RESUMEN

In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3-5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases.

Asunto(s)

Astrocitos/patología , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Gliosis/patología , Neuroprotección/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Animales , Encéfalo/citología , Encéfalo/inmunología , Movimiento Celular , Proliferación Celular , Quimiocina CCL1/inmunología , Quimiocina CCL20/inmunología , Interleucina-2/inmunología , Interleucina-33/inmunología , Interleucina-6/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/inmunología , Receptores CCR/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Factor de Transcripción STAT3/metabolismo , Serotonina/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismo

RESUMEN

Damage-associated molecular patterns (DAMPs) trigger sterile inflammation after tissue injury, but the mechanisms underlying the resolution of inflammation remain unclear. In this study, we demonstrate that common DAMPs, such as high-mobility-group box 1 (HMGB1), peroxiredoxins (PRXs), and S100A8 and S100A9, were internalized through the class A scavenger receptors MSR1 and MARCO in vitro. In ischemic murine brain, DAMP internalization was largely mediated by MSR1. An elevation of MSR1 levels in infiltrating myeloid cells observed 3 d after experimental stroke was dependent on the transcription factor Mafb. Combined deficiency for Msr1 and Marco, or for Mafb alone, in infiltrating myeloid cells caused impaired clearance of DAMPs, more severe inflammation, and exacerbated neuronal injury in a murine model of ischemic stroke. The retinoic acid receptor (RAR) agonist Am80 increased the expression of Mafb, thereby enhancing MSR1 expression. Am80 exhibited therapeutic efficacy when administered, even at 24 h after the onset of experimental stroke. Our findings uncover cellular mechanisms contributing to DAMP clearance in resolution of the sterile inflammation triggered by tissue injury.

Asunto(s)

Alarminas/inmunología , Encéfalo/inmunología , Infarto de la Arteria Cerebral Media/inmunología , Factor de Transcripción MafB/inmunología , Células Mieloides/inmunología , Receptores Inmunológicos/inmunología , Receptores Depuradores de Clase A/inmunología , Animales , Benzoatos/farmacología , Encéfalo/efectos de los fármacos , Isquemia Encefálica/inmunología , Sistemas CRISPR-Cas , Calgranulina A/inmunología , Calgranulina B/inmunología , Inmunoprecipitación de Cromatina , Proteína HMGB1/inmunología , Inflamación , Factor de Transcripción MafB/efectos de los fármacos , Factor de Transcripción MafB/genética , Ratones , Células Mieloides/metabolismo , Peroxirredoxinas/inmunología , Receptores Inmunológicos/genética , Receptores de Ácido Retinoico/agonistas , Receptores Depuradores de Clase A/efectos de los fármacos , Receptores Depuradores de Clase A/genética , Accidente Cerebrovascular/inmunología , Tetrahidronaftalenos/farmacología

RESUMEN

Histone H3 lysine 9 dimethylation (H3K9me2) is mainly regulated by the histone lysine methyltransferase G9a and is associated with the repression of transcription. However, both the role of G9a and the significance of H3K9me2 in hepatocellular carcinoma (HCC) cells remain unclear. In this study, we conducted loss-of-function assay of G9a using short-hairpin RNA and pharmacological interference. Knockdown of G9a reduced H3K9me2 levels and impaired both HCC cell growth and sphere formation. However, transforming growth factor ß1-induced epithelial mesenchymal transition (EMT) was not suppressed by G9a knockdown. Combined analyses of chromatin immunoprecipitation followed by sequencing and RNA-sequencing led to successful identification of 96 candidate epigenetic targets of G9a. Pharmacological inhibition of G9a by BIX-01294 resulted in both cell growth inhibition and induction of apoptosis in HCC cells. Intraperitoneal administration of BIX-01294 suppressed the growth of xenograft tumors generated by implantation of HCC cells in non-obese diabetic/severe combined immunodeficient mice. Immunohistochemical analyses revealed high levels of G9a and H3K9me2 in 36 (66.7%) and 35 (64.8%) primary HCC tissues, respectively. G9a expression levels were significantly positively correlated with H3K9me2 levels in tumor tissues. In contrast, in non-tumor tissues, G9a and H3K9me2 were only observed in biliary epithelial cells and periportal hepatocytes. In conclusion, G9a inhibition impairs anchorage-dependent and -independent cell growth, but not EMT in HCC cells. Our data indicate that pharmacological interference of G9a might be a novel epigenetic approach for the treatment of HCC.

