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1.
Allergol. immunopatol ; 47(5): 467-476, sept.-oct. 2019. tab, graf
Artigo em Inglês | IBECS | ID: ibc-186521

RESUMO

Background: House dust mite (Dermataphagoides pteronyssinus) is a widespread risk factor in the development of asthma. CD4+ T lymphocytes have an important role in the pathogenesis of allergic asthma by polarizing to Th2 cells. Objective: We aimed to evaluate the immunoregulatory effects of dental follicle mesenchymal stem cells with and without IFN-γ stimulation on peripheral blood mononuclear cells of house dust mite sensitive asthmatic patients, and compared those with Dexamethasone as a systemic steroid. Material and methods: PBMC of asthmatic patients and healthy individuals separately cultured with or without DF-MSCs in the presence and absence of IFN-γ or Der p1 or Dexamethasone for 72h. CD4+ T proliferation, cell viability, CD4+CD25+FoxP3+ Treg cell frequency and cytokine profiles of PBMC were evaluated via flow cytometry. Results: DF-MSCs suppressed proliferation of CD4+ T lymphocytes (pCDmix < 0.01, pDerp1 < 0.01, pIFN < 0.005) by increasing the number of FoxP3 expressing CD4 + CD25 + T regulatory cells (pCDmix < 0.005, pDerp1 < 0.01, pIFN < 0.001) and suppressed lymphocyte apoptosis (pCDmix < 0.05, pDerp1< 0.05, pIFN < 0.05), while Dexamethasone increased the apoptosis and decreased Treg cell frequency in asthmatic patients. IFN-γ stimulation increased the suppressive effect of DF-MSCs and also enhanced the frequency of FoxP3 expressing CD4+CD25 + T regulatory cells. The cytokine levels were regulated by DF-MSCs by reducing IL-4 cytokine levels (pCDmix < 0.01, pDerp1 < 0.05, pIFN < 0.05) and upregulating IFN-γ levels (pCDmix < 0.01, pDerp1< 0.05, pIFN < 0.005) in asthmatic patients. Conclusion: IFN-γ stimulated DF-MSCs were found to have a high modulatory effect on CD4 + T cell responses, while Dexamethasone had an apoptotic effect on CD4+ T cells in asthmatic patients. DF-MSCs may be a new cell-based therapy option for allergic diseases including asthma


No disponible


Assuntos
Humanos , Animais , Masculino , Feminino , Adulto Jovem , Adulto , Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Saco Dentário/patologia , Dermatophagoides pteronyssinus/imunologia , Interferon gama/imunologia , Células-Tronco Mesenquimais/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Células Cultivadas , Imunidade Celular , Imunização
2.
An Real Acad Farm ; 85(2): 189-197, abr.-jun. 2019. graf
Artigo em Inglês | IBECS | ID: ibc-186176

RESUMO

Previous studies have shown a key role of microglial cells in the neuroinflammatory processes associated with some neurodegenerative diseases, such as Alzheimer’s disease (AD). Microglia sense several types of diffusible molecules that regulate the multiple repertoire of microglial functions. Among them, extracellular nucleotides, acting on microglial P2 receptors, have central roles. In this sense, the ionotropic P2X7 receptor has gained recognition as a key regulator of microglial-mediated inflammatory responses. It is known that microglia releases ATP and other nucleotides to the extracellular medium. Although several mechanisms, such as release trough conexins or panexins, has been proposed, a vesicular origin for this released nucleotides, relying on the activity of the vesicular nucleotide transporter (VNUT), cannot be ruled out. In this work we evaluated whether the expression of VNUT and the P2X7 receptor, as well as the ATP release, could be modified in the reactive microglia. To achieve microglia activation we stimulated the cells with the lipopolysaccharide (LPS). Moreover, we analyzed the effect of the b-amyloid peptide b1-42, which is also able to activate the microglial cells, on the expression of VNUT and the ATP release in the microgli


Estudios previos han mostrado un papel clave de las células microgliales en los procesos neuroinflamatorios asociados con algunas enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA). La microglía detecta varios tipos de moléculas difusibles que regulan el múltiple repertorio de funciones microgliales. Entre ellos, los nucleótidos extracelulares, actuando sobre los receptores P2 microgliales, llevan a cabo un papel central. En este sentido, el receptor P2X7 ionotrópico ha sido reconocido como un regulador clave de las respuestas inflamatorias mediadas por la microglia. Se sabe que la microglía libera ATP y otros nucleótidos al medio extracelular. Aunque se han propuesto varios mecanismos, tales como la liberación a través de conexinas o panexinas, no se puede descartar un origen vesicular para estos nucleótidos liberados, basándose en la actividad del transportador vesicular de nucleótidos (VNUT). En este trabajo hemos analizado si la expresión de VNUT y el receptor P2X7, así como la liberación de ATP, podrían modificarse en la microglía reactiva. Para lograr la activación de la microglía estimulamos las células con el lipopolisacárido (LPS). Además, analizamos el efecto del péptido (R)1-amiloide, (R)1-42, que puede activar también las células microgliales, sobre la expresión de VNUT y la liberación de ATP en la microglía


Assuntos
Humanos , Peptídeos beta-Amiloides/fisiologia , Microglia/metabolismo , Microglia/patologia , Degeneração Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Reação em Cadeia da Polimerase em Tempo Real , Western Blotting , Imunofluorescência , Células Cultivadas , Transdução de Sinais
3.
Allergol. immunopatol ; 47(3): 234-240, mayo-jun. 2019. graf, tab
Artigo em Inglês | IBECS | ID: ibc-186483

