RESUMEN
Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diabetes Mellitus Tipo 1/congénito , Diarrea/genética , Factores de Transcripción Forkhead/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades del Sistema Inmune/congénito , Inflamación/genética , Linfocitos T Reguladores/fisiología , Alelos , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Análisis Mutacional de ADN , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diarrea/inmunología , Factores de Transcripción Forkhead/genética , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Humanos , Enfermedades del Sistema Inmune/genética , Enfermedades del Sistema Inmune/inmunología , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación Missense/genética , Especificidad de Órganos/genéticaRESUMEN
Magnesium oxide (MgO) is a widely used laxative. Because many antipsychotic drugs are lipophilic-basic-compounds, their solubility decreases with increasing pH and changes markedly as the pH of the solution approaches their pKa. It is highly important to clarify the effect of co-administration of MgO on the serum drug concentration for effective, safe, and appropriate medication therapy. However, the relationship between MgO administration and the serum concentration of antipsychotic drugs in patients with schizophrenia has not been reported. Therefore, in the present study, we investigated the effect of MgO administration on the concentration of antipsychotic drugs in the blood of patients with schizophrenia. The serum concentrations of biperiden, zotepine, and risperidone were assayed using an LC/MS system. The correlation between the daily dose of MgO and the relative-drug-concentration (rCp) in each patient was examined. As the MgO dose was increased, the risperidone concentration decreased. The correlation coefficient decreased for risperidone, zotepine, and biperiden, in the same order. To clarify the difference in the suppression potency of MgO on the three drugs, the relationship between the physical properties and the correlation coefficients of each drug was carefully examined. A strong correlation was observed between the pKa and the correlation coefficient. Patients with schizophrenia are often prescribed antipsychotic drugs, which have anticholinergic action and tend to suppress gastric acid secretion. We concluded that basic drug absorption might be suppressed due to an increase in the stomach pH following MgO administration. Therefore, MgO co-administration is better to avoid while taking antipsychotic drugs and anticholinergic drugs.
Asunto(s)
Antipsicóticos/farmacocinética , Laxativos/farmacología , Óxido de Magnesio/farmacología , Esquizofrenia/sangre , Adulto , Anciano , Anciano de 80 o más Años , Antipsicóticos/sangre , Biperideno/sangre , Biperideno/farmacocinética , Dibenzotiepinas/sangre , Dibenzotiepinas/farmacocinética , Interacciones Farmacológicas , Quimioterapia Combinada , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Persona de Mediana Edad , Risperidona/sangre , Risperidona/farmacocinética , Esquizofrenia/tratamiento farmacológicoRESUMEN
Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.
Asunto(s)
Diferenciación Celular/genética , Células Epiteliales , Receptores ErbB/genética , Factor de Transcripción STAT3/biosíntesis , Animales , Desarrollo Embrionario , Receptores ErbB/biosíntesis , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Ratones , Factor de Transcripción STAT3/genética , Transducción de Señal , Linfocitos T/metabolismo , Timocitos/metabolismo , Timo/crecimiento & desarrollo , Timo/metabolismoRESUMEN
The immune system is influenced by the vital zinc (Zn) status, and Zn deficiency triggers lymphopenia; however, the mechanisms underlying Zn-mediated lymphocyte maintenance remain elusive. Here we investigated ZIP10, a Zn transporter expressed in the early B-cell developmental process. Genetic ablation of Zip10 in early B-cell stages resulted in significant reductions in B-cell populations, and the inducible deletion of Zip10 in pro-B cells increased the caspase activity in parallel with a decrease in intracellular Zn levels. Similarly, the depletion of intracellular Zn by a chemical chelator resulted in spontaneous caspase activation leading to cell death. Collectively, these findings indicated that ZIP10-mediated Zn homeostasis is essential for early B-cell survival. Moreover, we found that ZIP10 expression was regulated by JAK-STAT pathways, and its expression was correlated with STAT activation in human B-cell lymphoma, indicating that the JAK-STAT-ZIP10-Zn signaling axis influences the B-cell homeostasis. Our results establish a role of ZIP10 in cell survival during early B-cell development, and underscore the importance of Zn homeostasis in immune system maintenance.