Asunto(s)

Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Neoplasias Hepáticas/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Azepinas/farmacología , Western Blotting , Carcinoma Hepatocelular/patología , Inmunoprecipitación de Cromatina , Metilación de ADN , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad , Histonas/genética , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Quinazolinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Irsogladine maleate (2,4-diamino-6-[2,5-dichlorophenyl]-s-triazine maleate; IM), an anti-peptic ulcer drug, may have a protective effect on the gastrointestinal mucosa. This study investigated the effects of IM on spontaneous colitis in interleukin-10 gene-deficient (IL-10(-/-)) mice. Five-week-old IL-10(-/-) mice were fed a control diet or one containing 100 ppm of IM for 10 weeks. Colonic tissues were evaluated morphologically and histologically. J774A.1 murine monocyte/macrophage cells were incubated with IM after lipopolysaccharide stimulation. mRNA expression was assessed by quantitative polymerase chain reaction (PCR) and protein concentration by enzyme-linked immunosorbent assay (ELISA). Colonic length, weight, and histological scores clearly demonstrated that spontaneous colitis was prevented in IL-10(-/-) mice fed a diet containing IM compared with those fed control diet. Levels of tumor necrosis factor-alpha (TNF-α) (-2.5-fold), IL-1ß (-5.4), interferon-gamma (IFN-γ) (-4.5), IL-17 (-113.0), IL-12p35 (-21.0), IL-12p40 (-3.4), and IL-23p19 (-4.2) mRNA expression were significantly decreased in the colonic tissues of IM-treated animals, suggesting that oral treatment with IM suppressed the T-helper (Th)1/Th17 immune response in the colonic mucosa. An in vitro study using monocyte/macrophage cells to clarify the pharmacological action of IM indicated that IL-12p40 and IL-23p19 mRNA expression levels were dose-dependently decreased by IM treatment. ELISA showed that IL-12p40 and IL-23 protein secretion were significantly decreased by IM in a dose-dependent manner. Oral treatment with IM prevented spontaneous colitis in IL-10(-/-) mice by suppressing the colonic mucosal Th1/Th17 immune response through inhibition of IL-12 and -23 production in monocyte/macrophage cells.

Asunto(s)

Colitis/prevención & control , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Macrófagos/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/metabolismo , Triazinas/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Colitis/etiología , Colitis/genética , Colitis/metabolismo , Colon/metabolismo , Colon/patología , Citocinas/genética , Citocinas/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-23/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Lipopolisacáridos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Triazinas/farmacología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Inflammasome activation has been implicated in various inflammatory diseases including post-ischaemic inflammation after stroke. Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1ß and IL-18, as well as a form of cell death called pyroptosis. In this study, we report that Bruton's tyrosine kinase (BTK) is an essential component of the NLRP3 inflammasome, in which BTK physically interacts with ASC and NLRP3. Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome. The FDA-approved BTK inhibitor ibrutinib (PCI-32765) efficiently suppresses infarct volume growth and neurological damage in a brain ischaemia/reperfusion model in mice. Ibrutinib inhibits maturation of IL-1ß by suppressing caspase-1 activation in infiltrating macrophages and neutrophils in the infarcted area of ischaemic brain. Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

Asunto(s)

Isquemia Encefálica/metabolismo , Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores

RESUMEN

BACKGROUND AND OBJECTIVES: Genome-wide association studies have revealed a link between autophagy-related (ATG) genes and susceptibility to Crohn's disease. This suggests underlying involvement of autophagy impairment in the pathogenesis of Crohn's disease. This study was performed to investigate the pathophysiological importance of autophagy impairment in intestinal epithelial cells exposed to TNF-α. METHODS: Human colonic epithelial cells (HT-29) and rat small intestinal epithelial cells (IEC-18) were used. Formation of phosphatidylethanolamine-conjugated microtubule-associated protein light chain 3 (LC3-II) was monitored as a marker of autophagy. Autophagy was inhibited using 3-methyladenine or short interfering RNA targeting ATG5 and ATG16L1. RESULTS: TNF-α treatment elicited a significant dose-dependent increase in LC3-II protein levels, thus autophagy is induced in the presence of TNF-α. Combined autophagy inhibition and TNF-α treatment induced a marked increase in the number of detached cells and a decrease in activated integrin ß1 protein levels. Trypan blue staining indicated 70-80 % of the detached cells were alive, suggesting that these cells became detached not because they were killed but because of dysfunction of cellular adhesion. CONCLUSIONS: This is the first study indicating that intestinal epithelial cells with impaired autophagy lose their adhesive capacity in the presence of TNF-α. These observations indicate that impairment of autophagy leads to disruption of the intestinal epithelial cell layers in TNF-α-rich environments.

