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Although recent advances in genome editing technology with homology-directed repair have enabled the insertion of various reporter genes into the genome of mammalian cells, the efficiency is still low due to the random insertion of donor vectors into the host genome. To efficiently select knocked-in cells without random insertion, we developed the "double-tk donor vector system," in which the expression units of the thymidine kinase of herpes simplex virus (HSV-tk) are placed on both outer sides of homology arms. This system is superior in enriching knocked-in human induced pluripotent stem cells (hiPSCs) than conventional donor vector systems with a single or no HSV-tk cassette. Using this system, we efficiently generated fluorescent reporter knockin hiPSCs targeting POU5F1 (OCT3/4), EEF1A1, H2BC21 (H2B clustered histone 21), ISL1, and MYH7 genes. These results indicate that the double-tk donor vector system enables efficient selection of knocked-in hiPSCs carrying reporter proteins.
Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Simplexvirus , Edição de Genes , Genes Homeobox , MamíferosRESUMO
Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.
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Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/efeitos adversos , Monofenol Mono-Oxigenase/genética , Hepatopatia Gordurosa não Alcoólica/etiologia , Mutação Puntual , Albinismo Oculocutâneo/complicações , Albinismo Oculocutâneo/enzimologia , Albinismo Oculocutâneo/genética , Animais , Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Lipoproteínas/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genéticaRESUMO
ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.
Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Proteínas Luminescentes , Proteína Vermelha FluorescenteRESUMO
Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro. Analysis of iRFP-expressing cells transplanted subcutaneously into NOG, nude, and ICR mice suggests that innate immunity was involved in the exclusion of the transplanted cells. We also evaluated the feasibility of this novel SeVdp vector for hemophilia A gene therapy. This system enabled insertion of full-length FVIII genes, and transduced cells secreted FVIII into the culture medium. Transient FVIII activity was detected in the plasma of mice after intraperitoneal transplantation of these FVIII-secreting cells. Further improvement in methods to evade immunity, such as simultaneous expression of immunomodulatory genes, would make this novel vector a very useful tool in regenerative medicine.
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Terapia Genética , Vetores Genéticos/genética , Hemofilia A/genética , Hemofilia A/terapia , Vírus Sendai/genética , Animais , Testes de Coagulação Sanguínea , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Modelos Animais de Doenças , Fator VIII/genética , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Knockout , Transdução Genética , TransgenesRESUMO
Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , ZigotoRESUMO
Exogenous gene expression is a fundamental and indispensable technique for testing gene function in neurons. Several ways to express exogenous genes in neurons are available, but each method has pros and cons. The lentivirus vector is useful for high efficiency gene transfer to neurons and stabilizes gene expression via genome integration, but this integration may destroy the host genome. The Epstein-Barr virus (EBV)-derived vector (EB vector) is an accessible and useful vector in human cell lines because the vector is not integrated into the host genome but stays in the nucleus as an episome. However, there has been no report on this process in rodent neurons. We examined the usefulness of the EB vector for testing gene function in neurons. We found that EB vector-derived exogenous proteins such as green fluorescent protein (GFP) and GFP-tagged actin were easily detectable even after three weeks of transfection. Second, a tetracycline-induced gene expression system in the EB vector was active after three weeks of transfection, indicating that plasmids were retained in neurons for up to three weeks. Third, we determined that only Family of repeat element of the plasmid vector is essential for its long-term presence in neurons. These results show that the modified EB vector is a useful tool for examining gene function in neurons.
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Uncoupling protein 1 (UCP1) is a mitochondrial protein that is expressed in both brown and beige adipocytes. UCP1 uncouples the mitochondrial electron transport chain from ATP synthesis to produce heat via non-shivering thermogenesis. Due to their ability to dissipate energy as heat and ameliorate metabolic disorders, UCP1-expressing adipocytes are considered as a potential target for anti-obesity treatment. To monitor the expression of UCP1 in live mice in a non-invasive manner, we generated the Ucp1-iRFP720 knock-in (Ucp1-iRFP720 KI) mice, in which the gene encoding a near-infrared fluorescent protein iRFP720 is inserted into the Ucp1 gene locus. Using the heterozygous Ucp1-iRFP720 KI mice, we observed robust iRFP fluorescence in the interscapular region where brown adipose tissue is located. Moreover, the iRFP fluorescence was clearly observable in inguinal white adipose tissues in live mice administered with ß3-adrenergic receptor agonist CL316,243. We also found that the homozygous Ucp1-iRFP720 KI mice, which are deficient in UCP1, displayed prominent iRFP fluorescence in the inguinal regions at the standard housing temperature. Consistent with this, the mice exhibited expanded populations of beige-like adipocytes in inguinal white adipose tissue, in which the Ucp1 promoter was dramatically activated. Thus, the Ucp1-iRFP720 KI mice provide a convenient model for non-invasive in vivo imaging of UCP1 expression in both brown and beige adipocytes in live mice.