Asunto(s)
Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/inmunología , Zinc/metabolismo , Animales , Linfocitos B/citología , Caspasas/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Diferenciación Celular , Supervivencia Celular/inmunología , Citocinas/metabolismo , Homeostasis , Humanos , Quinasas Janus/metabolismo , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Linfopenia/etiología , Linfopenia/inmunología , Linfopenia/metabolismo , Ratones , Ratones Noqueados , Modelos Inmunológicos , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Zinc/deficienciaRESUMEN
The humoral immune response, also called the antibody-mediated immune response, is one of the main adaptive immune systems. The essential micronutrient zinc (Zn) is known to modulate adaptive immune responses, and dysregulated Zn homeostasis leads to immunodeficiency. However, the molecular mechanisms underlying this Zn-mediated modulation are largely unknown. Here, we show that the Zn transporter SLC39A10/ZIP10 plays an important role in B-cell antigen receptor (BCR) signal transduction. Zip10-deficiency in mature B cells attenuated both T-cell-dependent and -independent immune responses in vivo. The Zip10-deficient mature B cells proliferated poorly in response to BCR cross-linking, as a result of dysregulated BCR signaling. The perturbed signaling was found to be triggered by a reduction in CD45R phosphatase activity and consequent hyperactivation of LYN, an essential protein kinase in BCR signaling. Our data suggest that ZIP10 functions as a positive regulator of CD45R to modulate the BCR signal strength, thereby setting a threshold for BCR signaling in humoral immune responses.
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Proteínas de Transporte de Catión/inmunología , Inmunidad Humoral , Receptores de Antígenos de Linfocitos B/metabolismo , Zinc/metabolismo , Inmunidad Adaptativa , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Diferenciación Celular/inmunología , Senescencia Celular/inmunología , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunología , Linfocitos T/inmunologíaRESUMEN
Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Receptores Notch/metabolismo , Células del Estroma/citología , Animales , Diferenciación Celular/fisiología , Técnicas In Vitro , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Transducción de Señal/fisiología , Células del Estroma/metabolismoRESUMEN
To provide safe dental care, it is important to minimize the pain associated with the initial injection of the local anesthetic. For this purpose, a topical anesthetic is preliminarily applied to the area where a needle will be inserted in a clinical setting. In this study, we prepared new topical anesthetic formulations with favorable intra-oral retentivity and an excellent anesthetic effect, and clinically evaluated their efficacy. We used 4% lidocaine solution as an anesthetic drug and gelatin, agar, and a food thickener as a base to prepare new topical anesthetic formulations. The subjects rested in a supine position on a chair for dental practice prior to the following experiments. Firstly, about 0.2 g of the sample was applied at a test site. One minute later, the sample was removed, and a 30 G dental injection needle was inserted into the test site. The agar/gelatin-based formulation containing gelatin of 2% and agar of 1% had a moderate solidity at 25°C and a moderate fluidity at 37°C. This formulation showed a significantly greater depth than any of the commercially available topical anesthetics. The results of the present study demonstrated that the agar/gelatin-based formulation showed an excellent analgesic effect against pain associated with needle insertion.
Asunto(s)
Agar , Gelatina , Lidocaína , Dolor/prevención & control , Adulto , Agar/química , Agar/uso terapéutico , Anestésicos Locales , Atención Odontológica , Método Doble Ciego , Femenino , Gelatina/química , Gelatina/uso terapéutico , Humanos , Inyecciones , Lidocaína/química , Lidocaína/uso terapéutico , Masculino , Agujas , Dimensión del DolorRESUMEN
There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T(H)2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RBâºiNKT cells are present in the thymic CD44âº/â» NK1.1â» population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RBâ»iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⺠and IL-17RBâ» subsets. The IL-17RBâºiNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⺠IL-17RBâºiNKT cells produce T(H)2 (IL-13), T(H)9 (IL-9 and IL-10), and T(H)17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4â» IL-17RBâºiNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⺠subset producing T(H)17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RBâºiNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RBâºiNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.
