Honors & Awards
Transformative RO1 award, NIH/NIDDK (2010)
One of 10 top pharmaceutical research executives, Nature Publications (2006)
M.D., Stanford University, Medicine (1983)
Ph.D., Stanford University, Structural Biology (1983)
Fellowship, University of California-San Francisco, Rheumatology (1989)
Residency, University of California-San Francisco, Internal Medicine (1985)
Board Certification, American Board of Internal Medicine, Internal Medicine, Rheumatology (1989)
Community and International Work
Computational Genetic Analysis of Biomedical Traits in Mice
Genetic Basis for Disease Susceptibility
International collaborations with many labs with mouse genetic models
Opportunities for Student Involvement
Current Research and Scholarly Interests
The laboratory develops and uses state of the art genomic methods to identify genetic factors affecting disease susceptibility, and to translate these findings into new treatments.
Computational mouse genetics: To reduce the cost and the time frame for genetic research, we developed a more efficient method for performing genetic analysis in mice. For this analysis, a property of interest is measured in ten or more inbred mouse strains; genetic factors are then computationally predicted by identifying genomic regions where the pattern of genetic variation correlates with the distribution of trait values among the inbred strains. This enables genetic analyses to be completed in far less time (days vs. years) and with far fewer personnel than conventional methods. In 2011, we developed a next generation version of this computational method, which analyzes whole genome sequence data for 20 inbred strains. This method has been used to analyze the genetic basis for 16 different biomedical traits. Clinical trials are currently underway that test the efficacy of 2 therapies that were generated by these genetic discoveries: a pilot study at Stanford to alleviate incisional pain after surgery and a multi-center NIH funded trial for preventing narcotic drug withdrawal in infants born to narcotic consuming mothers was begun in 2012.
Metabolomic analysis: We are developing novel methods for analyzing changes in cellular metabolites caused by drug treatment or in disease. The metabolomic results are integrated with the genetic data to identify new disease mechanisms and to develop new diagnostics.
Mice with human LIvers: This is a novel experimental in vivo platform that replaces mouse liver with functioning human liver tissue. The reconstituted liver was shown to be a mature and functioning human organ. It had zonal position-specific enzyme expression and bile duct function representative of mature human liver, and could generate a human-specific profile of drug metabolism. These features make this chimeric mouse the preferred experimental platform for in vivo analysis of drug metabolism or liver regeneration. The mice are maintained in a specialized barrier facility that was designed for housing these mice.The humanized mice are being used to: develop a novel platform for predicting human drug metabolism and human drug responses; for understanding stem cell development; and to develop a new method for liver transplantation that uses autologous cells without immunosuppression.
- Independent Studies (5)
Postdoctoral Faculty Sponsor
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.
American journal of drug and alcohol abuse
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.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.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.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.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.
View details for PubMedID 27712113
A Pharmacogenetic Discovery: Cystamine Protects Against Haloperidol-Induced Toxicity and Ischemic Brain Injury
2016; 203 (1): 599-?
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.
View details for DOI 10.1534/genetics.115.184648
View details for Web of Science ID 000376012100040
View details for PubMedID 26993135
View details for PubMedCentralID PMC4858802
The multiple PDZ domain protein Mpdz/MUPP1 regulates opioid tolerance and opioid-induced hyperalgesia
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.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.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.
View details for DOI 10.1186/s12864-016-2634-1
View details for Web of Science ID 000375079200001
View details for PubMedID 27129385
- Can Humanized Mice Predict Drug "Behavior" in Humans? ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, VOL 56 2016; 56: 323-338
Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.
2016; 17 (1): 45-?
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.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.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.
View details for DOI 10.1186/s13059-016-0900-9
View details for PubMedID 26956608
Humanized Thymidine Kinase-NOG Mice Can Be Used to Identify Drugs That Cause Animal-Specific Hepatotoxicity: A Case Study with Furosemide
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2015; 354 (1): 73-78
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.
View details for DOI 10.1124/jpet,115.223393
View details for Web of Science ID 000357096900009
View details for PubMedID 25962391
The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans.
2015; 12 (2)
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.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.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.
View details for DOI 10.1371/journal.pmed.1001782
View details for PubMedID 25647612
View details for PubMedCentralID PMC4315575
Ondansetron pharmacokinetics in pregnant women and neonates: towards a new treatment for neonatal abstinence syndrome.
Clinical pharmacology & therapeutics
2015; 97 (2): 167-176
Ondansetron is the drug of choice to prevent nausea in women undergoing cesarean surgery and can be used to prevent neonatal abstinence syndrome (NAS). The pharmacokinetics of ondansetron have not been characterized in pregnant women or in newborns. A nonlinear mixed-effects modeling approach was used to analyze plasma samples obtained from 20 nonpregnant and 40 pregnant women following a single administration of 4 or 8 mg ondansetron, from umbilical cord blood at delivery, and from neonates after birth. The analysis indicates that: ondansetron disposition is not affected by pregnancy (P > 0.05), but influenced by dose (P < 0.05), and is characterized by rapid transplacental transfer and longer elimination half-life in neonates compared to their mother. A dosing regimen for prevention of NAS was designed based on the model. The regimen involves IV administration of 4 mg to the mothers shortly before cord clamping, or oral administration of 0.07 mg/kg (or equivalently 0.04 mg/kg IV) to neonates.
View details for DOI 10.1002/cpt.5
View details for PubMedID 25670522
- The role of abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans. PLoS medicine 2015; 12 (2)
Chimeric TK-NOG Mice: A Predictive Model for Cholestatic Human Liver Toxicity.
journal of pharmacology and experimental therapeutics
2015; 352 (2): 274-280
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.
View details for DOI 10.1124/jpet.114.220798
View details for PubMedID 25424997
- The Netrin-1 receptor DCC is a regulator of maladaptive responses to chronic morphine administration BMC GENOMICS 2014; 15
- Fialuridine Induces Acute Liver Failure in Chimeric TK-NOG Mice: A Model for Detecting Hepatic Drug Toxicity Prior to Human Testing. PLoS medicine 2014; 11 (4)
Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.
2014; 11 (4)
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.
View details for DOI 10.1371/journal.pmed.1001628
View details for PubMedID 24736310
Enabling Autologous Human Liver Regeneration With Differentiated Adipocyte Stem Cells
2014; 23 (12): 1573-1584
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.
View details for DOI 10.3727/096368913X673432
View details for Web of Science ID 000346626300009
View details for PubMedID 24148223
Liquid chromatography/mass spectrometry methods for measuring dipeptide abundance in non-small-cell lung cancer.
Rapid communications in mass spectrometry : RCM
2013; 27 (18): 2091-2098
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.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.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.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. Copyright © 2013 John Wiley & Sons, Ltd.
View details for DOI 10.1002/rcm.6656
View details for PubMedID 23943330
- Acute and chronic phases of complex regional pain syndrome in mice are accompanied by distinct transcriptional changes in the spinal cord MOLECULAR PAIN 2013; 9
Metabolomic-derived novel cyst fluid biomarkers for pancreatic cysts: glucose and kynurenine.
2013; 78 (2): 295-302 e2
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.
View details for DOI 10.1016/j.gie.2013.02.037
View details for PubMedID 23566642
- Metabolomic-derived novel cyst fluid biomarkers for pancreatic cysts: glucose and kynurenine GASTROINTESTINAL ENDOSCOPY 2013; 78 (2): 295-?
Can 'humanized' mice improve drug development in the 21st century?
Trends in pharmacological sciences
2013; 34 (5): 255-260
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.
View details for DOI 10.1016/j.tips.2013.03.005
View details for PubMedID 23602782
Using Chimeric Mice with Humanized Livers to Predict Human Drug Metabolism and a Drug-Drug Interaction
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2013; 344 (2): 388-396
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.
View details for DOI 10.1124/jpet.112.198697
View details for Web of Science ID 000313745900008
View details for PubMedID 23143674
Human pharmacogenetic analysis in chimeric mice with 'humanized livers'
PHARMACOGENETICS AND GENOMICS
2013; 23 (2): 78-83
We investigated whether human pharmacogenetic factors could be characterized using chimeric NOG mice expressing a thymidine kinase transgene (TK-NOG) with 'humanized' livers.The rate of human-specific metabolism of two drugs was measured in chimeric mice reconstituted with human hepatocytes with different CYP2C19 and CYP2C9 genotypes.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.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.
View details for DOI 10.1097/FPC.0b013e32835cb2c7
View details for Web of Science ID 000313457400003
View details for PubMedID 23241944
OPIATE-INDUCED CHANGES IN BRAIN ADENOSINE LEVELS AND NARCOTIC DRUG RESPONSES
2013; 228: 235-242
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 twofold 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, 0.5mg/kg) or an A(2a) receptor (A(2a)) antagonist (SCH 58261, 1mg/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.
View details for DOI 10.1016/j.neuroscience.2012.10.031
View details for Web of Science ID 000313938700019
View details for PubMedID 23098802
- Reply to Bissig and Grompe. Trends in pharmacological sciences 2013
Identification of drug targets by chemogenomic and metabolomic profiling in yeast
PHARMACOGENETICS AND GENOMICS
2012; 22 (12): 877-886
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.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.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.
View details for DOI 10.1097/FPC.0b013e32835aa888
View details for Web of Science ID 000311031800006
View details for PubMedID 23076370
Diagnostic Utility of Metabolomic-Derived Biomarkers for Pancreatic Cysts
LIPPINCOTT WILLIAMS & WILKINS. 2012: 1394–94
View details for Web of Science ID 000310360500241
Computational genetic discoveries that could improve perioperative medicine
CURRENT OPINION IN ANESTHESIOLOGY
2012; 25 (4): 428-433
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.
View details for DOI 10.1097/ACO.0b013e32835561f9
View details for Web of Science ID 000306273900005
View details for PubMedID 22647490
Cd14 SNPs regulate the innate immune response
2012; 51 (2): 112-127
CD14 is a monocytic differentiation antigen that regulates innate immune responses to pathogens. Here, we show that murine Cd14 SNPs regulate the length of Cd14 mRNA and CD14 protein translation efficiency, and consequently the basal level of soluble CD14 (sCD14) and type I IFN production by murine macrophages. This has substantial downstream consequences for the innate immune response; the level of expression of at least 40 IFN-responsive murine genes was altered by this mechanism. We also observed that there was substantial variation in the length of human CD14 mRNAs and in their translation efficiency. sCD14 increased cytokine production by human dendritic cells (DCs), and sCD14-primed DCs augmented human CD4T cell proliferation. These findings may provide a mechanism for exploring the complex relationship between CD14 SNPs, serum sCD14 levels, and susceptibility to human infectious and allergic diseases.
View details for DOI 10.1016/j.molimm.2012.02.112
View details for Web of Science ID 000304687500002
View details for PubMedID 22445606
Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity
2012; 18 (4): 595-599
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.
View details for DOI 10.1038/nm.2710
View details for Web of Science ID 000302617800037
View details for PubMedID 22447075
A better prognosis for genetic association studies in mice
TRENDS IN GENETICS
2012; 28 (2): 62-69
Although inbred mouse strains have been the premier model organism used in biomedical research, multiple studies and analyses have indicated that genome-wide association studies (GWAS) cannot be productively performed using inbred mouse strains. However, there is one type of GWAS in mice that has successfully identified the genetic basis for many biomedical traits of interest: haplotype-based computational genetic mapping (HBCGM). Here, we describe how the methodological basis for a HBCGM study significantly differs from that of a conventional murine GWAS, and how an integrative analysis of its output within the context of other 'omic' information can enable genetic discovery. Consideration of these factors will substantially improve the prognosis for the utility of murine genetic association studies for biomedical discovery.
View details for DOI 10.1016/j.tig.2011.10.006
View details for Web of Science ID 000300532200002
View details for PubMedID 22118772
Next-Generation Computational Genetic Analysis: Multiple Complement Alleles Control Survival after Candida albicans Infection
INFECTION AND IMMUNITY
2011; 79 (11): 4472-4479
Candida albicans is a fungal pathogen that causes severe disseminated infections that can be lethal in immunocompromised patients. Genetic factors are known to alter the initial susceptibility to and severity of C. albicans infection. We developed a next-generation computational genetic mapping program with advanced features to identify genetic factors affecting survival in a murine genetic model of hematogenous C. albicans infection. This computational tool was used to analyze the median survival data after inbred mouse strains were infected with C. albicans, which provides a useful experimental model for identification of host susceptibility factors. The computational analysis indicated that genetic variation within early classical complement pathway components (C1q, C1r, and C1s) could affect survival. Consistent with the computational results, serum C1 binding to this pathogen was strongly affected by C1rs alleles, as was survival of chromosome substitution strains. These results led to a combinatorial, conditional genetic model, involving an interaction between C5 and C1r/s alleles, which accurately predicted survival after infection. Beyond applicability to infectious disease, this information could increase our understanding of the genetic factors affecting susceptibility to autoimmune and neurodegenerative diseases.
View details for DOI 10.1128/IAI.05666-11
View details for Web of Science ID 000296352400018
View details for PubMedID 21875959
Using 'Humanized' TK-NOG mice to predict human drug metabolism and drug-drug interactions
17th North American Regional International-Society-for-the-Study-of-Xenobiotics (ISSX) Meeting
INFORMA HEALTHCARE. 2011: 164–165
View details for Web of Science ID 000297056400313
H5N1 Influenza Virus Pathogenesis in Genetically Diverse Mice Is Mediated at the Level of Viral Load
2011; 2 (5)
The genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host's immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host's susceptibility to a given H5N1 virus.Highly pathogenic H5N1 influenza virus has circulated in Southeast Asia since 2003 but has been confirmed in relatively few individuals. It has been postulated that host genetic polymorphisms increase the susceptibility to infection and severe disease. The mechanisms and host proteins affected during severe disease are unknown. Inbred mouse strains vary considerably in their ability to resist H5N1 virus and were used to identify the primary mechanism determining disease severity. After inoculation with H5N1, resistant mouse strains had reduced amounts of virus in their lungs, which subsequently resulted in lower production of proinflammatory mediators and less pathology. We therefore conclude that the host genetic component controlling disease severity is primarily influencing viral replication. This is an important concept, as it emphasizes the need to limit virus replication through antiviral therapies and it shows that the hyperinflammatory environment is simply a reflection of more viral genetic material inducing a response.