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Expressão Gênica , Proteínas Luminescentes/genética , Imagem Molecular , Proteína Desacopladora 1/genética , Adipócitos Bege/metabolismo , Animais , Linhagem Celular , Marcação de Genes , Loci Gênicos , Genótipo , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Imagem Molecular/métodos , Espectroscopia de Luz Próxima ao Infravermelho , Proteína Desacopladora 1/metabolismo , Proteína Vermelha FluorescenteRESUMO
PURPOSE: Large inter-individual differences in warfarin maintenance dose are mostly due to the effect of genetic polymorphisms in multiple genes, including vitamin K epoxide reductase complex 1 (VKORC1), cytochromes P450 2C9 (CYP2C9), and cytochrome P450 4F2 (CYP4F2). Thus, several algorithms for predicting the warfarin dose based on pharmacogenomics data with clinical characteristics have been proposed. Although these algorithms consider these genetic polymorphisms, the formulas have different coefficient values that are critical in this context. In this study, we assessed the mutual validity among these algorithms by specifically considering racial differences. METHODS: Clinical data including actual warfarin dose (AWD) of 125 Japanese patients from our previous study (Eur J Clin Pharmacol 65(11):1097-1103, 2009) were used as registered data that provided patient characteristics, including age, sex, height, weight, and concomitant medications, as well as the genotypes of CYP2C9 and VKORC1. Genotyping for CYP4F2*3 was performed by the PCR method. Five algorithms that included these factors were selected from peer-reviewed articles. The selection covered four populations, Japanese, Chinese, Caucasian, and African-American, and the International Warfarin Pharmacogenetics Consortium (IWPC). RESULTS: For each algorithm, we calculated individual warfarin doses for 125 subjects and statistically evaluated its performance. The algorithm from the IWPC had the statistically highest correlation with the AWD. Importantly, the calculated warfarin dose (CWD) using the algorithm from African-Americans was less correlated with the AWD as compared to those using the other algorithms. The integration of CYP4F2 data into the algorithm did not improve the prediction accuracy. CONCLUSION: The racial difference is a critical factor for warfarin dose predictions based on pharmacogenomics.
Assuntos
Algoritmos , Anticoagulantes/administração & dosagem , Povo Asiático/genética , Citocromo P-450 CYP2C9/genética , Família 4 do Citocromo P450/genética , Vitamina K Epóxido Redutases/genética , Varfarina/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , FarmacogenéticaRESUMO
INTRODUCTION: To promote antimicrobial stewardship activity, an understanding of the incidence of antibiotic-associated adverse drug events (ADEs) is essential. In this study, we aimed to describe the occurrence of antibiotic-associated ADEs at our hospital. METHODS: We retrospectively searched the ADE registration system in Osaka University Hospital between 2010 and 2017. Registrations of ADEs were dependent on the patients' drug history and clinical course after hospitalization. We classified the data according to types of ADEs (gastrointestinal, hepatobiliary, renal, cardiac, respiratory, hematologic, neurologic, dermatologic, and musculoskeletal) and antibiotic class. RESULTS: During the study period, we found 707 cases of antibiotic-associated ADEs, accounting for 22.3% of all the cases. Beta-lactam antibiotics constitute more than half of the cases (51.3%). The most common ADE was dermatologic abnormalities (53.4%), followed by liver dysfunction (9.7%) and gastrointestinal symptoms (8.9%). Among all antibiotics, oral third-generation cephalosporins were frequently reported as offending drugs (107 cases), accounting for 29.5% of beta-lactam ADEs and 46.3% of cephem ADEs. CONCLUSION: Antibiotic-associated ADEs covered approximately 20% of all the ADEs at our hospital. We believe that the data would be helpful in ensuring patient safety by promoting antimicrobial stewardship in hospitals.
