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In this study, we developed active substrates consisting of Ag-decorated silicon nanowires on a Si substrate using a single-step Metal Assisted Chemical Etching (MACE) process, and evaluated their performance in the identification of low concentrations of Rhodamine 6G using surface-enhanced photoluminescence spectroscopy. Different structures with Ag-aggregates as well as Ag-dendrites were fabricated and studied depending on the etching parameters. Moreover, the addition of Au nanoparticles by simple drop-casting on the MACE-treated surfaces can enhance the photoluminescence significantly, and the structures have shown a Limit of Detection of Rhodamine 6G down to 10-12 M for the case of the Ag-dendrites enriched with Au nanoparticles.
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Photonic crystal structuring has emerged as an advanced method to enhance solar light harvesting by metal oxide photocatalysts along with rational compositional modifications of the materials' properties. In this work, surface functionalization of TiO2 photonic crystals by blue luminescent graphene quantum dots (GQDs), n-π* band at ca. 350 nm, is demonstrated as a facile, environmental benign method to promote photocatalytic activity by the combination of slow photon-assisted light trapping with GQD-TiO2 interfacial electron transfer. TiO2 inverse opal films fabricated by the co-assembly of polymer colloidal spheres with a hydrolyzed titania precursor were post-modified by impregnation in aqueous GQDs suspension without any structural distortion. Photonic band gap engineering by varying the inverse opal macropore size resulted in selective performance enhancement for both salicylic acid photocatalytic degradation and photocurrent generation under UV-VIS and visible light, when red-edge slow photons overlapped with the composite's absorption edge, whereas stop band reflection was attenuated by the strong UVA absorbance of the GQD-TiO2 photonic films. Photoelectrochemical and photoluminescence measurements indicated that the observed improvement, which surpassed similarly modified benchmark mesoporous P25 TiO2 films, was further assisted by GQDs electron acceptor action and visible light activation to a lesser extent, leading to highly efficient photocatalytic films.
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BACKGROUND/OBJECTIVES: Polyunsaturated fatty acid (PUFA) status during pregnancy has been suggested to influence offspring obesity and cardiometabolic health. We assessed whether prenatal PUFA exposure is associated with rapid infant growth, childhood BMI, and cardiometabolic profile. SUBJECTS/METHODS: In the Dutch MEFAB (n = 266) and Greek RHEA (n = 263) cohorts, we measured n-3 and n-6 PUFA concentrations in cord blood phospholipids, which reflect fetal exposure in late pregnancy. We defined rapid infant growth from birth to 6 months of age as an increase in weight z-score >0.67. We analyzed body mass index (BMI) as continuous and in categories of overweight/obesity at 4 and 6 years. We computed a cardiometabolic risk score at 6-7 years as the sum of waist circumference, non-high-density lipoprotein cholesterol and blood pressure z-scores. Associations of PUFAs with child health outcomes were assessed using generalized linear models for binary outcomes and linear regression models for continuous ones after adjusting for important covariates, and for the pooled estimates, a cohort indicator. RESULTS: In pooled analyses, we found no association of PUFA levels with rapid infant growth, childhood BMI (ß per SD increase in the total n-3:n-6 PUFA ratio = -0.04 SD; 99% CI: -0.15, 0.06; P = 0.65 at 4 years, and -0.05 SD; 99% CI: -0.18, 0.08; P = 0.78 at 6 years), and overweight/obesity. We also found no associations for clustered cardiometabolic risk and its individual components. The results were similar across cohorts. CONCLUSIONS: Our findings suggest that PUFA concentrations at birth are not associated with later obesity development and cardiometabolic risk in childhood.
