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TREM2 is among the most well-known Alzheimer’s disease (AD) risk genes, however, the functional roles of its AD-associated variants remain to be elucidated, and most known risk alleles are low-frequency variants whose investigation is challenging. Here, we utilized a splicing-guided aggregation method in which multiple low-frequency TREM2 variants were bundled together to investigate the functional impact of those variants on alternative splicing in AD. We analyzed whole genome sequencing (WGS) and RNA-seq data generated from cognitively normal elderly controls (CN) and AD patients in two independent cohorts, representing three regions in the frontal lobe of the human brain: the dorsolateral prefrontal cortex (CN = 213 and AD = 376), frontal pole (CN = 72 and AD = 175), and inferior frontal (CN = 63 and AD = 157). We observed an exon skipping event in the second exon of TREM2, with that exon tending to be more frequently skipped (p = 0.0012) in individuals having at least one low-frequency variant that caused loss-of-function for a splicing regulatory element. In addition, genes differentially expressed between AD patients with high vs. low skipping of the second exon (i.e., loss of a TREM2 functional domain) were significantly enriched in immune-related pathways. Our splicing-guided aggregation method thus provides new insight into the regulation of alternative splicing of the second exon of TREM2 by low-frequency variants and could be a useful tool for further exploring the potential molecular mechanisms of multiple, disease-associated, low-frequency variants.

Background and objectives Dilated cardiomyopathy can be the end-stage of severe cardiac disorders and directly affects the cardiac muscle, inducing cardiomegaly and heart failure (HF). Although a wide variety of animal models are available to study dilated cardiomyopathy, there is no model to assess dilated cardiomyopathy with non-invasive, simple, and large screening methods. Methods We developed a dilated cardiomyopathy model in zebrafish larvae using short duration terfenadine, a known cardiotoxic drug that induces ventricular size dilation. Fractional shortening of zebrafish hearts was calculated. Results We treated zebrafish with 5 to 10 μM terfenadine for 24 hours. In terfenadine-treated zebrafish, blood frequently pooled and clotted in the chamber, and circulation was remarkably reduced. Atria and ventricles were swollen, and fluid was deposited around the heart, mimicking edema. Cardiac contractility was significantly reduced, and ventricular area was significantly enlarged. Heart rate was markedly reduced even after terfenadine withdrawal. Acridine orange staining also showed that terfenadine increased cardiomyocyte apoptosis. A significant increase of natriuretic peptide B (NPPB) mRNA was found in terfenadine-treated zebrafish. A low dose of terfenadine (5-10 μM) did not show mortality in short-term treatment (24 hours). However, moderate dose (35-45 μM) terfenadine treatment reduced zebrafish survival within 1 hour. Conclusion With advantages of rapid sample preparation procedure and transparent observation of the live heart, this model can potentially be applied to large-scale drug screening and toxicity assays for non-ischemic HF.

Zinc oxide (ZnO) nanoparticles (NPs) are used in diverse applications ranging from paints and cosmetics to biomedicine and food. Although micron-sized ZnO is a traditional food supplement, ZnO NPs are an unknown public health risk because of their unique physicochemical properties. Herein, we studied the 13-week subchronic toxicity of ZnO NPs administered via the oral route according to Organization for Economic Cooperation and Development (OECD) test guideline 408. Well-dispersed ZnO NPs were administered to Sprague–Dawley (SD) rats (11/sex/group) at doses of 67.1, 134.2, 268.4 or 536.8 mg kg–1 per body weight over a 13-week period. The mean body weight gain in males given 536.8 mg kg–1 ZnO NPs was significantly lower than that of control male rats, whereas no significant differences were observed between the other treatment groups and the controls. Male and female rats dosed at 536.8 mg kg–1 ZnO NPs had significant changes in anemia-related hematologic parameters. Mild to moderate pancreatitis also developed in both sexes dosed at 536.8 mg kg–1, whereas no histological changes were observed in the other treatment groups. To evaluate the mechanism of toxicity, we performed a bio-persistence study and evaluated the effects of the ZnO NPs on cell proliferation. The treatment of a human gastric adenocarcinoma cell line with ZnO NPs resulted in a significant inhibition of cellular proliferation. The anti-proliferative effect of ZnO NPs or Zn2+ was effectively blocked by treatment with chelators. These results indicate that the bio-persistence of ZnO NPs after ingestion is key to their toxicity; the no-observed-adverse effect level (NOAEL) of ZnO NPs was found to be 268.4 mg kg–1 per day for both sexes. Copyright © 2013 John Wiley & Sons, Ltd.

