Tolerogenic nanoparticles suppress central nervous system inflammation.

Therapeutic approaches for the induction of immune tolerance remain an unmet clinical need for the treatment of autoimmune diseases, including multiple sclerosis (MS). Based on its role in the control of the immune response, the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is a candidate target for novel immunotherapies. Here, we report the development of AhR-activating nanoliposomes (NLPs) to induce antigen-specific tolerance. NLPs loaded with the AhR agonist ITE and a T cell epitope from myelin oligodendrocyte glycoprotein (MOG)35-55 induced tolerogenic dendritic cells and suppressed the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, in preventive and therapeutic setups. EAE suppression was associated with the expansion of MOG35-55-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells), concomitant with a reduction in central nervous system-infiltrating effector T cells (Teff cells). Notably, NLPs induced bystander suppression in the EAE model established in C57BL/6 × SJL F1 mice. Moreover, NLPs ameliorated chronic progressive EAE in nonobese diabetic mice, a model which resembles some aspects of secondary progressive MS. In summary, these studies describe a platform for the therapeutic induction of antigen-specific tolerance in autoimmune diseases.

Genetic structure of the dopamine receptor D4 gene (DRD4) and lack of association with schizophrenia in Japanese patients.

In order to investigate the contribution of genetic variation in the human dopamine receptor D4 gene (DRD4) to the risk of developing schizophrenia, we carried out a genetic analysis of 27 polymorphisms in 216 schizophrenic patients and 243 healthy controls from the Kyushu region of Japan. Twenty-two single nucleotide polymorphisms (SNPs) and five insertion/deletion polymorphisms were analyzed in this study, including four novel SNPs and a novel mononucleotide repeat. Linkage disequilibrium (LD) and haplotype analyses reveal weak LD across the DRD4 gene. In univariate analysis female individuals with allele -521C had a higher risk for schizophrenia. However, this finding was not significant after correction for multiple hypothesis testing. No other polymorphisms or haplotypes differed between schizophrenic patients and controls. Likewise, multivariate analyses did not reveal any statistically significant associations.

Analysis and exploration of the use of rule-based algorithms and consensus methods for the inferral of haplotypes.

The difficulty of experimental determination of haplotypes from phase-unknown genotypes has stimulated the development of nonexperimental inferral methods. One well-known approach for a group of unrelated individuals involves using the trivially deducible haplotypes (those found in individuals with zero or one heterozygous sites) and a set of rules to infer the haplotypes underlying ambiguous genotypes (those with two or more heterozygous sites). Neither the manner in which this "rule-based" approach should be implemented nor the accuracy of this approach has been adequately assessed. We implemented eight variations of this approach that differed in how a reference list of haplotypes was derived and in the rules for the analysis of ambiguous genotypes. We assessed the accuracy of these variations by comparing predicted and experimentally determined haplotypes involving nine polymorphic sites in the human apolipoprotein E (APOE) locus. The eight variations resulted in substantial differences in the average number of correctly inferred haplotype pairs. More than one set of inferred haplotype pairs was found for each of the variations we analyzed, implying that the rule-based approach is not sufficient by itself for haplotype inferral, despite its appealing simplicity. Accordingly, we explored consensus methods in which multiple inferrals for a given ambiguous genotype are combined to generate a single inferral; we show that the set of these "consensus" inferrals for all ambiguous genotypes is more accurate than the typical single set of inferrals chosen at random. We also use a consensus prediction to divide ambiguous genotypes into those whose algorithmic inferral is certain or almost certain and those whose less certain inferral makes molecular inferral preferable.

Dihydropyrimidine dehydrogenase and thymidylate synthase polymorphisms and their association with 5-fluorouracil/leucovorin chemotherapy in colorectal cancer.