Asunto(s)
Citocinas/metabolismo , Células T Asesinas Naturales/fisiología , Subgrupos de Linfocitos T/fisiología , Animales , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/patología , Hiperreactividad Bronquial/virología , Células Cultivadas , Citometría de Flujo , Perfilación de la Expresión Génica , Inmunofenotipificación , Hígado/patología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/patología , Especificidad de Órganos , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/patología , Bazo/patología , Subgrupos de Linfocitos T/metabolismo , Células Th17/patología , Células Th17/fisiología , Células Th2/patología , Células Th2/fisiología , Timo/patología , Técnicas de Cultivo de TejidosRESUMEN
Receptor activator of nuclear factor kappa-B ligand (RANKL) expression was examined during the development of mouse fetal peripheral lymphoid organs. A shift in the expression pattern was detected during the transition from lymphoid tissue inducer (LTi) cells to lymphoid tissue organizer (LTo) cells in the lymph node (LN) anlagen but not in the Peyer's patch anlagen. In order to understand the functional impact of these changes in the fetal expression of RANKL, the RANKL function was blocked by a blocking antibody. Excess anti-RANKL antibody was administered to pregnant mice between 13.5 and 16.5 dpc and was found to completely block LN anlagen development, suggesting that RANKL function during this period is critical for LN development. In addition, small amounts of anti-RANKL antibodies were injected directly into the amniotic space at 13.5 dpc, resulting in perturbed B-cell follicle formation and high endothelial venule differentiation after birth. These results suggest that RANKL expression on LTi cells during the early phase of LN development is critical for the development LN microarchitecture.
Asunto(s)
Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica , Ganglios Linfáticos/metabolismo , Ligando RANK/genética , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Microambiente Celular/efectos de los fármacos , Microambiente Celular/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Inmunohistoquímica , Ganglios Linfáticos/citología , Ganglios Linfáticos/embriología , Tejido Linfoide/citología , Tejido Linfoide/embriología , Tejido Linfoide/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Organogénesis/efectos de los fármacos , Organogénesis/genética , Ganglios Linfáticos Agregados/citología , Ganglios Linfáticos Agregados/embriología , Ganglios Linfáticos Agregados/metabolismo , Embarazo , Ligando RANK/inmunología , Ligando RANK/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hematopoietic lymphoid tissue inducer (LTi) cells are essential for the development of secondary lymphoid tissues including lymph nodes and Peyer's patches. Two transcription factors, the helix-loop-helix inhibitor Id2 and the retinoic acid-related orphan receptor γt (Rorγt), have been shown to be crucial for LTi cell development. However, it remains unclear how the specification of multipotent hematopoietic progenitor cells toward the LTi lineage is programmed. In this study, we report impaired lymphoid tissue organogenesis in mice in which the function of Runx1/Cbfß transcription factor complexes was attenuated by the loss of either the distal promoter-derived Runx1 or Cbfß2 variant protein. We found that LTi progenitors in fetal liver, defined previously as a lineage marker-negative α4ß7 integrin (α4ß7)(+) IL-7R α-chain (IL-7Rα)(+) population, can be subdivided into Rorγt-expressing IL-7Rα(high) cells and nonexpressing IL-7Rα(mid) cells. Whereas Id2 and Rorγt are required to direct α4ß7(+)IL-7Rα(mid) cells to become α4ß7(+)IL-7Rα(high) cells, Runx1/Cbfß2 complexes are necessary for the emergence of α4ß7(+)IL-7Rα(mid) cells. In addition, the loss of Cbfß2, but not P1-Runx1, resulted in an inefficient upregulation of Rorγt in residual α4ß7(+)IL-7Rα(+) LTi cells at anlagen. Our results thus revealed that Runx1/Cbfß2 complexes regulate the differentiation of LTi cells at two stages: an early specification of hematopoietic progenitors toward the LTi lineage and a subsequent activation of Rorγt expression at anlagen.