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Desarrollo Infantil/fisiología , Ácidos Grasos Insaturados/sangre , Obesidad Infantil/epidemiología , Adulto , Índice de Masa Corporal , Niño , Preescolar , Dieta , Femenino , Estudios de Seguimiento , Grecia , Humanos , Lactante , Recién Nacido , Exposición Materna , Madres , Países Bajos , Riesgo , Circunferencia de la Cintura/fisiologíaRESUMEN
Lower prenatal exposure to n-3 PUFA relative to n-6 PUFA has been hypothesised to influence allergy development, but evidence remains largely inconsistent. In the Dutch Maastricht Essential Fatty Acid Birth (MEFAB) (n 293) and Greek RHEA Mother-Child (n 213) cohorts, we investigated whether cord blood phospholipid PUFA concentrations are associated with symptoms of wheeze, asthma, rhinitis and eczema at the age of 6-7 years. Information on allergy-related phenotypes was collected using validated questionnaires. We estimated relative risks (RR) and 95 % CI for associations of PUFA with child outcomes using multivariable generalised linear regression models. In pooled analyses, higher concentration of the n-3 long-chain EPA and DHA and a higher total n-3:n-6 PUFA ratio were associated with lower risk of current wheeze (RR 0·61; 95 % CI 0·45, 0·82 per sd increase in EPA+DHA and 0·54; 95 % CI 0·39, 0·75 per unit increase in the n-3:n-6 ratio) and reduced asthma risk (RR 0·50; 95 % CI 0·31, 0·79 for EPA+DHA and 0·43; 95 % CI 0·26, 0·70 for the n-3:n-6 ratio). No associations were observed for other allergy-related phenotypes. The results were similar across cohorts. In conclusion, higher EPA and DHA concentrations and a higher n-3:n-6 fatty acid ratio at birth were associated with lower risk of child wheeze and asthma. Our findings suggest that dietary interventions resulting in a marked increase in the n-3:n-6 PUFA ratio, and mainly in n-3 long-chain PUFA intake in late gestation, may reduce the risk of asthma symptoms in mid-childhood.
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Ácidos Grasos Esenciales , Ácidos Grasos Insaturados/sangre , Sangre Fetal/química , Hipersensibilidad/sangre , Efectos Tardíos de la Exposición Prenatal , Adulto , Asma/epidemiología , Niño , Estudios de Cohortes , Eccema/epidemiología , Ácidos Grasos Omega-3/sangre , Ácidos Grasos Omega-6/sangre , Femenino , Estudios de Seguimiento , Grecia/epidemiología , Humanos , Recién Nacido , Masculino , Países Bajos/epidemiología , Fenotipo , Embarazo , Ruidos Respiratorios , RiesgoRESUMEN
Plants synthesize numerous specialized metabolites (also termed natural products) to mediate dynamic interactions with their surroundings. The complexity of plant specialized metabolism is the result of an inherent biosynthetic plasticity rooted in the substrate and product promiscuity of the enzymes involved. The pathway of carnosic acid-related diterpenes in rosemary and sage involves promiscuous cytochrome P450s whose combined activity results in a multitude of structurally related compounds. Some of these minor products, such as pisiferic acid and salviol, have established bioactivity, but their limited availability prevents further evaluation. Reconstructing carnosic acid biosynthesis in yeast achieved significant titers of the main compound but could not specifically yield the minor products. Specific production of pisiferic acid and salviol was achieved by restricting the promiscuity of a key enzyme, CYP76AH24, through a single-residue substitution (F112L). Coupled with additional metabolic engineering interventions, overall improvements of 24 and 14-fold for pisiferic acid and salviol, respectively, were obtained. These results provide an example of how synthetic biology can help navigating the complex landscape of plant natural product biosynthesis to achieve heterologous production of useful minor metabolites. In the context of plant adaptation, these findings also suggest a molecular basis for the rapid evolution of terpene biosynthetic pathways.
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Metabolismo de los Hidratos de Carbono , Diterpenos/metabolismo , Levaduras/metabolismo , Abietanos/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Diterpenos/química , Ingeniería Genética , Variación Genética , Genotipo , Redes y Vías Metabólicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
TGFß-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is considered a key intermediate in a multitude of innate immune signaling pathways. Yet, the specific role of TAK1 in the myeloid compartment during inflammatory challenges has not been revealed. To address this question, we generated myeloid-specific kinase-dead TAK1 mutant mice. TAK1 deficiency in macrophages results in impaired NF-κB and JNK activation upon stimulation with lipopolysaccharide (LPS). Moreover, TAK1-deficient macrophages and neutrophils show an enhanced inflammatory cytokine profile in response to LPS stimulation. Myeloid-specific TAK1 deficiency in mice leads to increased levels of circulating IL-1ß, TNF and reduced IL-10 after LPS challenge and sensitizes them to LPS-induced endotoxemia. These results highlight an antiinflammatory role for myeloid TAK1, which is essential for balanced innate immune responses and host survival during endotoxemia.
