RESUMEN
With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs.
Asunto(s)
Síndrome de QT Prolongado/inducido químicamente , Miocitos Cardíacos/efectos de los fármacos , Arritmias Cardíacas/inducido químicamente , Cardiotónicos/toxicidad , Electrodos , Expresión Génica , Guías como Asunto , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Activación del Canal Iónico/genética , Japón , Contracción Miocárdica/genética , Miocitos Cardíacos/fisiologíaRESUMEN
In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of drug-induced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9- to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia.
Asunto(s)
Arritmias Cardíacas/inducido químicamente , Diferenciación Celular , Canal de Potasio ERG1/antagonistas & inhibidores , Frecuencia Cardíaca/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Bloqueadores de los Canales de Potasio/toxicidad , Pruebas de Toxicidad/instrumentación , Potenciales de Acción , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Bioensayo , Cardiotoxicidad , Técnicas de Cultivo de Célula , Células Cultivadas , Relación Dosis-Respuesta a Droga , Canal de Potasio ERG1/metabolismo , Diseño de Equipo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Japón , Miocitos Cardíacos/metabolismo , Observación , Reproducibilidad de los Resultados , Medición de Riesgo , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad/normasRESUMEN
BACKGROUND: In Crohn's disease (CD), the involvement of food antigens in immune responses remains unclear. The objective of this study was to detect immune responses against food antigens in CD patients and examine the mechanism in a mouse model of colitis. METHODS: We enrolled 98 CD patients, 50 ulcerative colitis patients, and 52 healthy controls (HCs) to compare the levels of serum immunoglobulin (Ig)Gs against 88 foods. The presence of serum IgGs against foods was also examined in interleukin (IL)-10 knockout (KO) mice in which CD4(+) T cell activation by antigenic food protein was assessed. Mice transferred with IL-10 KO cells received diets with or without food antigens, and the development of colitis was evaluated. RESULTS: The prevalence of IgGs against various foods, especially vegetables, grains, and nuts, was significantly higher in CD patients than in HCs. Similarly, the prevalence of IgGs against food proteins was higher in IL-10 KO mice than in BALB/c mice. Beta-conglycinin, identified as an antigenic food proteins in IL-10 KO mice, induced CD4(+) T cell production of interferon-γ and IL-17 through dendritic cell antigen presentation. Elimination of the food antigens ameliorated the development of colitis in mice without altering the composition of their intestinal microbiota. CONCLUSIONS: In CD colitis mice, intestinal inflammation via CD4(+) T cell hyperactivation was induced by food antigens associated with high serum IgG levels and was ameliorated by the elimination of food antigens. This disrupted immunological tolerance to food antigen, which might act as an exacerbating factor, remains to be elucidated in CD patients.
Asunto(s)
Colitis/inmunología , Enfermedad de Crohn/inmunología , Hipersensibilidad a los Alimentos/inmunología , Adolescente , Adulto , Anciano , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Estudios de Casos y Controles , Células Cultivadas , Colitis/complicaciones , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/complicaciones , Modelos Animales de Enfermedad , Femenino , Hipersensibilidad a los Alimentos/complicaciones , Humanos , Inmunoglobulina G/sangre , Interleucina-10/deficiencia , Interleucina-10/genética , Activación de Linfocitos/inmunología , Masculino , Ratones Endogámicos BALB C , Ratones Noqueados , Persona de Mediana Edad , Adulto JovenRESUMEN
Skin surface bacteria contribute to body odor, especially axillary odor. We aimed to investigate anaerobic bacteria that had not been previously studied for axillary odor formation. A new anaerobic Anaerococcus sp. A20, that releases 3-hydroxy-3-metyl-hexanoic acid (HMHA, main component of axillary odor) from its glutamyl conjugate, was discovered from axillary isolates. This strain showed strong resistance to the antimicrobial agents, triclosan and 4-isopropyl-3-methylphenol; therefore, we screened plant extracts that inhibit the A20 strain. We discovered that pentagalloyl glucose (PGG) extracted from the Chinese Gall plant exhibited both antibacterial and inhibitory activities against HMHA release by the A20 strain. As the excellent antibacterial activity and inhibitory effect of PGG against HMHA release were seen in vitro, we conducted an open study to evaluate the deodorant effects of PGG on axillary odor. The sensory tests on odor strength showed that application of the PGG solution could reduce axillary odors in vivo. Although there was a small change in axillary microbiota, the microbial count of A20 significantly reduced. These results strongly indicate PGG as a new innovative deodorant material that only affects odor-releasing bacteria in the axillary microbiota.
