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1.
Nat Immunol ; 15(7): 676-86, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24908389

RESUMEN

The molecular checkpoints that drive inflammatory bowel diseases are incompletely understood. Here we found more T cells expressing the transcription factor PU.1 and interleukin 9 (IL-9) in patients with ulcerative colitis. In an animal model, citrine reporter mice had more IL-9-expressing mucosal T cells in experimental oxazolone-induced colitis. IL-9 deficiency suppressed acute and chronic colitis. Mice with PU.1 deficiency in T cells were protected from colitis, whereas treatment with antibody to IL-9 suppressed colitis. Functionally, IL-9 impaired intestinal barrier function and prevented mucosal wound healing in vivo. Thus, our findings suggest that the TH9 subset of helper T cells serves an important role in driving ulcerative colitis by regulating intestinal epithelial cells and that TH9 cells represent a likely target for the treatment of chronic intestinal inflammation.


Asunto(s)
Colitis/etiología , Mucosa Intestinal/inmunología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Interleucina-9/fisiología , Transducción de Señal/fisiología , Subgrupos de Linfocitos T/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Transactivadores/fisiología , Animales , Claudina-2/genética , Colitis/inmunología , Colitis Ulcerosa/inmunología , Humanos , Interleucina-9/inmunología , Ratones , Ratones Endogámicos BALB C , Células Th2/inmunología , Cicatrización de Heridas
2.
Gastroenterology ; 152(1): 176-192.e5, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27639807

RESUMEN

BACKGROUND & AIMS: GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. METHODS: We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. RESULTS: Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. CONCLUSIONS: Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.


Asunto(s)
Colitis/metabolismo , Colitis/prevención & control , ADN Catalítico/administración & dosificación , Factor de Transcripción GATA3/antagonistas & inhibidores , Factor de Transcripción GATA3/metabolismo , ARN Mensajero/análisis , Administración Rectal , Adolescente , Adulto , Anciano , Animales , Estudios de Casos y Controles , Niño , Colitis/inducido químicamente , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Colon/química , Colon/metabolismo , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor de Transcripción GATA3/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Oxazolona , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Linfocitos T/metabolismo , Ácido Trinitrobencenosulfónico , Adulto Joven
3.
J Immunol ; 195(12): 5787-94, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26561545

RESUMEN

IL-1R-associated kinase (IRAK) 1 is an important component of the IL-1R and TLR signaling pathways, which influence Th cell differentiation. In this study, we show that IRAK1 promotes Th17 development by mediating IL-1ß-induced upregulation of IL-23R and subsequent STAT3 phosphorylation, thus enabling sustained IL-17 production. Moreover, we show that IRAK1 signaling fosters Th1 differentiation by mediating T-bet induction and counteracts regulatory T cell generation. Cotransfer experiments revealed that Irak1-deficient CD4(+) T cells have a cell-intrinsic defect in generating Th1 and Th17 cells under inflammatory conditions in spleen, mesenteric lymph nodes, and colon tissue. Furthermore, IRAK1 expression in T cells was shown to be essential for T cell accumulation in the inflamed intestine and mesenteric lymph nodes. Transcriptome analysis ex vivo revealed that IRAK1 promotes T cell activation and induction of gut-homing molecules in a cell-intrinsic manner. Accordingly, Irak1-deficient T cells failed to upregulate surface expression of α4ß7 integrin after transfer into Rag1(-/-) mice, and their ability to induce colitis was greatly impaired. Lack of IRAK1 in recipient mice provided additional protection from colitis. Therefore, IRAK1 plays an important role in intestinal inflammation by mediating T cell activation, differentiation, and accumulation in the gut. Thus, IRAK1 is a promising novel target for therapy of inflammatory bowel diseases.


