Publications by authors named "Gary Peltz"

75 Publications

Transcription factor p73 regulates Th1 differentiation.

Nat Commun 2020 03 19;11(1):1475. Epub 2020 Mar 19.

Laboratory of Molecular Immunology and the Immunology Center, National Heart, Lung, and Blood Institute, Bethesda, MD, 20892-1674, USA.

Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to these response differences, here we analyze in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis indicates that the p53 family protein, p73, affects Th1 differentiation. In cells differentiated under Th1 conditions in vitro, p73 negatively regulates IFNγ production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes such as Ifng and Il12rb2. Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice have increased IFNγ production and less disease severity, whereas in an adoptive transfer model of inflammatory bowel disease, transfer of p73-deficient naïve CD4 T cells increases Th1 responses and augments disease severity. Our results thus identify p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease.
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http://dx.doi.org/10.1038/s41467-020-15172-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081339PMC
March 2020

The phosphatidylethanolamine biosynthesis pathway provides a new target for cancer chemotherapy.

J Hepatol 2020 04 22;72(4):746-760. Epub 2019 Nov 22.

Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305. Electronic address:

Background & Aims: Since human induced pluripotent stem cells (iPSCs) develop into hepatic organoids through stages that resemble human embryonic liver development, they can be used to study developmental processes and disease pathology. Therefore, we examined the early stages of hepatic organoid formation to identify key pathways affecting early liver development.

Methods: Single-cell RNA-sequencing and metabolomic analysis was performed on developing organoid cultures at the iPSC, hepatoblast (day 9) and mature organoid stage. The importance of the phosphatidylethanolamine biosynthesis pathway to early liver development was examined in developing organoid cultures using iPSC with a CRISPR-mediated gene knockout and an over the counter medication (meclizine) that inhibits the rate-limiting enzyme in this pathway. Meclizine's effect on the growth of a human hepatocarcinoma cell line in a xenotransplantation model and on the growth of acute myeloid leukemia cells in vitro was also examined.

Results: Transcriptomic and metabolomic analysis of organoid development indicated that the phosphatidylethanolamine biosynthesis pathway is essential for early liver development. Unexpectedly, early hepatoblasts were selectively sensitive to the cytotoxic effect of meclizine. We demonstrate that meclizine could be repurposed for use in a new synergistic combination therapy for primary liver cancer: a glycolysis inhibitor reprograms cancer cell metabolism to make it susceptible to the cytotoxic effect of meclizine. This combination inhibited the growth of a human liver carcinoma cell line in vitro and in a xenotransplantation model, without causing significant side effects. This drug combination was also highly active against acute myeloid leukemia cells.

Conclusion: Our data indicate that phosphatidylethanolamine biosynthesis is a targetable pathway for cancer; meclizine may have clinical efficacy as a repurposed anti-cancer drug when used as part of a new combination therapy.

Lay Summary: The early stages of human liver development were modeled using human hepatic organoids. We identified a pathway that was essential for early liver development. Based upon this finding, a novel combination drug therapy was identified that could be used to treat primary liver cancer and possibly other types of cancer.
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http://dx.doi.org/10.1016/j.jhep.2019.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085447PMC
April 2020

Laminin α1 is a genetic modifier of TGF-β1-stimulated pulmonary fibrosis.

JCI Insight 2018 09 20;3(18). Epub 2018 Sep 20.

Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, USA.

The pathogenetic mechanisms underlying the pathologic fibrosis in diseases such as idiopathic pulmonary fibrosis (IPF) are poorly understood. To identify genetic factors affecting susceptibility to IPF, we analyzed a murine genetic model of IPF in which a profibrotic cytokine (TGF-β1) was expressed in the lungs of 10 different inbred mouse strains. Surprisingly, the extent of TGF-β1-induced lung fibrosis was highly strain dependent. Haplotype-based computational genetic analysis and gene expression profiling of lung tissue obtained from fibrosis-susceptible and -resistant strains identified laminin α1 (Lama1) as a genetic modifier for susceptibility to IPF. Subsequent studies demonstrated that Lama1 plays an important role in multiple processes that affect the pulmonary response to lung injury and susceptibility to fibrosis, which include: macrophage activation, fibroblast proliferation, myofibroblast transformation, and the production of extracellular matrix. Also, Lama1 mRNA expression was significantly increased in lung tissue obtained from IPF patients. These studies identify Lama1 as the genetic modifier of TGF-β1 effector responses that significantly affects the development of pulmonary fibrosis.
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http://dx.doi.org/10.1172/jci.insight.99574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237225PMC
September 2018

A Flawed Design Produces Flawed Results.