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Endotoxemia/inmunología , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/inmunología , Macrófagos/inmunología , Animales , Citocinas/sangre , Inmunidad Innata , Proteínas Quinasas JNK Activadas por Mitógenos , Lipopolisacáridos/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/deficiencia , Ratones , Ratones Noqueados , FN-kappa B , Neutrófilos/inmunologíaRESUMEN
Τhe clinical significance of de novo post-transplant anti-HLA donor-specific antibodies (DSA) was evaluated using 4241 serum samples collected between 2000 and 2007 from 597 renal transplant recipients. Patients transplanted before December 1996 (n = 77) were included in the historic group and those transplanted thereafter (n = 520) were included in the study group. All recipients were negative for DSA before transplantation (Tx). Post-Tx, de novo DSA were detected in 92/597 (15.4%) patients, while 196 had third party anti-HLA antibodies (DSA-negative). DSA were more frequent in the historic group (33.8%) compared with the study group (12.7%) (P < 0.001). Anti-HLA class-II DSA predominated in both groups (84.6% vs. 69.7%). Recipients of HLA class II-incompatible grafts developed DSA more frequently than those receiving HLA class II-compatible grafts (17.9% vs.7.9%, P = 0.003), directed mainly against HLA-DQ graft molecules (64/446, 14.4%). DSA production was not different between presensitized and nonsensitized patients (P = 0.842). Graft survival was higher in patients without antibodies compared with DSA-positive (log-rank test, P = 0.002) and DSA-negative patients (log-rank test, P = 0.002). Univariate and multivariate analysis showed independent association for DSA class I (HR = 31.78), DSA class II (HR = 20.92) and non-DSA (HR = 5.94) and graft failure. We conclude that HLA class II incompatible graft transplantations need careful monitoring and should be avoided in high immunological risk cases.
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Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Trasplante de Riñón/inmunología , Donantes de Tejidos , Adulto , Anticuerpos/inmunología , Femenino , Estudios de Seguimiento , Rechazo de Injerto/inmunología , Antígenos HLA-DQ/inmunología , Prueba de Histocompatibilidad , Humanos , Isoanticuerpos/inmunología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Resultado del TratamientoRESUMEN
TNF plays a crucial role in the pathogenesis of Crohn disease. Dysregulated TNF production in mice that bear the genetic deletion of the TNF AU-rich regulatory elements (ARE) (Tnf(ΔARE/+) mice) results in TNF receptor I (TNFRI)-dependent spontaneous Crohn-like pathology. Current concepts consider intestinal epithelial cell (IEC) responses to TNF to be critical for intestinal pathology, but the potential contribution of IEC-derived TNF in disease pathogenesis has not been addressed. In this study we examined whether IEC are sufficient as cellular targets or sources of TNF in the development of intestinal pathology. Using IEC-specific reactivation of a hypomorphic Tnf(ΔAREneo) allele in mice, we show that selective chronic overproduction of TNF by IEC suffices to cause full development of Crohn-like pathology. Epithelial TNF overexpression leads to early activation of the underlying intestinal myofibroblast, a cell type previously identified as a sufficient target of TNF for disease development in the Tnf(ΔARE) model. By contrast, restricted TNFRI expression on IEC although sufficient to confer IEC apoptosis after acute exogenous TNF administration, fails to induce pathology following chronic specific targeting of IEC by endogenous TNF in Tnf(ΔARE/+) mice. Our results argue against IEC being early and sufficient responders to chronic TNF-mediated pathogenic signals and suggest that proinflammatory aberrations leading to chronic TNF production by IEC may initiate pathology in Crohn disease.