Asunto(s)
Colitis/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ratones Endogámicos C57BL/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Traslado Adoptivo , Animales , Diferenciación Celular , Movimiento Celular/genética , Citocinas/inmunología , Citocinas/metabolismo , Inflamación/inmunología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Integrinas/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/genética , Interleucina-17/inmunología , Intestinos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL/genética , Ratones Noqueados , Receptores Mensajeros de Linfocitos/genética , Transducción de Señal/inmunología , Linfocitos T Reguladores/trasplante , Células Th17/trasplante
4.
Nature ; 481(7379): 85-9, 2011 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-22158103

RESUMEN

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps. Although dissemination of tumour cells seems to be an early and frequent event, the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic of many tumour entities, suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.


Asunto(s)
Metástasis de la Neoplasia/patología , Células Madre Neoplásicas/patología , Nicho de Células Madre/fisiología , Animales , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Femenino , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/metabolismo , Células del Estroma/metabolismo , Vía de Señalización Wnt
5.
PLoS Comput Biol ; 11(3): e1004050, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25768678

RESUMEN

Angiogenesis plays a key role in tumor growth and cancer progression. TIE-2-expressing monocytes (TEM) have been reported to critically account for tumor vascularization and growth in mouse tumor experimental models, but the molecular basis of their pro-angiogenic activity are largely unknown. Moreover, differences in the pro-angiogenic activity between blood circulating and tumor infiltrated TEM in human patients has not been established to date, hindering the identification of specific targets for therapeutic intervention. In this work, we investigated these differences and the phenotypic reversal of breast tumor pro-angiogenic TEM to a weak pro-angiogenic phenotype by combining Boolean modelling and experimental approaches. Firstly, we show that in breast cancer patients the pro-angiogenic activity of TEM increased drastically from blood to tumor, suggesting that the tumor microenvironment shapes the highly pro-angiogenic phenotype of TEM. Secondly, we predicted in silico all minimal perturbations transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. In silico predicted perturbations were validated experimentally using patient TEM. In addition, gene expression profiling of TEM transitioned to a weak pro-angiogenic phenotype confirmed that TEM are plastic cells and can be reverted to immunological potent monocytes. Finally, the relapse-free survival analysis showed a statistically significant difference between patients with tumors with high and low expression values for genes encoding transitioning proteins detected in silico and validated on patient TEM. In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity. Results showed the successful in vitro reversion of such an activity by perturbation of in silico predicted target genes in tumor derived TEM, and indicated that targeting tumor TEM plasticity may constitute a novel valid therapeutic strategy in breast cancer.


Asunto(s)
Neoplasias de la Mama/fisiopatología , Modelos Biológicos , Monocitos/fisiología , Neovascularización Patológica/fisiopatología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Línea Celular , Biología Computacional , Citocinas/metabolismo , Citocinas/fisiología , Femenino , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Monocitos/química , Monocitos/clasificación , Neoplasias Experimentales , Fenotipo , Transducción de Señal/fisiología
6.
Proc Natl Acad Sci U S A ; 110(14): 5363-8, 2013 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-23479638

RESUMEN

Biomarker analysis is playing an essential role in cancer diagnosis, prognosis, and prediction. Quantitative assessment of immunohistochemical biomarker expression on tumor tissues is of clinical relevance when deciding targeted treatments for cancer patients. Here, we report a microfluidic tissue processor that permits accurate quantification of the expression of biomarkers on tissue sections, enabled by the ultra-rapid and uniform fluidic exchange of the device. An important clinical biomarker for invasive breast cancer is human epidermal growth factor receptor 2 [(HER2), also known as neu], a transmembrane tyrosine kinase that connotes adverse prognostic information for the patients concerned and serves as a target for personalized treatment using the humanized antibody trastuzumab. Unfortunately, when using state-of-the-art methods, the intensity of an immunohistochemical signal is not proportional to the extent of biomarker expression, causing ambiguous outcomes. Using our device, we performed tests on 76 invasive breast carcinoma cases expressing various levels of HER2. We eliminated more than 90% of the ambiguous results (n = 27), correctly assigning cases to the amplification status as assessed by in situ hybridization controls, whereas the concordance for HER2-negative (n = 31) and -positive (n = 18) cases was 100%. Our results demonstrate the clinical potential of microfluidics for accurate biomarker expression analysis. We anticipate our technique will be a diagnostic tool that will provide better and more reliable data, onto which future treatment regimes can be based.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Inmunohistoquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Receptor ErbB-2/metabolismo , Biomarcadores de Tumor/genética , Femenino , Fluorescencia , Humanos , Procesamiento de Imagen Asistido por Computador , Receptor ErbB-2/genética
7.
Am J Pathol ; 182(6): 2155-67, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23583421