The development of novel alternative testing methods is required to identify the sensitizing capacity of chemicals as a replacement for animal experimentation. We aimed to evaluate in vitro assays as screening tools for detecting skin sensitizers. The murine epidermal keratinocyte cell line HEL-30 was exposed to 16 relevant skin sensitizers and 6 skin irritants. The dose causing 75% cell viability (CV(75)) measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was chosen as a highest dose and three more doses (0.5×, 0.1×, and 0.01× of CV(75)) were tested. As parameters, levels of interleukin 1α (IL-1α), macrophage inflammatory protein 2 (MIP-2), IL-6, and IL-18 production were measured using 4 different doses. The accuracy of detecting sensitizers or irritants by IL-1α or MIP-2 alone was exactly same: 75% (12 out of 16) for sensitizers, 83% (5 out of 6) for irritants, and overall 77% (17 out of 22). However, combination of IL-1α and MIP-2 showed better accuracy: 94% (15 out of 16), 67% (4 out of 6), and overall 86% (19 out of 22). IL-6 and IL-18 could not differentiate sensitizers from irritants. This study suggests that the combination of pro-inflammatory cytokines IL-1α and MIP-2 in murine HEL-30 cells can be a reliable in vitro method for identifying chemicals that may act as skin sensitizers.

In an effort to explore the use of alternative methods to animal testing for the evaluation of the ocular irritancy of medical devices, we evaluated representative contact lenses with the bovine corneal opacity and permeability test (BCOP) and an in vitro eye irritation test using the three-dimensionally-reconstructed human corneal epithelium (RhCE) models, EpiOcular™ and MCTT HCE™. In addition, we compared the obtained results with the ISO standard in vivo rabbit eye irritation test (ISO10993-10). Along with the positive controls (benzalkonium chloride, BAK, 0.02, 0.2, and 1%), the extracts of 4 representative contact lenses (soft, disposable, hard, and colored lenses) and 2 reference lenses (dye-eluting and BAK-coated lenses) were tested. All the lenses, except for the BAK-coated lens, were determined non-irritants in all test methods, while the positive controls yielded relevant results. More importantly, BCOP, EpiOcular™, and MCTT HCE™ yielded a consistent decision for all the tested samples, with the exception of 0.2% BAK in BCOP, for which no prediction could be made. Overall, all the in vitro tests correlated well with the in vivo rabbit eye irritation test, and furthermore, the combination of in vitro tests as a tiered testing strategy was able to produce results similar to those seen in vivo. These observations suggest that such methods can be used as alternative assays to replace the conventional in vivo test method in the evaluation of the ocular irritancy of ophthalmic medical devices, although further study is necessary.

Mycobacterium massiliense (M. mass), belonging to the M. abscessus complex, is a rapidly growing mycobacterium that is known to cause tuberculous-like lesions in humans. To better understand the interaction between host cells and M. mass, we used a recently developed in vitro model of early granuloma-like cell aggregates composed of human peripheral blood mononuclear cells (PBMCs). PBMCs formed granuloma-like, small and rounded cell aggregates when infected by live M. mass. Microscopic examination showed monocytes and macrophages surrounded by lymphocytes, which resembled cell aggregation induced by M. tuberculosis (M. tb). M. mass-infected PBMCs exhibited higher expression levels of HLA-DR, CD86 and CD80 on macrophages, and a significant decrease in the populations of CD4+ and CD8+ T cells. Interestingly, low doses of M. mass were sufficient to infect PBMCs, while active host cell death was gradually induced with highly increased bacterial loads, reflecting host destruction and dissemination of virulent rapid-growing mycobacteria (RGM). Collectively, this in vitro model of M. mass infection improves our understanding of the interplay of host immune cells with mycobacteria, and may be useful for developing therapeutics to control bacterial pathogenesis., Summary: An in vitro model of granuloma-like cell aggregates infected with Mycobacterium massiliense improves our understanding of the interplay of host immune cells with mycobacteria.