View details for DOI 10.1128/mBio.00171-11
View details for Web of Science ID 000296844300008
View details for PubMedID 21896679
The reconstituted 'humanized liver' in TK-NOG mice is mature and functional
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2011; 405 (3): 405-410
To overcome the limitations of existing models, we developed a novel experimental in vivo platform for replacing mouse liver with functioning human liver tissue. To do this, a herpes simplex virus type 1 thymidine kinase (HSVtk) transgene was expressed within the liver of highly immunodeficient NOG mice (TK-NOG). Mouse liver cells expressing this transgene were ablated after a brief exposure to a non-toxic dose of ganciclovir (GCV), and transplanted human liver cells are stably maintained within the liver (humanized TK-NOG) without exogenous drug. The reconstituted liver was shown to be a mature and functioning "human organ" that had zonal position-specific enzyme expression and a global gene expression pattern representative of mature human liver; and could generate a human-specific profile of drug metabolism. The 'humanized liver' could be stably maintained in these mice with a high level of synthetic function for a prolonged period (8 months). This novel in vivo system provides an optimized platform for studying human liver physiology, including drug metabolism, toxicology, or liver regeneration.
View details for DOI 10.1016/j.bbrc.2011.01.042
View details for Web of Science ID 000287996500014
View details for PubMedID 21238430
In silico and in vitro pharmacogenetics: aldehyde oxidase rapidly metabolizes a p38 kinase inhibitor
2011; 11 (1): 15-24
The clinical development of a candidate p38 kinase inhibitor was terminated because of its unexpectedly rapid clearance in human subjects. Its short half-life and metabolic profile in human beings were vastly different from that in rats, dogs, and monkeys characterized during routine pre-clinical studies. Mice generated the predominant drug (4-hydroxylated) metabolite produced in human beings, which was not found in other species. The data from a murine in vitro drug biotransformation assay that used liver extracts from 14 inbred mouse strains were analyzed by haplotype-based computational genetic analysis. This led to the identification of aldehyde oxidase-1 (AOX1) as the enzyme responsible for the rapid metabolism of this drug. Specific enzyme inhibitors and expressed recombinant enzymes were used to confirm that AOX catalyzed the formation of the 4-hydroxylated drug metabolite in mouse and man. Genetic variation within Aox1 regulated the level of hepatic Aox1 mRNA, AOX1 protein, and enzyme activity among the inbred strains. Thus, computational murine pharmacogenetic analysis can facilitate the identification and characterization of drug metabolism pathways that are differentially utilized by humans and other species.
View details for DOI 10.1038/tpj.2010.8
View details for Web of Science ID 000286589600002
View details for PubMedID 20177421
The Role of Interleukin-1 in Wound Biology. Part I: Murine In Silico and In Vitro Experimental Analysis
ANESTHESIA AND ANALGESIA
2010; 111 (6): 1525-1533
Wound healing is a multistep, complex process that involves the coordinated action of multiple cell types. Conflicting results have been obtained when conventional methods have been used to study wound biology. Therefore, we analyzed the wound response in a mouse genetic model.We analyzed inflammatory mediators produced within incisional wounds induced in 16 inbred mouse strains. Computational haplotype-based genetic analysis of inter-strain differences in the level of production of 2 chemokines in wounds was performed. An in vitro experimental analysis system was developed to investigate whether interleukin (IL)-1 could affect chemokine production by 2 different types of cells that are present within wounds.The level of 2 chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 1α, exhibited very large (75- and 463-fold, respectively) interstrain differences within wound tissue across this inbred strain panel. Genetic variation within Nalp1, an inflammasome component that regulates IL-1 production, correlated with the interstrain differences in KC and macrophage inhibitory protein 1α production. Consistent with the genetic correlation, IL-1β was shown to stimulate KC production by murine keratinocyte and fibroblast cell lines in vitro.Genetic variation within Nalp1 could contribute to interstrain differences in wound chemokine production by altering the amount of IL-1 produced.
View details for DOI 10.1213/ANE.0b013e3181f5ef5a
View details for Web of Science ID 000284973300032
View details for PubMedID 20889942
The Role of Interleukin-1 in Wound Biology. Part II: In Vivo and Human Translational Studies
ANESTHESIA AND ANALGESIA
2010; 111 (6): 1534-1542
In the accompanying paper, we demonstrate that genetic variation within Nalp1 could contribute to interstrain differences in wound chemokine production through altering the amount of interleukin (IL)-1 produced. We further investigate the role of IL-1 in incisional wound biology and its effect on wound chemokine production in vivo and whether this mechanism could be active in human subjects.A well-characterized murine model of incisional wounding was used to assess the in vivo role of IL-1 in wound biology. The amount of 7 different cytokines/chemokines produced within an experimentally induced skin incision on a mouse paw and the nociceptive response was analyzed in mice treated with an IL-1 inhibitor. We also investigated whether human IL-1β or IL-1α stimulated the production of chemokines by primary human keratinocytes in vitro, and whether there was a correlation between IL-1β and chemokine levels in 2 experimental human wound paradigms.Administration of an IL-1 receptor antagonist to mice decreased the nociceptive response to an incisional wound, and reduced the production of multiple inflammatory mediators, including keratinocyte-derived chemokine (KC) and macrophage inhibitory protein (MIP)-1α, within the wounds. IL-1α and IL-1β stimulated IL-8 and GRO-α (human homologues of murine keratinocyte-derived chemokine) production by primary human keratinocytes in vitro. IL-1β levels were highly correlated with IL-8 in human surgical wounds, and at cutaneous sites of human ultraviolet B-induced sunburn injury.IL-1 plays a major role in regulating inflammatory mediator production in wounds through a novel mechanism; by stimulating the production of multiple cytokines and chemokines, it impacts clinically important aspects of wound biology. These data suggest that administration of an IL-1 receptor antagonist within the perioperative period could decrease postsurgical wound pain.
View details for DOI 10.1213/ANE.0b013e3181f691eb
View details for Web of Science ID 000284973300033
View details for PubMedID 20889944
Genetic Susceptibility to the Delayed Sequelae of Neonatal Respiratory Syncytial Virus Infection Is MHC Dependent
JOURNAL OF IMMUNOLOGY
2010; 185 (9): 5384-5391
Respiratory syncytial virus (RSV) is a major cause of respiratory morbidity, resulting in hospitalization for bronchiolitis in some infected infants that is associated with wheeze in later life. Genetic factors are known to affect the severity of the sequelae after RSV infection, but the complexity of the temporal and genetic effects makes it difficult to analyze this response in studies in man. Therefore, we developed a murine genetic model to analyze the sequelae occurring after RSV infection in early life. Haplotype-based genetic analysis of interstrain differences in severity identified the MHC as an important genetic determinant. This was confirmed by analysis of responses in congenic mice with different MHC haplotypes. We also found that susceptible strains had high CD8 levels during secondary infection. Analysis of first filial generation, second filial generation, and back-cross progeny produced by intercrossing resistant (H-2(k), C3H/HeN) and sensitive (H-2(b), BALB/c) strains indicated that susceptibility to sequelae after RSV infection was dominantly inherited but also segregated in a non-MHC-dependent manner. Thus, MHC haplotype and its effect on CD8 cell response is an important determinant of the outcome of neonatal RSV infection.
View details for DOI 10.4049/jimmunol.1001594
View details for Web of Science ID 000283248700051
View details for PubMedID 20921522
Special article: an optimistic prognosis for the clinical utility of laboratory test data.
Anesthesia and analgesia
2010; 111 (4): 1026-1035
It is hoped that anesthesiologists and other clinicians will be able to increasingly rely upon laboratory test data to improve the perioperative care of patients. However, it has been suggested that in order for a laboratory test to have clinically useful diagnostic performance characteristics (sensitivity and specificity), its performance must be considerably better than those that have been evaluated in most etiologic or epidemiologic studies. This pessimism about the clinical utility of laboratory tests is based upon the untested assumption that laboratory data are normally distributed within case and control populations. We evaluated the data distribution for 700 commonly ordered laboratory tests, and found that the vast majority (99%) do not have a normal distribution. The deviation from normal was most pronounced at extreme values, which had a large quantitative effect on laboratory test performance. At the sensitivity and specificity values required for diagnostic utility, the minimum required odds ratios for laboratory tests with a nonnormal data distribution were significantly smaller (by orders of magnitude) than for tests with a normal distribution. By evaluating the effect that the data distribution has on laboratory test performance, we have arrived at the more optimistic outlook that it is feasible to produce laboratory tests with diagnostically useful performance characteristics. We also show that moderate errors in the classification of outcome variables (e.g., death vs. survival at a specified end point) have a small impact on test performance, which is of importance for outcomes research that uses anesthesia information management systems. Because these analyses typically seek to identify factors associated with an undesirable outcome, the data distributions of the independent variables need to be considered when interpreting the odds ratios obtained from such investigations.
View details for DOI 10.1213/ANE.0b013e3181efff0c
View details for PubMedID 20810674
METABOLOMIC PROFILING OF LUNG ADENOCARCINOMA
LIPPINCOTT WILLIAMS & WILKINS. 2010: S51–S52
View details for Web of Science ID 000277242900122
MULTIPLE MICRORNA ARE DYSREGULATED IN MESOTHELIOMA
LIPPINCOTT WILLIAMS & WILKINS. 2010: S105–S106
View details for Web of Science ID 000277242900264
Discovery of potent and bioavailable GSK-3 beta inhibitors
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2010; 20 (5): 1693-1696
Here we report on the discovery of a series of maleimides which have high potency and good selectivity for GSK-3beta. The incorporation of polar groups afforded compounds with good bioavailability. The most potent compound 34 has an IC(50) of 0.6nM for GSK-3beta, over 100-fold selectivity against a panel of other kinases, and shows efficacy in rat osteoporosis models. The X-ray structure of GSK-3beta protein with 34 bound revealed the binding mode of the template and provided insights for future optimization opportunities.
View details for DOI 10.1016/j.bmcl.2010.01.038
View details for Web of Science ID 000274714600052
View details for PubMedID 20138512
Expression genetics identifies spinal mechanisms supporting formalin late phase behaviors
Formalin injection into rodent hind paws is one of the most commonly employed pain assays. The resulting nocifensive behaviors can be divided into two phases differing in timing, duration and underlying mechanisms. Spinal sensitization has long been felt to participate in the second phase of this response, although this sensitization is incompletely understood. By using correlative analysis between spinal gene expression and mouse strain-dependent intensity of late phase behavior, we hypothesized genes participating in variability of the response could be identified.Late phase formalin behavior scores among 10 inbred mouse strains were correlated with a spinal cord gene expression database constructed using expression arrays. Messenger RNA levels for several genes were highly correlated with the late phase behavioral responses. Most of these genes had already been implicated in mechanisms regulating pain and analgesia. One of the most strongly correlated genes, Mapk8 coding for c-Jun N-terminal kinase 1 (JNK1), was chosen for further analysis. Studies using additional strains of mice confirmed that spinal cord mRNA expression levels of Mapk8 followed the pattern predicted by strain-specific levels of formalin behavior. Interestingly, spinal cord JNK1 protein levels displayed an inverse relationship with mRNA measurements. Finally, intrathecal injections of the selective JNK inhibitor, SP600125, selectively reduced late phase licking behavior.Wide differences in pain behaviors, including those resulting from the injection of formalin, can be observed in inbred strains of mice suggesting strong genetic influences. Correlating levels of gene expression in tissues established to be mechanistically implicated in the expression of specific behaviors can identify genes involved in the behaviors of interest. Comparing formalin late phase behavior levels with spinal cord gene expression yielded several plausible gene candidates, including the Mapk8 gene. Additional molecular and pharmacologic evidence confirmed a functional role for this gene in supporting formalin late phase responses.
View details for DOI 10.1186/1744-8069-6-11
View details for Web of Science ID 000275468100001
View details for PubMedID 20149257
An integrative genomic analysis identifies Bhmt2 as a diet-dependent genetic factor protecting against acetaminophen-induced liver toxicity
2010; 20 (1): 28-35
Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.
View details for DOI 10.1101/gr.097212.109
View details for Web of Science ID 000273249500004
View details for PubMedID 19923254
View details for PubMedCentralID PMC2798828
Genetic discovery: the prescription for chronic pain.
2010; 2 (11): 82-?
A recent publication that combined rat gene expression data and a human genetic association study has identified the first genetic risk factor for chronic pain in humans. In four of the five cohorts studied, there was a significant association of an allele within a gene (KCNS1) encoding a potassium channel (Kv9.1) with an increased risk for chronic pain. Identification of genetic risk factors for chronic pain could catalyze new advances in this difficult clinical area that has become a major public health problem. Genomic-medicine-based advances for chronic pain could include the development of a mechanism-based classification system for chronic pain, new treatment options, improved methods for treatment selection and targeted prevention strategies for high-risk individuals.
View details for DOI 10.1186/gm203
View details for PubMedID 21092146
- Genetic discovery: the prescription for chronic pain GENOME MEDICINE 2010; 2
Computational Genetic Mapping in Mice: The Ship Has Sailed
SCIENCE TRANSLATIONAL MEDICINE
2009; 1 (3)
Computational haplotype-based genetic mapping can be used to discover new biological mechanisms, disease-related pathways, and unexpected uses for existing drugs. Here we discuss the benefits and limitations of this methodology, its impact on translational medicine, and its future course.
View details for DOI 10.1126/scitranslmed.3000377
View details for Web of Science ID 000277196700004
View details for PubMedID 20368166
The beta(3) subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test
2009; 144 (3): 294-302
It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the beta3 subunit of the Na+,K+-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, beta3 protein expression, and biophysical properties of the Na+,K+ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the beta3 subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the beta3 subunit of the Na+,K+-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.