RESUMEN

Inflammatory bowel diseases are commonly complicated by weight and bone loss. We hypothesized that IL-15, a pro-inflammatory cytokine expressed in colitis and an osteoclastogenic factor, could play a central role in systemic and skeletal complications of inflammatory bowel diseases. We evaluated the effects of an IL-15 antagonist, CRB-15, in mice with chronic colitis induced by oral 2% dextran sulfate sodium for 1 week, followed by another 1% for 2 weeks. During the last 2 weeks, mice were treated daily with CRB-15 or an IgG2a control antibody. Intestinal inflammation, disease severity, and bone parameters were evaluated at days 14 and 21. CRB-15 improved survival, early weight loss, and colitis clinical score, although colon damage and inflammation were prevented in only half the survivors. CRB-15 also delayed loss of femur bone mineral density and trabecular microarchitecture. Bone loss was characterized by decreased bone formation, but increased bone marrow osteoclast progenitors and osteoclast numbers on bone surfaces. CRB-15 prevented the suppression of osteoblastic markers of bone formation, and reduced osteoclast progenitors at day 14, but not later. However, by day 21, CRB-15 decreased tumor necrosis factor α and increased IL-10 expression in bone, paralleling a reduction of osteoclasts. These results delineate the role of IL-15 on the systemic and skeletal manifestations of chronic colitis and provide a proof-of-concept for future therapeutic developments.


Asunto(s)
Colitis/prevención & control , Interleucina-15/antagonistas & inhibidores , Osteoporosis/prevención & control , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Peso Corporal/efectos de los fármacos , Densidad Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Enfermedad Crónica , Colitis/inducido químicamente , Colitis/complicaciones , Colitis/fisiopatología , Citocinas/metabolismo , Sulfato de Dextran , Evaluación Preclínica de Medicamentos/métodos , Femenino , Fémur/patología , Fémur/fisiopatología , Mediadores de Inflamación/metabolismo , Interleucina-15/farmacología , Interleucina-15/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteoporosis/etiología , Osteoporosis/patología , Osteoporosis/fisiopatología , Proteínas Recombinantes de Fusión/farmacología , Índice de Severidad de la Enfermedad , Análisis de Supervivencia
8.
Pathobiology ; 81(2): 86-93, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24457836

RESUMEN

OBJECTIVE: Macrophages play a critical role in wound repair. However, the specific role of the different macrophage subtypes in wound repair remains incompletely understood. The aim of this study was to compare the wound repair activities of undifferentiated macrophages (M0), classically activated macrophages (M1) and alternatively activated (M2) macrophages. METHODS: The macrophage repair activities of intestinal wounds were evaluated using in vitro and in vivo models. RESULTS: All three macrophage subtypes enhanced wound closure in vitro, with the M2 macrophages demonstrating greater repair activities than the M0 and M1 macrophages. Injection of M0 and M2 macrophages into mice with experimental dextran sodium sulfate-induced colitis significantly enhanced ulcer repair when compared to control mice. In contrast, injection of M1 macrophages did not affect ulcer repair. CONCLUSIONS: These results underscore the wound repair capacity of different macrophage subsets. Notably, wound repair activity is not restricted to M2 macrophages, as the current literature suggests.