Background With high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs. Methods We injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer’s solution containing carboplatin and melphalan after the injection of retinoblastoma cells. Results The degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP. Conclusions This orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma.

Zinc oxide (ZnO) nanoparticles (NPs) are used in diverse applications ranging from paints and cosmetics to biomedicine and food. Although micron-sized ZnO is a traditional food supplement, ZnO NPs are an unknown public health risk because of their unique physicochemical properties. Herein, we studied the 13-week subchronic toxicity of ZnO NPs administered via the oral route according to Organization for Economic Cooperation and Development (OECD) test guideline 408. Well-dispersed ZnO NPs were administered to Sprague-Dawley (SD) rats (11/sex/group) at doses of 67.1, 134.2, 268.4 or 536.8 mg kg(-1) per body weight over a 13-week period. The mean body weight gain in males given 536.8 mg kg(-1) ZnO NPs was significantly lower than that of control male rats, whereas no significant differences were observed between the other treatment groups and the controls. Male and female rats dosed at 536.8 mg kg(-1) ZnO NPs had significant changes in anemia-related hematologic parameters. Mild to moderate pancreatitis also developed in both sexes dosed at 536.8 mg kg(-1) , whereas no histological changes were observed in the other treatment groups. To evaluate the mechanism of toxicity, we performed a bio-persistence study and evaluated the effects of the ZnO NPs on cell proliferation. The treatment of a human gastric adenocarcinoma cell line with ZnO NPs resulted in a significant inhibition of cellular proliferation. The anti-proliferative effect of ZnO NPs or Zn(2+) was effectively blocked by treatment with chelators. These results indicate that the bio-persistence of ZnO NPs after ingestion is key to their toxicity; the no-observed-adverse effect level (NOAEL) of ZnO NPs was found to be 268.4 mg kg(-1) per day for both sexes.

Spontaneous basal cell carcinoma (BCC) is very rare in rats, with an incidence rate of only 0.14% reported in aged animals. A spontaneous BCC occurred in a 7-week-old Sprague-Dawley rat housed in a specific-pathogen-free animal facility. The tumor was a single, well-delineated reddish-brown subcutaneous mass measuring 2 × 2 cm and located in the left inguinal region. Microscopically, the tumor consisted of basaloid cells in lobular and cribriform growth patterns and with a high mitotic rate. Immunohistochemically, cytokeratin 14 (an indicator for basal keratinocytes of the epidermis) showed strong reactions throughout the whole tumor, and cytokeratin 18 showed weak but positive reaction in the majority of nested tumor cells. To the authors' knowledge, this is the first report of spontaneous BCC occurrence in young Sprague-Dawley rats.

Chemotherapy is often futile in systemic listeriosis, translating to being a peril to public health. There is, thus, an imperative need for novel antilisterial compounds, possibly acting through mechanisms dissimilar to those of existing drugs. The present study describes one such agent-the non-steroidal anti-inflammatory drug (NSAID) diclofenac sodium (Dc). The National Committee for Clinical Laboratory Standards (NCCLS) minimum inhibitory concentration (MIC), mode of action, and two mechanisms of action, i.e., on bacterial DNA and membrane, have been characterized with respect to Dc. The drug showed noteworthy inhibitory action (MIC90 = 50 microg/ml) against Listeria strains, demonstrated cidal (minimum bactericidal concentration [MBC]=100 microg/ml) activity, inhibited listerial DNA synthesis (45.48%; incorporation of [methyl-3H] thymidine), and possessed bacterial membrane-damaging activity (37.33%; BacLight assay). Dc could be used as a lead compound for the synthesis of new, more active agents perhaps devoid of side effects. Further, quantitative structure-activity relationship (QSAR) studies will contribute to a new generation of promising adjuvants to existing antilisterial drugs.