View details for DOI 10.1016/j.pain.2009.04.028
View details for Web of Science ID 000268442300014
View details for PubMedID 19464798
From mouse to man: the 5-HT3 receptor modulates physical dependence on opioid narcotics
PHARMACOGENETICS AND GENOMICS
2009; 19 (3): 193-205
Addiction to opioid narcotics represents a major public health challenge. Animal models of one component of addiction, physical dependence, show this trait to be highly heritable. The analysis of opioid dependence using contemporary in-silico techniques offers an approach to discover novel treatments for dependence and addiction.In these experiments, opioid withdrawal behavior in 18 inbred strains of mice was assessed. Mice were treated for 4 days with escalating doses of morphine before the administration of naloxone allowing the quantification of opioid dependence. After haplotypic analysis, experiments were designed to evaluate the top gene candidate as a modulator of physical dependence. Behavioral studies as well as measurements of gene expression on the mRNA and protein levels were completed. Finally, a human model of opioid dependence was used to quantify the effects of the 5-HT3 antagonist ondansetron on signs and symptoms of withdrawal.The Htr3a gene corresponding to the 5-HT3 receptor emerged as the leading candidate. Pharmacological studies using the selective 5-HT3 antagonist ondansetron supported the link in mice. Morphine strongly regulated the expression of the Htr3a gene in various central nervous system regions including the amygdala, dorsal raphe, and periaqueductal gray nuclei, which have been linked to opioid dependence in previous studies. Using an acute morphine administration model, the role of 5-HT3 in controlling the objective signs of withdrawal in humans was confirmed.These studies show the power of in-silico genetic mapping, and reveal a novel target for treating an important component of opioid addiction.
View details for DOI 10.1097/FPC.0b013e328322e73d
View details for Web of Science ID 000264169500002
View details for PubMedID 19214139
Establishment of a humanized model of liver using NOD/Shi-scid IL2Rg(null) mice
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2008; 377 (1): 248-252
Severely immunodeficient NOD/Shi-scid IL2Rg(null) (NOG) mice are used as recipients for human tissue transplantation, which produces chimeric mice with various types of human tissue. NOG mice expressing transgenic urokinase-type plasminogen activator in the liver (uPA-NOG) were produced. Human hepatocytes injected into uPA-NOG mice repopulated the recipient livers with human cells. The uPA-NOG model has several advantages over previously produced chimeric mouse models of human liver: (1) the severely immunodeficient NOG background enables higher xenogeneic cell engraftment; (2) the absence of neonatal lethality enables mating of homozygotes, which increased the efficacy of homozygote production; and (3) donor xenogeneic human hepatocytes could be readily transplanted into young uPA-NOG mice, which provide easier surgical manipulation and improved recipient survival.
View details for DOI 10.1016/j.bbrc.2008.09.124
View details for Web of Science ID 000260738500048
View details for PubMedID 18840406
Genetic Variation in Candidate Osteoporosis Genes, Bone Mineral Density, and Fracture Risk: The Study of Osteoporotic Fractures
CALCIFIED TISSUE INTERNATIONAL
2008; 83 (3): 155-166
Candidate osteoporosis gene variants were examined for associations with fracture risk and bone mineral density (BMD). A total of 9,704 white women were recruited at four U.S. clinical centers and enrolled into the Study of Osteoporotic Fractures, a longitudinal cohort study. Genotyping of 31 polymorphisms from 18 candidate osteoporosis genes was performed in 6,752 women. Incident radiographic fractures were identified at the third and eighth examinations compared with the baseline examination. BMD was measured at the total hip by dual-energy X-ray absorptiometry. Analyses were adjusted for age, clinic site, and self-reported ethnicity. During a mean follow-up of 14.5 years, a total of 849 hip, 658 vertebral, and 2,496 nonhip/nonvertebral fractures occurred in 6,752 women. Women carrying the ALOX15_G48924T T/T genotype had a higher rate of hip fracture (hazard ratio [HR] = 1.33;95% confidence interval [95% CI] = 1.00-1.77) compared with the G/G genotype. Compared with those carrying the PRL_T228C T/T genotype, women with either the C/C (HR = 0.80; 95% CI = 0.67-0.95) or C/T (HR = 0.81; 95% CI = 0.68-0.97) genotype had a lower rate of nonvertebral/nonhip fractures. Women carrying the BMP2_A125611G G/G genotype had a higher rate of vertebral fracture (odds ratio [OR] = 1.51; 95% CI = 1.03-2.23) compared with the A/A genotype. Women with the ESR1_C1335G G/G genotype had a higher rate of vertebral fracture (OR = 1.64; 95% CI = 1.07-2.50) compared with the C/C genotype. Compared with those with the MMP2_C595T C/C genotype, women with the C/T (OR = 0.79; 95% CI = 0.65-0.96) or T/T (OR = 0.44; 95% CI = 0.27-0.72) genotype had a lower rate of vertebral fracture. In conclusion, polymorphisms in several candidate genes were associated with hip, vertebral, and nonhip/nonvertebral fractures but not with total hip BMD in this large population based cohort study.
View details for DOI 10.1007/s00223-008-9165-y
View details for Web of Science ID 000260119500001
View details for PubMedID 18787887
Plasminogen Alleles Influence Susceptibility to Invasive Aspergillosis
2008; 4 (6)
Invasive aspergillosis (IA) is a common and life-threatening infection in immunocompromised individuals. A number of environmental and epidemiologic risk factors for developing IA have been identified. However, genetic factors that affect risk for developing IA have not been clearly identified. We report that host genetic differences influence outcome following establishment of pulmonary aspergillosis in an exogenously immune suppressed mouse model. Computational haplotype-based genetic analysis indicated that genetic variation within the biologically plausible positional candidate gene plasminogen (Plg; Gene ID 18855) correlated with murine outcome. There was a single nonsynonymous coding change (Gly110Ser) where the minor allele was found in all of the susceptible strains, but not in the resistant strains. A nonsynonymous single nucleotide polymorphism (Asp472Asn) was also identified in the human homolog (PLG; Gene ID 5340). An association study within a cohort of 236 allogeneic hematopoietic stem cell transplant (HSCT) recipients revealed that alleles at this SNP significantly affected the risk of developing IA after HSCT. Furthermore, we demonstrated that plasminogen directly binds to Aspergillus fumigatus. We propose that genetic variation within the plasminogen pathway influences the pathogenesis of this invasive fungal infection.
View details for DOI 10.1371/journal.pgen.1000101
View details for Web of Science ID 000260410300015
View details for PubMedID 18566672
Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease
2008; 14 (6): 633-640
To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of chronic lung disease with pathology that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after an infection with a common type of respiratory virus is cleared to only trace levels of noninfectious virus. Chronic inflammatory disease is generally thought to depend on an altered adaptive immune response. However, here we find that this type of disease arises independently of an adaptive immune response and is driven instead by interleukin-13 produced by macrophages that have been stimulated by CD1d-dependent T cell receptor-invariant natural killer T (NKT) cells. This innate immune axis is also activated in the lungs of humans with chronic airway disease due to asthma or COPD. These findings provide new insight into the pathogenesis of chronic inflammatory disease with the discovery that the transition from respiratory viral infection into chronic lung disease requires persistent activation of a previously undescribed NKT cell-macrophage innate immune axis.
View details for DOI 10.1038/nm1770
View details for Web of Science ID 000256468700029
View details for PubMedID 18488036
Osteoprotegerin Lys3Asn polymorphism and the risk of fracture in older women
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
2008; 93 (5): 2002-2008
Osteoprotegerin (OPG) is a soluble decoy receptor for receptor activator nuclear factor kappa-beta that blocks osteoclastic bone resorption.We investigated the association between a Lys3Asn polymorphism in the OPG gene and bone mineral density (BMD), and the risk of fracture in 6695 women aged 65 yr and older participating in the Study of Osteoporotic Fractures.BMD was measured using either single-photon absorptiometry (Osteon Osteoanalyzer; Dove Medical Group, Los Angeles, CA) or dual-energy x-ray absorptiometry (Hologic QDR 1000; Hologic, Inc., Bedford, MA). Incident fractures were confirmed by physician adjudication of radiology reports. Genotyping was performed using an immobilized probe-based assay.Women who were homozygous for the minor G (Lys) allele had significantly lower BMD at the intertrochanter, distal radius, lumbar spine, and calcaneus than those with the C (Asn) allele. There were 701 incident hip fractures during 13.6-yr follow-up (91,249 person-years), including 362 femoral neck and 333 intertrochanteric hip fractures. Women with the C/C (Asn-Asn) genotype had a 51% higher risk of femoral neck fracture (95% confidence interval, 1.13-2.02) and 26% higher risk of hip fracture (95% confidence interval, 1.02-1.54) than those with the G/G (Lys-Lys) genotype. These associations were independent of BMD. Intertrochanteric fractures were not associated with the Lys3Asn polymorphism.These results require confirmation but suggest a role for the OPG Lys3Asn polymorphism in the genetic susceptibility to hip fractures among older white women.
View details for DOI 10.1210/jc.2007-1019
View details for Web of Science ID 000255663100067
View details for PubMedID 18319311
Quantitative trait locus and computational mapping identifies Kcnj9 (GIRK3) as a candidate gene affecting analgesia from multiple drug classes
PHARMACOGENETICS AND GENOMICS
2008; 18 (3): 231-241
Interindividual differences in analgesic drug response complicate the clinical management of pain. We aimed to identify genetic factors responsible for variable sensitivity to analgesic drugs of disparate neurochemical classes.Quantitative trait locus mapping in 872 (C57BL/6x129P3)F2 mice was used to identify genetic factors contributing to variability in the analgesic effect of opioid (morphine), alpha2-adrenergic (clonidine), and cannabinoid (WIN55,212-2) drugs against thermal nociception. A region on distal chromosome 1 showing significant linkage to analgesia from all three drugs was identified. Computational (in silico) genetic analysis of analgesic responses measured in a panel of inbred strains identified a haplotype block within this region containing the Kcnj9 and Kcnj10 genes, encoding the Kir3.3 (GIRK3) and Kir4.1 inwardly rectifying potassium channel subunits. The genes are differentially expressed in the midbrain periaqueductal gray of 129P3 versus C57BL/6 mice, owing to cis-acting genetic elements. The potential role of Kcnj9 was confirmed by the demonstration that knockout mice have attenuated analgesic responses.A single locus is partially responsible for the genetic mediation of pain inhibition, and genetic variation associated with the potassium channel gene, Kcnj9, is a prime candidate for explaining the variable response to these analgesic drugs.
View details for Web of Science ID 000253446400008
View details for PubMedID 18300945
Bone microstructure and its associated genetic variability in 12 inbred mouse strains: mu T study and in silico genome scan
2008; 42 (2): 439-451
MicroCT analysis of 12 inbred strains of mice identified 5 novel chromosomal regions influencing skeletal phenotype. Bone morphology varied in a compartment- and site-specific fashion across strains and genetic influences contributed to the morphometric similarities observed in femoral and vertebral bone within the trabecular bone compartment.Skeletal development is known to be regulated by both heritable and environmental factors, but whether genetic influence on peak bone mass is site- or compartment-specific is unknown. This study examined the genetic variation of cortical and trabecular bone microarchitecture across 12 strains of mice.MicroCT scanning was used to measure trabecular and cortical bone morphometry in the femur and vertebra of 12 strains of 4-month-old inbred male mice. A computational genome mapping technique was used to identify chromosomal intervals associated with skeletal traits.Skeletal microarchitecture varied in a compartment- and site-specific fashion across strains. Genome mapping identified 13 chromosomal intervals associated with skeletal traits and 5 of these intervals were novel. Trabecular microarchitecture in different bone sites correlated across strains and most of the chromosomal intervals associated with these trabecular traits were shared between skeletal sites. Conversely, no chromosomal intervals were shared between the trabecular and cortical bone compartments in the femur, even though there was a strong correlation for these different bone compartments across strains, suggesting site-specific regulation by environmental or intrinsic factors.In summary, these data confirm that there are distinct genetic determinants that define the skeletal phenotype at the time when peak bone mass is being acquired, and that genomic regulation of bone morphology is specific for skeletal compartment.
View details for DOI 10.1016/j.bone.2007.09.041
View details for Web of Science ID 000252914600025
View details for PubMedID 17967568
View details for PubMedCentralID PMC2704123
A genomic "Roadmap" to "Better" drugs
DRUG METABOLISM REVIEWS
2008; 40 (2): 225-239
Utilization of pharmacogenomic information has the potential to significantly improve treatment outcome and markedly reduce the rate of attrition of drugs in clinical development. A major gap that limits our ability to utilize pharmacogenomic information in drug discovery, drug development or clinical practice is that we often do not know the genetic variants responsible for inter-individual differences in drug metabolism or drug response. We examine emerging genomic methods that can fill this gap; these methods can be used to generate new information about drug metabolism or mechanism of action, or to identify predictors of drug response. Although they have not yet had their full impact, a wider application of these emerging genomic technologies has the potential to significantly improve the safety of drugs, the quality of patient care and the efficiency of clinical drug development.
View details for DOI 10.1080/03602530801952815
View details for Web of Science ID 000255688000003
View details for PubMedID 18464044
In silico and in vitro pharmacogenetic analysis in mice
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (45): 17735-17740
Combining the experimental efficiency of a murine hepatic in vitro drug biotransformation system with in silico genetic analysis produces a model system that can rapidly analyze interindividual differences in drug metabolism. This model system was tested by using two clinically important drugs, testosterone and irinotecan, whose metabolism was previously well characterized. The metabolites produced after these drugs were incubated with hepatic in vitro biotransformation systems prepared from the 15 inbred mouse strains were measured. Strain-specific differences in the rate of 16 alpha-hydroxytestosterone generation and irinotecan glucuronidation correlated with the pattern of genetic variation within Cyp2b9 and Ugt1a loci, respectively. These computational predictions were experimentally confirmed using expressed recombinant enzymes. The genetic changes affecting irinotecan metabolism in mice mirrored those in humans that are known to affect the pharmacokinetics and incidence of adverse responses to this medication.