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Enfermedad de Crohn/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Intestinos/citología , Intestinos/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Enfermedad de Crohn/metabolismo , Células Epiteliales/citología , Humanos , Inflamación/metabolismo , Inflamación/patología , Mucosa Intestinal/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVE: To investigate the role of invariant natural killer T (iNKT) cells in TNF(DeltaARE/+) mice, an animal model of spondylarthritis (SpA) with both gut and joint inflammation. METHODS: The frequency and activation of iNKT cells were analyzed on mononuclear cells from the lymph nodes and livers of mice, using flow cytometry with alpha-galactosylceramide/CD1d tetramers and quantitative polymerase chain reaction for the invariant V(alpha)14-J(alpha)18 rearrangement. Bone marrow-derived dendritic cells (DCs) were obtained by expansion of primary cells with granulocyte-macrophage colony-stimulating factor followed by coculture with iNKT cell hybridomas, and interleukin-2 release into the cocultures was then measured by enzyme-linked immunosorbent assay (ELISA). Cytokine levels were determined by ELISA or cytometric bead array analyses of freshly isolated DCs and iNKT cells in mixed cocultures. TNF(DeltaARE/+) mice were backcrossed onto J(alpha)18(-/-) and CD1d(-/-) mice, and disease onset was evaluated by clinical scoring, positron emission tomography, and histology. CD1d levels were analyzed on mononuclear cells in paired blood and synovial fluid samples from patients with SpA compared with healthy control subjects. RESULTS: In the absence of iNKT cells, symptoms of gut and joint inflammation in TNF(DeltaARE/+)mice were aggravated. Invariant NKT cells were activated during the course of the disease. This was linked to an enrichment of inflammatory DCs, characterized by high levels of CD1d, particularly at draining sites of inflammation. A similar increase in CD1d levels was observed on DCs from patients with SpA. Inflammatory DCs from TNF(DeltaARE/+) mice stimulated iNKT cells to produce immunomodulatory cytokines, in the absence of exogenous stimulation. Prolonged, continuous exposure, but not short-term exposure, to tumor necrosis factor (TNF) was found to be responsible for the enhanced DC-NKT cell crosstalk. CONCLUSION: This mode of iNKT cell activation represents a natural counterregulatory mechanism for the dampening of TNF-driven inflammation.
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Células Asesinas Naturales/inmunología , Espondiloartritis/inmunología , Animales , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Citometría de Flujo , Reordenamiento Génico , Homeostasis/inmunología , Inflamación/genética , Inflamación/inmunología , Artropatías/genética , Artropatías/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Espondiloartritis/genética , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The TNF/TNF receptor (TNFR) system has a prominent role in the pathogenesis of chronic inflammatory and autoimmune disorders. Extensive research in animal models with deregulated TNF expression has documented that TNF may initiate or sustain inflammatory pathology, while at the same time may exert immunomodulatory or disease-suppressive activities. The TNF/TNFR system encompassing both the soluble and the transmembrane form of TNF with differential biological activities, as well as the differential usage of its receptors, mediating distinct functions, appears to confer complexity but also specificity in the action of TNF. The inherent complexity in TNF-mediated pathophysiology highlights the requirement to address the role of TNF taking into account both proinflammatory tissue-damaging and immunomodulatory functions in a cellular and receptor-specific manner. In this review, we discuss our current understanding of the involvement of TNF in chronic inflammation and autoimmunity, focusing on TNF-mediated cellular pathways leading to the pathogenesis or progression of joint and intestinal inflammatory pathology. Knowledge of the mechanisms by which TNF either initiates or contributes to disease pathology is fundamentally required for the design of safe and effective anti-TNF/TNFR therapies for human inflammatory and autoimmune disorders.
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Autoinmunidad/inmunología , Inflamación/inmunología , Modelos Inmunológicos , Factor de Necrosis Tumoral alfa/inmunología , Animales , Artritis Reumatoide/etiología , Artritis Reumatoide/inmunología , Autoinmunidad/genética , Células Dendríticas Foliculares/inmunología , Modelos Animales de Enfermedad , Humanos , Inflamación/etiología , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/inmunología , Mesodermo/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
The analysis of amino acids by gas chromatography mass spectrometry (GC-MS) after their derivatization with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide was investigated as an alternative approach for the determination of free (FAA) and combined amino acids (CAA) in aerosols. This technique showed excellent linearity with r(2) values ranging from 0.9029 to 0.9995 and instrumental limits of detection ranging from 0.3 to 46pg for the different amino acids. The quality of water used for sample extraction was found to be of utmost importance for achieving low blank levels of FAA and CAA. The addition of isopropanol during the extraction of aerosols was also shown to minimize the coextraction of inorganic salts that interfered with the analysis of FAA, Moreover, the ascorbic acid was found to be the most effective reagent for preventing the oxidative destruction of CAA during the hydrolysis process. By the analysis of spiked aerosol samples, the average recoveries determined for FAA and CAA were higher than 60% and the associated relative standard deviation was lower than 10% for the majority of amino acids. The application of the adopted method in background aerosols of the eastern Mediterranean enabled the unambiguous identification and quantification of 20 amino acids. The total concentration of FAA and CAA in aerosols ranged from 13 to 34ngm(-3) and from 29 to 79ngm(-3), respectively. The GC-MS based method is proposed to overcome several analytical difficulties usually encountered with the conventional HPLC-fluoresence technique.