Cyp1A1 transcription is induced by polycyclic aromatic hydrocarbons (PAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC), through the AHR pathway. Ligand-activated aryl hydrocarbon receptor (AHR) interacts with aryl hydrocarbon receptor nuclear translocator (ARNT), and this heterodimeric complex binds to aryl hydrocarbon response elements (AHRE) in the regulatory region of the Cyp1A1 gene, thereby inducing Cyp1A1 transcription. In hypoxic conditions, accumulated hypoxia-induced factor-1α (HIF-1α) interacts with ARNT, and this dimer binds to the regulatory region of these target genes and activates their expression. Because ARNT plays a critical role in both the AHR- and HIF-1α-mediated pathways, activation of HIF-1α may sequester ARNT, thus inhibiting the AHR-mediated pathway. To determine whether the 3MC-induced AHR pathway can be inhibited by the hypoxia-inducing agent, cobalt chloride, fertilized zebrafish embryos, and embryos (F 4 ) of stable transgenic zebrafish containing the EGFP reporter gene driven by AHRE were treated with vehicle (0.1% DMSO) or 3MC (60 μM). Immediately, 3 MC-treated embryos were transferred to media in either normoxic or hypoxic conditions induced by cobalt chloride. 3MC-induced Cyp1A1 mRNA expression was significantly inhibited by cobalt chloride treatment. 3MC-induced EGFP expression in the liver and head area of stable transgenic zebrafish embryos was reduced by cobalt chloride treatment. In this study, we have shown that the hypoxic condition induced by cobalt chloride inhibited 3MC-induced Cyp1A1 transcription in zebrafish embryos. This might be due to the sequestering of ARNT by HIF-1α or another mechanism.

Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1 beta and TNF-alpha in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1 beta expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis.

Increased fat intake is known to be a major cause of prostate cancer. In this study, we investigated the effect of dietary high fat on prostate intraepithelial neoplasia using transgenic adenocarcinoma mouse prostate (TRAMP) mice. Six-week-old male TRAMP mice were fed AIN93G (control group, 4.0 kcal/kg, n=6) and AIN93G-HFD (experimental group, 4.8 kcal/kg, n=7) for 10 weeks. Prostate histopathology, urogenital tract (UGT) weight, epididymal white adipose tissue weight, argyrophilic nucleolar organizer regions (AgNORs) counts, and serum leptin levels were examined. AIN93G-HFD fed group showed progressed neoplastic lesions in the prostate (P

The Draize eye irritation test has been changed to alternative methods based on 3 Rs (Replacement, Reduction, Refinement) of animal experiments. The bovine corneal opacity and permeability (BCOP) assay is well established as one of the alternative methods to the Draize test but the supply of bovine eyes from a slaughterhouse is not sufficient in actuality. The porcine corneal opacity and permeability(PCOP) assay have been proposed as a reliable alternative to the BCOP assay and the Draize test because porcine corneas have numerous advantages including structural similarities with human corneas and the stable capacity for a corneal supply. In this study, three cosmetics and seven chemicals that are already known as irritants at Draize score and the bovine corneal opacity(BCO) assay were tested by the PCOP assay for the first time in Korea. The porcine corneal opacity was measured by quantifying the ability of light to pass through a cornea using an opacitometer. The permeability was measured at 10 and 30 min time points after chemical exposure. The PCOP assay showed the high correlation with the Draize score and BCO assay. These results showed that the PCOP assay can be used as an alternative method for the BCO assay and the Draize test although it needs further studies about insoluble chemicals and others to increase reliability.