View details for DOI 10.1073/pnas.0700724104
View details for Web of Science ID 000250897600036
View details for PubMedID 17978195
View details for PubMedCentralID PMC2077071
2D NMR metabonomic analysis: a novel method for automated peak alignment
2007; 23 (21): 2926-2933
Comparative metabolic profiling by nuclear magnetic resonance (NMR) is showing increasing promise for identifying inter-individual differences to drug response. Two dimensional (2D) (1)H (13)C NMR can reduce spectral overlap, a common problem of 1D (1)H NMR. However, the peak alignment tools for 1D NMR spectra are not well suited for 2D NMR. An automated and statistically robust method for aligning 2D NMR peaks is required to enable comparative metabonomic analysis using 2D NMR.A novel statistical method was developed to align NMR peaks that represent the same chemical groups across multiple 2D NMR spectra. The degree of local pattern match among peaks in different spectra is assessed using a similarity measure, and a heuristic algorithm maximizes the similarity measure for peaks across the whole spectrum. This peak alignment method was used to align peaks in 2D NMR spectra of endogenous metabolites in liver extracts obtained from four inbred mouse strains in the study of acetaminophen-induced liver toxicity. This automated alignment method was validated by manual examination of the top 50 peaks as ranked by signal intensity. Manual inspection of 1872 peaks in 39 different spectra demonstrated that the automated algorithm correctly aligned 1810 (96.7%) peaks.Algorithm is available upon request.
View details for DOI 10.1093/bioinformatics/btm427
View details for Web of Science ID 000251197700017
View details for PubMedID 17846038
beta-Catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone
2007; 25 (11): 2827-2836
The subventricular zone (SVZ) is the largest germinal zone in the mature rodent brain, and it continuously produces young neurons that migrate to the olfactory bulb. Neural stem cells in this region generate migratory neuroblasts via highly proliferative transit-amplifying cells. The Wnt/beta-catenin signaling pathway partially regulates the proliferation and neuronal differentiation of neural progenitor cells in the embryonic brain. Here, we studied the role of beta-catenin signaling in the adult mouse SVZ. beta-Catenin-dependent expression of a destabilized form of green fluorescent protein was detected in progenitor cells in the adult SVZ of Axin2-d2EGFP reporter mice. Retrovirus-mediated expression of a stabilized beta-catenin promoted the proliferation of Mash1+ cells and inhibited their differentiation into neuroblasts. Conversely, the expression of Dkk1, an inhibitor of Wnt signaling, reduced the proliferation of Mash1+ cells. In addition, an inhibitor of GSK3 beta promoted the proliferation of Mash1+ cells and increased the number of new neurons in the olfactory bulb 14 days later. These results suggest that beta-catenin signaling plays a role in the proliferation of progenitor cells in the SVZ of the adult mouse brain.
View details for DOI 10.1634/stemcells.2007-0177
View details for Web of Science ID 000250642200016
View details for PubMedID 17673525
Mouse chromosome 7 harbors a quantitative trait locus for isoflurane minimum alveolar concentration
ANESTHESIA AND ANALGESIA
2007; 105 (2): 381-385
The minimum alveolar concentration (MAC) of isoflurane is a quantitative trait because it varies continuously in a population. The location on the genome of genes or other genetic elements controlling quantiative traits is called quantitative trait loci (QTLs). In this study we sought to detect a quantitative trait locus underlying isoflurane MAC in mice.To accomplish this, two inbred mouse strains differing in isoflurane MAC, the C57BL/6J and LP/J mouse strains, were bred through two generations to produce genetic recombination. These animals were genotyped for microsatellite markers. We also applied an independent, computational method for identifying QTL-regulating differences in isoflurane MAC. In this approach, the isoflurane MAC was measured in a panel of 19 inbred strains, and computationally searched for genomic intervals where the pattern of genetic variation, based on single nucleotide polymorphisms, correlated with the differences in isoflurane MAC among inbred strains.Both methods of genetic analysis identified a QTL for isoflurane MAC that was located on the proximal part of mouse chromosome 7.
View details for DOI 10.1213/01.ane.0000261277.653.13.4e
View details for Web of Science ID 000248343400018
View details for PubMedID 17646494
Genetic mapping in mice identifies DMBT1 as a candidate modifier of mammary tumors and breast cancer risk
AMERICAN JOURNAL OF PATHOLOGY
2007; 170 (6): 2030-2041
Low-penetrance breast cancer susceptibility alleles seem to play a significant role in breast cancer risk but are difficult to identify in human cohorts. A genetic screen of 176 N2 backcross progeny of two Trp53(+/-) strains, BALB/c and C57BL/6, which differ in their susceptibility to mammary tumors, identified a modifier of mammary tumor susceptibility in an approximately 25-Mb interval on mouse chromosome 7 (designated SuprMam1). Relative to heterozygotes, homozygosity for BALB/c alleles of SuprMam1 significantly decreased mammary tumor latency from 70.7 to 61.1 weeks and increased risk twofold (P = 0.002). Dmbt1 (deleted in malignant brain tumors 1) was identified as a candidate modifier gene within the SuprMam1 interval because it was differentially expressed in mammary tissues from BALB/c-Trp53(+/-) and C57BL/6-Trp53(+/-) mice. Dmbt1 mRNA and protein was reduced in mammary glands of the susceptible BALB/c mice. Immunohistochemical staining demonstrated that DMBT1 protein expression was also significantly reduced in normal breast tissue from women with breast cancer (staining score, 1.8; n = 46) compared with cancer-free controls (staining score, 3.9; n = 53; P < 0.0001). These experiments demonstrate the use of Trp53(+/-) mice as a sensitized background to screen for low-penetrance modifiers of cancer. The results identify a novel mammary tumor susceptibility locus in mice and support a role for DMBT1 in suppression of mammary tumors in both mice and women.
View details for DOI 10.2353/ajpath.2007.060512
View details for Web of Science ID 000246955300022
View details for PubMedID 17525270
Association of the VDR translation start site polymorphism and fracture risk in older women
JOURNAL OF BONE AND MINERAL RESEARCH
2007; 22 (5): 730-736
We evaluated the association between the VDR translation start site polymorphism and osteoporotic phenotypes among 6698 older white women. Women with the C/C genotype had lower wrist BMD and an increased risk of wrist and all non-spine/low-trauma fractures. The high frequency of this variant confers a population attributable risk that is similar to several established risk factors for fracture.The vitamin D receptor (VDR) is a nuclear receptor that regulates bone formation, bone resorption, and calcium homeostasis. A common C to T polymorphism in exon 2 of the VDR gene introduces a new translation start site and a protein that differs in length by three amino acids (T = 427aa, C = 424aa; rs10735810).We conducted genetic association analyses of this polymorphism, BMD, and fracture outcomes in a prospective cohort of 6698 white American women >or=65 years of age. Incident fractures were confirmed by physician adjudication of radiology reports. There were 2532 incident nontraumatic/nonvertebral fractures during 13.6 yr of follow-up including 509 wrist and 703 hip fractures.Women with the C/C genotype had somewhat lower distal radius BMD compared with those with the T/T genotype (CC=0.358 g/cm(2), CT=0.361 g/cm(2), TT=0.369 g/cm(2), p=0.003). The C/C genotype was also associated with increased risk of non-spine, low traumatic fractures (HR: 1.18; 95% CI: 1.04, 1.33) and wrist fractures (HR: 1.33; 95% CI: 1.01, 1.75) compared with the T/T genotype in age-adjusted models. Further adjustments for distal radius BMD only slightly attenuated these associations. The VDR polymorphism was not associated with hip fracture. The population attributable risk (PAR) of the C/C genotype for incident fractures was 6.1%. The PAR for established risk factors for fracture were: low femoral neck BMD (PAR=16.3%), maternal history of fracture (PAR=5.1%), low body weight (PAR=5.3%), corticosteroid use (PAR=1.3%), and smoking (PAR=1.6%). Similar PAR results were observed for wrist fractures.The common and potentially functional VDR translation start site polymorphism confers a modestly increased relative risk of fracture among older white women. However, the high frequency of this variant confers a population attributable risk that is similar to or greater than several established risk factors for fracture.
View details for DOI 10.1359/JBMR.070201
View details for Web of Science ID 000245934000012
View details for PubMedID 17280526
Mammary tumor modifiers in BALB/cJ mice heterozygous for p53
2007; 18 (5): 300-309
BALB/c mice are predisposed to developing spontaneous mammary tumors, which are further increased in a p53 heterozygous state. C57BL/6J mice are resistant to induced mammary tumors and develop less than 1% mammary tumors in both wild-type and p53+/- states. To map modifiers of mammary tumorigenesis, we have established F1 and F2 crosses and backcrosses to BALB/cJ (N2-BALB/cJ) and C57BL/6J (N2-C57BL/6J) strains. All cohorts developed mammary carcinomas in p53+/- females, suggesting that multiple loci dominantly and recessively contributed to mammary tumorigenesis. We mapped two modifiers of mammary tumorigenesis in the BALB/cJ strain. Mtsm1 (mammary tumor susceptibility modifier), a dominant-acting modifier, is located on chromosome 7. Mtsm1 is suggestive for linkage to mammary tumorigenesis (p = 0.001). We have analyzed the Mtsm1 region to locate candidate genes by comparing it to a rat modifier region, Mcs3, which shares syntenic conservation with Mtsm1. Expression data and SNPs were also taken into account. Five potential candidate genes within Mtsm1 are Aldh1a3, Chd2, Nipa2, Pcsk6, and Tubgcp5. The second modifier mapped is Mtsm2, a recessive-acting modifier. Mtsm2 is located on chromosome X and is significantly linked to mammary tumorigenesis (p = 1.03 x 10(-7)).
View details for DOI 10.1007/s00335-007-9028-2
View details for Web of Science ID 000248328800002
View details for PubMedID 17557176
Genetic variants of the P-glycoprotein gene Abcb1b modulate opioid-induced hyperalgesia, tolerance and dependence
PHARMACOGENETICS AND GENOMICS
2006; 16 (11): 825-835
Opioid-induced hyperalgesia (OIH) is a state of paradoxically increased nociceptive sensitivity seen in both humans and rodents following the resolution of the acute opioid antinociceptive effects or during periods of chronic opioid administration. Using the power of genetic analysis, we hoped to discover novel mechanisms modulating this trait.The degree of opioid-induced hyperalgesia displayed in response to a thermal stimulus applied to the hind paw was measured in 16 strains of inbred mice after 4 days of morphine administration. The degree of thermal sensitization was then used in a recently developed in silico haplotypic mapping algorithm along with a haplotypic map constructed from a database containing 209,000 single nucleotide polymorphisms.Analysis of the data resulted in the identification of several haplotype blocks strongly associated with the thermal opioid-induced hyperalgesia trait. The most strongly associated block was located within the Abcb1b P-glycoprotein drug transporter gene. Experiments using the P-glycoprotein inhibitor cyclosporine A and P-glycoprotein null mutant mice supported the hypothesis that a functional association exists between P-glycoprotein transporters and opioid-induced hyperalgesia. The observation of a correlation between morphine brain concentrations and the development of opioid-induced hyperalgesia was consistent with this hypothesis as well. In addition, P-glycoprotein gene deletion and pharmacological inhibition altered morphine ED50, tolerance and physical dependence.We conclude that the use of haplotypic mapping to identify novel mechanisms controlling complex traits is a viable approach. Variants of the Abcb1b gene may explain some portion of the interstrain differences in OIH and perhaps other consequences of chronic opioid administration.
View details for Web of Science ID 000241971500008
View details for PubMedID 17047491
Understanding our drugs and our diseases.
Proceedings of the American Thoracic Society
2006; 3 (5): 409-412
Analysis of mouse genetic models of human disease-associated traits has provided important insight into the pathogenesis of human disease. As one example, analysis of a murine genetic model of osteoporosis demonstrated that genetic variation within the 15-lipoxygenase (Alox15) gene affected peak bone mass, and that treatment with inhibitors of this enzyme improved bone mass and quality in rodent models. However, the method that has been used to analyze mouse genetic models is very time consuming, inefficient, and costly. To overcome these limitations, a computational method for analysis of mouse genetic models was developed that markedly accelerates the pace of genetic discovery. It was used to identify a genetic factor affecting the rate of metabolism of warfarin, an anticoagulant that is commonly used to treat clotting disorders. Computational analysis of a murine genetic model of narcotic drug withdrawal suggested a potential new approach for treatment of narcotic drug addiction. Thus, the results derived from computational mouse genetic analysis can suggest new treatment strategies, and can provide new information about currently available medicines.
View details for PubMedID 16799083
Genetic segregation of airway disease traits despite redundancy of calcium-activated chloride channel family members
2006; 25 (3): 502-513
Complex airway diseases such as asthma and chronic obstructive pulmonary disease exhibit stereotyped traits (especially airway hyperreactivity and mucous cell metaplasia) that are variably expressed in each patient. Here, we used a mouse model for virus-induced long-term expression of these traits to determine whether individual traits can be genetically segregated and thereby linked to separate determinants. We showed that an F2 intercross population derived from susceptible and nonsusceptible mouse strains can manifest individual phenotypic extremes that exhibit one or the other disease trait. Functional genomic analysis of these extremes further indicated that a member of the calcium-activated chloride channel (CLCA) gene family designated mClca3 was inducible with mucous cell metaplasia but not airway hyperreactivity. In confirmation of this finding, we found that mClca3 gene transfer to mouse airway epithelium was sufficient to induce mucous cell metaplasia but not airway hyperreactivity. However, newly developed mClca3(-/-) mice exhibited the same degree of mucous cell metaplasia and airway hyperreactivity as wild-type mice. Bioinformatic analysis of the Clca locus led to the identification of mClca5, and gene transfer indicated that mClca5 also selectively drives mucous cell metaplasia. Thus, in addition to the capacity of CLCA family members to exhibit diverse functional activities, there is also preserved function so that more than one family member mediates mucous cell metaplasia. Nonetheless, Clca expression appears to be a selective determinant of mucous cell metaplasia so that shared homologies between CLCA family members may still represent a useful target for focused therapeutic intervention in hypersecretory airway disease.
