Skin fibrotic disorders are understood to develop under the influence of various cytokines, such as transforming growth factor(TGF)-beta1, connective tissue growth factor(CTGF) and vascular endothelial growth factor (VEGF). To establish an appropriate animal model of skin fibrosis by exogenous application of growth factors, the author investigated the in vivo effects of growth factors by injecting recombinant TGF-beta1 protein and pCMV- Flag5-CTGF into the subcutaneous tissue of Sprague- Dawley rats. A single application of TGF-beta1 protein and CTGF DNA resulted in the formation of transient granulation tissue. Immunohistochemical finding showed increased expression of TGF-beta1 protein after injection of pCMV-Flag5-CTGF. In situ hybridization analysis revealed the expression of CTGF mRNA after injection of TGF-beta1 protein. VEGF expression was not affected by the TGF-beta1 and CTGF injection. These findings suggest TGF-beta1 and CTGF are deeply related with skin fibrosis and it appears that TGF-beta1 may cause the induction of CTGF expression. The animal model on skin fibrosis by exogenous application of TGF-beta1 protein and CTGF DNA developed in this study may be useful for future studies on fibrotic disorders.
Animals ; Cicatrix* ; Connective Tissue ; Connective Tissue Growth Factor ; Cytokines ; DNA ; Fibrosis ; Granulation Tissue ; In Situ Hybridization ; Intercellular Signaling Peptides and Proteins ; Models, Animal ; Rats* ; RNA, Messenger ; Skin ; Subcutaneous Tissue ; Transforming Growth Factor beta1* ; Vascular Endothelial Growth Factor A*

Ochratoxin A is a natural contaminant of mouldy food and feed, which is produced by Penicillium and Aspergillus, and is suspected of being one of the etiological agents responsible for Balkan endemic nephropathy and the associated urinary tract tumors. For evaluation of the mutagenicity of ochratoxin A, we performed in vitro chromosome aberration tests using Chinese hamster lung fibroblast cells (CHL cells) and monkey kidney cells (VERO cells), in vivo micronueleus tests using ddY mouse bone marrow cells and somatic mutation and recombination tests (SMART) using Drosophila melanogaster. The results of chromosome aberration tests in CHL cells showed no incidence of increased structural and numerical aberrations regardless of metabolic activation, while in VERO cells treated with 2.0, 1.0, 0.5, 0.3 ug/ml of ochratoxin A showed significant increase of structural aberrations without metabolic activation. Aspartame and-phenylalanine, structural analogs of ochratoxin A, didn't affect the chromosome aberrations induced by ochratoxin A. The in vivo induction of micronucleated polychromatic erythrocytes were measured in bone marrows of ddY mice treated with 10.0, 5.0, 2.5mg/kg/10ml of ochratoxin A through intraperitoneal route once. At 24 and 48 hours after treatment, ochratoxin A didn't induce micronuclei in bone marrows of ddY mice. And at the concentration of 40, 20, 10 ug/ml of ochratoxin A, which was administered by feeding to larvae of Drosophila melanogaster, showed no incidence of increased multiple wing hairs and flares. Summarizing all results, we concluded that ochratoxin A is a kidney cell specific direct genotoxicant.
Animals ; Asian Continental Ancestry Group* ; Aspartame ; Aspergillus ; Balkan Nephropathy ; Biotransformation ; Bone Marrow ; Bone Marrow Cells ; Chromosome Aberrations ; Cricetinae ; Cricetulus* ; Drosophila melanogaster* ; Drosophila* ; Erythrocytes ; Fibroblasts ; Hair ; Haplorhini ; Humans ; Incidence ; Kidney ; Larva ; Lung* ; Mice* ; Penicillium ; Recombination, Genetic ; Urinary Tract ; Vero Cells*