The causes of interpatient variation in severe toxicity resulting from treatment with weekly 5-fluorouracil (5-FU)/ leucovorin (LV) are poorly understood. This study was undertaken to examine the contribution of commonly occurring polymorphisms in the dihydropyrimidine dehydrogenase (DPYD) gene to interpatient variability in 5-FU pharmacokinetics and toxicity. Patients with stage III/IV colorectal cancer were treated by bolus intravenous (I.V.) injection with 500 mg/m2 doses of 5-FU and LV once every week. The pharmacokinetics of 5-FU was determined on weeks 1 and 4. Genotyping assays were developed for 8 polymorphisms in the DPYD gene. A well-characterized functional polymorphism in the 5' untranslated region of the thymidylate synthase (TS) gene was also analyzed. A cohort of 22 patients (15 male, 7 female) with a median age of 61 years was evaluated. Although there was no relationship between the area under the plasma concentration time curve (AUC) for the first dose of 5-FU and worst-grade toxicity during the first cycle of therapy, 3 of the 4 patients in whom the AUC on week 4 was more than equal to 5 microgram/h/mL greater than the value for the first dose experienced grade 3/4 toxicity during subsequent treatment. Among the 8 polymorphisms in the DPYD gene, 7 were found to vary in the study population but none were significantly associated with the AUC of 5-FU. There was no relationship between the DPYD and TS genotypes examined and 5-FU toxicity. Extensive polymorphism in the DPYD gene was observed; however, no conclusive correlations existed between the DPYD and TS genotype and 5-FU pharmacokinetics or toxicity. Decreases in 5-FU clearance in certain patients may provide insight into the increased toxicity following repetitive cycles of treatment with weekly I.V. bolus 5-FU. The present study offers useful themes for undertaking larger prospective pharmacogenetic studies in the future.

Pharmacogenetic study of statin therapy and cholesterol reduction.

CONTEXT: Polymorphisms in genes involved in cholesterol synthesis, absorption, and transport may affect statin efficacy. OBJECTIVE: To evaluate systematically whether genetic variation influences response to pravastatin therapy. DESIGN, SETTING, AND POPULATION: The DNA of 1536 individuals treated with pravastatin, 40 mg/d, was analyzed for 148 single-nucleotide polymorphisms (SNPs) within 10 candidate genes related to lipid metabolism. Variation within these genes was then examined for associations with changes in lipid levels observed with pravastatin therapy during a 24-week period. MAIN OUTCOME MEASURE: Changes in lipid levels in response to pravastatin therapy. RESULTS: Two common and tightly linked SNPs (linkage disequilibrium r2 = 0.90; heterozygote prevalence = 6.7% for both) were significantly associated with reduced efficacy of pravastatin therapy. Both of these SNPs were in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the target enzyme that is inhibited by pravastatin. For example, compared with individuals homozygous for the major allele of one of the SNPs, individuals with a single copy of the minor allele had a 22% smaller reduction in total cholesterol (-32.8 vs -42.0 mg/dL [-0.85 vs -1.09 mmol/L]; P =.001; absolute difference, 9.2 mg/dL [95% confidence interval [CI], 3.8-14.6 mg/dL]) and a 19% smaller reduction in low-density lipoprotein (LDL) cholesterol (-27.7 vs -34.1 mg/dL [-0.72 vs -0.88 mmol/L]; P =.005; absolute difference, 6.4 mg/dL [95% CI, 2.2-10.6 mg/dL]). The association for total cholesterol reduction persisted even after adjusting for multiple tests on all 33 SNPs evaluated in the HMG-CoA reductase gene as well as for all 148 SNPs evaluated was similar in magnitude and direction among men and women and was present in the ethnically diverse total cohort as well as in the majority subgroup of white participants. No association for either SNP was observed for the change in high-density lipoprotein (HDL) cholesterol (P>.80) and neither was associated with baseline lipid levels among those actively treated or among those who did not receive the drug. Among the remaining genes, less robust associations were found for squalene synthase and change in total cholesterol, apolipoprotein E and change in LDL cholesterol, and cholesteryl ester transfer protein and change in HDL cholesterol, although none of these met our conservative criteria for purely pharmacogenetic effects. CONCLUSION: Individuals heterozygous for a genetic variant in the HMG-CoA reductase gene may experience significantly smaller reductions in cholesterol when treated with pravastatin.

Association analysis of adenosine A1 receptor gene (ADORA1) polymorphisms with schizophrenia in a Japanese population.