Authors:
Gary Peltz

J Addict Med 2018 May/Jun;12(3):252

Stanford University School of Medicine Stanford, CA.

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http://dx.doi.org/10.1097/ADM.0000000000000394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978761PMC
March 2020

The Neurobiology of Opioid Addiction and the Potential for Prevention Strategies.

JAMA 2018 05;319(20):2071-2072

Department of Molecular & Cellular Physiology, Howard Hughes Medical Institute, Stanford, California.

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http://dx.doi.org/10.1001/jama.2018.3394DOI Listing
May 2018

Dynamic landscape and regulation of RNA editing in mammals.

Nature 2017 10;550(7675):249-254

Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.
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http://dx.doi.org/10.1038/nature24041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723435PMC
October 2017

Human hepatic organoids for the analysis of human genetic diseases.

JCI Insight 2017 09 7;2(17). Epub 2017 Sep 7.

Department of Anesthesia.

We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct-like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.
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http://dx.doi.org/10.1172/jci.insight.94954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621886PMC
September 2017

Profiling of ARDS pulmonary edema fluid identifies a metabolically distinct subset.

Am J Physiol Lung Cell Mol Physiol 2017 05 3;312(5):L703-L709. Epub 2017 Mar 3.

Departments of Medicine and Anesthesia, Cardiovascular Research Institute, University of California, San Francisco, California.

There is considerable biological and physiological heterogeneity among patients who meet standard clinical criteria for acute respiratory distress syndrome (ARDS). In this study, we tested the hypothesis that there exists a subgroup of ARDS patients who exhibit a metabolically distinct profile. We examined undiluted pulmonary edema fluid obtained at the time of endotracheal intubation from 16 clinically phenotyped ARDS patients and 13 control patients with hydrostatic pulmonary edema. Nontargeted metabolic profiling was carried out on the undiluted edema fluid. Univariate and multivariate statistical analyses including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were conducted to find discriminant metabolites. Seven-hundred and sixty unique metabolites were identified in the pulmonary edema fluid of these 29 patients. We found that a subset of ARDS patients (6/16, 38%) presented a distinct metabolic profile with the overrepresentation of 235 metabolites compared with edema fluid from the other 10 ARDS patients, whose edema fluid metabolic profile was indistinguishable from those of the 13 control patients with hydrostatic edema. This "high metabolite" endotype was characterized by higher concentrations of metabolites belonging to all of the main metabolic classes including lipids, amino acids, and carbohydrates. This distinct group with high metabolite levels in the edema fluid was also associated with a higher mortality rate. Thus metabolic profiling of the edema fluid of ARDS patients supports the hypothesis that there is considerable biological heterogeneity among ARDS patients who meet standard clinical and physiological criteria for ARDS.
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http://dx.doi.org/10.1152/ajplung.00438.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451591PMC
May 2017

Palonosetron and hydroxyzine pre-treatment reduces the objective signs of experimentally-induced acute opioid withdrawal in humans: a double-blinded, randomized, placebo-controlled crossover study.

Am J Drug Alcohol Abuse 2017 01 11;43(1):78-86. Epub 2016 Aug 11.

a Department of Anesthesia , Stanford University School of Medicine , Stanford , CA , USA.

Background: Treatments for reducing opioid withdrawal are limited and prone to problematic side effects. Laboratory studies, clinical observations, and limited human trial data suggest 5-HT3-receptor antagonists and antihistamines may be effective.

Objectives: This double-blind, crossover, placebo-controlled study employing an acute physical dependence model evaluated whether (i) treatment with a 5-HT3-receptor antagonist (palonosetron) would reduce opioid withdrawal symptoms, and (ii) co-administration of an antihistamine (hydroxyzine) would enhance any treatment effect.

Methods: At timepoint T = 0, healthy (non-opioid dependent, non-substance abuser) male volunteers (N = 10) were pre-treated with either a) placebo, b) palonosetron IV (0.75 mg), or c) palonosetron IV (0.75 mg) and hydroxyzine PO (100 mg) in a crossover study design. This was followed at T = 30 by intravenous morphine (10 mg/70kg). At T = 165, 10 mg/70kg naloxone IV was given to precipitate opioid withdrawal. The objective opioid withdrawal score (OOWS) and subjective opioid withdrawal score (SOWS) were determined 5 and 15 minutes after naloxone administration (T = 170, 180, respectively). Baseline measurements were recorded at T = -30 and T = -15.

