Mikio Aoki

Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28.

Although embryonic stem (ES) cell–like induced pluripotent stem (iPS) cells have potential therapeutic applications in humans, they are also useful for creating genetically modified human disease models in nonhuman primates. In this study, we generated common marmoset iPS cells from fetal liver cells via the retrovirus‐mediated introduction of six human transcription factors: Oct‐3/4, Sox2, Klf4, c‐Myc, Nanog, and Lin28. Four to five weeks after introduction, several colonies resembling marmoset ES cells were observed and picked for further expansion in ES cell medium. Eight cell lines were established, and validation analyses of the marmoset iPS cells followed. We detected the expression of ES cell–specific surface markers. Reverse transcription‐PCR showed that these iPS cells expressed endogenous Oct‐3/4, Sox2, Klf4, c‐Myc, Nanog and Lin28 genes, whereas all of the transgenes were silenced. Karyotype analysis showed that two of three iPS cell lines retained a normal karyotype after a 2‐month culture. Both embryoid body and teratoma formation showed that marmoset iPS cells had the developmental potential to give rise to differentiated derivatives of all three primary germ layers. In summary, we generated marmoset iPS cells via the transduction of six transcription factors; this provides a powerful preclinical model for studies in regenerative medicine.

Prediction of siRNA functionality using generalized string kernel and support vector machine

Small interfering RNAs (siRNAs) are becoming widely used for sequence‐specific gene silencing in mammalian cells, but designing an effective siRNA is still a challenging task. In this study, we developed an algorithm for predicting siRNA functionality by using generalized string kernel (GSK) combined with support vector machine (SVM). With GSK, siRNA sequences were represented as vectors in a multi‐dimensional feature space according to the numbers of subsequences in each siRNA, and subsequently classified with SVM into effective or ineffective siRNAs. We applied this algorithm to published siRNAs, and could classify effective and ineffective siRNAs with 90.6%, 86.2% accuracy, respectively.

Amyloid β-peptide levels in laser capture microdissected cornu ammonis 1 pyramidal neurons of Alzheimerʼs brain

Deposition of the amyloid &bgr;-peptide (A&bgr;) is a pathophysiological event associated with Alzheimers disease. Although much is known about the molecular composition of extracellular A&bgr; deposits, the role of the intracellular pool of A&bgr; is not fully understood. We investigated whether A&bgr; levels are increased in cornu ammonis 1 pyramidal neurons of Alzheimers disease hippocampus, using laser capture microdissection to isolate the neurons and enzyme-linked immunosorbent assay for quantification. Our results showed increased A&bgr;42 levels and an elevated A&bgr;42/A&bgr;40 ratio in neurons from sporadic as well as from familial cases of Alzheimers disease, whereas A&bgr;40 levels remain unchanged between the cases and controls. We speculate that intracellular accumulation of A&bgr;42 increase vulnerability of cornu ammonis 1 pyramidal neurons in Alzheimers disease.

Effects of DMSO on gene expression in human and rat hepatocytes

Dimethyl sulfoxide (DMSO) is a very common organic solvent used for dissolving lipophilic substances, for example for in vitro cell-based assays. At the same time, DMSO is known to be cytotoxic at high concentrations. Therefore, it is important to define threshold concentrations of DMSO for cells but relevant data at the molecular level are very limited. We have focused on conducting microarray analyses of human and rat hepatocytes treated with more than 100 chemicals in attempts to identify candidate biomarker genes. In the present study, the effects of DMSO on gene expression and cytotoxicity were assessed in human cryopreserved hepatocytes and rat primary cultured hepatocytes. A cytotoxicity test with lactate dehydrogenase (LDH) activity demonstrated DMSO to be noncytotoxic up to a concentration of 2% (v/v) in both cases and there were only few effects on the gene expression profiles up to 0.5% (v/v). The observed differences from controls were considered to be of little toxicological importance, but still need to be taken into account in interpretation of findings when DMSO is used at high concentration.

Biological characterization of a novel class of toll‐like receptor 7 agonists designed to have reduced systemic activity

BACKGROUND AND PURPOSE Toll‐like receptor 7 (TLR7) agonists have potential in the treatment of allergic diseases. However, the therapeutic utility of current low molecular weight TLR7 agonists is limited by their systemic activity, resulting in unwanted side effects. We have developed a series of TLR7‐selective ‘antedrugs’, including SM‐324405 and AZ12441970, which contain an ester group rapidly cleaved in plasma to reduce systemic exposure.