PURPOSE: Chemotherapies for breast cancer generally have strong cellular cytotoxicity and severe side effects. Thus, significant emphasis has been placed on combinations of naturally occurring chemopreventive agents. Silibinin is a major bioactive flavonolignan extracted from milk thistle with chemopreventive activity in various organs including the skin, prostate, and breast. However, the mechanism underlying the inhibitory action of silibinin in breast cancer has not been completely elucidated. Therefore, we investigated the effect of silibinin in MCF-7 human breast cancer cells and determined whether silibinin enhances ultraviolet (UV) B-induced apoptosis. METHODS: The effects of silibinin on MCF-7 cell viability were determined using the MTT assay. The effect of silibinin on PARP cleavage, as the hallmark of apoptotic cell death, and p53 protein expression in MCF-7 cells was analyzed using Western blot. The effect of silibinin on UVB-induced apoptosis in MCF-7 cells was analyzed by flow cytometry. RESULTS: A dose- and time-dependent reduction in viability was observed in MCF-7 cells treated with silibinin. Silibinin strongly induced apoptotic cell death in MCF-7 cells, and induction of apoptosis was associated with increased p53 expression. Moreover, silibinin enhanced UVB-induced apoptosis in MCF-7 cells. CONCLUSION: Silibinin induced a loss of cell viability and apoptotic cell death in MCF-7 cells. Furthermore, the combination of silibinin and UVB resulted in an additive effect on apoptosis in MCF-7 cells. These results suggest that silibinin might be an important supplemental agent for treating patients with breast cancer.
Apoptosis ; Blotting, Western ; Breast ; Breast Neoplasms ; Cell Death ; Cell Survival ; Humans ; MCF-7 Cells ; Milk Thistle ; Prostate ; Silymarin ; Skin

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their inter-assay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.
Citric Acid* ; Colitis ; Cytochrome P-450 CYP1A2 ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; Drug Discovery ; Enzyme Induction* ; Fireflies ; Hepatocytes ; Humans* ; Isoenzymes ; Liver ; Luciferases ; Luminescence ; Luminescent Measurements* ; Mass Screening ; Mesalamine* ; Metabolism ; RNA, Messenger

PURPOSE: Transglutaminase type 2 (TG2) is an extracellular matrix crosslinking enzyme with a pivotal role in kidney fibrosis. We tested whether quantification of urinary TG2 may represent a noninvasive method to estimate the severity of kidney allograft fibrosis. METHODS: We prospectively collected urine specimens from 18 deceased donor kidney transplant recipients at 1-day, 7-day, 1-month, 3-month, and 6-month posttransplant. In addition, kidney allograft tissue specimens at 0-day and 6-month posttransplant were sampled to analyze the correlation of urinary TG2 and kidney allograft fibrosis. RESULTS: Thirteen recipients had increased interstitial fibrosis and tubular atrophy (IFTA) scores at the 6-month protocol biopsy (IFTA group). The mean level of urinary TG2 in the IFTA group was higher compared to that of 5 other recipients without IFTA (no IFTA group). Conversely, the mean level of urinary syndecan-4 in the IFTA group was lower than levels in patients without IFTA. In the IFTA group, double immunofluorescent staining revealed that TG2 intensity was significantly upregulated and colocalizations of TG2/heparin sulfate proteoglycan and nuclear syndecan-4 were prominent, usually around tubular structures. CONCLUSION: Urinary TG2 in early posttransplant periods is a potent biomarker for kidney allograft inflammation or fibrosis.
Allografts ; Atrophy ; Biomarkers ; Biopsy ; Extracellular Matrix ; Fibrosis ; Humans ; Inflammation ; Kidney Transplantation ; Kidney ; Methods ; Prospective Studies ; Proteoglycans ; Syndecan-4 ; Tissue Donors ; Transplant Recipients

PURPOSE: Population studies have reported that sensitization to inhalant allergens is rare in young children; however, most subjects in those studies had little or no symptoms or signs highly suggestive of allergic diseases. The aim of the present study was to assess the prevalence of sensitization to inhalant allergens in young children with symptoms and/or signs of allergic disease. METHODS: We analyzed the results of all specific IgE tests performed at our hospital laboratory in children younger than 6 years presenting with symptoms and/or signs highly suggestive of allergic diseases between 2008 and 2013. Specific IgE tests for Dermatophagoides pteronyssinus, Dermatophagoides farinae, Alternaria alternata, German cockroach, cat dander, egg white or egg yolk, milk, peanut, and soybean were performed on 295 children; a specific IgE concentration > or =0.35 or > or =0.2 IU/mL was considered positive. We also compared allergen sensitization rates using the two cutoff values. RESULTS: One hundred eighty-one children (61.4%) were positive to at least 1 allergen tested and 53 children (18.9%) were positive to at least 1 inhalant allergen when a specific IgE concentration > or =0.35 IU/mL was considered positive. The children were more likely to have asthma or allergic rhinitis when they were sensitized to any inhalant allergen, particularly house dust mites. The prevalence of sensitization to inhalant allergens increased with age (P<0.001). There was no significant difference in the prevalence of polysensitization among different age groups, but sensitization to both inhalant and food allergens significantly increased with age. CONCLUSION: Our results suggest that specific IgE tests to common inhalant allergens, particularly the house dust mites, may be considered when performing blood screening tests for young children presenting with symptoms and/or signs of allergic diseases.
Allergens* ; Alternaria ; Animals ; Asthma ; Blattellidae ; Cats ; Child* ; Dander ; Dermatophagoides farinae ; Dermatophagoides pteronyssinus ; Egg White ; Egg Yolk ; Humans ; Immunoglobulin E ; Laboratories, Hospital ; Mass Screening ; Milk ; Prevalence ; Pyroglyphidae ; Rhinitis ; Soybeans