Asunto(s)
Diferenciación Celular/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Tejido Linfoide/inmunología , Animales , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Subunidad beta del Factor de Unión al Sitio Principal/genética , Variación Genética/inmunología , Proteína 2 Inhibidora de la Diferenciación/biosíntesis , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/fisiología , Hígado/embriología , Hígado/inmunología , Hígado/patología , Tejido Linfoide/citología , Tejido Linfoide/embriología , Ratones , Ratones Mutantes , Células Madre Multipotentes/citología , Células Madre Multipotentes/inmunología , Células Madre Multipotentes/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/fisiologíaRESUMEN
BACKGROUND: Despite the potent antitumor activity of CPT-11, late-onset diarrhea owing to enterohepatic circulation of SN-38 is a critical issue. METHODS: We aimed to evaluate the inhibitory potency of gefitinib against the ABCB1- or ABCG2-mediated excretion of CPT-11 and its active metabolite SN-38 in vitro and in vivo. RESULTS: Gefitinib dose-dependently enhanced the antiproliferation activity of SN-38 in vitro by inhibiting ABCG2. The inhibitory effect of gefitinib on ABCB1 was marginal. When both CPT-11 and gefitinib were administered orally to nude mice bearing human lung cancer PC-6 cells, tumor growth was markedly suppressed. By gefitinib coadministration, the lactone forms of both CPT-11 and SN-38 in the tumor tissue increased more than 2-fold. CONCLUSIONS: Gefitinib significantly enhances the antitumor efficacy of CPT-11 and its tumor distribution in vivo. Coadministration of gefitinib may provide a new means to reduce the dose of CPT-11 and to circumvent the gastrointestinal toxicity risk.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Camptotecina/análogos & derivados , Proteínas de Neoplasias/metabolismo , Quinazolinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Administración Oral , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Camptotecina/efectos adversos , Camptotecina/farmacología , Camptotecina/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Diarrea/etiología , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Gefitinib , Células HEK293 , Humanos , Irinotecán , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Quinazolinas/efectos adversos , Quinazolinas/uso terapéutico , ARN Mensajero/metabolismo , Trasplante HeterólogoRESUMEN
During the course of inflammation and its resolution, macrophages are exposed to various cytotoxic materials, including reactive oxygen species. Thus, macrophages require a protective machinery against oxidative stress to survive at the inflammatory site. Here, we showed that xCT, a component of transport system x(c)(-), was significantly up-regulated in activated infiltrating cells, including macrophages and neutrophils at the inflammatory site. System x(c)(-) mediates the uptake of extracellular L-cystine and is consequently responsible for maintenance of intracellular glutathione levels. We established a loss-of-function mouse mutant line of xCT by N-ethyl-N-nitrosourea mutagenesis. Macrophages from xCT(mu/mu) mice showed cell death in association with the excessive release of high mobility group box chromosomal protein 1 upon stimulation with LPS, suggesting that xCT deficiency causes unremitting inflammation because of the impaired survival of activated macrophages at the inflammatory site. Subcutaneous injection of 3-methylcholanthrene (3-MCA) induced the generation of fibrosarcoma in association with inflammation. When 3-MCA was injected s.c. into mice, xCT mRNA was up-regulated in situ. In xCT(mu/mu) mice, inflammatory cytokines (such as IL-1beta and TNFalpha) were overexpressed, and the generation of 3-MCA-induced fibrosarcoma was accelerated. These results clearly indicate that the defect of the protective system against oxidative stress impaired survival of activated macrophages and subsequently enhanced tumorigenecity.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Sistema de Transporte de Aminoácidos y+/deficiencia , Sistema de Transporte de Aminoácidos y+/inmunología , Animales , Muerte Celular , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Fibrosarcoma/inducido químicamente , Fibrosarcoma/inmunología , Regulación Neoplásica de la Expresión Génica , Interleucina-1beta/inmunología , Metilcolantreno , Ratones , Ratones Noqueados , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia ArribaRESUMEN