The Application of Mass Spectrometry to Proteomics and Metabolomics in Biomarker Discovery and Drug Development

Drugs are launched to market after the lengthy process of development. Despite careful preclinical assessment, there is still a significant risk that a drug candidate may elicit adverse effects or display a low level of efficacy during clinical trials. If a drug candidate fails in the latter stages of the clinical process, the overall loss, both in terms of time and money, is enormous. A major concern for the pharmaceutical companies is to improve the drug development process to make it faster and more cost-effective by adoption of new technologies. Biomarkers are emerging as a key tool in identifying potential drug failures at an early stage or in helping to make go/no-go decisions, which should significantly accelerate drug development. Omics technologies play an important role in biomarker discovery as well as in other stages of the drug discovery and development (e.g. target discovery, mechanism of action or predicting toxicity). In particular, recent progress in mass spectrometry techniques such as selected reaction monitoring (SRM) and novel high-resolution features have helped facilitate the realization of the inherent power of proteomics and metabolomics in biomarker discovery, validation and qualification. In this manuscript, we review the current state of proteomics and metabolomics in conjunction with recent technical advances in mass spectrometry with some examples of applications in biomarker research. In addition, we discuss the possible impact of biomarker research with these technologies in drug discovery and development.

Identification of a novel set of biomarkers for evaluating phospholipidosis-inducing potential of compounds using rat liver microarray data measured 24-h after single dose administration

Phospholipid accumulation manifests as an adverse effect of cationic amphiphilic drugs in particular. Detection, however, by histopathology examination is time-consuming and may require repeated administration of compounds for several weeks. To eliminate compounds with potential for inducing phospholipidosis from the discovery pipeline, we have identified and validated a set of biomarkers for predicting the phospholipidosis-inducing potential utilizing a comprehensive rat transcriptome microarray database created by the Japanese Toxicogenomics and Toxicogenomics Informatics Projects (TGP/TGP2) together with in-house data. The set of biomarkers comprising 25 Affymetrix GeneChip probe sets was identified using genetic algorithm optimization on 24-h time-point microarray data from rats treated with single doses of hepatotoxic compounds including amiodarone, clomipramine, haloperidol, hydroxyzine, imipramine, and perhexiline. The set of novel biomarkers represents an early time-point gene-expression pattern characteristic for a condition eventually leading to phospholipidosis. This implies significant advantages in terms of time and resources over currently published biomarkers derived using repeated-dosing late time-point data. The biomarker set was validated by 11 independent compounds. Accuracy, sensitivity, and specificity values were 82%, 67%, and 100%, respectively and the area under the receiver operating characteristic curve was 0.97. These results show that the biomarker set possesses a high classification accuracy for novel compounds. Pathway analysis was carried out for the biomarkers and the detection of pathways related to lipid-metabolism was statistically significant. These pathways most probably reflect lipid metabolism changes associated with phospholipidosis supporting the validity of our novel biomarkers.

A simple scoring system using type IV collagen 7S and aspartate aminotransferase for diagnosing nonalcoholic steatohepatitis and related fibrosis

BackgroundRecently we reported novel noninvasive scoring systems for diagnosing nonalcoholic steatohepatitis (NASH) and related fibrosis, namely FM-NASH index and FM-fibro index. They are highly accurate, however, they contain some items not widely used in clinical practice and require six or more items to diagnose both NASH and related fibrosis. By focusing on widely used items, we tried to identify convenient markers in common with the both diagnoses.MethodsTo explore the markers for NASH and related fibrosis in nonalcoholic fatty liver disease (NAFLD) patients, we used data of 24 clinical items in our previous report. By logistic regression analysis, we identified items suitable for the both diagnoses. We then evaluated their accuracies by area under the receiver operator characteristic curves (AUROCs) on independent validation data.ResultsWe identified the combination of type IV collagen 7S and aspartate aminotransferase (AST) as the predictor both for NASH and related fibrosis. We developed a scoring system based on the combination and evaluated the prediction accuracy: the AUROCs for training/validation data sets are 0.857/0.769 for NASH and 0.918/0.842 for NASH-related fibrosis. The former was higher than that of NAFIC score, and the latter was higher than those of existing fibrosis markers: BARD score, FIB-4 index and NAFLD fibrosis score but lower than FM-fibro index.ConclusionsThe scoring system using type IV collagen 7S and AST named CA index can predict both NASH and related fibrosis in NAFLD patients with sufficient accuracy and could be a convenient diagnostic and screening tool for NASH and related fibrosis. The scoring system needs to be validated in independent larger populations from multiple clinical centers.