OBJECTIVE: The human adenosine A1 receptor gene (ADORA1) localizes to chromosome 1q32 is 76.8 kbp in length and contains six exons. ADORA1 is ubiquitously expressed in the central nervous system and clinical and pharmacological evidence suggest the involvement of adenosine neurotransmission in the pathogenesis of schizophrenia. Therefore, we investigated the contribution of genetic variations of ADORA1 to the pathophysiological mechanisms of Japanese schizophrenia patients. METHODS: We performed genetic analysis of 29 polymorphic markers in 200 schizophrenic patients and 210 healthy controls from the Kyushu region of Japan. In statistical analysis, we performed the univariate analysis with genotypes and allele frequencies, linkage disequilibrium (LD) analyses, multivariate analysis, haplotype analysis, and sliding window haplotype analysis. RESULTS: In univariate analysis, no statistical difference was shown, after Bonferroni correction. By LD analysis, however, we could not find any LD blocks. In haplotype analysis, a total of 359 haplotypes were estimated. In multivariate analysis, we found three statistically different markers. In sliding window haplotype analysis, there were four statistically different haplotypes. CONCLUSION: This is the first study describing the involvement of ADORA1 polymorphisms in the pathophysiological mechanisms of schizophrenia in a Japanese population. These results corroborate our previous pharmacological and neurochemical studies in the rat that have suggested an association between ADORA1 neurotransmission and the schizophrenic effects of the N-methyl-D-aspartate receptor antagonist phencyclidine. Thus, ADORA1 polymorphisms may represent good candidate markers for schizophrenia research and ADORA1 may be involved in the pathophysiological mechanisms of schizophrenia in Japanese populations.

Sequence-specific dinucleotide cleavage promoted by synergistic interactions between neighboring modified nucleotides in DNA.

Sequence-specific cleavage of DNA by restriction endonucleases has been an indispensable tool in modern molecular biology. However, many potential applications are yet to be realized because of the limited number of naturally available restriction specificities. Efforts to expand this repertoire through protein engineering have met considerable challenges and only brought forth modest success. Taking an alternative approach, we developed a methodology to generate modified DNA susceptible to specific cleavage at selected dinucleotide sequences. This method requires the incorporation of two deoxyribonucleotide analogues by a DNA polymerase: a ribonucleotide and a 5'-amino-2',5'-dideoxyribonucleotide, each of which contains a different base. When linked in a 5' to 3' geometry, the two modified nucleotides act synergistically to promote cleavage at the phosphoramidate linkage, thus providing sequence specificity. Using the transferrin receptor gene as an example, we demonstrate that this dinucleotide cleavage generates discrete DNA fragments that can be either visualized by gel electrophoresis or detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A genotyping strategy based on incorporation and cleavage of chemically modified nucleotides.

Aiming to facilitate the analysis of human genetic variations in the context of disease susceptibility and varied drug response, we have developed a genotyping method that entails incorporation of a chemically labile nucleotide by PCR followed by specific chemical cleavage of the resulting amplicon at the modified bases. The identity of the cleaved fragments determines the genotype of the DNA. This method, termed Incorporation and Complete Chemical Cleavage, utilizes modified nucleotides 7-deaza-7-nitro-dATP, 7-deaza-7-nitro-dGTP, 5-hydroxy-dCTP, and 5-hydroxy-dUTP, which have increased chemical reactivity but are able to form standard Watson-Crick base pairs. Thus one analog is substituted for the corresponding nucleotide during PCR, generating amplicons that contain nucleotide analogs at each occurrence of the selected base throughout the target DNA except for the primer sequences. Subsequent treatment with an oxidant followed by an organic base results in chemical cleavage at each site of modification, which produces fragments of different lengths and/or molecular weights that reflect the genotype of the original DNA sample at the site of interest. This incorporation and cleavage chemistry are widely applicable to many existing nucleic acid analysis platforms, including gel electrophoresis and mass spectrometry. By combining DNA amplification and analog incorporation into one step, this strategy eliminates preamplification, DNA-strand separation, primer extension, and purification procedures associated with traditional chain-termination chemistry and therefore presents significant advantages in terms of speed, cost, and simplicity of genotyping.