Results: Comparison of average baseline OOWS scores with OOWS scores obtained 15 minutes after naloxone was significant (p = 0.0001). Scores from 15 minutes post-naloxone infusion showed significant differences in OOWS scores between treatment groups: placebo, 3.7 ± 2.4; palonosetron, 1.5 ± 0.97; and palonosetron with hydroxyzine, 0.2 ± 0.1333.

Conclusions: Pretreatment with palonosetron significantly reduced many signs of experimentally-induced opioid withdrawal. Co-administration with hydroxyzine further reduced opioid withdrawal severity. These results suggest that 5-HT3 receptor antagonists, alone or in combination with an antihistamine, may be useful in the treatment of opioid withdrawal.
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http://dx.doi.org/10.1080/00952990.2016.1210614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728104PMC
January 2017

Treating Liver Fibrosis: (Re)Programmed to Succeed.

Cell Stem Cell 2016 06;18(6):683-684

Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Two papers (Rezvani et al., 2016; Song et al., 2016) in this issue of Cell Stem Cell use transcription-factor-mediated reprogramming to convert liver myofibroblasts into hepatocyte-like cells in mice. Moreover, murine models of fibrotic and cholestatic liver injury were used to demonstrate that this approach has potential for treatment of liver cirrhosis.
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http://dx.doi.org/10.1016/j.stem.2016.05.007DOI Listing
June 2016

The multiple PDZ domain protein Mpdz/MUPP1 regulates opioid tolerance and opioid-induced hyperalgesia.

BMC Genomics 2016 Apr 29;17:313. Epub 2016 Apr 29.

Anesthesiology Service, Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave., Anesthesiology, 112A, Palo Alto, CA, 94304, USA.

Background: Opioids are a mainstay for the treatment of chronic pain. Unfortunately, therapy-limiting maladaptations such as loss of treatment effect (tolerance), and paradoxical opioid-induced hyperalgesia (OIH) can occur. The objective of this study was to identify genes responsible for opioid tolerance and OIH.

Results: These studies used a well-established model of ascending morphine administration to induce tolerance, OIH and other opioid maladaptations in 23 strains of inbred mice. Genome-wide computational genetic mapping was then applied to the data in combination with a false discovery rate filter. Transgenic mice, gene expression experiments and immunoprecipitation assays were used to confirm the functional roles of the most strongly linked gene. The behavioral data processed using computational genetic mapping and false discovery rate filtering provided several strongly linked biologically plausible gene associations. The strongest of these was the highly polymorphic Mpdz gene coding for the post-synaptic scaffolding protein Mpdz/MUPP1. Heterozygous Mpdz +/- mice displayed reduced opioid tolerance and OIH. Mpdz gene expression and Mpdz/MUPP1 protein levels were lower in the spinal cords of low-adapting 129S1/Svlm mice than in high-adapting C57BL/6 mice. Morphine did not alter Mpdz expression levels. In addition, association of Mpdz/MUPP1 with its known binding partner CaMKII did not differ between these high- and low-adapting strains.

Conclusions: The degrees of maladaptive changes in response to repeated administration of morphine vary greatly across inbred strains of mice. Variants of the multiple PDZ domain gene Mpdz may contribute to the observed inter-strain variability in tolerance and OIH by virtue of changes in the level of their expression.
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http://dx.doi.org/10.1186/s12864-016-2634-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850636PMC
April 2016

A Pharmacogenetic Discovery: Cystamine Protects Against Haloperidol-Induced Toxicity and Ischemic Brain Injury.