Assuntos
Sistemas de Notificação de Reações Adversas a Medicamentos/estatística & dados numéricos , Antibacterianos/efeitos adversos , Gestão de Antimicrobianos/estatística & dados numéricos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Hospitais/estatística & dados numéricos , Hospitais de Ensino , Humanos , Incidência , Japão/epidemiologia , Estudos RetrospectivosRESUMO
The gut microbiota has been causally linked to cancer, yet how intestinal microbes influence progression of extramucosal tumors is poorly understood. Here we provide evidence implying that Prevotella heparinolytica promotes the differentiation of Th17 cells colonizing the gut and migrating to the bone marrow (BM) of transgenic Vk*MYC mice, where they favor progression of multiple myeloma (MM). Lack of IL-17 in Vk*MYC mice, or disturbance of their microbiome delayed MM appearance. Similarly, in smoldering MM patients, higher levels of BM IL-17 predicted faster disease progression. IL-17 induced STAT3 phosphorylation in murine plasma cells, and activated eosinophils. Treatment of Vk*MYC mice with antibodies blocking IL-17, IL-17RA, and IL-5 reduced BM accumulation of Th17 cells and eosinophils and delayed disease progression. Thus, in Vk*MYC mice, commensal bacteria appear to unleash a paracrine signaling network between adaptive and innate immunity that accelerates progression to MM, and can be targeted by already available therapies.
Assuntos
Eosinófilos/imunologia , Microbioma Gastrointestinal/imunologia , Interleucina-17/imunologia , Mieloma Múltiplo/imunologia , Células Th17/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Progressão da Doença , Eosinófilos/metabolismo , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Prevotella/imunologia , Células Th17/metabolismoRESUMO
By using near-infrared fluorescent protein (iRFP)-expressing hematopoietic cells, we established a novel, quantitative, in vivo, noninvasive atherosclerosis imaging system. This murine atherosclerosis imaging approach targets macrophages expressing iRFP in plaques. Low-density lipoprotein receptor-deficient (LDLR-/-) mice transplanted with beta-actin promoter-derived iRFP transgenic (TG) mouse bone marrow (BM) cells (iRFP â LDLR-/-) were used. Atherosclerosis was induced by a nonfluorescent 1.25% cholesterol diet (HCD). Atherosclerosis was compared among the three differently induced mouse groups. iRFP â LDLR-/- mice fed a normal diet (ND) and LDLR-/- mice transplanted with wild-type (WT) BM cells were used as controls. The in vivo imaging system (IVIS) detected an enhanced iRFP signal in the thoracic aorta of HCD-fed iRFP â LDLR-/- mice, whereas iRFP signals were not observed in the control mice. Time-course imaging showed a gradual increase in the signal area, which was correlated with atherosclerotic plaque progression. Oil red O (ORO) staining of aortas and histological analysis of plaques confirmed that the detected signal was strictly emitted from plaque-positive areas of the aorta. Our new murine atherosclerosis imaging system can noninvasively image atherosclerotic plaques in the aorta and generate longitudinal data, validating the ability of the system to monitor lesion progression.
Assuntos
Doenças da Aorta/diagnóstico por imagem , Aterosclerose/diagnóstico por imagem , Medições Luminescentes/métodos , Proteínas Luminescentes/análise , Imagem Óptica/métodos , Placa Aterosclerótica/diagnóstico por imagem , Actinas/genética , Animais , Doenças da Aorta/genética , Aterosclerose/etiologia , Aterosclerose/genética , Compostos Azo , Transplante de Medula Óssea , Colesterol na Dieta/toxicidade , Corantes , Citometria de Fluxo , Genes Reporter , Genes Sintéticos , Proteínas Luminescentes/genética , Macrófagos Peritoneais/química , Macrófagos Peritoneais/ultraestrutura , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Receptores de LDL/deficiência , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaRESUMO
The acquisition of resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) is one of the major problems in the pharmacotherapy against non-small cell lung cancers; however, molecular mechanisms remain to be fully elucidated. Here, using a newly-established erlotinib-resistant cell line, PC9/ER, from PC9 lung cancer cells, we demonstrated that the expression of translation-related molecules, including eukaryotic translation initiation factor 3 subunit C (eIF3c), was upregulated in PC9/ER cells by proteome analyses. Immunoblot analyses confirmed that eIF3c protein increased in PC9/ER cells, compared with PC9 cells. Importantly, the knockdown of eIF3c with its siRNAs enhanced the drug sensitivity in PC9/ER cells. Mechanistically, we found that LC3B-II was upregulated in PC9/ER cells, while downregulated by the knockdown of eIF3c. Consistently, the overexpression of eIF3c increased the number of autophagosomes, proposing the causality between eIF3c expression and autophagy. Moreover, chloroquine, an autophagy inhibitor, restored the sensitivity to erlotinib. Finally, immunohistochemical analyses of biopsy samples showed that the frequency of eIF3c-positive cases was higher in the patients with EGFR-TKI resistance than those prior to EGFR-TKI treatment. Moreover, the eIF3c-positive cases exhibited poor prognosis in EGFR-TKI treatment. Collectively, the upregulation of eIF3c could impair the sensitivity to EGFR-TKI as a novel mechanism of the drug resistance.