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Contaminantes Atmosféricos/química , Aminoácidos/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Aerosoles/química , Límite de DetecciónRESUMEN
In normal conditions intestinal epithelial cells (IECs) constitutively stimulate regulatory CD4(+) T cells. However, in Crohn's disease (CD), this major histocompatibility complex (MHC) class II-restricted antigen presentation results in stimulation of proinflammatory CD4(+) T cells. We hypothesized that these alternative functions might be mediated by differential sorting and processing of antigens into distinct MHC II-enriched compartments (MIICs). Accordingly, we analysed the endocytic pathways of lumenally applied ovalbumin (OVA) in IECs of the jejunum and ileum of wild-type (WT) and TNFDeltaARE/WT mice that develop a CD-resembling ileitis. Using quantitative reverse transcription polymerase chain reaction, we found that messenger RNA levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-17 and interleukin-10 were significantly up-regulated in the inflamed ileum of TNFDeltaARE/WT mice, confirming CD-like inflammation. Fluorescence and immunoelectron microscopy revealed the presence of MHC II and invariant chain throughout the late endocytic compartments, with most molecules concentrated in the multivesicular bodies (MVB). OVA was targeted into MVB and, in contrast to other MIICs, accumulated in these structures within 120 min of exposure. The IEC-specific A33 antigen localized to internal vesicles of MVB and A33/class II-bearing exosomes were identified in intercellular spaces. Remarkably, the expression pattern of MHC II/invariant chain molecules and the trafficking of OVA were independent of mucosal inflammation and the specific region in the small intestine. MVB seem to be principally responsible for class II-associated antigen processing in IECs and to constitute the origin of MHC II-loaded exosomes. The distinctive functions of IECs in antigen presentation to CD4(+) T cells might arise as a result of differential processing within the MVB identified here.
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Endosomas/inmunología , Exosomas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Mucosa Intestinal/inmunología , Animales , Presentación de Antígeno , Antígenos de Diferenciación de Linfocitos B/análisis , Transporte Biológico , Enfermedad de Crohn/inmunología , Citocinas/análisis , Citocinas/genética , Modelos Animales de Enfermedad , Endosomas/ultraestructura , Células Epiteliales/inmunología , Células Epiteliales/ultraestructura , Antígenos de Histocompatibilidad Clase II/análisis , Íleon , Mucosa Intestinal/ultraestructura , Ratones , Ratones Noqueados , Microscopía Inmunoelectrónica , Ovalbúmina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Renal osteodystrophy is very common in hemodialysis (HD) patients. HD is a chronic inflammatory state. Studies in other pathological entities have shown an impact of chronic inflammation on bone metabolism. In the present study, the impact of chronic inflammation on bone turnover in HD patients was evaluated. PATIENTS AND METHODS: Thirty-three anuric HD patients free of other pathological conditions or medications that affect immune system or bone metabolism and 30 healthy volunteers enrolled into the study. Intact parathyroid hormone (iPTH), the markers of inflammation IL-6 and CRP, as well as the markers of bone turnover osteocalcin (OCN) and beta-isomerized C-terminal cross-linked peptide of collagen type I (beta-CTx) were measured in the serum. RESULTS: All evaluated factors were increased in HD patients. In the HD group, the serum marker of osteoblastic activity OCN was related inversely to patients' age (r = -0.469, p = 0.006), CRP (rho = -0.460, p = 0.007), and IL-6 (r = -0.485, p = 0.004) but positively to iPTH (r = 0.707, p < 0.001). Similarly, the serum marker of osteoclastic activity beta-CTx was related inversely to patients' age (r = -0.383, p = -0.028), CRP (rho = -0.466, p = 0.006), and IL-6 (r = -0.460, p = 0.007) but positively to iPTH (r = 0.657, p < 0.001). Multiple linear regression analysis revealed that IL-6 affects bone turnover independently of PTH and to the opposite direction. CONCLUSION: Chronic inflammation has a negative impact on bone turnover in HD patients. Certainly, further research and large clinical trials are needed for definite conclusions and for clarifying the exact molecular mechanisms implicated in the interaction between the immune system and bone metabolism in HD patients.