Asunto(s)
Antibacterianos/farmacología , Axila/microbiología , Bacterias Grampositivas/efectos de los fármacos , Taninos Hidrolizables/farmacología , Piel/microbiología , Caproatos/metabolismo , Cresoles/farmacología , Farmacorresistencia Bacteriana , Femenino , Genes Bacterianos , Bacterias Grampositivas/genética , Bacterias Grampositivas/aislamiento & purificación , Bacterias Grampositivas/metabolismo , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Tipificación Molecular , Odorantes , Filogenia , ARN Ribosómico 16S/genética , Sudor/microbiología , Triclosán/farmacologíaRESUMEN
Previous work has demonstrated that intestinal bacteria, such as Fusobacterium varium (F. varium), contribute to the clinical activity in ulcerative colitis (UC); thus, an antibiotic combination therapy (amoxicillin, tetracycline, and metronidazole (ATM)) against F. varium can induce and maintain UC remission. Therefore, we investigated whether ATM therapy induces a long-term alteration of intestinal microbiota in patients with UC. Patients with UC were enrolled in a multicenter, randomized, double-blind, placebo-controlled study. Biopsy samples at the beginning of the trial and again at 3 months after treatment completion were randomly obtained from 20 patients. The terminal restriction fragment length polymorphism (T-RFLP) in mucosa-associated bacterial components was examined to assess the alteration of the intestinal microbiota. Profile changes of T-RFLP in mucosa-associated bacterial components were found in 10 of 12 patients in the treatment group and in none of 8 in the placebo group. Dice similarity coefficients using the unweighted pair group method with arithmetic averages (Dice-UPGMA) confirmed that the similarity of mucosal microbiota from the descending colon was significantly decreased after the ATM therapy, and this change was maintained for at least 3 months. Moreover, at 3 months after treatment completion, the F. varium/ß-actin ratio, examined by real-time PCR using nested PCR products from biopsy samples, was reduced less than 40% in 8 of 12 treated patients, which was higher, but not significantly, than in 4 of 8 patients in the placebo group. Together, these results suggest that ATM therapy induces long-term alterations in the intestinal microbiota of patients with UC, which may be associated, at least in part, with clinical effects of the therapy.
Asunto(s)
Colitis Ulcerosa/microbiología , Infecciones por Fusobacterium/microbiología , Fusobacterium/genética , Mucosa Intestinal/microbiología , Microbiota/genética , Actinas/metabolismo , Adolescente , Adulto , Anciano , Amoxicilina/uso terapéutico , Antibacterianos/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Método Doble Ciego , Quimioterapia Combinada , Femenino , Fusobacterium/aislamiento & purificación , Infecciones por Fusobacterium/tratamiento farmacológico , Humanos , Mucosa Intestinal/efectos de los fármacos , Masculino , Metronidazol/uso terapéutico , Persona de Mediana Edad , Tetraciclina/uso terapéuticoRESUMEN
BACKGROUND: Intestinal microbiota contributes to the pathogenesis of Crohn's disease. Elemental diet and total parenteral nutrition are effective therapies for Crohn's disease; however, changes of microbiota as a result of both treatments have not been fully elucidated. AIM: To elucidate changes of faecal microbiota in Crohn's disease patients treated with elemental diet and total parenteral nutrition. METHODS: Stool samples were collected from 33 active Crohn's disease patients and 17 healthy subjects, and recollected after elemental diet (8 patients) and total parenteral nutrition (9 patients). Terminal restriction fragment length polymorphism analysis of bacterial 16srDNA was performed to evaluate the whole microbiota. Specific quantitative PCR was then used to determine populations of predominant bacterial groups. RESULTS: In Crohn's disease patients, the number of terminal restriction fragments, which reflects bacterial species, was significantly lower. Populations of total bacteria and Bifidobacterium were significantly lower and the ratio of Enterococcus was higher. The number of terminal restriction fragments was significantly decreased after total parenteral nutrition, but not after elemental diet. Population of Bacteroides fragilis significantly decreased after elemental diet, while population of Enterococcus significantly increased after total parenteral nutrition. CONCLUSION: Faecal microbiota in Crohn's disease patients was markedly different from healthy subjects. Species diversity was reduced by total parenteral nutrition, but not by elemental diet.
Asunto(s)
Enfermedad de Crohn/microbiología , Enfermedad de Crohn/terapia , Heces/microbiología , Alimentos Formulados , Nutrición Parenteral Total , Adolescente , Adulto , Anciano , Bacteroides fragilis/genética , Bifidobacterium/genética , Estudios de Casos y Controles , Clostridium/genética , Enfermedad de Crohn/dietoterapia , ADN Bacteriano/análisis , Enterococcus/genética , Femenino , Humanos , Masculino , Metagenoma/genética , Persona de Mediana Edad , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
Rapid enumeration of Escherichia coli strains by quantitative real-time PCR targeting the uidA gene was developed and confirmed for minced beef, tuna and raw oyster. Higher sensitivity (1 CFU/g of E. coli in all three food samples) was obtained by incubating for 7 h in TSB. Colony-directed E. coli specific TaqMan PCR assay could effectively distinguish colonies grown on various selective media within 1.5-h. Inspection of E. coli in food testing laboratories is important, and our rapid E. coli detection strategy will contribute to quality control in food industries.
Asunto(s)
Recuento de Colonia Microbiana/métodos , Escherichia coli/aislamiento & purificación , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa/métodos , Animales , ADN Bacteriano/genética , Proteínas de Escherichia coli/genética , Humanos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Ammocidins B, C and D were isolated from the culture broth of Saccharothrix sp. AJ9571, an ammocidin A-producing strain. Their structures were determined by a detailed spectroscopic analysis and by a comparison of their NMR data with those of ammocidin A. Ammocidins A and B showed potent anti-proliferative activities against human cancer cell lines.