Asunto(s)
Activación de Macrófagos/fisiología , Macrófagos/citología , Cicatrización de Heridas/fisiología , Animales , Células 3T3 BALB , Diferenciación Celular , Colitis/patología , Modelos Animales de Enfermedad , Femenino , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
9.
Gastroenterology ; 142(2): 335-45, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22057112

RESUMEN

BACKGROUND & AIMS: Priming of T cells by dendritic cells (DCs) in the intestinal mucosa and associated lymphoid tissues helps maintain mucosal tolerance but also contributes to the development of chronic intestinal inflammation. Chemokines regulate the intestinal immune response and can contribute to pathogenesis of inflammatory bowel diseases. We investigated the role of the chemokine CCL17, which is expressed by conventional DCs in the intestine and is up-regulated during colitis. METHODS: Colitis was induced by administration of dextran sodium sulfate (DSS) to mice or transfer of T cells to lymphopenic mice. Colitis activity was monitored by body weight assessment, histologic scoring, and cytokine profile analysis. The direct effects of CCL17 on DCs and the indirect effects on differentiation of T helper (Th) cells were determined in vitro and ex vivo. RESULTS: Mice that lacked CCL17 (Ccl17(E/E) mice) were protected from induction of severe colitis by DSS or T-cell transfer. Colonic mucosa and mesenteric lymph nodes from Ccl17-deficient mice produced lower levels of proinflammatory cytokines. The population of Foxp3(+) regulatory T cells (Tregs) was expanded in Ccl17(E/E) mice and required for long-term protection from colitis. CCR4 expression by transferred T cells was not required for induction of colitis, but CCR4 expression by the recipients was required. CCL17 promoted Toll-like receptor-induced secretion of interleukin-12 and interleukin-23 by DCs in an autocrine manner, promoted differentiation of Th1 and Th17 cells, and reduced induction of Foxp3(+) Treg cells. CONCLUSIONS: The chemokine CCL17 is required for induction of intestinal inflammation in mice. CCL17 has an autocrine effect on DCs that promotes production of inflammatory cytokines and activation of Th1 and Th17 cells and reduces expansion of Treg cells.


Asunto(s)
Quimiocina CCL17/metabolismo , Colitis Ulcerosa/inmunología , Células Dendríticas/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Células Cultivadas , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Ratones , Ratones Transgénicos , Receptores CCR4/metabolismo
10.
Mod Pathol ; 26(3): 336-42, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23041831

RESUMEN

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.


Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/cirugía , Proliferación Celular , Mastectomía , Mitosis , Índice Mitótico , Biopsia , Neoplasias de la Mama/química , Carcinoma Ductal de Mama/química , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Modelos Lineales , Clasificación del Tumor , Invasividad Neoplásica , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Factores de Tiempo , Fijación del Tejido
12.
Breast Cancer Res Treat ; 132(2): 621-4, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22189628

RESUMEN

Intraoperative examination of sentinel axillary lymph nodes can be done by imprint cytology, frozen section, or, most recently, by PCR-based amplification of a cytokeratin signal. Using this technique, benign epithelial inclusions, representing mammary tissue displaced along the milk line, will likely generate a positive PCR signal and lead to a false-positive diagnosis of metastatic disease. To better appreciate the incidence of ectopic epithelial inclusions in axillary lymph nodes, we have performed an autopsy study, examining on 100 µm step sections 3,904 lymph nodes obtained from 160 axillary dissections in 80 patients. The median number of lymph nodes per axilla was 23 (15, 6, and 1 in levels 1, 2, and 3, respectively). A total of 30,450 hematoxylin-eosin stained slides were examined, as well as 8,825 slides immunostained with pan-cytokeratin antibodies. Despite this meticulous work-up, not a single epithelial inclusion was found in this study, suggesting that the incidence of such inclusions is much lower than the assumed 5% reported in the literature.


