokine that we showed to be upregulated at the mRNA and protein levels in spinal cord tissue in our model. We confirmed its association with the nociceptive sensitization displayed in this model by demonstrating that the spinal but not peripheral administration of a CCR2 antagonist (RS504393) in CRPS animals could decrease mechanical allodynia. The spinal administration of CcL2 itself resulted in mechanical allodynia in control mice.
Our data provide a global look at the transcriptional changes in the spinal cord that accompany the acute and chronic phases of CRPS as modeled in mice. Furthermore, it follows up on one of the top-regulated genes coding for CcL2 and validates its role in regulating nociception in the fracture/cast model of CRPS.
[Show abstract][Hide abstract] ABSTRACT: We investigated microRNA expression profiles of malignant pleural mesothelioma (MPM) specimens to identify novel microRNA that are potentially involved in the oncogenic transformation of human pleural cells.
microRNA microarray transcriptional profiling studies of 25 MPM primary tumors were performed. We used normal pleural from an unmatched patient cohort as normal comparators. To confirm microarray data, we used real-time quantitative PCR. Representative cell lines H513 and H2052 were used in functional analyses of microRNA-1.
In addition to several novel MPM-associated microRNAs, we observed that the expression level of microRNA-1 was significantly lower in tumors as compared to normal pleural specimens. Subsequently, pre-mir of microRNA-1 was introduced into MPM cell lines to overexpress this microRNA. Phenotypic changes of these altered cells were assayed. The cellular proliferation rate was significantly inhibited after overexpression of microRNA-1. Early and late apoptosis was increased markedly in microRNA-1-transfected cell lines. Taken together, these data suggested that overexpression of microRNA-1 induced apoptosis in these MPM cell lines, acting as a tumor suppressor. We confirmed our observations by assessing in the transduced MPM cells cell cycle-related genes, pro-apoptotic and anti-apoptotic genes, which all showed coordinated, significant changes characteristic of the apoptotic phenotype.
Thus, further investigation and validation of our microRNA database of MPM may elucidate previously unrecognized molecular pathways and/ or mechanisms by identifying novel microRNAs that are involved in malignant transformation. Our study has now found microRNA-1 to be one of these MPM-associated microRNAs, with potential pathogenic and therapeutic significance.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Better pancreatic cyst fluid biomarkers are needed. OBJECTIVE: To determine whether metabolomic profiling of pancreatic cyst fluid would yield clinically useful cyst fluid biomarkers. DESIGN: Retrospective study. SETTING: Tertiary-care referral center. PATIENTS: Two independent cohorts of patients (n = 26 and n = 19) with histologically defined pancreatic cysts. INTERVENTION: Exploratory analysis for differentially expressed metabolites between (1) nonmucinous and mucinous cysts and (2) malignant and premalignant cysts was performed in the first cohort. With the second cohort, a validation analysis of promising identified metabolites was performed. MAIN OUTCOME MEASUREMENTS: Identification of differentially expressed metabolites between clinically relevant cyst categories and their diagnostic performance (receiver operating characteristic [ROC] curve). RESULTS: Two metabolites had diagnostic significance-glucose and kynurenine. Metabolomic abundances for both were significantly lower in mucinous cysts compared with nonmucinous cysts in both cohorts (glucose first cohort P = .002, validation P = .006; and kynurenine first cohort P = .002, validation P = .002). The ROC curve for glucose was 0.92 (95% confidence interval [CI], 0.81-1.00) and 0.88 (95% CI, 0.72-1.00) in the first and validation cohorts, respectively. The ROC for kynurenine was 0.94 (95% CI, 0.81-1.00) and 0.92 (95% CI, 0.76-1.00) in the first and validation cohorts, respectively. Neither could differentiate premalignant from malignant cysts. Glucose and kynurenine levels were significantly elevated for serous cystadenomas in both cohorts. LIMITATIONS: Small sample sizes. CONCLUSION: Metabolomic profiling identified glucose and kynurenine to have potential clinical utility for differentiating mucinous from nonmucinous pancreatic cysts. These markers also may diagnose serous cystadenomas.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: We investigated whether human pharmacogenetic factors could be characterized using chimeric NOG mice expressing a thymidine kinase transgene (TK-NOG) with 'humanized' livers. MATERIALS AND METHODS: The rate of human-specific metabolism of two drugs was measured in chimeric mice reconstituted with human hepatocytes with different CYP2C19 and CYP2C9 genotypes. RESULTS: The rate of generation of human-predominant drug metabolites for S-mephenytoin and diclofenac in the chimeric mice was correlated with the CYP2C19 (n=9 donors, P=0.0005) or CYP2C9 (n=7 donors, P=0.0394) genotype, respectively, of the transplanted human hepatocytes. CONCLUSION: This study suggests that TK-NOG mice reconstituted with hepatocytes obtained from a relatively small number (3-10 per genotype) of human donors may be a promising model to identify human pharmacogenetic factors affecting the metabolism of clinically important drugs. For certain compounds, this innovative model system enables pharmacogenetic analyses to be efficiently performed in vivo within a human context and with control of all confounding environmental variables.