Genetics 2016 05 18;203(1):599-609. Epub 2016 Mar 18.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California

Haloperidol is an effective antipsychotic agent, but it causes Parkinsonian-like extrapyramidal symptoms in the majority of treated subjects. To address this treatment-limiting toxicity, we analyzed a murine genetic model of haloperidol-induced toxicity (HIT). Analysis of a panel of consomic strains indicated that a genetic factor on chromosome 10 had a significant effect on susceptibility to HIT. We analyzed a whole-genome SNP database to identify allelic variants that were uniquely present on chromosome 10 in the strain that was previously shown to exhibit the highest level of susceptibility to HIT. This analysis implicated allelic variation within pantetheinase genes (Vnn1 and Vnn3), which we propose impaired the biosynthesis of cysteamine, could affect susceptibility to HIT. We demonstrate that administration of cystamine, which is rapidly metabolized to cysteamine, could completely prevent HIT in the murine model. Many of the haloperidol-induced gene expression changes in the striatum of the susceptible strain were reversed by cystamine coadministration. Since cystamine administration has previously been shown to have other neuroprotective actions, we investigated whether cystamine administration could have a broader neuroprotective effect. Cystamine administration caused a 23% reduction in infarct volume after experimentally induced cerebral ischemia. Characterization of this novel pharmacogenetic factor for HIT has identified a new approach for preventing the treatment-limiting toxicity of an antipsychotic agent, which could also be used to reduce the extent of brain damage after stroke.
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http://dx.doi.org/10.1534/genetics.115.184648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858802PMC
May 2016

Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.

Genome Biol 2016 Mar 8;17:45. Epub 2016 Mar 8.

Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA, 94304, USA.

Background: Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae.

Results: We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets.

Conclusions: Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.
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http://dx.doi.org/10.1186/s13059-016-0900-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784398PMC
March 2016

Can Humanized Mice Predict Drug "Behavior" in Humans?

Authors:
Dan Xu Gary Peltz

Annu Rev Pharmacol Toxicol 2016 22;56:323-38. Epub 2015 Oct 22.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California 94305; email:

Most of what we know about a drug prior to human clinical studies is derived from animal testing. Because animals and humans have substantial differences in their physiology and in their drug metabolism pathways, we do not know very much about the pharmacokinetic and pharmacodynamic behavior of a drug in humans until after it is administered to many people. Hence, drug-induced liver injury has become a significant public health problem, and we have a very inefficient drug development process with a high failure rate. Because the human liver is at the heart of these problems, chimeric mice with humanized livers could be used to address these issues. We examine recent evidence indicating that drug testing in chimeric mice could provide better information about a drug's metabolism, disposition, and toxicity (i.e., its "behavior") in humans and could aid in developing personalized medicine strategies, which would improve drug efficacy and safety.
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http://dx.doi.org/10.1146/annurev-pharmtox-010715-103644DOI Listing
September 2016

Long-Acting Opioids for Treating Neonatal Abstinence Syndrome: A High Price for a Short Stay?

JAMA 2015 Nov;314(19):2023-4

Department of Pediatrics and Anesthesia, Stanford University School of Medicine, Stanford, California.

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http://dx.doi.org/10.1001/jama.2015.13537DOI Listing
November 2015

Humanized thymidine kinase-NOG mice can be used to identify drugs that cause animal-specific hepatotoxicity: a case study with furosemide.

J Pharmacol Exp Ther 2015 Jul 11;354(1):73-8. Epub 2015 May 11.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California (D.X., M.Z., M.W., G.P.); Department of Pathology, Stanford University, Stanford, California (S.A.M.); and In Vivo Sciences International, Sunnyvale, California (S.T.)

Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)-NOG mice with humanized livers could prevent this unfortunate outcome (i.e., "rescue" a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide.
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http://dx.doi.org/10.1124/jpet.115.224493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468429PMC
July 2015

The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans.

PLoS Med 2015 Feb 3;12(2):e1001782. Epub 2015 Feb 3.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California, United States of America.

Background: We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study.

Methods And Findings: A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered.

Conclusions: ABCB5 alleles alter susceptibility to HIT in mouse and humans. This discovery leads to a new model that (at least in part) explains inter-individual differences in susceptibility to a drug-induced CNS toxicity.
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http://dx.doi.org/10.1371/journal.pmed.1001782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315575PMC
February 2015

Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity.

J Pharmacol Exp Ther 2015 Feb 25;352(2):274-80. Epub 2014 Nov 25.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California (D.X., M.W., T.N., M.Z., Yu.G., G.P.); Center for the Advancement of Health and Bioscience, Sunnyvale, California (S.N., T.N.); Central Institute for Experimental Animals, Kawasaki, Japan (T.N.); Department of Pathology, Stanford University, Stanford, California (S.A.M.); Bruker CAM & LSC7, Fremont, California (Z.Y., A.J.Y.); Department of Drug Disposition, Eli Lilly and Company, Indianapolis, Indiana (J.S.D., K.M.H., Yi.G.); and In Vivo Sciences International, Sunnyvale, California (S.T.T.)