Asunto(s)
Células Epiteliales , Cuerpos de Inclusión , Ganglios Linfáticos/citología , Biopsia del Ganglio Linfático Centinela , Anciano , Anciano de 80 o más Años , Autopsia , Axila , Biomarcadores/análisis , Células Epiteliales/química , Femenino , Alemania , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/química , Queratinas/análisis , Ganglios Linfáticos/química , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos
13.
Am J Physiol Regul Integr Comp Physiol ; 302(9): R1025-33, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22403797

RESUMEN

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.


Asunto(s)
Enfermedad de la Arteria Coronaria/fisiopatología , Desoxicorticosterona , Hipertrofia Ventricular Izquierda/inducido químicamente , Hipertrofia Ventricular Izquierda/fisiopatología , Miocardio/metabolismo , Proteoma/metabolismo , Cloruro de Sodio Dietético/metabolismo , Animales , Enfermedad de la Arteria Coronaria/complicaciones , Citocinas/metabolismo , Regulación de la Expresión Génica , Hipertensión/complicaciones , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mineralocorticoides
14.
Dig Dis ; 30 Suppl 1: 82-90, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23075874

RESUMEN

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation due to dysregulation of the mucosal immune system. The cytokines IL-1ß and IL-18 appear early in intestinal inflammation and their pro-forms are processed via the caspase-1-activating multiprotein complex, the Nlrp3 inflammasome. Previously, we reported that the uptake of dextran sodium sulfate (DSS) by macrophages activates the Nlrp3 inflammasome and that Nlrp3(-/-) mice are protected in the acute DSS colitis model. Of note, other groups have reported opposing effects in regards to DSS susceptibility in Nlrp3(-/-) mice. Recently, mice lacking inflammasomes were found to develop a distinct intestinal microflora. METHODS: To reconcile the contradicting observations, we investigated the role of Nlrp3 deficiency in two different IBD models: acute DSS colitis and TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis. In addition, we investigated the impact of the intestinal flora on disease severity by performing cohousing experiments of wild-type and Nlrp3(-/-) mice, as well as by antibiotic treatment. RESULTS: Nlrp3(-/-) mice treated with either DSS or TNBS exhibited attenuated colitis and lower mortality. This protective effect correlated with an increased frequency of CD103+ lamina propria dendritic cells expressing a tolerogenic phenotype in Nlrp3(-/-) mice in steady state conditions. Interestingly, after cohousing, Nlrp3(-/-) mice were as susceptible as wild-type mice, indicating that transmission of endogenous bacterial flora between the two mouse strains might increase susceptibility of Nlrp3(-/-) mice towards DSS-induced colitis. Accordingly, treatment with antibiotics almost completely prevented colitis in the DSS model. CONCLUSIONS: The composition of the intestinal microflora significantly influences disease severity in IBD models comparing wild-type and Nlrp3(-/-) mice. This observation may - at least in part - explain contradictory results concerning the role of the inflammasome in different labs. Further studies are required to define the role of the Nlrp3 inflammasome in noninflamed mucosa under steady state conditions and in IBD.


Asunto(s)
Proteínas Portadoras/metabolismo , Ambiente , Predisposición Genética a la Enfermedad , Inflamasomas/metabolismo , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Sustancias Protectoras/metabolismo , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Colitis/inmunología , Colitis/patología , Colitis/fisiopatología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Tolerancia Inmunológica/inmunología , Inflamasomas/deficiencia , Inflamasomas/genética , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/patología , Intestinos/inmunología , Intestinos/microbiología , Intestinos/patología , Metagenoma , Ratones , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Proteína con Dominio Pirina 3 de la Familia NLR , Fenotipo , Ácido Trinitrobencenosulfónico
15.
Sci Transl Med ; 14(661): eaax8933, 2022 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-36070364

