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Class of 2017 Abstract / Objectives: To determine the cost-effectiveness of proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors with high-intensity statins compared to high-intensity statins alone for the treatment of heterozygous familial hypercholesterolemia (HeFH). Methods: A Markov model was built through TreeAge Pro to model two groups: patients taking PCSK9 inhibitors with high-intensity statins or high-intensity statins alone. For each group, there were five health states that patients could be in: well, unstable angina, myocardial infarction, ischemic stroke, or death. The data used in the model were extracted from published clinical trials evaluating PCSK9 inhibitors and statins. Results: For the primary analysis, the overall cost and effectiveness was $31,390.93 and 23.01 for the statin alone group and $362,798.50 and 24.32 for the PCSK9 with statin group, respectively. The incremental cost, incremental QALY, and incremental cost-effectiveness ratio (ICER) was $331,407.60, 1.31 QALYs, and $252,833.60/QALY, respectively. Conclusions: Since the calculated ICER was higher than the pre-established threshold of $150,000, the results from our primary analysis suggest that treatment of patients with HeFH with a PCSK9 inhibitor and a high-intensity statin is not cost effective, compared to treatment with a high-intensity statin alone. However, when certain parameters (cost of PSCK9 and mortality rate) were adjusted in the secondary analyses, these agents appear to be cost-effective.

In the past ten years, synthetic cannabinoids (SC) have emerged as drugs of abuse. Unlike D9-tetrahydrocannabinol (THC), many SCs are associated with serious health complications and death. One way in which THC and SCs differ lies with their enhanced potency and efficacy at the CB1 receptor. No current methods exist to measure efficacy at the CB1 receptor in vivo, and the abuse-related properties of SC cannabinoids are not well explored. Here, we utilized CB1 wild type (WT), heterozygous (HET), and knockout (KO) mice. By employing CB1 ligands which differ in efficacy we have developed a method to explore the relationship between efficacy and the ability to produce cannabimimetic (catalepsy, hypothermia, and antinociception) effects when CB1 expression was reduced by half. Additionally, the intracranial self-stimulation procedure (ICSS) was utilized to investigate the effects of enhanced efficacy at CB1 upon reward processes using representative SC CP55,940. As predicted, the potency shift between WT and HET mice inversely correlated with the efficacy of the test drug for both hypothermia and antinociception, but not catalepsy. This efficacy stratification was correlated with the agonist-stimulated [35S]GTPgS binding assay, demonstrating this model as an effective tool to ascertain in vivo efficacy differences at CB1. In ICSS, CP55,940 elicited only rate-decreasing effects acutely, although tolerance developed following repeated dosing, with no evidence for spontaneous or rimonabant-precipitated withdrawal. Together, these data indicate that highly efficacious cannabinoid ligands require few receptors to produce cannabimimetic effects, and that the model provides an effective means to quickly ascertain differences in efficacy.

Dehydropyrrolizidine alkaloids (DHPAs) are a large group of globally important plant-derived pro-toxins that can contaminate or are naturally present in animal feed and the human food supply as well as herbal supplements. Their bioactive metabolites are potentially hepatotoxic, pneumotoxic, genotoxic and carcinogenic. Due to the difficulty in obtaining sufficient quantities of purified DHPAs, toxicity studies have largely relied on single intraperitoneal injections in rodent models, and carcinogenicity studies have been limited to a small handful of the hundreds of isolated DHPAs. To assess the relative toxicity of structurally diverse DHPAs in a more biologically relevant manner, male California White chicks were dosed orally with 0.01, 0.04, 0.13, or 0.26 mmol of seven different DHPAs and three DHPA N-oxides kg-1 bodyweight for 7 days. DHPAs were grouped in relation to their toxicity based on clinical, serum biochemical, and histopathological evaluations as well as tissue adduct accumulation rates. Using the same model, a reduced extract from comfrey, a commonly used DHPA containing herb, was compared to its two major constituent DHPAs, intermedine and lycopsamine. Based on the same parameters, the comfrey extract was more toxic than pure lycopsamine or intermedine. Addressing the need for a more sensitive carcinogenicity model, male heterozygous p53 knockout mice were treated with riddelliine 5, 15 or 45 mg kg–1 bodyweight day-1 by oral gavage for 14 days, or given a long-term treatment of riddelliine 1 mg kg-1 bodyweight day–1 in pelleted feed for 12 months. Exposure to riddelliine increased the odds of tumor development in a dose-responsive manner (odds ratio 2.05 and Wald 95% confidence limits between 1.2 and 3.4). The most common neoplasm was hepatic hemangiosarcoma, which is consistent with previously published lifetime rodent studies. The results of this research demonstrate that the California White chick model is sensitive for comparison of DHPA toxicity, and data obtained from this research can be used to validate previous DHPA toxicity research. It also demonstrates that comfrey toxicity may have been previously underestimated. The heterozygous p53 knockout mouse model is beneficial for further investigation of comparative carcinogenesis of structurally and toxicologically different DHPAs and their N-oxides.

High-throughput DNA sequencing technologies are currently revolutionizing the fields of biology and medicine by elucidating the structure and function of the components of life. Modern DNA sequencing machines typically produce relatively short reads of DNA which are then assembled by software in an attempt to produce a representation of the entire genome. Due to the complex structure of all but the smallest genomes, especially the abundant presence of exact or almost exact repeats, all genome assemblers introduce errors into the final sequence and output a relatively large set of contigs instead of full-length chromosomes (a contig is a DNA sequence built from the overlaps between many reads). These problems are dramatically worse when homologous copies of the same chromosome differ substantially. Currently such genomes are usually avoided as assembly targets and, when they are not avoided, they generally produce assemblies of relatively low quality. An improved algorithm for the assembly of such data would dramatically improve our understanding of the genetics of a large class of organisms. We present a unique algorithm for the assembly of diploid genomes which have a high degree of variation between homologous chromosomes. The approach uses coverage, graph patterns and machine-learning classification to identify haplotype-specific sequences in the input reads. It then uses these haplotype-specific markers to guide an improved assembly. We validate the approach with a large experiment that isolates and elucidates the effect of single nucleotide polymorphisms (SNPs) on genome assembly more clearly than any previous study. The experiment conclusively demonstrates that the Bioluminescence heterozygous genome assembler produces dramatically longer contigs with fewer haplotype-switch errors than competing algorithms under conditions of high heterozygosity.