View details for DOI 10.1152/physiolgenomics.00321.2005
View details for Web of Science ID 000238375600016
View details for PubMedID 16569774
In silico pharmacogenetics of warfarin metabolism
2006; 24 (5): 531-536
Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.
View details for DOI 10.1038/nbt1195
View details for Web of Science ID 000237331300027
View details for PubMedID 16680137
A genetic analysis of opioid-induced hyperalgesia in mice
2006; 104 (5): 1054-1062
Opioid-induced hyperalgesia (OIH) is a syndrome of increased sensitivity to noxious stimuli, seen after both the acute and chronic administration of opioids, that has been observed in humans and rodent models. This syndrome may reduce the clinical utility of opioids in treating acute and chronic pain.In these studies, the authors measured the propensity of 15 strains of inbred mice to develop mechanical manifestations of OIH. These data were subjected to in silico genetic analysis, which resulted in the association of haplotypic blocks within or near several known genes. Both pharmacologic agents and transgenic mice were used to confirm the functional association of the most strongly linked gene with OIH.Both baseline mechanical nociceptive thresholds and the percentage changes in these thresholds after 4 days of morphine treatment were found to be highly strain dependent. The haplotypic blocks most strongly associated with the mechanical OIH data were located within the beta2 adrenergic receptor gene (beta2-AR). Using the selective beta2-AR antagonist butoxamine, the authors observed a dose-dependent reversal of OIH. Furthermore, deletion of the beta2-AR gene sharply reduced the mechanical allodynia present after morphine treatment in the wild-type mouse strain. Analysis of the associated beta2-AR haplotypic block identified single nucleotide polymorphisms potentially explaining in part the strain specific differences in OIH.Genetic variants of the beta2-AR gene seem to explain some part of the differences between various strains of mice to develop OIH. The association of this gene with OIH suggests specific pharmacologic strategies for reducing the impact of OIH on patients consuming opioids.
View details for Web of Science ID 000237375400022
View details for PubMedID 16645459
Chronic pain and genetic background interact and influence opioid analgesia, tolerance, and physical dependence
2006; 121 (3): 232-240
Opioids are commonly used in the treatment of moderate to severe pain. However, their chronic use is limited by analgesic tolerance and physical dependence. Few studies have examined how chronic pain affects the development of tolerance or dependence, and essentially no studies have looked at the role of both genetics and pain together. For these studies we used 12 strains of inbred mice. Groups of mice from each strain were tested at baseline for morphine analgesic sensitivity, mechanical nociceptive threshold, and thermal nociceptive threshold. Mice were then given morphine in a 4-day escalating morphine administration paradigm followed by reassessment of the morphine dose-response relationship. Finally, physical dependence was measured by administering naloxone. Parallel groups of mice underwent hind paw injection of complete Freund's adjuvant (CFA) to induce chronic hind paw inflammation 7 days prior to the beginning of testing. The data showed that CFA treatment tended to lower baseline ED(50) values for morphine and enhanced the degree of analgesic tolerance observed after 4 days of morphine treatment. In addition, the degree of jumping behavior indicative of physical dependence was often altered if mice had been treated with CFA. The influence of background strain was substantial for all traits measured. In silico haplotypic mapping of the tolerance and physical dependence data demonstrated that CFA pretreatment altered the pattern of the predicted associations and greatly reduced their statistical significance. We conclude that chronic inflammatory pain and genetics interact to modulate the analgesic potency of morphine, tolerance, and physical dependence.
View details for DOI 10.1016/j.pain.2005.12.026
View details for Web of Science ID 000236896900009
View details for PubMedID 16516386
Pharmacogenomics and drug development
2005; 6 (8): 857-864
It is generally anticipated that pharmacogenomic information will have a large impact on drug development and will facilitate individualized drug treatment. However, there has been relatively little quantitative modeling to assess how pharmacogenomic information could be best utilized in clinical practice. Using a quantitative model, this review demonstrates that efficacy is increased and toxicity is reduced when a genetically-guided dose adjustment strategy is utilized in a clinical trial. However, there is limited information available regarding the genetic variables affecting the disposition or mechanism of action of most commonly used medications. These genetic factors must be identified to enable pharmacogenomic testing to be routinely used in the clinic. A recently described murine haplotype-based computational genetic analysis method provides one strategy for identifying genetic factors regulating the pharmacokinetics and pharmacodynamics of commonly used medications.
View details for DOI 10.2217/14622422.214.171.1247
View details for Web of Science ID 000233692000010
View details for PubMedID 16296948
Computational genetics: from mouse to human?
TRENDS IN GENETICS
2005; 21 (9): 526-532
In this article, we describe a novel computational-analysis method that rapidly identified the genetic basis for several trait differences among inbred mouse strains. This approach enables researchers to identify a causative genetic factor by correlating a pattern of observable physiological or pathological differences among selected inbred strains with a pattern of genetic variation. Compared with conventional methods used for mouse genetic analysis, which require many years to produce results, this haplotype-based computational analysis can be rapidly performed. We discuss the factors affecting the performance and precision of this computational method. Although it currently can analyze traits of limited genetic complexity in mouse, the potential application of this genetic-analysis method to other experimental organisms, and possibly humans, is evaluated.
View details for DOI 10.1016/j.tig.2005.06.010
View details for Web of Science ID 000231783300011
View details for PubMedID 16009447
Tumor necrosis factor-alpha polymorphism, bone strength phenotypes, and the risk of fracture in older women
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
2005; 90 (6): 3491-3497
TNFalpha is a proinflammatory cytokine that promotes osteoclastic bone resorption. We evaluated the association between a G-308A polymorphism (rs1800629) at the TNFA locus and osteoporosis phenotypes in 4306 older women participating in the Study of Osteoporotic Fractures. Femoral neck bone mineral density (BMD) and structural geometry were measured using dual-energy x-ray absorptiometry and hip structural analysis. Incident fractures were confirmed by physician adjudication of radiology reports. Despite similar femoral neck BMD, women with the A/A genotype had greater subperiosteal width (P = 0.01) and endocortical diameter (P = 0.03) than those with the G/G genotype. The net result of these structural differences was that there was a greater distribution of bone mass away from the neutral axis of the femoral neck in women with the A/A genotype, resulting in greater indices of bone bending strength (cross-sectional moment of inertia: P = 0.004; section modulus: P = 0.003). Among 376 incident hip fractures during 12.1 yr of follow-up, a 22% decrease in the risk of hip fracture was seen per copy of the A allele (relative risk 0.78; 95% confidence interval 0.63, 0.96), which was not influenced by adjustments for potential confounding factors, BMD, or bone strength indices. The G-308A polymorphism was not associated with a reduced risk of other fractures. These results suggest a potential role of genetic variation in TNFalpha in the etiology of osteoporosis.
View details for DOI 10.1210/jc.2004-2235
View details for Web of Science ID 000229351000052
View details for PubMedID 15797957
From mouse genetics to human therapeutics
CURRENT OPINION IN DRUG DISCOVERY & DEVELOPMENT
2005; 8 (2): 253-261
This review examines how and where genomic and genetic research will impact pharmaceutical research and development, and emphasizes how mouse genomics and genetics can improve the understanding of human disease pathobiology and drug metabolism, and identify new targets for therapeutic intervention. Although important discoveries can be made from mouse genetic analysis, its utility has been limited by the high cost and long time lines required for such research. A recently developed computational method that markedly accelerates the pace of genetic discovery and reduces its cost is also described.
View details for Web of Science ID 000227463000010
View details for PubMedID 15782548
In silico genetics: Identification of a functional element regulating H2-E alpha gene expression
2004; 306 (5696): 690-695
Computational tools can markedly accelerate the rate at which murine genetic models can be analyzed. We developed a computational method for mapping phenotypic traits that vary among inbred strains onto haplotypic blocks. This method correctly predicted the genetic basis for strain-specific differences in several biologically important traits. It was also used to identify an allele-specific functional genomic element regulating H2-Ealpha gene expression. This functional element, which contained the binding sites for YY1 and a second transcription factor that is probably serum response factor, is located within the first intron of the H2-Ealpha gene. This computational method will greatly improve our ability to identify the genetic basis for a variety of phenotypic traits, ranging from qualitative trait information to quantitative gene expression data, which vary among inbred mouse strains.
View details for DOI 10.1126/science.1100636
View details for Web of Science ID 000224756700053
View details for PubMedID 15499019
Ozone-induced acute pulmonary injury in inbred mouse strains
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY
2004; 31 (1): 69-77
To determine if host factors influence the time course and extent of lung injury after acute inhalation of ozone (O3), we evaluated the physiologic and biologic response of nine genetically diverse inbred strains of mice (C57BL/6J, 129/SvIm, BTBR, BALB/cJ, DBA/2J, A/J, FVB/NJ, CAST/Ei, and C3H/HeJ) exposed to O3 (2.0 ppm x 3 h). Whole lung lavage determined that 129/Svlm, BTBR, DBA/2J, and FVB/NJ had a peak increase in polymorphonuclear cells (PMNs) at 6 h, whereas C57BL/6J and CAST/Ei had a peak increase at 24 h after exposure; airway PMNs were minimally elevated in A/J and C3H/HeJ; BALB/cJ had a predominant lymphocytic influx. Interleukin-6 concentration in the lavage fluid was associated with the influx of PMNs, whereas the total protein in the lavage fluid did not always correlate with lavage cellularity. Respiratory responses were monitored using whole body plethysmography and enhanced pause index. C57BL/6J, BALB/cJ, 129/SvIm, and BTBR were highly sensitive to O3 and exhibited significant increases in enhanced pause to methacholine aerosol stimulation at 6 and 24 h after exposure to O3. In contrast, DBA/2J, A/J, FVB/NJ, CAST/Ei, and C3H/HeJ strains had demonstrated increases in sensitivity to MCh at 6 h after exposure, but responses had returned to near baseline by 24 h after exposure to O3. Epithelial cell proliferation as assessed by proliferating cell nuclear antigen staining was evident at 24 h after exposure to O3. C57BL/6J and A/J showed 4% proliferating cell nuclear antigen-positive cells; 129/SvIm, DBA/2J, and FVB/NJ had 1-3%; and BTBR, BALB/cJ, CAST/Ei, and C3H/HeJ had < 1%. Phenotypic measurements in six inbred strains were used for an in silico genome analysis based on the Roche mouse database. Consistent loci on chromosomes 1, 7, and 15 were among those identified to have a significant association with the phenotypes studied. In aggregate, our approach has identified O3-resistant (C3H/HeJ and A/J) and -vulnerable (C57BL/6J and 129/SvIm) strains of mice, and determined novel genomic loci, suggesting a clear genetic basis for the lung response to inhaled O3.
View details for DOI 10.1165/rcmb.2003-0001OC
View details for Web of Science ID 000222492300011
View details for PubMedID 14975936
Regulation of bone mass in mice by the lipoxygenase gene Alox15
2004; 303 (5655): 229-232
The development of osteoporosis involves the interaction of multiple environmental and genetic factors. Through combined genetic and genomic approaches, we identified the lipoxygenase gene Alox15 as a negative regulator of peak bone mineral density in mice. Crossbreeding experiments with Alox15 knockout mice confirmed that 12/15-lipoxygenase plays a role in skeletal development. Pharmacologic inhibitors of this enzyme improved bone density and strength in two rodent models of osteoporosis. These results suggest that drugs targeting the 12/15-lipoxygenase pathway merit investigation as a therapy for osteoporosis.
View details for Web of Science ID 000187908500048
View details for PubMedID 14716014
A polymorphism in the TCF7 gene, C883A, is associated with type 1 diabetes
2003; 52 (6): 1579-1582
Type 1 diabetes is an autoimmune disease with a Th1 phenotype in which insulin-producing beta-cells in the pancreas are destroyed. The T-cell-specific transcription factor TCF7 activates genes involved in immune regulation and is a candidate locus for genetic susceptibility to type 1 diabetes. A nonsynonymous single nucleotide polymorphism (SNP) (Pro to Thr) in the TCF7 gene, C883A, was examined in samples from 282 Caucasian multiplex type 1 diabetic families. HLA-DRB1 and -DQB1 genotypes were previously determined for these samples, allowing data stratification based on HLA-associated risk. The transmission disequilibrium test showed significant overtransmission of the A allele from fathers (64.1%, P < 0.007) and nonsignificant overtransmission (57.4%, P < 0.06) of the A allele to patients who do not carry the highest-risk HLA-DR3/DR4 genotype. Elliptical sib pair analysis showed significant associations of the A allele with type 1 diabetes in paternal transmissions (P < 0.03), transmissions to male children (P < 0.04), and in the non-DR3/DR4 group (P < 0.04). These data also suggest that TCF7 C883A may affect age of disease onset. Analysis of genotype data from surrounding SNPs suggests that this TCF7 polymorphism may itself represent a risk factor for type 1 diabetes.
View details for Web of Science ID 000183167600037
View details for PubMedID 12765974
A polymorphism in the TCF7 locus is associated with type I diabetes in Caucasians.
CELL PRESS. 2001: 226–26
View details for Web of Science ID 000171648900255
Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus
2001; 15 (3): 435-443
The Nba2 locus is a major genetic contribution to disease susceptibility in the (NZB x NZW)F(1) mouse model of systemic lupus. We generated C57BL/6 mice congenic for this NZB locus, and these mice produced antinuclear autoantibodies characteristic of lupus. F(1) offspring of congenic and NZW mice developed high autoantibody levels and severe lupus nephritis similar to (NZB x NZW)F(1) mice. Expression profiling with oligonucleotide microarrays revealed only two differentially expressed genes, interferon-inducible genes Ifi202 and Ifi203, in congenic versus control mice, and both were within the Nba2 interval. Quantitative PCR localized increased Ifi202 expression to splenic B cells and non-T/non-B cells. These results, together with analyses of promoter region polymorphisms, strain distribution of expression, and effects on cell proliferation and apoptosis, implicate Ifi202 as a candidate gene for lupus.