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Remodelación Ósea/fisiología , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/etiología , Inflamación/diagnóstico , Interleucina-6/sangre , Fallo Renal Crónico/complicaciones , Diálisis Renal/efectos adversos , Anciano , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Enfermedad Crónica , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/fisiopatología , Progresión de la Enfermedad , Femenino , Historia Antigua , Humanos , Inflamación/etiología , Mediadores de Inflamación/sangre , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/terapia , Modelos Lineales , Cuidados a Largo Plazo , Masculino , Persona de Mediana Edad , Análisis Multivariante , Probabilidad , Pronóstico , Diálisis Renal/métodos , Medición de Riesgo , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Análisis de SupervivenciaRESUMEN
BACKGROUND & AIMS: In the present work, we address the requirement for intestinal-specific homing molecules, the chemokine/chemokine receptor pair CCL25/CCR9 and beta7 integrin, in the pathogenesis of the CD8(+) T cell-dependent Tnf(DeltaARE) mouse model of Crohn's-like inflammatory bowel disease. METHODS: We investigated by flow cytometry lymphocyte recruitment in the intestinal epithelium and lamina propria (LP); cytokine production by intraepithelial and LP lymphocytes; and peripheral expression of CCR9, alpha4beta7, and alphaEbeta7 integrin. The functional significance of CCL25/CCR9 and beta7 integrin in inflammatory lymphocyte recruitment and intestinal disease development was assessed in Tnf(DeltaARE) mice genetically lacking these molecules. RESULTS: Intestinal inflammation in the Tnf(DeltaARE) mice is associated with early reduction of CD8alphaalpha-expressing intraepithelial lymphocytes, decreased T helper cell 1 and increased T helper cell 17 responses by LP CD4(+) lymphocytes, increased alphaEbeta7 integrin expression in peripheral activated/memory intestinal-homing CD8alphabeta lymphocytes, and predominance of tumor necrosis factor/interferon-gamma-producing CD8alphabeta lymphocytes in the epithelium. Although CCL25/CCR9 have been strongly implicated in T-lymphocyte recruitment to the small intestine, inflammatory pathology develops unperturbed in the genetic absence of CCL25/CCR9. Furthermore, CD8alphabeta lymphocyte recruitment in the intestinal epithelium and inflammatory infiltration in the LP are not impaired in CCR9- or CCL25-deficient Tnf(DeltaARE) mice. In contrast, genetic ablation of beta7 integrin results in complete amelioration of intestinal pathology. CONCLUSIONS: Our findings demonstrate that development of intestinal inflammation in the Tnf(DeltaARE) mice is critically dependent on beta7 integrin-mediated T-lymphocyte recruitment, whereas the function of the CCL25/CCR9 axis appears dispensable in this model.