Spontaneous mutant mice that showed high levels of serum IgE and an atopic dermatitis (AD)-like skin disease were found in a colony of the KOR inbred strain that was derived from Japanese wild mice. No segregation was observed between hyper-IgE-emia and dermatitis in (BALB/c x KOR mutant) N(2) mice, suggesting that the mutation can be attributed to a single recessive locus, which we designated adjm (atopic dermatitis from Japanese mice). All four adjm congenic strains in different genetic backgrounds showed both hyper-IgE-emia and dermatitis, although the disease severity varied among strains. Linkage analysis using (BALB/c x KOR-adjm/adjm) N(2) mice restricted the potential adjm locus to the 940 kb between D10Stm216 and D10Stm238 on chromosome 10. Sequence analysis of genes located in this region revealed that the gene AI429613, which encodes the mouse homologue of the human TNFR-associated factor 3-interacting protein 2 (TRAF3IP2) protein (formerly known as NF-kappaB activator 1/connection to IkappaB kinase and stress-activated protein kinase/Jun kinase), carried a single point mutation leading to the substitution of a stop codon for glutamine at amino acid position 214. TRAF3IP2 has been shown to function as an adaptor protein in signaling pathways mediated by the TNFR superfamily members CD40 and B cell-activating factor in epithelial cells and B cells as well as in the IL-17-mediated signaling pathway. Our results suggest that malfunction of the TRAF3IP2 protein causes hyper-IgE-emia through the CD40- and B cell-activating factor-mediated pathway in B cells and causes skin inflammation through the IL-17-mediated pathway. This study demonstrates that the TRAF3IP2 protein plays an important role in AD and suggests the protein as a therapeutic target to treat AD.
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Proteínas Adaptadoras Transductoras de Señales/genética , Inmunoglobulina E/sangre , Mutación Puntual , Enfermedades de la Piel/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Western Blotting , Dermatitis Atópica/sangre , Dermatitis Atópica/genética , Dermatitis Atópica/inmunología , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad , Interleucina-17/sangre , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Linaje , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades de la Piel/sangre , Enfermedades de la Piel/inmunología , Análisis de SupervivenciaRESUMEN
Calcium-independent group VIA phospholipase A(2) (iPLA(2)beta), encoded by PLA2G6, has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Mutations in the PLA2G6 gene have been recently identified in patients with infantile neuroaxonal dystrophy (INAD). Subsequently, it was reported that similar neurological impairment occurs in gene-targeted mice with a null mutation of iPLA(2)beta, whose disease onset became apparent approximately 1 to 2 years after birth. Here, we report the establishment of an improved mouse model for INAD that bears a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethyl-N-nitrosourea mutagenesis. These mutant mice developed severe motor dysfunction, including abnormal gait and poor performance in the hanging grip test, as early as 7 to 8 weeks of age, in a manner following Mendelian law. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. Because of the significantly early onset of the disease, this mouse mutant (Pla2g6-inad) could be highly useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration.
Asunto(s)
Modelos Animales de Enfermedad , Fosfolipasas A2 Grupo VI/genética , Distrofias Neuroaxonales/genética , Distrofias Neuroaxonales/patología , Edad de Inicio , Animales , Secuencia de Bases , Western Blotting , Ratones , Ratones Mutantes , Microscopía Electrónica de Transmisión , Mutación Puntual , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Graft-versus host disease (GVHD) is a complication of stem cell transplantation associated with significant morbidity and mortality. Non-specific immune-suppression, the mainstay of treatment, may result in immune-surveillance dysfunction and disease recurrence. METHODS: We created humanised mice model for chronic GVHD (cGVHD) by injecting cord blood (CB)-derived human CD34+CD38-CD45RA- haematopoietic stem/progenitor cells (HSPCs) into hIL-6 transgenic NOD/SCID/Il2rgKO (NSG) newborns, and compared GVHD progression with NSG newborns receiving CB CD34- cells mimicking acute GVHD. We characterised human immune cell subsets, target organ infiltration, T-cell repertoire (TCR) and transcriptome in the humanised mice. FINDINGS: In cGVHD humanised mice, we found activation of T cells in the spleen, lung, liver, and skin, activation of macrophages in lung and liver, and loss of appendages in skin, obstruction of bronchioles in lung and portal fibrosis in liver recapitulating cGVHD. Acute GVHD humanised mice showed activation of T cells with skewed TCR repertoire without significant macrophage activation. INTERPRETATION: Using humanised mouse models, we demonstrated distinct immune mechanisms contributing acute and chronic GVHD. In cGVHD model, co-activation of human HSPC-derived macrophages and T cells educated in the recipient thymus contributed to delayed onset, multi-organ disease. In acute GVHD model, mature human T cells contained in the graft resulted in rapid disease progression. These humanised mouse models may facilitate future development of new molecular medicine targeting GVHD.