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21253号 / 医博第4371号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 浅野 雅秀, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Leptin and its receptor are essential for regulating food intake, energy expenditure, glucose homeostasis and fertility. Mutations within leptin or the leptin receptor cause early-onset obesity and hyperphagia, as described in human and animal models. The effect of both heterozygous and homozygous variants is much more investigated than compound heterozygous ones. Recently, we discovered a spontaneous compound heterozygous mutation within the leptin receptor, resulting in a considerably more obese phenotype than described for the homozygous leptin receptor deficient mice. Accordingly, we focus on compound heterozygous mutations of the leptin receptor and their effects on health, as well as possible therapy options in human and animal models in this review.

Heterozygous mutations in the glucocerebrosidase (GBA) gene represent the most common risk factor for Parkinson’s disease (PD), a disease in which midbrain dopaminergic neurons are preferentially vulnerable. However, the mechanisms underlying this association are still unknown, mostly due to the lack of an appropriate model of study. In this thesis, we aimed at elucidating the role of heterozygous GBA mutations in PD using a specific human induced pluripotent stem cell (hiPSC)-based model of disease. First we developed a protocol for the efficient differentiation of hiPSCs into dopaminergic cultures, and extensively characterized the derived dopaminergic neurons which expressed multiple midbrain relevant markers and produced dopamine. Next we screened a clinical cohort of PD patients to identify carriers of GBA mutations of interest. Using for the first time hiPSCs generated from PD patients heterozygous for a GBA mutation (together with idiopathic cases and control individuals) we were able to efficiently derive dopaminergic cultures and identify relevant disease mechanisms. Upon differentiation into dopaminergic neuronal cultures, we observed retention of mutant glucocerebrosidase (GCase) protein in the endoplasmic reticulum (ER) with no change in protein levels, leading to upregulation of ER stress machinery and resulting in increased autophagic demand. At the lysosomal level, we found a reduction of GCase activity in dopaminergic neuronal cultures, and the enlargement of the lysosomal compartment in identified dopaminergic neurons suggesting a decreased capacity for protein clearance. Together, these perturbations of cellular homeostasis resulted in increased release of α-synuclein and could likely represent critical early cellular phenotypes of Parkinson's disease and explain the high risk of heterozygous GBA mutations for PD.

Au cours de ce travail, nous avons utilisé des souris ayant une délétion totale (KO) ou partielle (hétérozygotes) du gène codant la protéine STOP. La souris KO STOP constitue un modèle reconnu pour l’étude de la physiopathologie de la schizophrénie (SCH). Ce travail a permis d’étendre les données disponibles en mettant en évidence chez la souris KO STOP des troubles comportementaux qui miment les symptômes apparentés à l’ensemble des dimensions de la SCH. La comparaison des déficits chez des souris KO STOP générées à partir de différentes souches a permis de montrer que la délétion du gène STOP induit un phénotype robuste. De plus, notre travail permet de suggérer que, de par leur construction et leur phénotype, la souris hétérozygote STOP évoque la vulnérabilité à la SCH et pourrait constituer un modèle animal pertinent pour étudier les facteurs qui, interagissant avec une vulnérabilité génétique, favoriserait la décompensation psychotique. Dans cette optique, nous avons étudié l’influence de différents types de stress chroniques appliqués à différents moments clés du développement sur le phénotype de nos modèles animaux. Aucun des stress utilisés (isolement, stress chronique multiple, privation maternelle) n’a modifié le phénotype des souris hétérozygotes STOP à l’âge adulte, ni dans le sens de l’émergence ni dans celui d’une aggravation de troubles apparentés à la symptomatologie de la SCH. Ces résultats posent la question du type de stress, de son intensité et de la fenêtre temporelle où il doit être appliqué qui, associé à une altération génique donnée, validerait le modèle physiopathologique actuel basé sur une interaction délétère entre vulnérabilité et stress / In this work, we used mice with total (STOP null) or partial deletion (heterozygous) for the gene encoding the STOP protein. STOP null mice represent a recognized model for studying the physiopathology of schizophrenia (SCH). This work allowed us to extend the available phenotype by showing that STOP null mice exhibit various behavioural impairments mimicking symptoms corresponding to all SCH dimensions. By comparing the alterations present in STOP null mice generated from various strains, we showed that the STOP deletion induces a robust phenotype linked to SCH. In addition, through their construct and phenotype validity, the present work allows us to suggest that the STOP heterozygous mice evoke SCH vulnerability and could be a relevant model for studying the parameters which could favour SCH when interacting with a genetic vulnerability. In this context, the effects of chronic stress applied at different key steps of the mouse development were studied on the phenotype of our animal models. Any stress paradigm used (short-term isolation, chronic multiple stress, maternal deprivation) has been not able to alter the phenotype of STOP heterozygous mice measured in adulthood, neither by inducing nor by worsening alterations linked to SCH. According to the current SCH physiopathology hypothesis based on a deleterious interaction between susceptibility to SCH and stress, our findings raise the question regarding the type of stress paradigm, including stress intensity and the time window where it is applied, that could induce frank psychosis when interacting with a specific genetic alteration