View details for Web of Science ID 000171211800010
View details for PubMedID 11567633
In silico mapping of complex disease-related traits in mice
2001; 292 (5523): 1915-1918
Experimental murine genetic models of complex human disease show great potential for understanding human disease pathogenesis. To reduce the time required for analysis of such models from many months down to milliseconds, a computational method for predicting chromosomal regions regulating phenotypic traits and a murine database of single nucleotide polymorphisms were developed. After entry of phenotypic information obtained from inbred mouse strains, the phenotypic and genotypic information is analyzed in silico to predict the chromosomal regions regulating the phenotypic trait.
View details for Web of Science ID 000169200700050
View details for PubMedID 11397946
Quantitative trait loci controlling allergen-induced airway hyperresponsiveness in inbred mice
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY
2000; 23 (4): 537-545
Identification of the genetic loci underlying asthma in humans has been hampered by variability in clinical phenotype, uncontrolled environmental influences, and genetic heterogeneity. To circumvent these complications, the genetic regulation of asthma-associated phenotypes was studied in a murine model. We characterized the strain distribution patterns for the asthma-related phenotypes airway hyperresponsiveness (AHR), lung eosinophils, and ovalbumin (OVA)-specific serum immunoglobulin (Ig) E induced by allergen exposure protocols in A/J, AKR/J, BALB/cJ, C3H/HeJ, and C57BL/6J inbred strains and in (C3H/HeJ x A/J)F1 mice. Expression of AHR differed between strains and was sometimes discordant with lung eosinophils or serum IgE. Furthermore, we identified two distinct quantitative trait loci (QTL) for susceptibility to allergen-induced AHR, Abhr1 (allergen-induced bronchial hyperresponsiveness) (lod = 4. 2) and Abhr2 (lod = 3.7), on chromosome 2 in backcross progeny from A/J and C3H/HeJ mice. In addition, a QTL on chromosome 7 was suggestive of linkage to this trait. These QTL differ from those we have previously found to control noninflammatory AHR in the same crosses. Elucidation of the genes underlying these QTL will facilitate the identification of biochemical pathways regulating AHR in animal models of asthma and may provide insights into the pathogenesis of human disease.
View details for Web of Science ID 000089888300017
View details for PubMedID 11017920
Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma
2000; 1 (3): 221-226
The prevalence and severity of allergic asthma continue to rise, lending urgency to the search for environmental triggers and genetic substrates. Using microarray analysis of pulmonary gene expression and single nucleotide polymorphism-based genotyping, combined with quantitative trait locus analysis, we identified the gene encoding complement factor 5 (C5) as a susceptibility locus for allergen-induced airway hyperresponsiveness in a murine model of asthma. A deletion in the coding sequence of C5 leads to C5-deficiency and susceptibility. Interleukin 12 (IL-12) is able to prevent or reverse experimental allergic asthma. Blockade of the C5a receptor rendered human monocytes unable to produce IL-12, mimicking blunted IL-12 production by macrophages from C5-deficient mice and providing a mechanism for the regulation of susceptibility to asthma by C5. The role of complement in modulating susceptibility to asthma highlights the importance of immunoregulatory events at the interface of innate and adaptive immunity in disease pathogenesis.
View details for Web of Science ID 000089815100020
View details for PubMedID 10973279
Tpm1, a locus controlling IL-12 responsiveness, acts by a cell-autonomous mechanism
JOURNAL OF IMMUNOLOGY
1999; 162 (3): 1339-1347
Th phenotype development is controlled not only by cytokines but also by other parameters including genetic background. One site of genetic variation between murine strains that has direct impact on Th development is the expression of the IL-12 receptor. T cells from B10.D2 and BALB/c mice show distinct control of IL-12 receptor expression. When activated by Ag, B10.D2 T cells express functional IL-12 receptors and maintain IL-12 responsiveness. In contrast, under the same conditions, BALB/c T cells fail to express IL-12 receptors and become unresponsive to IL-12, precluding any Th1-inducing effects if subsequently exposed to IL-12. Previously, we identified a locus, which we termed T cell phenotype modifier 1 (Tpm1), on murine chromosome 11 that controls this differential maintenance of IL-12 responsiveness. In this study, we have produced a higher resolution map around Tpm1. We produced and analyzed a series of recombinants from a first-generation backcross that significantly narrows the genetic boundaries of Tpm1. This allowed us to exclude from consideration certain previous candidates for Tpm1, including IFN-regulatory factor-1. Also, cellular analysis of F1(B10.D2 x BALB/c) T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner, rather than through influencing the extracellular milieu. This result strongly implies that despite the proximity of our locus to the IL-13/IL-4 gene cluster, these cytokines are not candidates for Tpm1.
View details for Web of Science ID 000078261000017
View details for PubMedID 9973388
- Loci influencing development of Th responses. Identification from in vitro analysis MICROBES AND INFECTION 1999; 1 (1): 79-88
GATA-3-dependent enhancer activity in IL-4 gene regulation.
Journal of immunology
1998; 161 (8): 3822-3826
Previously, we analyzed the proximal IL-4 promoter in directing Th2-specific activity. An 800-base pair proximal promoter conferred some Th2-selective expression in transgenic mice. However, this region directed extremely low reporter mRNA levels relative to endogenous IL-4 mRNA, suggesting that full gene activity requires additional enhancer elements. Here, we analyzed large genomic IL-4 regions for enhancer activity and interaction with transcription factors. The proximal IL-4 promoter is only moderately augmented by GATA-3, but certain genomic regions significantly enhanced GATA-3 promoter transactivation. Some enhancing regions contained consensus, GATA sites that bound Th2-specific complexes. However, retroviral transduction of GATA-3 into developing T cells induced IL-5 to full Th2 levels, but only partially restored IL-4 production. Thus, we propose that GATA-3 is permissive, but not sufficient, for full IL-4 enhancement and may act through GATA elements surrounding the IL-13/IL-4 gene locus.
View details for PubMedID 9780146
Cutting edge: GATA-3-dependent enhancer activity in IL-4 gene regulation
JOURNAL OF IMMUNOLOGY
1998; 161 (8): 3822-3826
View details for Web of Science ID 000076343300006
Transcription factors in immune-mediated disease
CURRENT OPINION IN BIOTECHNOLOGY
1997; 8 (4): 467-473
A large amount of detailed information about the intracellular proteins regulating NF-kappa B activation and the cellular response to NF-kappa B activation has emerged recently. Several small molecules, an antisense oligonucleotide, and gene therapeutic agents that inhibit NF-kappa b activation have been described. Despite this, there are still significant gaps in our understanding of this process and its consequences. In contrast, the characterization of transcription factors selectively regulating cytokine production by CD4+ T cell subsets is at a very early stage. Three interacting proteins have recently been shown to contribute to subset-restricted expression of the IL-4 gene. There are other elements regulating IL-4 gene expression, however, and the relative importance of these recently identified proteins has yet to be determined.
View details for Web of Science ID A1997XN33900012
View details for PubMedID 9265727
Induction of alloantigen-specific T cell tolerance through the treatment of human T lymphocytes with wortmannin
JOURNAL OF IMMUNOLOGY
1997; 158 (6): 2745-2755
Signaling through the CD28 molecule on T cells by its natural ligand, B7, on APCs has recently been shown to require the presence of an active phosphatidylinositol 3-kinase pathway to mediate some of its costimulatory activities (1-7). Using the phosphatidylinositol 3-kinase inhibitor, wortmannin (WN) (8), on human and murine T cells, we have inhibited B7-1-mediated T cell activation and induced Ag-specific tolerance. The addition of WN and/or the B7-1 antagonist, CTLA4Ig, to primary human T cell cultures stimulated with B7-1-transfected allogeneic melanoma cell lines inhibited the generation of alloantigen-specific proliferative and cytolytic responses in vitro. Subsequent examination of these WN- and CTLA4Ig-treated primary T cell cultures revealed that these lymphocyte populations were tolerized to rechallenge with the priming alloantigens in secondary cultures in the absence of additional inhibitor(s). However, reactivity to a third party allogeneic stimulator remained intact. This WN-induced tolerance was reversed by the addition of high dose IL-2, but not IL-4 or IL-7, to the primary cultures, indicating that T cell anergy, not deletion, was responsible for this phenomenon. In vivo studies using a murine graft-vs-host disease (GVHD) model demonstrated that WN treatment of allogeneic donor lymphocytes in vitro failed to generate a significant GVHD in irradiated mouse recipients compared with control allogeneic donor lymphocytes. These findings suggest potentially novel therapeutic strategies for the prevention of GVHD.
View details for Web of Science ID A1997WM43500031
View details for PubMedID 9058809
CD45 isoforms on human CD4(+) T-cell subsets
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
1996; 98 (2): 433-440
We have examined the level of surface expression and functional properties of leukocyte function associated antigen-1, very late antigen-4, and CD45 isoforms on a panel of human CD4+ T-cell clones representative of TH0, TH1, and TH2 cells. There were no qualitative differences in the expression of these antigens among the three types of CD4+ T-cell clones. However, CD45RB was the only CD45 isoform that provided a costimulatory signal in a solid-phase antibody-induced cellular proliferation assay. Additionally, the antigen-induced proliferative response of T-cell clones was inhibited by soluble anti-CD45RO and anti-CD45RB antibodies. Our results suggest that CD45 isoforms differentially provide costimulatory signals to T cells. However, the ability of each CD45 isoform to provide a costimulatory signal does not differ among the TH0, TH1, or TH2 T-cell populations.
View details for Web of Science ID A1996VC99500023
View details for PubMedID 8757221
PATHOGEN ANTIGEN-REACTIVE AND SUPERANTIGEN-REACTIVE SYNOVIAL-FLUID T-CELLS IN REACTIVE ARTHRITIS
JOURNAL OF INFECTIOUS DISEASES
1995; 172 (5): 1290-1297
Analysis of pathogen-reactive T cell clones (CD3+4+8-TCR alpha beta +), isolated from the synovial fluid of 2 HLA-B27-positive patients with Yersinia enterocolitica-triggered reactive arthritis, has provided important information about the cellular immune response to this disease-inciting pathogen. This study demonstrates that the proteins secreted by Y. enterocolitica, including a protein with tyrosine phosphatase activity (YopH), are potent immunogens stimulating CD4+ cells within the inflamed joint. The pathogen-reactive T cell clones preferentially utilized a limited set of T cell receptor variable region gene segments. A purified Yersinia superantigen triggered a proliferative response in most of the antigen-reactive T cell clones tested. These results suggest that the activity of this pathogen's superantigen influences the cellular immune response to its antigens.
View details for Web of Science ID A1995TA55200016
View details for PubMedID 7594666
CD28-B7 INTERACTIONS PROMOTE T-CELL ADHESION
EUROPEAN JOURNAL OF IMMUNOLOGY
1995; 25 (11): 3087-3093
CD28 activation by antibody-mediated ligation has been shown to provide an important co-stimulatory signal for T cell adhesion to purified protein ligands. However, the effect of CD28 ligation by one of its natural ligands, B7.1, on T cell adhesion to other cells has not been studied. Therefore, in the present manuscript, we characterized the adhesive interactions between human T cells and B7.1-transfected major histocompatibility complex class II+ and class II- melanoma cells. In our studies, human T cells and T cell clones adhered to B7.1-transfected melanoma cells, but not to untransfected parental cells. The adhesive reaction in this model was rapid, occurring within 15 min, and was inhibited by anti-B7.1 antibody and soluble CTLA-4 immunoglobulin. Antibody inhibition studies demonstrated that adhesion between T cells and B7.1-transfected melanoma cells was mediated by interactions between LFA-1:ICAM-1 and CD2:LFA-3. Inhibition by pharmacological agents demonstrated that the CD28-induced adhesion required specific intracellular signaling events. A protein kinase C inhibitor, staurosporin, significantly inhibited T cell binding to transfected melanoma cells, while cyclosporin A and wortmannin, an inhibitor of phosphatidylinositol-3-kinase, did not. These results suggest that the presence of B7 on various cell populations may activate lymphocytes to adhere better, thus promoting activation, cytolysis, and migration.
View details for Web of Science ID A1995TE64700014
View details for PubMedID 7489747
MODULATION OF THE GRB2-ASSOCIATED PROTEIN COMPLEX IN HUMAN CD4(+) T-CELLS BY RECEPTOR ACTIVATION
JOURNAL OF IMMUNOLOGY
1995; 155 (8): 3815-3822
A panel of human CD4+ T cell clones was utilized to dissect and analyze the biochemical consequences of activation of CD3 or CD28. To molecularly characterize receptor-activated proximal signaling events, tyrosine-phosphorylated proteins co-precipitating with a Grb2 fusion protein after receptor activation were analyzed. Ligation of CD28, but not other costimulatory molecules, induced the tyrosine phosphorylation of two previously identified Grb2 binding proteins (pp76 and pp116). A third Grb2 binding protein (pp36) was extensively tyrosine phosphophorylated in response to combined CD3 and CD28 activation, but not in response to ligation of either receptor alone. cAMP and co-ligation of CD45 affected the receptor-activated tyrosine phosphorylation of Grb2-associated proteins. Furthermore, we demonstrated that two signaling molecules, Vav and phosphatidylinositol 3'-kinase (PI(3)K), also interacted with the Grb2 protein complex. The activity of PI(3)K was required for T cell activation, because wortmannin, a PI(3)K inhibitor, blocked T cell proliferation and cytokine production induced by ligation of CD3 and CD28. In conclusion, we demonstrate that in activated human T cell clones, the composition of Grb2 protein complex is modulated by costimulatory signals and cAMP, which may be important for the regulation of intracellular signal transduction.