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Enfermedad Injerto contra Huésped/inmunología , Interleucina-6/genética , Macrófagos/inmunología , Linfocitos T/inmunología , Enfermedad Aguda , Animales , Animales Recién Nacidos , Enfermedad Crónica , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Tasa de Supervivencia , Linfocitos T/metabolismo , TranscriptomaRESUMEN
Silencing of transduced genes hampers production of transgenic mice using retroviral vectors. We show stable expression of the enhanced green fluorescent protein (EGFP) gene in chimeric mice generated from retrovirally transduced embryonic stem cells. The vector was a murine stem cell virus-typed retroviral vector (GCDsap) in which the long terminal repeat and primer-binding site were derived from a PCC4 cell-passaged myeloproliferative sarcoma virus and the endogenous retrovirus dl587rev, respectively. To increase the viral titer, the vector was packaged with vesicular stomatitis virus G protein, which allowed concentration of the virus into pellets followed by resuspension in serum-free medium. In chimeric mice, EGFP was detected in various tissues including hematopoietic cells, neurons, cardiac muscle, and intestine. Furthermore, high expression was maintained in the progeny of these mice, suggesting successful germline transmission of active proviruses. Although the proportion of EGFP-expressing cells and the mean intensity of EGFP expression varied among tissues and mice, 100% of peripheral blood leukocytes expressed EGFP in mice carrying a single provirus copy, as well as in their progeny. Therefore, the gene transfer system described here provides a useful tool not only to generate transgenic animals but also to manipulate human embryonic stem cells..
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Células Madre Embrionarias/metabolismo , Expresión Génica/genética , Retroviridae/genética , Transgenes/genética , Animales , Quimera , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Genes Reporteros/genética , Vectores Genéticos/genética , Ratones , Ratones Endogámicos C57BL , Familia de Multigenes , Trasplante de Células MadreRESUMEN
BACKGROUND: Gefitinib competes with topoisomerase I inhibitors at drug efflux pumps in vitro. We evaluated the effects of oral gefitinib on pharmacokinetic parameters of orally coadministered irinotecan. METHODS: We measured the serum pharmacokinetic parameters and biliary excretion of irinotecan, SN-38 and its glucuronide after irinotecan (50 or 100 mg/kg) was orally administered with or without gefitinib 100 mg/kg to rats. We measured the concentrations of irinotecan and SN-38 in the small intestine, liver, lungs and kidneys in each rat. RESULTS: The plasma area under the curve (0-24 h) of irinotecan and SN-38 was increased significantly, while the apparent elimination constant of irinotecan was decreased significantly. Gefitinib significantly increased the biliary cumulative amounts of irinotecan, but not of SN-38, and also influenced the enterohepatic circulation of irinotecan, but not of SN-38. CONCLUSIONS: Gefitinib increased the serum concentrations of irinotecan and SN-38 following oral administration of irinotecan without increasing the biliary excretion of SN-38 in vivo.