RESUMEN

Brain metastasis is a complication of increasing incidence in patients with breast cancer at advanced disease stage. It is a severe condition characterized by a rapid decline in quality of life and poor prognosis. There is a critical clinical need to develop effective therapies to prevent and treat brain metastases. Here, we describe a unique and robust spontaneous preclinical model of breast cancer metastasis to the brain (4T1-BM2) in mice that has been instrumental in uncovering molecular mechanisms guiding metastatic dissemination and colonization of the brain. Key experimental findings were validated in the additional murine D2A1-BM2 model and in human MDA231-BrM2 model. Gene expression analyses and functional studies, coupled with clinical transcriptomic and histopathological investigations, identified connexins (Cxs) and focal adhesion kinase (FAK) as master molecules orchestrating breast cancer colonization of the brain. Cx31 promoted homotypic tumor cell adhesion, heterotypic tumor-astrocyte interaction, and FAK phosphorylation. FAK signaling prompted NF-κB activation inducing Lamc2 expression and laminin 332 (laminin 5) deposition, α6 integrin-mediated adhesion, and sustained survival and growth within brain parenchyma. In the MDA231-BrM2 model, the human homologous molecules CX43, LAMA4, and α3 integrin were involved. Systemic treatment with FAK inhibitors reduced brain metastasis progression. In conclusion, we report a spontaneous model of breast cancer metastasis to the brain and identified Cx-mediated FAK-NF-κB signaling as a mechanism promoting cell-autonomous and microenvironmentally controlled cell survival for brain colonization. Considering the limited therapeutic options for brain metastatic disease in cancer patients, we propose FAK as a therapeutic candidate to further pursue in the clinic.


Asunto(s)
Neoplasias Encefálicas , Neoplasias de la Mama , Animales , Encéfalo/metabolismo , Neoplasias de la Mama/genética , Conexinas/metabolismo , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Melanoma , Ratones , FN-kappa B/metabolismo , Calidad de Vida , Neoplasias Cutáneas , Melanoma Cutáneo Maligno
16.
Eur J Cancer ; 173: 10-19, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35839597

RESUMEN

BACKGROUND: Recently, novel antibody--drug conjugates (ADCs) showed clinical activity in a subset of advanced human epidermal growth factor receptor 2 (HER2)-negative patients. We investigated the prognostic significance of HER2-low and HER2-zero tumours. PATIENTS AND METHODS: The retrospective cohort study included 410 consecutive node-negative breast cancer patients without adjuvant systemic therapy treated between 1985 and 2000 (median follow-up: 16.73 [IQR 8.58-23.45] years). 351 (85.6%) were HER-2 negative and subdivided into HER2-zero (immunohistochemistry [IHC] score 0) and HER2-low (IHC score 1+ or 2+/in situ hybridisation [ISH]-negative). HER2 gene expression was available in 170 (48.4%) patients. Differences in HER2 status for immunohistochemistry, gene expression and clinico-pathologic parameters were assessed using Fisher's exact test, Pearson's correlation and Mann-Whitney test. Prognosis was investigated using the Kaplan-Meier method and Cox regression analyses. RESULTS: Of the 351 HER2-negative patients, 198 (56.4%) had HER2-low tumours and 153 (43.6%) were HER2-zero. Significant differences between HER2-zero and HER2-low tumours were found in histologic grading (P = 0.001), Ki-67 (P = 0.013) and HER2 gene expression (P = 0.002). HER2-low patients had significantly longer disease-free survival (DFS) (15-year rate: 67.5% [95% CI 61.0-74.7] vs. 47.3% [95% CI 39.9-56.1], P < 0.001) and overall survival (OS) (15-year rate: 75.4% [95% CI 69.4-81.9] vs. 66.8% [95% CI 59.5-74.9], P = 0.009). The OS difference was observed in hormone receptor (HR)-positive (P = 0.039) but not HR-negative (P = 0.086) tumours. The results of multivariable analyses confirmed the independent prognostic significance of HER2 status (DFS: HR, 0.546; 95% CI, 0.402-0.743; P < 0.001; OS: HR, 0.653; 95% CI, 0.458-0.932; P = 0.019). CONCLUSION: HER2-low patients had a better survival than HER2-zero patients.