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Linfocitos T CD8-positivos/inmunología , Quimiocinas CC/metabolismo , Quimiotaxis de Leucocito , Colon/inmunología , Enfermedad de Crohn/inmunología , Cadenas beta de Integrinas/metabolismo , Receptores CCR/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antígenos CD8/metabolismo , Quimiocinas CC/genética , Colon/patología , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Citometría de Flujo , Cadenas beta de Integrinas/genética , Integrinas/metabolismo , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores CCR/genética , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Patients suffering from Crohn's disease (CD) show increased incidence of low bone mineral density. Investigating this complication is difficult because the exact etiology of CD remains elusive. Mice carrying a deletion in the tumor necrosis factor (TNF) AU-rich elements (ARE) are reported as a model for human CD and are characterized by elevated TNF-alpha levels and inflammations in the terminal ileum. To evaluate whether these mice have a Ca(2+) handling problem, this study analyzed the Ca(2+) homeostasis in heterozygous TNF(DeltaARE) mice (TNF(DeltaARE/+)) in comparison to wildtype littermates. METHODS: Beside serum Ca(2+) and vitamin D levels, the expression of Ca(2+) transporters was analyzed in intestine, kidney and bone using quantitative real-time PCR, Western blot and immunohistochemistry. Bone scans were performed to measure bone parameters. RESULTS: Ca(2+) transporters in duodenum (TRPV6, calbindin-D(9K), PMCA1b) and kidney (TRPV5, calbindin-D(28K), NCX1) showed significantly reduced mRNA expression levels in TNP(DeltaARE/+) mice, except for renal TRPV5. In bone, only calbindin-D(9K) mRNA displayed a significant down-regulation. These findings were supported by declined duodenal calbindin-D(9K) and renal calbindin-D(28K) protein values. Likely, this down-regulation of Ca(2+) transporters in TNP(DeltaARE/+) mice is mediated by the 58 +/- 9% reduction in serum 1,25(OH)(2)D(3) levels. Diminished expression of Ca(2+) transporters combined with unchanged serum Ca(2+) levels assumes Ca(2+) loss from bone to compensate for the body's overall Ca(2+) shortage. Indeed, microcomputed tomography scanning demonstrated reduced trabecular and corticol bone thickness and volume in TNF(DeltaARE/+) mice. This finding is further supported by increased total deoxypyridinoline in serum. CONCLUSIONS: Our results imply that TNF(DeltaARE/+) mice have a disturbed Ca(2+) homeostasis characterized by reduced duodenal and renal Ca(2+) transporters, diminished 1,25(OH)(2)D(3) levels, and increased bone resorption associated with profound bone abnormalities.
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Calcio/metabolismo , Enfermedad de Crohn/metabolismo , Modelos Animales de Enfermedad , Mucosa Intestinal/metabolismo , Factor de Necrosis Tumoral alfa/genética , Animales , Resorción Ósea/metabolismo , Calcitriol/sangre , Enfermedad de Crohn/genética , Femenino , Homeostasis , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Tumor necrosis factor (TNF) is key to the pathogenesis of various arthritic diseases and inflammatory bowel disease (IBD). Anti-TNF therapies have proved successful in the clinical treatment of these diseases, but a mechanistic understanding of TNF function is still lacking. We have investigated early cellular mechanisms of TNF function in these diseases using an established TNF transgenic model, which develops a spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis, and Crohn's-like IBD. Bone marrow grafting experiments demonstrated that development of arthritis requires TNF receptor I (TNFRI) expression in the radiation-resistant compartment, which is also known to be a sufficient target of TNF in the development of Crohn's-like IBD in the same model. Early activation of synovial fibroblasts and intestinal myofibroblasts could also be demonstrated by perturbed expression of matrix metalloproteases and their inhibitors. Notably, selective Cre/loxP-mediated TNFRI expression in mesenchymal cells resulted in a fully arthritic-spondyloarthritic and intestinal phenotype, indicating that mesenchymal cells are primary and sufficient targets of TNF in these pathologies. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate early common cellular pathways that may also explain the often observed synovial-gut axis in human disease.
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Artritis/inmunología , Enfermedades Intestinales/inmunología , Células Madre Mesenquimatosas/inmunología , Factores de Necrosis Tumoral/inmunología , Animales , Artritis/patología , Trasplante de Médula Ósea , Células Cultivadas , Fibroblastos/inmunología , Íleon/patología , Inflamación/inmunología , Enfermedades Intestinales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Articulación Sacroiliaca/patología , Factores de Necrosis Tumoral/genéticaRESUMEN
Atmospheric input was studied and found to be the major source of PAHs in the eastern Mediterranean open marine ecosystem. Dry and wet atmospheric deposition, air-sea exchange, and sediment trap fluxes of polycyclic aromatic hydrocarbons (PAHs) in the eastern Mediterranean basin were estimated from November 2000 to July 2002. Seven dry and four wet deposition samples were analyzed in total and PAH concentrations were determined. Airsea exchange fluxes based on air-water concentration gradientwere drawn from five air and water samples collected concurrently from a coastal area in the eastern Mediterranean. Total annual average deposition fluxes of dry, wet, and air-sea exchange sigma35PAHs were 58.0, 165.7, and -706.4 microg m(-2) y(-1), respectively. Only 1.1 and 0.7% of the total atmospheric deposition flux of PAHs was measured in the sediment traps at 280 and 1440 m depth, respectively.