Basic NeuroscienceShort communicationA novel approach for Aβ1–40 quantification using immuno-PCR

Several lines of evidence suggest that aggregation of the amyloid β-peptide (Aβ) in the brain is a trigger of Alzheimers disease (AD). Thus, quantification of Aβ in several different types of samples from brain is fundamental for understanding AD pathogenesis. For analysis of the low levels of Aβ present in microdissected neurons, a more sensitive system than the ELISAs used today would be helpful. Here, we report a novel immuno-PCR (IPCR) system in which the lowest quantitative level of Aβ₁₋₄₀ is 2 attomol/μL. We use the novel IPCR to quantify the intracellular Aβ₁₋₄₀ levels in pyramidal neurons microdissected from human brain. We show that the level of Aβ₁₋₄₀ is around 10 attomol/neuron, and thus, only 3 neurons are needed for analysis.

Abstract 2571: Sustained induction of TRAIL and granzyme B as well as intratumor infiltration of cytotoxic T lymphocytes (CTL) by a novel TLR7 agonist, DSR-6434, after systemic administration

Although immediate responses after treatment of TLR7 agonist have been extensively characterized the genes that are directly responsible for the antitumor activity of TLR7 agonists have not been well identified. Moreover, the immediate responses to TLR7 agonist stimulation, such as induction of interferon-alpha, IP-10 and IL-1RA, generally diminish rapidly, and therefore, cannot be used as optimal pharmacodynamic (PD) biomarkers to monitor longer term PD effects. This study was undertaken to investigate the antitumor mechanism of TLR7 agonists and to identify novel PD biomarkers. To identify PD biomarkers, mice were inoculated with mouse renal carcinoma cell line, Renca, and were administered intravenously with DSR-6434 at 0.1 mg/kg once a week, 3 times. Total RNA samples were collected 2 hours or 5 days after final dose from tumor or blood. Global gene expression profiles in tumor or blood were analyzed using GeneChip Mouse Genome 430 2.0 array (Affymetrix). Differentially expressed genes by DSR-6434 were selected compared to vehicle, and pathway analysis was performed using the genes. Expression of TRAIL and granzyme B and its time course after the administration was investigated in peripheral blood leukocytes. In tumor of mouse model, 312 and 53 genes were upregulated and downregulated, respectively, more than 2 fold at 2 hours after DSR-6434 administration. The upregulated genes included those related to immune cell activation and apoptosis pathways. The upregulated immune-related genes included T-cell marker genes (Cd3g, Cd3e), marker genes of cytotoxic T-cells (CTL) (Cd8a), lymphocyte activation (Cd69), dendritic cells activation (Cd83 and Cd86) and cytotoxic factors against tumor (TRAIL and granzyme B). These results suggested that DSR-6434 induced infiltration and activation of CTL and production of cytotoxic factors at tumor site. At 5 days after the administration of TLR7 agonist DSR-6434, 4 genes, including TRAIL, remained upregulated in tumor. Surprisingly, upregulation of TRAIL and granzyme B in blood sustained for 3 or 6 days, respectively. In conclusion, unbiased gene expression profiling has revealed that TLR7 agonist DSR-6434 triggers CD8+ cells infiltration into tumor and sustained increase of TRAIL and granzyme B in blood for at least 3 days, thus offering novel PD biomarkers that could potentially predict antitumor activity of TLR7 agonists. Citation Format: Ryosaku Inagaki, Mikio Aoki, Toru Kimura, Yuko Hirose, Hiroki Umehara, Erina Koga, Masashi Murata, Robert W. Wilkinson, David T. Robinson, Philip J. Jewsbury, Chiang J. Li. Sustained induction of TRAIL and granzyme B as well as intratumor infiltration of cytotoxic T lymphocytes (CTL) by a novel TLR7 agonist, DSR-6434, after systemic administration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2571. doi:10.1158/1538-7445.AM2014-2571