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Sulforaphane (SFN) plays an important role in preventing oxidative stress by activating the nuclear factor (erythroid derived 2)-like 2 (Nrf2) signalling pathway. SFN may improve exercise endurance capacity by counteracting oxidative stress-induced damage during exercise. We assessed running ability based on an exhaustive treadmill test (progressive-continuous all-out) and examined the expression of markers for oxidative stress and muscle damage. Twelve- to 13-week-old Male wild-type mice (Nrf2 +/+) and Nrf2-null mice (Nrf2 -/-) on C57BL/6J background were intraperitoneally injected with SFN or vehicle prior to the test. The running distance of SFN-injected Nrf2 +/+ mice was significantly greater compared with that of uninjected mice. Enhanced running capacity was accompanied by upregulation of Nrf2 signalling and downstream genes. Marker of oxidative stress in SFN-injected Nrf2 +/+ mice were lower than those in uninjected mice following the test. SFN produced greater protection against muscle damage during exhaustive exercise conditions in Nrf2 +/+ mice than in Nrf2 -/- mice. SFN-induced Nrf2 upregulation, and its antioxidative effects, might play critical roles in attenuating muscle fatigue via reduction of oxidative stress caused by exhaustive exercise. This in turn leads to enhanced exercise endurance capacity. These results provide new insights into SFN-induced upregulation of Nrf2 and its role in improving exercise performance.
Assuntos
Músculo Esquelético/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Resistência Física , Adenosina Trifosfatases/metabolismo , Animais , Metabolismo Energético/efeitos dos fármacos , Glutationa/metabolismo , Isotiocianatos/farmacologia , Luciferases/metabolismo , Luminescência , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Músculo Esquelético/efeitos dos fármacos , Biogênese de Organelas , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Condicionamento Físico Animal , Sulfóxidos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The in vivo imaging of mice makes it possible to analyze disease progress non-invasively through reporter gene expression. As the removal of hair improves the accuracy of in vivo imaging, gene-modified mice with a reporter gene are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous Hrhr mutation that exhibit a hair loss phenotype. However, it is time consuming to produce mice carrying both the reporter gene and mutant Hrhr gene by mating. In addition, there is a risk that genetic background of the gene-modified mice would be altered by mating. To resolve these issues, we established a simple method to generate hairless mice maintaining the original genetic background by CRISPR technology. First, we constructed the pX330 vector, which targets exon 3 of Hr. This DNA vector (5 ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced Hr gene mutations were found in many founders (76.1%) and these mutations were heritable. Next, we performed in vivo imaging using these gene-modified hairless mice. As expected, luminescent objects in their body were detected by in vivo imaging. This study clearly showed that hairless mice could be simply generated by the CRISPR/Cas9 system, and this method may be useful for in vivo imaging studies with various gene-modified mice.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Diagnóstico por Imagem/métodos , Camundongos Pelados/genética , Terapia de Substituição Mitocondrial/métodos , Mutação , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , DNA/genética , Genes Reporter/genética , Vetores Genéticos , Camundongos Endogâmicos C57BL , Microinjeções , FenótipoRESUMO
Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.