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Monitoreo del Ambiente/métodos , Contaminantes Ambientales , Sedimentos Geológicos , Aire , Movimientos del Aire , Atmósfera , Hidrocarburos/química , Región Mediterránea , Modelos Estadísticos , Hidrocarburos Policíclicos Aromáticos , Estaciones del Año , Agua , Movimientos del Agua , Contaminantes Químicos del AguaRESUMEN
p38 mitogen-activated protein kinases (MAPKs) are activated primarily in response to inflammatory cytokines and cellular stress, and inhibitors which target the p38alpha and p38beta MAPKs have shown potential for the treatment of inflammatory disease. Here we report the generation and initial characterization of a knockout of the p38beta (MAPK11) gene. p38beta-/- mice were viable and exhibited no apparent health problems. The expression and activation of p38alpha, ERK1/2, and JNK in response to cellular stress was normal in embryonic fibroblasts from p38beta-/- mice, as was the activation of p38-activated kinases MAPKAP-K2 and MSK1. The transcription of p38-dependent immediate-early genes was also not affected by the knockout of p38beta, suggesting that p38alpha is the predominant isoform involved in these processes. The p38beta-/- mice also showed normal T-cell development. Lipopolysaccharide-induced cytokine production was also normal in the p38beta-/- mice. As p38 is activated by tumor necrosis factor, the p38beta-/- mice were crossed onto a TNFDeltaARE mouse line. These mice overexpress tumor necrosis factor, which results in development symptoms similar to rheumatoid arthritis and inflammatory bowel disease. The progression of these diseases was not however moderated by knockout of p38beta. Together these results suggest that p38alpha, and not p38beta, is the major p38 isoform involved in the immune response and that it would not be necessary to retain activity against p38beta during the development of p38 inhibitors.
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Eliminación de Gen , Proteína Quinasa 11 Activada por Mitógenos/deficiencia , Proteína Quinasa 11 Activada por Mitógenos/metabolismo , Animales , Artritis/genética , Artritis/metabolismo , Artritis/patología , Diferenciación Celular , Células Cultivadas , Enfermedad Crónica , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Citocinas/biosíntesis , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Isoenzimas/genética , Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Transgénicos , Proteína Quinasa 11 Activada por Mitógenos/genética , Transducción de Señal/genética , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Escherichia coli heat-labile enterotoxin has unique immunogenic and adjuvant properties when administered mucosally to mice. These properties have revealed the potential for its use in the development of mucosal vaccines, an area of increasing interest. However, the inherent toxicity mediated by the A subunit precludes its widespread use. This problem has led to attempts to dissociate toxicity from adjuvant function by use of the B subunit. The ability of the B subunit of E. coli heat-labile enterotoxin (EtxB) to enhance responses against antigens coadministered intranasally is demonstrated here with the use of the DO11.10 adoptive-transfer model, in which ovalbumin (OVA)-specific adoptively transferred T cells can be monitored directly by flow cytometry. Intranasal delivery of OVA with EtxB resulted in increased T-cell proliferative and systemic antibody responses against antigens. The increased Th2 cytokine production detected following in vitro restimulation of splenocyte and cervical lymph node (CLN) cells from the immunized mice correlated with increased OVA-specific immunoglobulin G1 antibody production. Flow cytometric analysis of T cells from mice early after immunization directly revealed the ability of EtxB to support antigen-specific clonal expansion and differentiation. Furthermore, while responses were first detected in the CLNs, they rapidly progressed to the spleen, where they were further sustained. Examination of CD69 expression on dividing cells supported the notion that activation induced by the presence of antigens is not sufficient to drive T-cell differentiation. Furthermore, a lack of CD25 expression on dividing cells suggested that EtxB-mediated T-cell clonal expansion may occur without a sustained requirement for interleukin 2.
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Toxinas Bacterianas/inmunología , Diferenciación Celular/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli , Linfocitos T/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Antígenos de Superficie/inmunología , Toxinas Bacterianas/farmacología , Biomarcadores , División Celular/inmunología , Enterotoxinas/farmacología , Escherichia coli , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (Tnf(Delta)(ARE) mouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon gamma and requires the function of CD8(+) T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell-derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor-deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow-derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase-deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.