Asunto(s)

Autofagia , Mucosa Intestinal/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Adhesión Celular , Enfermedad de Crohn/etiología , Células Epiteliales/citología , Proteínas de Unión al GTP/metabolismo , Células HT29 , Humanos , Integrina beta1/metabolismo , Mucosa Intestinal/citología , Ratas , Proteínas de Transporte Vesicular

RESUMEN

Deficiency of Smad3, an intracellular mediator of TGF-ß, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3(ex8/ex8) C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3(-/-) mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3(-/-) mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear ß-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3(-/-) mice in accordance with nuclear ß-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3(-/-) mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3(-/-) mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a "proliferative zone" at the bottom of colonic crypts in the normal colon.

Asunto(s)

Colon/fisiología , Mucosa Intestinal/fisiología , Receptores de la Familia Eph/biosíntesis , Proteína smad3/fisiología , Animales , Proliferación Celular , Células Cultivadas , Colon/citología , Mucosa Intestinal/citología , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Biosíntesis de Proteínas , Receptor EphA2/biosíntesis , Receptor EphA3/biosíntesis , Proteína smad3/genética , Factor de Crecimiento Transformador beta/fisiología

RESUMEN

BACKGROUND: Two main functionally distinct monocytes phenotypes are known: the CD14(hi)CD16(-) "classical" and the CD14(+)CD16(+) "proinflammatory" phenotypes. The latter phenotype is elevated in patients with ulcerative colitis (UC) and is suspected to have a major role in the immunopathogenesis of UC. AIM: To selectively deplete circulating proinflammatory CD14(+)CD16(+) monocyte phenotype. METHODS: Seven corticosteroid-naïve patients with UC (clinical activity index = 8.7 +/- 1.3) and seven healthy subjects were included. In patients with UC, granulocyte/monocyte adsorption (GMA) was done with an Adacolumn that selectively adsorbs leucocytes of the myeloid lineage. Blood from all subjects at baseline and from the patients immediately after the first GMA session was processed. Isolated monocytes were subjected to fluorescence-activated cell sorter analyses. RESULTS: The seven UC patients achieved remission (CAI

Asunto(s)

Colitis Ulcerosa/sangre , Colitis Ulcerosa/terapia , Integrina alfa4/inmunología , Leucaféresis/métodos , Receptores de Lipopolisacáridos/inmunología , Receptores CXCR3/inmunología , Adolescente , Adsorción , Adulto , Estudios de Casos y Controles , Colitis Ulcerosa/fisiopatología , Femenino , Citometría de Flujo , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Inmunosupresores/uso terapéutico , Integrina alfa4/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Probabilidad , Receptores CXCR3/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Estadísticas no Paramétricas , Adulto Joven

RESUMEN

Fibrocytes contribute to wound healing and are uniquely defined by coexpression of hematopoietic and mesenchymal cell markers. In this study, trafficking of fibrocytes was determined in a murine model of colitis induced by administering 3% dextran sodium sulfate (DSS) for seven days. Colonic tissues were immunostained for CD45, collagen type I (Col I), and alpha-SMA. On day 0, there were no CD45(+)Col I(+) cells in colonic tissues. However, on day 7 when inflammatory cells showed remarkable accumulation, oval-shaped CD45(+)Col I(+) fibrocytes were obvious in the submucosal layer. On day 14 when colonic tissues were in the healing phase, numerous spindle-shaped CD45(+)Col I(+) fibrocytes were observed. Emergence of CD45(+)Col I(+) fibrocytes preceded the appearance of alpha-SMA(+) myofibroblasts. Oval-shaped fibrocytes recruited as early as the inflammatory phase of colitis are likely to differentiate into spindle-shaped fibrocytes in the healing phase, suggesting that fibrocytes may promote wound healing in inflamed colonic tissues.