Assuntos
Carcinoma Pulmonar de Lewis/diagnóstico por imagem , Células Endoteliais/fisiologia , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Fisiológica , Proteínas Recombinantes de Fusão/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Fluorescência , Luciferases/genética , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia Confocal , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas Recombinantes de Fusão/genética , Imagem com Lapso de Tempo/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Previous Japanese trials of the docetaxel, cisplatin, and 5-fluorouracil regimen for oesophageal cancer have demonstrated that a large proportion of patients also develop grade IV neutropenia. Our aim was to examine the risk factors for neutropenia in patients treated with this regimen. METHODS: We retrospectively analysed the risk factors for developing grade IV neutropenia in 66 patients with oesophageal cancer using a multivariate analysis. RESULTS: After administering the docetaxel, cisplatin, and 5-fluorouracil regimen, 49 patients (74.2%) developed grade IV neutropenia. Grade IV neutropenia was significantly associated with platelet count (p < 0.01), alanine transaminase level (p = 0.05), and proton-pump inhibitor administration (p < 0.05). Receiver operating characteristic curve analysis confirmed a platelet count of 290 × 103/µL as the optimal diagnostic cut-off value for grade IV neutropenia. The receiver operating characteristic area for grade IV neutropenia was increased by including patients that were administered a proton-pump inhibitor and alanine transaminase level (updated model; sensitivity and specificity, 75.5 and 88.2%, respectively). CONCLUSIONS: Our findings suggest that a platelet count is the most significant predictor of grade IV neutropenia.
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Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Fluoruracila/efeitos adversos , Neutropenia/etiologia , Taxoides/efeitos adversos , Idoso , Alanina Transaminase/sangue , Antineoplásicos/uso terapêutico , Área Sob a Curva , Plaquetas/citologia , Cisplatino/uso terapêutico , Docetaxel , Esquema de Medicação , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Feminino , Fluoruracila/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Inibidores da Bomba de Prótons/administração & dosagem , Curva ROC , Estudos Retrospectivos , Fatores de Risco , Taxoides/uso terapêuticoRESUMO
OBJECTIVE: Drug therapy is occasionally accompanied by an idiosyncratic severe toxicity, which occurs very rarely, but can lead to patient mortality. Methazolamide, an anti-glaucomatous agent, could cause severe skin eruptions called Stevens-Johnson syndrome/toxic epidermal necrolyis (SJS/TEN). Its precise etiology is still uncertain. In this study, the metabolism of methazolamide was investigated in immortalized human keratinocytes to reveal the possible mechanism which causes SJS/TEN. METHODS: The metabolism of methazolamide was studied using immortalized human keratinocytes, HaCaT cells. HPLC was used to isolate a metabolite from the culture medium. Mass spectrometry (LCMS/ MS) was employed for its characterization. Three typical chemical inducers were assessed for the inducibility of cytochrome P450, and methimazole was used as the inhibitor of flavin-containing monooxygenase (FMO). RESULTS: A sulfonic acid, N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) was identified as the final metabolite. Dexamethasone and ß-naphthoflavone behaved as an inducer of cytochrome P450 in the metabolism, but isoniazid did not. The effect of methimazole was not consistent. We did not detect any glucuronide nor any mercapturic acid (N-acetylcysteine conjugate). CONCLUSION: N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) is not considered to be a direct product of an enzymatic reaction, but rather an auto-oxidation product of N-[3-methyl-5- sulfe-1,3,4-thiadiazol-2(3H)-ylidene]acetamide, a chemically unstable sulfenic acid, which is produced by cytochrome P450 from the ß-lyase product of cysteine conjugate of methazolamide. MSO is considered to be susceptible to glutathione and to return to glutathione conjugate of methazolamide, forming a futile cycle. A hypothetical scenario is presented as to the onset of the disease.