View details for Web of Science ID A1995RY58100017
View details for PubMedID 7561087
MOLECULAR-CLONING OF SLP-76, A 76-KDA TYROSINE PHOSPHOPROTEIN ASSOCIATED WITH GRB2 IN T-CELLS
JOURNAL OF BIOLOGICAL CHEMISTRY
1995; 270 (13): 7029-7032
The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-gamma 1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.
View details for Web of Science ID A1995QQ43100005
View details for PubMedID 7706237
COSTIMULATORY SIGNALS CAN SELECTIVELY MODULATE CYTOKINE PRODUCTION BY SUBSETS OF CD4(+) T-CELLS
JOURNAL OF IMMUNOLOGY
1995; 154 (4): 1684-1690
Analysis of experimental animal models and human clinical samples has indicated that the selective activation of CD4+ T cell subsets with distinct profiles of cytokine production plays an important role in the pathogenesis of human inflammatory and allergic diseases. The possibility that differential activation of costimulatory pathways is a mechanism for selectively modulating cytokine production by CD4+ T cells was tested. The proliferative response and cytokines secreted by a panel of human CD4+ T cell clones, representative of Th1 or Th2/0 cells, in response to activation of different costimulatory pathways was measured. CD28-mediated costimulatory signals induced proliferation and IFN-gamma secretion by Th1 cells. Although CD28-ligation induced Th2/0 cells to proliferate, it did not trigger IL-4 production. Ligation of LFA-1 and CD45 isoforms also generated costimulatory signals activating cytokine secretion by the different types of T cell clones. Th1 cells secreted the same profile of cytokines, irrespective of which costimulatory pathway was engaged. However, the cytokine secreted by a subset of Th2/0 cells varied, depending upon which costimulatory pathways were activated. These results suggest that the costimulatory pathways activated by APCs can selectively influence cytokine production by CD4+ T cell subsets.
View details for Web of Science ID A1995QG20800017
View details for PubMedID 7836752
- IMMUNOPATHOGENESIS OF HUMAN INFLAMMATORY ARTHRITIS - LESSONS FROM LYME AND REACTIVE ARTHRITIS JOURNAL OF INFECTIOUS DISEASES 1994; 170 (4): 978-985
GENES WITHIN THE HLA CLASS-II REGION CONFER BOTH PREDISPOSITION AND RESISTANCE TO PRIMARY BILIARY-CIRRHOSIS
1994; 43 (2): 71-77
Nonradioactive sequence-specific oligonucleotide probes for the polymorphic HLA class II genes have been used to type samples from 51 Caucasian patients with the autoimmune liver disease, primary biliary cirrhosis, and 240 Caucasian controls. Although the allelic distribution at the DPB1 locus showed no significant variation between patients and controls, there was heterogeneity in the distribution of DR-DQ haplotypes where the frequency of the DRB1*0801-DQA1*0401/0601-DQB1*04 haplotype was significantly increased in the patients, suggesting it confers susceptibility to this disease. Two other haplotypes, DRB1*1501-DQA1*0102-DQB1*0602 and DRB1*1302-DQA1*0102-DQB1*0604, were significantly reduced in the patients, suggesting they confer protection. Tests of the individual loci show that resistance to this disease is most strongly associated with the DQA1*0102 allele shared by both protective haplotypes. Due to linkage disequilibrium it is unclear whether multiple genes or a single locus on the susceptible DR8 haplotype are needed for predisposition. These data show that distinct HLA class II alleles confer both predisposition and resistance to PBC and provide insight into the role that these genes may play in the immunopathogenesis of this disease.
View details for Web of Science ID A1994NA46800002
View details for PubMedID 8016844
Inflammatory disease: where immunology and adhesion meet?
Current opinion in biotechnology
1993; 4 (6): 745-750
Our understanding of the basic mechanisms regulating the entry of leukocytes into inflamed tissues has increased dramatically over the past few years. It is anticipated that increased understanding of this process will promote the design and discovery of agents capable of selectively modulating the recruitment of leukocyte subsets to foci of inflammation. Work is currently underway to develop a novel class of drugs influencing leukocyte adhesive interactions that have therapeutic potential in a wide variety of human chronic inflammatory diseases.
View details for PubMedID 7764475
PREFERENTIAL USAGE OF T-CELL ANTIGEN RECEPTOR-V REGION GENE SEGMENT V-BETA-5.1 BY BORRELIA-BURGDORFERI ANTIGEN-REACTIVE T-CELL CLONES ISOLATED FROM A PATIENT WITH LYME-DISEASE
JOURNAL OF IMMUNOLOGY
1993; 150 (9): 4125-4135
Forty-three CD3+4+8- TCR alpha beta+ Borrelia burgdorferi-reactive T cell clones isolated from the peripheral blood of a single patient with clinically active chronic Lyme arthritis were characterized. The spirochetal Ag recognized by 16 of these T cell clones was determined by reactivity with a panel of recombinant spirochetal Ag, which included the OspA, OspB, flagellin, Hsp60 and Hsp70 proteins. All three T cell clones reactive with heat shock proteins recognized a non-cross-reactive epitope unique to the spirochetal Ag. Analysis of the TCR V regions revealed preferential usage of V beta 5.1; 5 of 15 T cell clones that recognized an unidentified spirochetal Ag utilized this V beta gene segment. Most of the T cell clones recognized a given spirochetal Ag exclusively within the context of one HLA class II allele. However, two T cell clones, which recognized an unidentified Ag in the spirochetal lysate within the context of different HLA class II alleles, were both TCR V beta 5.1+, although each displayed a distinct alpha-chain. Moreover, in vitro incubation of this patient's PBMC with B. burgdorferi Ag resulted in a specific increase in the percentage of T cells expressing TCR V beta 5.1. These results indicate that B. burgdorferi has a V beta-selective factor influencing the cellular immune response in a patient with clinically active Lyme disease.
View details for Web of Science ID A1993KY45200047
View details for PubMedID 7682589
CHARACTERIZATION OF A BORRELIA-BURGDORFERI-DNAJ HOMOLOG
INFECTION AND IMMUNITY
1992; 60 (11): 4965-4968
The gene encoding a Borrelia burgdorferi DnaJ homolog, located immediately 3' of the hsp70 gene, was characterized. Although there is a single copy of the dnaJ gene on the spirochetal chromosome, two distinct dnaJ transcripts are detected in B. burgdorferi RNA. RNA blot analysis indicates that the dnaJ gene can be transcribed alone or as part of a larger transcript containing the hsp70 homolog.
View details for Web of Science ID A1992JV76300071
View details for PubMedID 1383161
BORRELIA-BURGDORFERI HSP70 HOMOLOG - CHARACTERIZATION OF AN IMMUNOREACTIVE STRESS PROTEIN
INFECTION AND IMMUNITY
1992; 60 (9): 3704-3713
The gene encoding an immunoreactive Borrelia burgdorferi HSP70 homolog was isolated and characterized. The predicted amino acid sequence of this spirochetal protein confirms that this gene encodes a member of the HSP70 family of proteins. Although there appears to be a single copy of this gene on the spirochetal chromosome, two distinct transcripts hybridizing to the hsp70 probe are detected in RNA isolated from B. burgdorferi. The amount of spirochetal HSP70 RNA transcripts is shown to be thermally regulated. Antibodies in the serum of three Lyme arthritis patients and cloned T-cell lines isolated from one patient with Lyme arthritis recognize the expressed recombinant HSP70, indicating that it is an immunologically important spirochetal antigen. Antibodies in a rabbit antiserum, as well as antibodies in the serum of two of three Lyme arthritis patients examined, bound to expressed truncated recombinant HSP70s with 250 amino acids deleted from either the amino or carboxy terminus of the protein. However, antibodies in the serum of three Lyme arthritis patients, which were reactive with spirochetal HSP70, did not cross-react with human HSP70 proteins.
View details for Web of Science ID A1992JJ86100034
View details for PubMedID 1379988
YERSINIA-ENTEROCOLITICA ACTIVATES A T-HELPER TYPE-1-LIKE T-CELL SUBSET IN REACTIVE ARTHRITIS
JOURNAL OF IMMUNOLOGY
1992; 148 (10): 3079-3085
The profile of lymphokines secreted by 14 T cell clones and 24 T cell lines reactive with Yersinia Ag isolated from the synovial fluid cells of two HLA-B27+ patients with Yersinia-triggered reactive arthritis was characterized. In response to Ag-specific or -nonspecific stimulation, all of the Yersinia-reactive T cell clones and lines had a pattern of lymphokine secretion resembling that of murine (Th1) cells. A total of 50% of T cell lines and clones randomly isolated from a reactive arthritis patient, without prior in vitro stimulation with Yersinia Ag, also exhibited a Th1-like profile of cytokine secretion upon nonspecific activation. This indicates that the selective expansion of this subset of T cells had already occurred in vivo. The possibility that the predominance of Th1-like T cells was an artefact generated by the T cell cloning procedure was excluded; 50% of the randomly isolated T cell clones and lines produced IL-4, IL-5, or both cytokines upon nonspecific activation. These results indicate that Yersinia Ag selectively activate a Th1-like subset of T cells in patients with Yersinia-triggered reactive arthritis. Accumulation of such cells in the synovial tissue of patients with reactive arthritis may play a key role in the pathogenesis of this disease.
View details for Web of Science ID A1992HU68600015
View details for PubMedID 1533651
EPITOPES ON THE OUTER SURFACE PROTEIN-A OF BORRELIA-BURGDORFERI RECOGNIZED BY ANTIBODIES AND T-CELLS OF PATIENTS WITH LYME-DISEASE
JOURNAL OF IMMUNOLOGY
1992; 148 (1): 218-224
We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease. Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined. The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids. Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope. One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules. In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein. Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells.
View details for Web of Science ID A1992GX16200034
View details for PubMedID 1370170
- A ROLE FOR CD4+ T-CELL SUBSETS PRODUCING A SELECTIVE PATTERN OF LYMPHOKINES IN THE PATHOGENESIS OF HUMAN CHRONIC INFLAMMATORY AND ALLERGIC DISEASES IMMUNOLOGICAL REVIEWS 1991; 123: 23-35
BORRELIA-BURGDORFERI ACTIVATES A T-HELPER TYPE-1 LIKE T-CELL SUBSET IN LYME ARTHRITIS
JOURNAL OF EXPERIMENTAL MEDICINE
1991; 174 (3): 593-601
18 cloned T cell lines reactive with Borrelia burgdorferi proteins, all CD3+4+8-TCR-alpha/beta+ and restricted by HLA class II proteins, were isolated from four patients with chronic Lyme arthritis. Analysis of these T cell clones indicated that the T cell response to the Lyme disease spirochete is not oligoclonally restricted; yet all produced the same pattern of lymphokines, resembling that of murine type 1 T helper cells, after antigen-specific or nonspecific stimulation. Therefore, a subset of human CD4+ T cells, with a distinct profile of lymphokine secretion, is selectively activated by the pathogen inciting this chronic inflammatory disease.
View details for Web of Science ID A1991GC96000010
View details for PubMedID 1831490
T-CELL AND ANTIBODY REACTIVITY WITH THE BORRELIA-BURGDORFERI 60-KDA HEAT-SHOCK PROTEIN IN LYME ARTHRITIS
JOURNAL OF IMMUNOLOGY
1991; 146 (11): 3985-3992
The reactivity of cloned T cells and serum antibodies, obtained from patients with chronic Lyme arthritis, with expressed recombinant B. burgdorferi 60-kDa heat shock protein homologue (HSP60) was analyzed. The expressed recombinant Borrelia burgdorferi HSP60 was bound by antibodies in the sera of patients with Lyme arthritis, but not by control sera. A T cell clone (CR253), isolated from one of four patients examined, exhibited an HLA-DR2 restricted proliferative response to the expressed recombinant B. burgdorferi HSP60. This T cell clone specifically recognized the HSP60 of B. burgdorferi and did not proliferate in response to the human, mycobacterial, or Escherichia coli HSP60 homologues. The epitope recognized by this cloned T cell, located between amino acids 260 and 274, is in a region of the spirochetal HSP60 that is not conserved between bacteria and eukaryotes.
View details for Web of Science ID A1991FN05400043
View details for PubMedID 1709664
IMMUNOREACTIVE EPITOPES ON AN EXPRESSED RECOMBINANT FLAGELLAR PROTEIN OF BORRELIA-BURGDORFERI
INFECTION AND IMMUNITY
1991; 59 (2): 514-520
A recombinant Borrelia burgdorferi flagellin protein expressed in Escherichia coli is bound by a murine monoclonal antiflagellin antibody (H9724) and by antibodies in the sera of patients with Lyme disease. Immunoreactive epitopes on the flagellar protein were identified by immunoblot analysis of antibody binding to expressed truncated flagellar proteins. The epitope recognized by the murine monoclonal antibody is within the central heterologous region of the flagellar protein (amino acids 90 to 266). However, antiflagellin antibodies in the sera of patients with Lyme arthritis bound an epitope entirely within, or whose conformation was partly formed by, the 90 NH2-terminal amino acids of the flagellar protein. The binding of antibodies in the sera of patients with Lyme arthritis to the NH2-terminal region of the flagellar protein, a region with sequence homology to the flagellar proteins of other bacterial species, suggests the possibility that antigenic mimicry contributes to the immunopathogenesis of Lyme disease. The fact that human antibodies bind to a highly conserved and hence shared portion of the flagellin reduces the specificity of serological assays for the diagnosis of Lyme disease which use the flagellar protein as antigen.