Asunto(s)

Colitis/patología , Fibroblastos/patología , Actinas/metabolismo , Animales , Colitis/inmunología , Colitis/metabolismo , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibroblastos/inmunología , Fibroblastos/metabolismo , Estudios de Seguimiento , Inmunohistoquímica , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal

RESUMEN

The developmental competence of in vitro cultured embryos vitrified-warmed at an early cleavage stage (2- or 4, 8-cell stage) was examined by both direct transfer into recipient animals and after in vitro manipulation for chimeric mice production using embryonic stem (ES) cells. Vitrified-warmed embryos transferred at the morulae and blastocyst stages showed fetus development comparable to control embryos, although blastocyst development of vitrified-warmed embryos was significantly slower than that of controls. When vitrified-warmed early cleavage stage embryos were used for chimeric mouse production using ES cells, 1 to 10% of the injected or aggregated embryos developed into chimeric neonates and germ-line chimeric mice were obtained from all ES cell lines. This study indicates that embryos developed in vitro from vitrified-warmed embryos have equivalent competence with unvitrified embryos irrespective of stage of vitrification and that these vitrified-warmed embryos maintain adequate viability even after in vitro manipulation such as aggregation and microinjection with ES cells.

Asunto(s)

Quimera , Embrión de Mamíferos/citología , Animales , Ratones

RESUMEN

The developmental response of in vitro fertilized embryos to oxygen and amino acids were compared between in-house bred C57BL/6JNrs (Nrs) and commercially available C57BL/6JSlc (Slc) mice. Under 20% oxygen, the percentage of embryos that developed to blastocysts and expanded blastocysts, and nuclear numbers were lower in Slc embryos than in Nrs embryos. Moreover, the nuclear number did not increase in Slc embryos during 72-96 h culture. Effects of amino acids were beneficial on development of Slc embryos under 20% oxygen, but inhibitory on blastocoel formation at 78 h under 5% oxygen. On the other hand effects of amino acids on Nrs embryos were observed in nuclear number at 72 and 78 h culture under 5% oxygen. Because the present study showed differences in sensitivity to culture conditions between the C57BL/6J substrains, care must be taken in embryo manipulation of this inbred strain in studies of embryo development or other studies.

Asunto(s)

Aminoácidos/farmacología , Desarrollo Embrionario y Fetal/efectos de los fármacos , Fertilización In Vitro , Ratones Endogámicos C57BL , Oxígeno/farmacología , Animales , Blastocisto/efectos de los fármacos , Células Cultivadas , Femenino , Masculino , Ratones , Factores de Tiempo

RESUMEN

OBJECTIVE: The purpose of this study was to evaluate the prognostic and clinical significance of newly acquired complete right bundle branch block (CRBBB) in airline pilots. PATIENTS: This study included pilots with acquired CRBBB, identified from a group of over 2,700 Japan Airline pilots. When the pilots applied for employment, a past medical history, physical examination, electrocardiogram, and chest radiograph were obtained. The pilots with ECG abnormality including CRBBB were not included in the study because of hiring requirements. RESULTS: Thirty-six pilots with CRBBB were identified between 1983 and 2002. All pilots with CRBBB were evaluated for the presence of ischemic heart disease by treadmill exercise testing, echocardiogram and exercise thallium scintigraphy. Twelve individuals underwent coronary angiography. The mean age of pilots was 44.4 +/- 5.8 years. The mean observation period was 10.9 +/- 5.7 years. For each of the 36 study subjects, Holter electrocardiogram and echocardiogram were obtained every 6 months after the CRBBB was detected. Exercise stress testing was performed every year. Exercise thallium scintigraphy was performed every 2 years to detect ischemic heart disease. During the observation period, two pilots stopped flying temporarily because of frequent ventricular premature beats and one pilot stopped flying permanentaly because of atrial fibrillation. During the follow-up period, no cardiovascular events were observed in pilots with CRBBB who had no underlying ischemic heart disease. CONCLUSION: Acquired CRBBB does not confer a poor prognosis, particularly in young men working as a pilot if there is no evidence of ischemia on exercise stress testing, echocardiography and exercise thallium scintigraphy.

Asunto(s)

Bloqueo de Rama/etiología , Adulto , Medicina Aeroespacial , Aeronaves , Bloqueo de Rama/diagnóstico , Bloqueo de Rama/fisiopatología , Enfermedad Coronaria/complicaciones , Enfermedad Coronaria/diagnóstico , Humanos , Japón , Masculino , Persona de Mediana Edad , Pronóstico , Factores de Riesgo