Pharmacogenetics and Genomics 12/2012; · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inter-species differences in drug metabolism have made it difficult to use pre-clinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDI) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite prior to human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, since the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was co-administered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of pre-clinical drug assessment.
Journal of Pharmacology and Experimental Therapeutics 11/2012; · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have very little information about the metabolomic changes that mediate neurobehavioral responses, including addiction. It was possible that opioid-induced metabolomic changes in brain could mediate some of the pharmacodynamic effects of opioids. To investigate this, opiate-induced brain metabolomic responses were profiled using a semi-targeted method in C57BL/6 and 129Sv1 mice, which exhibit extreme differences in their tendency to become opiate dependent. Escalating morphine doses (10-40 mg/kg) administered over a 4-day period selectively induced a two-fold decrease (p<0.00005) in adenosine abundance in the brainstem of C57BL/6 mice, which exhibited symptoms of narcotic drug dependence; but did not decrease adenosine abundance in 129Sv1 mice, which do not exhibit symptoms of dependence. Based on this finding, the effect of adenosine on dependence was investigated in genetically engineered mice with alterations in adenosine tone in the brain and in pharmacologic experiments. Morphine withdrawal behaviors were significantly diminished (P<0.0004) in genetically engineered mice with reduced adenosine tone in the brainstem, and by treatment with an adenosine receptor(1) (A(1)) agonist (2-chloro-N6-cyclopentyladenosine, 0.5 mg/kg) or an A(2a) receptor (A(2a)) antagonist (SCH 58261 1 mg/kg). These results indicate that adenosine homeostasis plays a crucial role in narcotic drug responses. Opiate-induced changes in brain adenosine levels may explain many important neurobehavioral features associated with opiate addiction and withdrawal.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: To advance our understanding of disease biology, the characterization of the molecular target for clinically proven or new drugs is very important. Because of its simplicity and the availability of strains with individual deletions in all of its genes, chemogenomic profiling in yeast has been used to identify drug targets. As measurement of drug-induced changes in cellular metabolites can yield considerable information about the effects of a drug, we investigated whether combining chemogenomic and metabolomic profiling in yeast could improve the characterization of drug targets. BASIC METHODS: We used chemogenomic and metabolomic profiling in yeast to characterize the target for five drugs acting on two biologically important pathways. A novel computational method that uses a curated metabolic network was also developed, and it was used to identify the genes that are likely to be responsible for the metabolomic differences found. RESULTS AND CONCLUSION: The combination of metabolomic and chemogenomic profiling, along with data analyses carried out using a novel computational method, could robustly identify the enzymes targeted by five drugs. Moreover, this novel computational method has the potential to identify genes that are causative of metabolomic differences or drug targets.
Pharmacogenetics and Genomics 10/2012; · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The review examines the rationale and translational utility of computational genetic studies using murine models of biomedical traits.
Computational genetic mapping studies have identified the genetic basis for biomedical trait differences in 16 different murine models, including several that are of importance to perioperative medicine.
The results have generated new treatments for alleviating incisional pain and narcotic drug withdrawal symptoms, which are now in clinical trials. A recent study identified allelic differences affecting chronic pain responses in mice and humans, which may enable a new 'personalized' approach to treating chronic pain.
Current opinion in anaesthesiology 05/2012; 25(4):428-33.
[Show abstract][Hide abstract] ABSTRACT: Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.
Nature medicine 03/2012; 18(4):595-9. · 27.14 Impact Factor