Due to the substantial interspecies differences in drug metabolism and disposition, drug-induced liver injury (DILI) in humans is often not predicted by studies performed in animal species. For example, a drug (bosentan) used to treat pulmonary artery hypertension caused unexpected cholestatic liver toxicity in humans, which was not predicted by preclinical toxicology studies in multiple animal species. In this study, we demonstrate that NOG mice expressing a thymidine kinase transgene (TK-NOG) with humanized livers have a humanized profile of biliary excretion of a test (cefmetazole) drug, which was shown by an in situ perfusion study to result from interspecies differences in the rate of biliary transport and in liver retention of this drug. We also found that readily detectable cholestatic liver injury develops in TK-NOG mice with humanized livers after 1 week of treatment with bosentan (160, 32, or 6 mg/kg per day by mouth), whereas liver toxicity did not develop in control mice after 1 month of treatment. The laboratory and histologic features of bosentan-induced liver toxicity in humanized mice mirrored that of human subjects. Because DILI has become a significant public health problem, drug safety could be improved if preclinical toxicology studies were performed using humanized TK-NOG.
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http://dx.doi.org/10.1124/jpet.114.220798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293443PMC
February 2015

The Netrin-1 receptor DCC is a regulator of maladaptive responses to chronic morphine administration.

BMC Genomics 2014 May 8;15:345. Epub 2014 May 8.

Anesthesiology Service, Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, USA.

Background: 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.

Results: Whole genome-HBCGM was used to analyze phenotypic data on morphine-induced tolerance, dependence and hyperalgesia obtained from 23 inbred strains. The robustness of the genetic mapping results was analyzed using strain subsets. In addition, the results of analyzing all of the opioid-related traits together were examined. To characterize the functional role of the leading candidate gene, we analyzed transgenic animals, mRNA and protein expression in behaviorally divergent mouse strains, and immunohistochemistry in spinal cord tissue. Our mapping procedure identified the allelic pattern within the netrin-1 receptor gene (Dcc) as most robustly associated with OIH, and it was also strongly associated with the combination of the other maladaptive opioid traits analyzed. Adult mice heterozygous for the Dcc gene had significantly less tendency to develop OIH, become tolerant or show evidence of dependence after chronic exposure to morphine. The difference in opiate responses was shown not to be due to basal or morphine-stimulated differences in the level of Dcc expression in spinal cord tissue, and was not associated with nociceptive neurochemical or anatomical alterations in the spinal cord or dorsal root ganglia in adult animals.

Conclusions: Whole-genome HBCGM is a powerful tool for identifying genes affecting biomedical traits such as opioid maladaptations. We demonstrate that Dcc affects tolerance, dependence and OIH after chronic opioid exposure, though not through simple differences in expression in the adult spinal cord.
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http://dx.doi.org/10.1186/1471-2164-15-345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038717PMC
May 2014

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

PLoS Med 2014 Apr 15;11(4):e1001628. Epub 2014 Apr 15.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California, United States of America.

Background: 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.

Methods And Findings: 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.

Conclusions: 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.
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http://dx.doi.org/10.1371/journal.pmed.1001628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988005PMC
April 2014

Enabling autologous human liver regeneration with differentiated adipocyte stem cells.

Cell Transplant 2014 21;23(12):1573-84. Epub 2013 Oct 21.

Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA.

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. 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.
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http://dx.doi.org/10.3727/096368913X673432DOI Listing
September 2015

Liquid chromatography/mass spectrometry methods for measuring dipeptide abundance in non-small-cell lung cancer.

Rapid Commun Mass Spectrom 2013 Sep;27(18):2091-2098

Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Stanford University School of Medicine.

Rationale: Metabolomic profiling is a promising methodology of identifying candidate biomarkers for disease detection and monitoring. Although lung cancer is among the leading causes of cancer-related mortality worldwide, the lung tumor metabolome has not been fully characterized.