Sibutramine is an anorectic that has been banned since 2010 due to cardiovascular safety issues. However, counterfeit drugs or slimming products that include sibutramine are still available in the market. It has been reported that illegal sibutramine-contained pharmaceutical products induce cardiovascular crisis. However, the mechanism underlying sibutramine-induced cardiovascular adverse effect has not been fully evaluated yet. In this study, we performed cardiovascular safety pharmacology studies of sibutramine systemically using by hERG channel inhibition, action potential duration, and telemetry assays. Sibutramine inhibited hERG channel current of HEK293 cells with an IC50 of 3.92 muM in patch clamp assay and increased the heart rate and blood pressure (76 Deltabpm in heart rate and 51 DeltammHg in blood pressure) in beagle dogs at a dose of 30 mg/kg (per oral), while it shortened action potential duration (at 10 muM and 30 muM, resulted in 15% and 29% decreases in APD50, and 9% and 17% decreases in APD90, respectively) in the Purkinje fibers of rabbits and had no effects on the QTc interval in beagle dogs. These results suggest that sibutramine has a considerable adverse effect on the cardiovascular system and may contribute to accurate drug safety regulation.
Action Potentials ; Animals ; Blood Pressure ; Cardiovascular System ; Counterfeit Drugs ; Dogs ; Heart Rate ; HEK293 Cells ; Inhibitory Concentration 50 ; Pharmaceutical Preparations ; Pharmacology* ; Purkinje Fibers ; Rabbits ; Telemetry

PURPOSE: Childhood asthma has a considerable social impact and economic burden, especially in severe asthma. This study aimed to identify the proportion of childhood asthma severity and to evaluate associated factors for greater asthma severity. METHODS: This study was performed on 667 children aged 5–15 years with asthma from the nationwide 19 hospitals in the Korean childhood Asthma Study (KAS). Asthma was classified as mild intermittent, mild persistent, and moderate/severe persistent groups according to the National Asthma Education and Prevention Program recommendations. Multinomial logistic regression models were used to identify the associated factors for greater asthma severity. RESULTS: Mild persistent asthma was most prevalent (39.0%), followed by mild intermittent (37.6%), moderate persistent (22.8%), and severe persistent asthma (0.6%). Onset later than 6 years of age (adjusted odds ratio [aOR], 1.69 for mild persistent asthma; aOR, 1.92 for moderate/severe persistent asthma) tended to increase asthma severity. Exposure to environmental tobacco smoke (aOR, 1.53 for mild persistent asthma; aOR, 1.85 for moderate/severe persistent asthma), and current dog ownership with sensitization to dog dander (aOR, 5.86 for mild persistent asthma; aOR, 6.90 for moderate/severe persistent asthma) showed increasing trends with greater asthma severity. Lower maternal education levels (aOR, 2.32) and no usage of an air purifier in exposure to high levels of outdoor air pollution (aOR, 1.76) were associated with moderate/severe persistent asthma. CONCLUSIONS: Modification of identified environmental factors associated with greater asthma severity might help better control childhood asthma, thereby reducing the disease burden due to childhood asthma.
Air Filters ; Air Pollution ; Animals ; Asthma ; Child ; Dander ; Dogs ; Education ; Environmental Exposure ; Humans ; Logistic Models ; Odds Ratio ; Ownership ; Risk Factors ; Smoke ; Social Change ; Tobacco