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Sistema Biliar , Camptotecina/análogos & derivados , Quinazolinas/farmacología , Administración Oral , Animales , Sistema Biliar/metabolismo , Camptotecina/administración & dosificación , Camptotecina/metabolismo , Gefitinib , Irinotecán , Masculino , Ratas , Ratas WistarRESUMEN
Contrast-induced nephropathy (CIN) is one of the serious side effects of contrast media. A few studies have suggested that N-acetylcysteine (NAC) is effective to prevent CIN, but the efficacy remains unclear in Japanese. Therefore, we retrospectively studied the preventive effect of NAC on CIN in Sakakibara Heart Institute. Patients who had been administered NAC for the purpose of preventing CIN before coronary intervention between February 2005 and November 2006 were included in the NAC group. In addition, age- and rate of diabetes mellitus-matched controls were randomly extracted. We retrieved and analyzed patient data including demographics, NAC dosage, and serum creatinine concentrations (Scr). NAC group (n=16) showed significantly higher baseline Scr (p<0.01) and a tendency toward a lower dose of contrast media (p=0.068) compared with controls (n=48). Since the occurrence of CIN was low, there was no significant difference in the proportion of CIN between the groups (NAC: 6%, controls: 4%). NAC group trended toward a decrease in Scr after the use of contrast media, while controls increased (-0.04+/-0.25 versus +0.03+/-0.36 mg/dl, p=0.096). The multivariate analysis showed that the dosage of NAC is inversely correlated with Scr independent of baseline Scr and dosage of contrast media. Despite higher baseline Scr (i.e., high-risk with CIN) in the NAC group, the real Scr value reflected a lower trend on average. In addition, this finding suggests that a larger dose of NAC results in a lower Scr value, we consider that the NAC dosage more likely prevented CIN.
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Acetilcisteína/administración & dosificación , Medios de Contraste/efectos adversos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/prevención & control , Anciano , Angioplastia , Biomarcadores/sangre , Angiografía Coronaria , Creatinina/sangre , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Enfermedades Renales/diagnóstico , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Camptotecina/análogos & derivados , Carcinoma de Células Pequeñas/tratamiento farmacológico , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo , Neoplasias de la Mama/metabolismo , Camptotecina/farmacología , Carcinoma de Células Pequeñas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Factor de Crecimiento Epidérmico/biosíntesis , Gefitinib , Humanos , Concentración 50 Inhibidora , Irinotecán , Neoplasias Pulmonares/metabolismo , Mitoxantrona/farmacología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Topotecan/farmacocinética , Topotecan/farmacologíaRESUMEN
BACKGROUND: Administration of valproic acid (VPA) is complicated with approximately 0.9% of patients developing hyperammonemia, but the pathogenesis of this adverse effect remains to be clarified. The aim of the present study was to search for mechanisms associated with VPA-induced hyperammonemia in the light of changes in serum amino acids concentrations associated with the urea cycle of schizophrenic patients. METHOD: Blood samples (10 mL) were obtained from 37 schizophrenic patients receiving VPA for the prevention of violent behaviors in the morning after overnight fast. Blood concentrations of ammonia, VPA, free carnitine, acyl-carnitine, and 40 amino acids including glutamate and citrulline were measured for each patient. Univariate and multivariate regression analyses were performed to identify amino acids or concomitantly administered drugs that were associated with variability in the blood concentrations of ammonia. RESULT: The blood ammonia level was positively correlated with the serum glutamate concentration (r = 0.44, p < 0.01) but negatively correlated with glutamine (r = -0.41, p = 0.01), citrulline (r = -0.42, p = 0.01), and glycine concentrations (r = -0.54, p < 0.01). It was also revealed that the concomitant administration of the mood stabilizers (p = 0.04) risperidone (p = 0.03) and blonanserin (p < 0.01) was positively associated with the elevation of the blood ammonia level. CONCLUSION: We hypothisized that VPA would elevate the blood ammonia level of schizophrenic patients. The observed changes in serum amino acids are compatible with urea cycle dysfunction, possibly due to reduced carbamoyl-phosphate synthase 1 (CPS1) activity. We conclude that VPA should be prudently prescribed to schizophrenic patients, particularly those receiving mood stabilizers or certain antipsychotics.