Methods: We utilized a targeted metabolomic approach to analyze discrete groups of related metabolites. We adopted a dansyl [5-(dimethylamino)-1-naphthalene sulfonamide] derivatization with liquid chromatography/mass spectrometry (LC/MS) to analyze changes of metabolites from paired tumor and normal lung tissues. Identification of dansylated dipeptides was confirmed with synthetic standards. A systematic analysis of retention times was required to reliably identify isobaric dipeptides. We validated our findings in a separate sample cohort.

Results: We produced a database of the LC retention times and MS/MS spectra of 361 dansyl dipeptides. Interpretation of the spectra is presented. Using this standard data, we identified a total of 279 dipeptides in lung tumor tissue. The abundance of 90 dipeptides was selectively increased in lung tumor tissue compared to normal tissue. In a second set of validation tissues, 12 dipeptides were selectively increased.

Conclusions: A systematic evaluation of certain metabolite classes in lung tumors may identify promising disease-specific metabolites. Our database of all possible dipeptides will facilitate ongoing translational applications of metabolomic profiling as it relates to lung cancer.
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http://dx.doi.org/10.1002/rcm.6656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755500PMC
September 2013

Acute and chronic phases of complex regional pain syndrome in mice are accompanied by distinct transcriptional changes in the spinal cord.

Mol Pain 2013 Aug 8;9:40. Epub 2013 Aug 8.

Background: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1186/1744-8069-9-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751593PMC
August 2013

Reply to bissig and grompe.

Authors:
Gary Peltz

Trends Pharmacol Sci 2013 Aug 11;34(8):426. Epub 2013 Jul 11.

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http://dx.doi.org/10.1016/j.tips.2013.06.003DOI Listing
August 2013

Can 'humanized' mice improve drug development in the 21st century?

Authors:
Gary Peltz

Trends Pharmacol Sci 2013 May 19;34(5):255-60. Epub 2013 Apr 19.

Department of Anesthesia, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.

Chimeric mice, which have human hepatocytes engrafted in their liver, have been used to study human drug metabolism and pharmacodynamic responses for nearly 20 years. However, there are very few examples where their use has prospectively impacted the development of a candidate medication. Here, three different chimeric mouse models and their utility for pharmacology studies are evaluated. Several recent studies indicate that using these chimeric mouse models could help to overcome traditional (predicting human-specific metabolites and toxicities) and 21st century problems (strategies for personalized medicine and selection of optimal combination therapies) in drug development. These examples suggest that there are many opportunities in which the use of chimeric mice could significantly improve the quality of preclinical drug assessment.
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http://dx.doi.org/10.1016/j.tips.2013.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682766PMC
May 2013

Metabolomic-derived novel cyst fluid biomarkers for pancreatic cysts: glucose and kynurenine.

Gastrointest Endosc 2013 Aug 6;78(2):295-302.e2. Epub 2013 Apr 6.

Department of Medicine, Stanford University, Stanford, California, USA.

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.
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http://dx.doi.org/10.1016/j.gie.2013.02.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3780566PMC
August 2013

Human pharmacogenetic analysis in chimeric mice with 'humanized livers'.

Pharmacogenet Genomics 2013 Feb;23(2):78-83

Department of Anesthesia, Stanford University School of Medicine, Stanford, California 94305, USA.

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.
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http://dx.doi.org/10.1097/FPC.0b013e32835cb2c7DOI Listing
February 2013

Using chimeric mice with humanized livers to predict human drug metabolism and a drug-drug interaction.

J Pharmacol Exp Ther 2013 Feb 8;344(2):388-96. Epub 2012 Nov 8.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California, USA.

Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) 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 before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because 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 coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.
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http://dx.doi.org/10.1124/jpet.112.198697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558826PMC
February 2013

Identification of drug targets by chemogenomic and metabolomic profiling in yeast.

Pharmacogenet Genomics 2012 Dec;22(12):877-86

Department of Anesthesia, Stanford University School of Medicine, Stanford University, Stanford, California 94305, USA.

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.
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http://dx.doi.org/10.1097/FPC.0b013e32835aa888DOI Listing
December 2012

Computational genetic discoveries that could improve perioperative medicine.

Curr Opin Anaesthesiol 2012 Aug;25(4):428-33

Department of Anesthesia, Stanford University, School of Medicine, Stanford, California, USA.

Purpose Of Review: The review examines the rationale and translational utility of computational genetic studies using murine models of biomedical traits.

Recent Findings: 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.

Summary: 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.
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http://dx.doi.org/10.1097/ACO.0b013e32835561f9DOI Listing
August 2012