Asunto(s)
Neoplasias de la Mama , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Pronóstico , Receptor ErbB-2/metabolismo , Estudios Retrospectivos
17.
J Hepatol ; 55(2): 322-7, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21147186

RESUMEN

BACKGROUND & AIMS: Recent studies have described a major impact of genetic variations near the IL28B gene on the natural course and outcome of antiviral therapy in chronic hepatitis C. We therefore, aimed to explore the impact of donor and recipient genotypes of these polymorphisms on hepatitis C virus (HCV) liver graft reinfection. METHODS: Donor and recipient genotypes of IL28B rs12979860C>T single nucleotide polymorphism were determined in 91 patients with HCV liver graft reinfection, 47 of whom were treated with pegylated interferon-α (PEG-IFN-α) and ribavirin. IL28B genetic polymorphisms were correlated with the natural course and treatment outcome of recurrent hepatitis C. RESULTS: Patients requiring liver transplantation due to end-stage chronic hepatitis C appeared to be selected toward the adverse genotypes rs12979860 CT/TT compared to non-transplanted HCV-infected patients (p=0.046). Patients with the donor genotype rs12979860 CC had higher peak ALT and HCV RNA serum concentrations than those with CT/TT (p=0.04 and 0.06, respectively). No association was observed between ALT/HCV RNA serum concentrations and recipient genotypes (p>0.3). More important, donor IL28B rs12979860 CC vs. CT/TT genotypes were associated with rapid, complete early, and sustained virologic response (RVR, cEVR, SVR) to treatment with PEG-IFN-α and ribavirin (p=0.003, 0.0012, 0.008, respectively), but weaker associations of recipient genotypes with RVR, cEVR, and SVR were observed as well (p=0.0046, 0.115, 0.118, respectively). CONCLUSIONS: We provide evidence for a dominant, but not exclusive impact of the donor rather than the recipient IL28B genetic background on the natural course and treatment outcome of HCV liver graft reinfection.


Asunto(s)
Hepatitis C Crónica/genética , Hepatitis C Crónica/cirugía , Interleucinas/genética , Trasplante de Hígado/efectos adversos , Adulto , Anciano , Alanina Transaminasa/sangre , Antivirales/administración & dosificación , Femenino , Genotipo , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/fisiopatología , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferones , Cirrosis Hepática/etiología , Trasplante de Hígado/fisiología , Masculino , Persona de Mediana Edad , Polietilenglicoles/administración & dosificación , Polimorfismo de Nucleótido Simple , ARN Viral/sangre , Proteínas Recombinantes/administración & dosificación , Recurrencia , Ribavirina/administración & dosificación , Donantes de Tejidos , Resultado del Tratamiento
18.
Lasers Surg Med ; 43(7): 696-704, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22057497

RESUMEN

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) affects vascular barrier function and thus increases vessel permeability. This phenomenon may be exploited to facilitate targeted drug delivery and may lead to a new clinical application of photodynamic therapy. Here, we investigate the role of leukocyte recruitment for PDT-induced vascular permeabilization. STUDY DESIGN/MATERIAL AND METHODS: Fluorescein isothiocyanate dextran (FITC-D, 2,000 kDa) was injected intravenously 120 minutes after focal PDT on striated muscle in nude mice bearing dorsal skinfold chambers (Visudyne® 800 µg/kg, fluence rate 300 mW/cm2 , light dose of 200 J/cm2). Leukocyte interaction with endothelial cells was inhibited by antibodies functionally blocking adhesion molecules ("MABS-PDT" group, n = 5); control animals had PDT but no antibody injection (group "PDT", n = 7). By intravital microscopy, we monitored leukocyte rolling and sticking in real-time before, 90 and 180 minutes after PDT. The extravasation of FITC-D from striated muscle vessels into the interstitial space was determined in vivo during 45 minutes to assess treatment-induced alterations of vascular permeability. RESULTS: PDT significantly increased the recruitment of leukocytes and enhanced the leakage of FITC-D. Neutralization of adhesion molecules before PDT suppressed the rolling of leukocytes along the venular endothelium and significantly reduced the extravasation of FITC-D as compared to control animals (156 ± 27 vs. 11 ± 2 (mean ± SEM, number of WBC/30 seconds mm vessel circumference; P < 0.05) at 90 minutes after PDT and 194 ± 21 vs. 14 ± 4 at 180 minutes after PDT). In contrast, leukocyte sticking was not downregulated by the antibody treatment. CONCLUSION: Leukocyte recruitment plays an essential role in the permeability-enhancing effect of PDT.


Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Leucocitos/efectos de los fármacos , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Animales , Dextranos/farmacocinética , Sistemas de Liberación de Medicamentos , Femenino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Colorantes Fluorescentes/farmacocinética , Ratones , Ratones Desnudos , Microscopía Fluorescente , Verteporfina
19.
Gut ; 59(9): 1192-9, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20442201

RESUMEN

BACKGROUND: The proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. Here, the role of the NLRP3 inflammasome in experimental colitis induced by dextran sodium sulfate (DSS) was examined. METHODS: IL-1beta production in response to DSS was studied in macrophages of wild-type, caspase-1(-/-), NLRP3(-/-), ASC(-/-), cathepsin B(-/-) or cathepsin L(-/-) mice. Colitis was induced in C57BL/6 and NLRP3(-/-) mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. RESULTS: Macrophages incubated with DSS in vitro secreted high levels of IL-1beta in a caspase-1-dependent manner. IL-1beta secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1beta secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3(-/-) mice developed a less severe colitis than wild-type mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection comparable with NLRP3 deficiency. CONCLUSIONS: The NLRP3 inflammasome was identified as a critical mechanism of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for patients with IBD.


Asunto(s)
Proteínas Portadoras/fisiología , Colitis/fisiopatología , Animales , Caspasa 1/fisiología , Colitis/inducido químicamente , Colitis/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/fisiopatología , Interleucina-1beta/metabolismo , Lisosomas/fisiología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología
20.
Microcirculation ; 17(1): 69-78, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20141602

RESUMEN

BACKGROUND: This study was designed to explore the effect of transient inducible nitric oxide synthase (iNOS) overexpression via cationic liposome-mediated gene transfer on cardiac function, fibrosis, and microvascular perfusion in a porcine model of chronic ischemia. METHODS AND RESULTS: Chronic myocardial ischemia was induced using a minimally invasive model in 23 landrace pigs. Upon demonstration of heart failure, 10 animals were treated with liposome-mediated iNOS-gene-transfer by local intramyocardial injection and 13 animals received a sham procedure to serve as control. The efficacy of this iNOS-gene-transfer was demonstrated for up to 7 days by reverse transcriptase-polymerase chain reaction in preliminary studies. Four weeks after iNOS transfer, magnetic resonance imaging showed no effect of iNOS overexpression on cardiac contractility at rest and during dobutamine stress (resting ejection fraction: control 27%, iNOS 26%; P = ns). Late enhancement, infarct size, and the amount of fibrosis were similar between groups. Although perfusion and perfusion reserve in response to adenosine and dobutamine were not significantly modified by iNOS-transfer, both vessel number and diameter were significantly increased in the ischemic area in the iNOS-treated group versus control (point score: control 15.3, iNOS 34.7; P < 0.05). CONCLUSIONS: Our findings demonstrate that transient iNOS overexpression does not aggravate cardiac dysfunction or postischemic fibrosis, while potentially contributing to neovascularization in the chronically ischemic heart.


Asunto(s)
Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Arteriolas/patología , Arteriolas/fisiopatología , Femenino , Fibrosis , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Liposomas , Imagen por Resonancia Magnética , Masculino , Isquemia Miocárdica/genética , Neovascularización Patológica , Óxido Nítrico/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sus scrofa , Función Ventricular Izquierda
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