Assuntos
Inibidores da Anidrase Carbônica/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Metazolamida/metabolismo , Síndrome de Stevens-Johnson/etiologia , Ácidos Sulfônicos/toxicidade , Acetilcisteína/metabolismo , Inibidores da Anidrase Carbônica/uso terapêutico , Inibidores da Anidrase Carbônica/toxicidade , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/metabolismo , Dexametasona/farmacologia , Glaucoma/tratamento farmacológico , Glucuronídeos/metabolismo , Humanos , Isoniazida/farmacologia , Queratinócitos , Liases/metabolismo , Metazolamida/uso terapêutico , Metazolamida/toxicidade , Metimazol/farmacologia , Oxirredução , Oxigenases/antagonistas & inibidores , Ácidos Sulfênicos/metabolismo , Ácidos Sulfônicos/metabolismo , Espectrometria de Massas em Tandem/métodos , beta-Naftoflavona/farmacologiaRESUMO
BACKGROUND: Hand-foot syndrome (HFS) is a common side effect that has a high occurrence rate with capecitabine (Cape) chemotherapy. However, little is known about the risk factors of developing HFS under the Cape regimen. Our aim was to examine these risk factors. METHODS: A univariate analysis was used to determine the risk factors associated with developing HFS, and we calculated the effect sizes between the patients who developed HFS compared to those who did not. RESULTS: Of the 52 patients enrolled in our research, 24 (46.2%) developed HFS. This group was significantly associated with hemoglobin (Hb) values (p < 0.001), and the effect size (1.21) was more than moderate. The receiver operating characteristic curve analysis confirmed 12 mg/dl Hb as the best diagnostic cut-off value for developing HFS. The sensitivity and specificity were 75.5 and 88.2%, respectively. Patients who had Hb values of 12 or below who developed HFS had longer median times without HFS compared to patients with high Hb values (115 vs. 75 days, p = 0.30, hazard ratio = 1.42, 95% CI 0.73-2.76) and a greater area under the Kaplan-Meier curves (p < 0.05). CONCLUSION: This research suggests that the Hb value is an important factor for developing HFS.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Capecitabina/efeitos adversos , Síndrome Mão-Pé/etiologia , Hemoglobinas/análise , Antimetabólitos Antineoplásicos/uso terapêutico , Capecitabina/uso terapêutico , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Razão de Chances , Modelos de Riscos Proporcionais , Fatores de RiscoRESUMO
In this study, we generated transgenic (Tg) mice, which overexpressed transforming growth factor (TGF)-ß stimulated clone-22 (TSC-22), and investigate the functional role of TSC-22 on their development and pathogenesis. We obtained 13 Tg-founders (two mice from C57BL6/J and 11 mice from BDF1). Three of 13 Tg-founders were sterile, and the remaining Tg-founders also could generate only a limited number of the F1 generation. We obtained 32 Tg-F1 mice. Most of the Tg-mice showed marked obesity. Histopathological examination could be performed on 31 Tg-mice; seventeen mice died by some disease in their entire life and 14 mice were killed for examination. Most of the Tg-mice examined showed splenic abnormality, in which marked increase of the megakaryocytes, unclearness of the margin of the red pulp and the white pulp, and the enlargement of the white pulp was observed. B cell lymphoma was developed in 10 (71%) of 14 disease-died F1 mice. These results indicate that constitutive over-expression of TSC-22 might disturb the normal embryogenesis and the normal lipid metabolism, and induce the oncogenic differentiation of hematopoietic cells.
Assuntos
Linfoma de Células B/etiologia , Obesidade/etiologia , Proteínas Repressoras/fisiologia , Baço/patologia , Animais , Células Cultivadas , Feminino , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/metabolismo , Obesidade/patologia , Baço/metabolismoRESUMO
Postsurgical tissue adhesion formation caused by inflammation and oxidative stress is one of the serious issues because it induces severe clinical disorders. In this study, we designed redox injectable gel (RIG) which covalently possesses nitroxide radicals as a reactive oxygen species (ROS) scavenger for high performance anti-adhesion agent. The redox flower micelles exhibiting gelation under physiological conditions were prepared by a polyion complex (PIC) between polyamine-PEG-polyamine triblock copolymer possessing nitroxide radicals as a side chain of polyamine segments and poly(acrylic acid). RIG showed prolonged local retention in the abdominal cavity of the mice, which was monitored by in vivo imaging system (IVIS). Compared with a commercial anti-adhesion agent (Seprafilm(®), Genzyme, Cambridge, MA), RIG dramatically inhibited the formation of tissue adhesions via a combination of physical separation and biological elimination of generated ROS in talc-induced adhesion model mice. Treatment with RIG suppressed inflammatory cytokines and neutrophil invasion, suppressing the increase in peritoneal membrane thickness. It is also emphasized that RIG suppressed the increase of white blood cells level, indicating that the present RIG treatment effectively prevents diffusion of local inflammation to entire body. These findings indicate that RIG has a great potential as a high performance anti-adhesion agent.