View details for Web of Science ID A1991EU97300007
View details for PubMedID 1702766
ANALYSIS OF LYMPHOCYTES-T CLONED FROM THE SYNOVIAL-FLUID AND BLOOD OF A PATIENT WITH LYME ARTHRITIS
1990; 2 (11): 1081-1089
Cloned T lymphocytes reactive with Borrelia burgdorferi proteins were isolated from a patient with chronic Lyme arthritis. All of the T cell clones which proliferated in response to Borrelia proteins were CD3 + CD4 + CD8 - TCR alpha beta + and HLA-DR restricted. One T cell clone (GN30) exhibited HLA-DR-restricted cytotoxic activity against antigen-presenting cells pulsed with Borrelia antigen. In response to Borrelia antigen, the T cell clones produced TNF-alpha, INF-gamma, and GM-CSF. There are at least three distinct spirochetal proteins recognized by the four T cell clones analyzed. Purified Borrelia proteins triggered the HLA-DR-restricted proliferative and cytotoxic responses, as well as lymphokine secretion by two of the T cell clones. The spirochetal protein which triggered the HLA-DR-restricted proliferative and cytotoxic activities of the T cell clone (GN30) isolated from synovial fluid is the 41 kd flagellar protein.
View details for Web of Science ID A1990EH39600009
View details for PubMedID 2083228
CHARACTERIZATION OF BORRELIA-BURGDORFERI PROTEINS REACTIVE WITH ANTIBODIES IN SYNOVIAL-FLUID OF A PATIENT WITH LYME ARTHRITIS
INFECTION AND IMMUNITY
1990; 58 (7): 2404-2407
Four Borrelia burgdorferi proteins reactive with antibodies in the synovial fluid of a patient with Lyme arthritis were characterized. Homology between amino acid sequences of immunoreactive spirochetal proteins and human proteins, including members of the Escherichia coli GroEL protein family, suggests that antigenic mimicry may play a role in the pathogenesis of Lyme arthritis.
View details for Web of Science ID A1990DL13400059
View details for PubMedID 2365463
REACTIVITY OF CLONED, EXPRESSED HUMAN FC-GAMMA-RIII ISOFORMS WITH MONOCLONAL-ANTIBODIES WHICH DISTINGUISH CELL-TYPE-SPECIFIC AND ALLELIC FORMS OF FC-GAMMA-RIII
1990; 2 (4): 303-310
We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were digested with N-glycanase, deglycosylated neutrophil Fc gamma RIII had a more rapid electrophoretic mobility than NK cell Fc gamma RIII, as is observed for the human Fc gamma RIII isoforms on normal cells. The neutrophil and NK cell Fc gamma RIII isoforms apparently result from cell-type specific expression of different forms of Fc gamma RIII mRNA. A TaqI RFLP was also found for human Fc gamma RIII. Monoclonal antibodies which have been used to distinguish the neutrophil and NK cell Fc gamma RIII isoforms and the NA1 and NA2 alleles of human neutrophil Fc gamma RIII were employed to study the expression of two Fc gamma RIII cDNA clones derived from neutrophils and NK cells. Fc gamma RIII encoded by the neutrophil-derived cDNA reacts with the monoclonal antibody CLBgran11, while the NK-cell Fc gamma RIII cDNA expresses the Fc receptor which carries an antigenic determinant recognized by the antibody GRM1. Characterization of hybrid Fc gamma RIII constructed by interchange of restriction fragments between the neutrophil and NK cell cDNAs allowed localization of antigenic determinants recognized by the monoclonal antibodies.
View details for Web of Science ID A1990DK02100003
View details for PubMedID 1703781
THE POLYMORPHIC FC-GAMMA-RECEPTOR-II GENE MAPS TO HUMAN CHROMOSOME-1Q
1989; 29 (5): 331-339
Human receptors for IgG (Fc gamma R) play important roles in the immune response. Expression of the human Fc gamma RII gene may be relevant in immune complex related disorders such as systemic lupus erythematosus and Sjogren's syndrome. We have used spot blot analysis of dual laser-sorted human chromosomes to localize the Fc gamma RII gene to human chromosome 1. Spot blot analysis of sorted derivative chromosomes sublocalized the gene to the chromosome 1 long arm (1q12----q25.1). This subchromosomal localization involved reassigning a reciprocal chromosome translocation breakpoint. We also identified Xmn I and Taq I Fc gamma RII polymorphic restriction sites that arose before the races diverged. These common Xmn I and Taq I polymorphisms are predicted to be informative for segregation analysis with human diseases in 85% of all matings.
View details for Web of Science ID A1989U360300007
View details for PubMedID 2565886
HUMAN FC-GAMMA-RIII - CLONING, EXPRESSION, AND IDENTIFICATION OF THE CHROMOSOMAL LOCUS OF 2 FC-RECEPTORS FOR IGG
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1989; 86 (3): 1013-1017
A cDNA clone encoding a human receptor for the Fc portion of IgG (Fc gamma R), Fc gamma RIII or CD16, was isolated from a human leukocyte library by a transient expression-immunoselection procedure. This cDNA (pGP5) encodes a 46-kDa phosphatidylinositol-linked cell surface protein with CD16 determinants and affinity for human IgG. The deduced protein sequence is most homologous to the murine receptor Fc gamma RII alpha, with slightly less homology to the human receptors Fc gamma RII and Fc epsilon RI. The cDNA hybridizes to a 2.2 kilobase mRNA in human leukocytes and a cloned human natural killer cell line. Fc gamma RIII is mapped to chromosome 1 by spot-blot analysis of sorted human chromosomes. Hybridization of Fc gamma RII and Fc gamma RIII probes to restriction digests of human genomic DNA separated by pulsed-field gel electrophoresis demonstrates physical linkage of the two genes within a maximum distance of 200 kilobases. The results identify a locus for at least two Fc gamma R genes on human chromosome 1.
View details for Web of Science ID A1989T144400056
View details for PubMedID 2521732
CLONED AND EXPRESSED HUMAN FC RECEPTOR FOR IGG MEDIATES ANTI-CD3-DEPENDENT LYMPHOPROLIFERATION
JOURNAL OF IMMUNOLOGY
1988; 141 (6): 1891-1896
We have utilized gene transfer experiments to investigate the role of a human monocyte receptor for IgG (Fc gamma RII) in mouse IgG1 anti-CD3 (Leu 4)-induced lymphoproliferation in vitro. Mouse Ltk- cells expressing human Fc gamma RII or a mutant of Fc gamma RII lacking the entire cytoplasmic domain of the receptor mediate anti-CD3-induced lymphoproliferation in cultures of adherent cell-depleted human PBMC. Expression of an Fc gamma RII mutant lacking transmembrane and cytoplasmic domains (soluble Fc gamma RII) in COS7 cells yielded a secreted receptor which retained affinity for IgG, even in the absence of the mutant receptor's N-linked oligosaccharides. Soluble Fc gamma RII inhibits rosette formation by human IgG-sensitized RBC and the Fc gamma RII-bearing cell line K562, but does not sitmulate anti-CD3-induced lymphoproliferation under the conditions tested.
View details for Web of Science ID A1988Q102200014
View details for PubMedID 2971717
CHARACTERIZATION OF THE HUMAN MONOCYTE HIGH-AFFINITY FC RECEPTOR (HU FCRI)
1988; 25 (3): 243-250
The high affinity Fc receptor (FcRI) of a human monocytic cell line, U937, was further characterized using a previously described murine monoclonal antibody, FcRmAb32. This antibody immunoprecipitated a 70 K cell surface glycoprotein. A solid phase ligand binding assay and a solid phase immunoprecipitation assay were combined to confirm that the 70 K cell surface glycoprotein immunoprecipitated by FcRmAb32 is an IgG binding protein. N-glycanase digestion shows that at least 20% of the relative mobility of the 70 K FcRI glycoprotein is due to N-linked carbohydrate. FcRmAb32 immunoprecipitated a 70 K glycoprotein from biosynthetically labelled U937 cells that co-migrated with the surface iodinated glycoprotein on 2-dimensional gel electrophoresis. A 50 K protein, that is biosynthetically labelled but not accessible to surface iodination, which, bound to control antibodies was also present in FcRmAb32 immunoprecipitates. FcRmAb32 only bound the mature fully glycosylated form of FcRI. The 70 K FcRI was not phosphorylated constitutively nor when U937 cells were stimulated by PMA.
View details for Web of Science ID A1988M746900003
View details for PubMedID 2967428
MONOCLONAL-ANTIBODY IMMUNOPRECIPITATION OF CELL-MEMBRANE GLYCOPROTEINS
1987; 167 (2): 239-244
Procedural modifications facilitating the immunoprecipitation of cell surface-associated glycoproteins by monoclonal antibodies are presented. The use of complexes of antibodies coupled to protein A-Sepharose in place of antibodies directly coupled to Sepharose, and the inclusion of ATP and salt in the lysis buffer, is shown to markedly reduce the nonspecific binding of aggregated cytoskeletal proteins. These modifications result in low backgrounds while the specific membrane-associated proteins are still quantitatively immunoprecipitated.
View details for Web of Science ID A1987L350700005
View details for PubMedID 3442319
MONOCLONAL-ANTIBODIES AGAINST 7 SITES ON THE HEAD AND TAIL OF DICTYOSTELIUM MYOSIN
JOURNAL OF CELL BIOLOGY
1985; 100 (4): 1016-1023
Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.
View details for Web of Science ID A1985AEQ9400003
View details for PubMedID 2579955
SITE-SPECIFIC INHIBITION OF MYOSIN-MEDIATED MOTILITY INVITRO BY MONOCLONAL-ANTIBODIES
JOURNAL OF CELL BIOLOGY
1985; 100 (4): 1024-1030
Monoclonal antibodies directed against seven different sites on Dictyostelium myosin (Peltz, G., J. A. Spudich, and P. Parham, 1985, J. Cell Biol., 100: 1016-1023) were tested for their ability to inhibit movement of myosin in vitro, using the Nitella-based myosin-mediated bead movement assay (Sheetz, M. P., R. Chasan, and J. A. Spudich, 1984, J. Cell Biol., 99: 1867-1871). To complement this functional assay, we located the binding sites of these antibodies by electron microscopy, using the rotary shadowing technique. One antibody bound to the 18,000-dalton light chain and inhibited movement completely. All of the remaining antibodies bound to various positions along the rod portion of the myosin molecule, which is approximately 1,800 A long. Antibodies that bound to the rod about 470, 680, and 1400 A from the head-tail junction did not alter myosin movement. One antibody appeared to bind very close to the head-tail junction and to inhibit movement 50%. Surprisingly, three antibodies that bound about 1,200 A from the head-tail junction inhibited movement completely. This inhibition did not depend on using intact IgG, since Fab' fragments had the same effect.
View details for Web of Science ID A1985AEQ9400004
View details for PubMedID 3980577
- A QUANTITATIVE ASSAY FOR THE ACTIVATION OF PLASMINOGEN BY TRANSFORMED-CELLS INSITU AND BY UROKINASE BIOCHEMISTRY 1981; 20 (15): 4307-4314
- A NEW ACTIVE-SITE TITRANT OF SERINE PROTEASES METHODS IN ENZYMOLOGY 1981; 80: 414-424
SYNTHESIS AND CHARACTERIZATION OF A NEW FLUOROGENIC ACTIVE-SITE TITRANT OF SERINE PROTEASES
1981; 20 (15): 4298-4306
The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.
View details for Web of Science ID A1981LZ80400010
View details for PubMedID 6456760
DICTYOSTELIUM MYOSIN - CHARACTERIZATION OF CHYMOTRYPTIC FRAGMENTS AND LOCALIZATION OF THE HEAVY-CHAIN PHOSPHORYLATION SITE
JOURNAL OF CELL BIOLOGY
1981; 89 (1): 104-108
Chymotrypsin cleaves Dictyostelium myosin in half, splitting the heavy chain (210,000 daltons) into two fragments of 105,000 daltons each. One of the two major fragments is soluble at low ionic strength and has a native molecular weight of 130,000. As judged by SDS polyacrylamide gel electrophoresis, this soluble fragment consists of the two intact myosin light chains of 18,000 and 16,000 daltons and a 105,000-dalton polypeptide derived from the myosin heavy chain. The soluble fragment retains actin-activated ATPase activity and the ability to bind to actin in an ATP-dissociable fashion. The maximal velocity of the actin-activated ATPase activity of the soluble fragment is 80% of that of uncleaved myosin, although its apparent Km for actin is 12-fold greater than that of myosin. In addition to the major soluble 105,000-dalton fragment discussed above, chymotryptic cleavage of the Dictyostelium myosin also generates fragments that are insoluble at low ionic strength. The major insoluble fragment is 105,000 daltons on an SDS polyacrylamide gel and forms thick filaments that are devoid of myosin heads. A less prevalent insoluble fragment has a molecular weight of 83,000 and is probably a subfragment of the insoluble 105,000-dalton fragment. The heavy chain of myosin is phosphorylated in vivo and the phosphorylation site has been localized to the insoluble fragments, which derive from the tail portion of the myosin molecule.
View details for Web of Science ID A1981LH41600013
View details for PubMedID 7228895
New assay for the plasminogen activator activity of transformed cells.
Cold Spring Harbor symposia on quantitative biology
1980; 44: 669-680
View details for PubMedID 6448727
SENSITIVE ASSAY FOR PLASMINOGEN-ACTIVATOR OF TRANSFORMED-CELLS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
1980; 77 (7): 3796-3800
A sensitive in situ assay for the plasminogen activator of transformed cells is described; it uses the fluorogenic molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-(N'-4-carboxylphenyl)thioureidospiro[isobenz ofuran-1(3H),9'-[9H]xanthen]-3-one. This fluorescein derivative is an excellent active-site titrant of the esterase activity of plasmin. When transformed cells are incubated with purified plasminogen and the resulting plasmin is titrated with the fluorogenic substrate, the amount of plasmin formed is linearly proportional to time and cell number. The assay is sensitive enough to detect quantitatively the plasminogen activator activity of as few as 250 transformed cells. This substrate should be useful in studying quantitatively the correlation between increased levels of plasminogen activator activity and cellular transformation and as a general active site titrant of serine proteases.
View details for Web of Science ID A1980KB88900008
View details for PubMedID 6449002
NEW ASSAY FOR THE PLASMINOGEN-ACTIVATOR ACTIVITY OF TRANSFORMED-CELLS
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
1979; 44: 669-680
View details for Web of Science ID A1979KQ93600070