RESUMO

Introduction and objectives: Allergic rhinitis (AR) is a classic Th2-mediated disease, with important contributions to the pathology of interleukins 4, 5, and 13. The co-stimulatory molecule of OX40 and its ligand interaction participate in the immune response by regulation of Th1/Th2 cells balance. Considering the paucity of information on the relation between OX40 ligand (OX40L) and AR, this study aimed to examine its expression on B lymphocytes. Patients and methods: This case-control study consisted of 20 AR patients and 20 healthy subjects. The serum level of total immunoglobulin E (IgE) was measured using the electro-chemiluminescence (ECL) technology. The percentage of B-lymphocytes expressing OX40L was assessed by flow cytometry. The amounts of IL-4 in CD4+ T cells culture supernatant was also measured by the enzyme-linked immunosorbent assay (ELISA). Results: OX40L expression on B lymphocytes of patients was significantly higher than the control group (44.32 ± 19.21% vs. 2.79 ± 2.48% respectively, p < 0.001). In AR patients, OX40L expression correlated positively with the levels of serum total IgE and IL-4 produced by CD4+ T lymphocytes (p < 0.01 - p < 0.05) respectively. Conclusions: Collectively, the findings of this work suggest that there is a relationship between the OX40L expression level on B lymphocytes and allergic markers such as IgE and IL-4 in patients with allergic rhinitis


No disponible


Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Linfócitos B/imunologia , Biomarcadores/sangue , Imunoglobulina E/sangue , Interleucina-4/sangue , Ligante OX40/metabolismo , Rinite Alérgica/imunologia , Células Th2/imunologia , Antígenos CD4/metabolismo , Estudos de Casos e Controles , Regulação para Cima , Células Cultivadas
4.
Allergol. immunopatol ; 47(2): 172-178, mar.-abr. 2019. graf, tab
Artigo em Inglês | IBECS | ID: ibc-180806

RESUMO

Background: Common variable immunodeficiency (CVID) is the most common symptomatic form of primary immunodeficiency (PID). LPS-responsive beige-like anchor protein (LRBA) deficiency is an autosomal recessive disease characterized by a CVID-like phenotype. T cell abnormality was reported in patients with CVID and LRBA deficiency. The study's aim was to evaluate IL-4, IL-5, IL-10 and GATA3 expression in patients with LRBA deficiency and CVID with no known monogenic disease, and further evaluate its relevance with immunological futures and clinical complications of patients. Methods: The study population comprised patients with CVID, LRBA deficiency and age-sex matched healthy controls. Mutation analysis was done by whole exome sequencing in CVID patients to rule out monogenic PIDs. After CD4+ T cell stimulation with anti-CD3 and anti-CD28 monoclonal antibodies, gene expression of IL-4, IL-5, IL-10 and transcription factor GATA3 was evaluated by real-time polymerase chain reaction. The protein of mentioned cytokines was assessed by enzyme-linked immunosorbent assay. Results: The main clinical presentations of CVID patients were infections only and lymphoproliferations phenotypes, but in LRBA patients were autoimmune and enteropathy phenotype. The frequencies of CD4+ T cells were significantly reduced in LRBA and CVID patients. There were no statistically significant differences among GATA3, IL4, and IL5 gene expressions by CD4+ T cells of patients and controls, however, the IL10 expressions in CVID patients was significantly lower than in LRBA patients and HCs. As compared with HCs, CVID patients showed a prominent decrease in IL-4 and IL-10 production by CD4+ T cells. Conclusions: Our findings demonstrated that patients with CVID and LRBA deficiency (even with severe infectious and inflammatory complications) have not imbalance in Th2 response, which is in parallel with lower frequency of allergy and asthma in these patients


No disponible


Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto Jovem , Adulto , Proteínas Adaptadoras de Transdução de Sinal/genética , Linfócitos T CD4-Positivos/fisiologia , Imunodeficiência de Variável Comum/genética , Fator de Transcrição GATA3/genética , Interleucina-10/genética , Interleucina-4/genética , Interleucina-5/genética , Autoimunidade , Análise Mutacional de DNA , Células Cultivadas , Análise Mutacional de DNA , Progressão da Doença
5.
Allergol. immunopatol ; 47(2): 185-193, mar.-abr. 2019. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-180808

RESUMO

Introduction: Asthma is a chronic inflammatory disease of the airways. In this study, we evaluated the anti-inflammatory effects of myrtenol on the inflammatory indices in the pulmonary parenchyma and airways and on the inflammatory and oxidative indices of the bronchoalveolar lavage fluid (BALF) of asthmatic rats. Methods: The allergic asthma was induced by sensitization (two weeks) followed by the inhalation of ovalbumin (four weeks). Animals were divided into two main groups: (1) Histopathology, and (2) measurement of inflammatory and oxidative biomarkers in the BALF. Each main group was subdivided into four subgroups: Control, Asthma, Asthma+Dexamethasone and Asthma + Myrtenol. (-)-Myrtenol (50 mg/kg) or Dexamethasone (2.5 mg/kg) was administered intraperitoneally once a day for one week, at the end of the inhalation period. On day 50, lung histopathologic parameters and inflammatory indices in BALF including INF-gamma, IL-10, IL-1Beta, and TNF-alfa and oxidative stress biomarkers (MDA, SOD, and GPX) were measured. Result: In the Asthma group, leukocyte infiltration, the thickness of smooth muscle and epithelium of airways wall and the number of goblet cells increased. Myrtenol reduced all of the above-mentioned indices except the epithelium thickness. It also inhibited the increase in BALF IL-1Beta, TNF-alfa and MDA and increased the levels of INF-gamma, IL-10 and SOD. Conclusion: Our results suggest that myrtenol reduced damage caused by experimental asthma by reducing the inflammatory indices, normalizing the level of interleukins and balancing oxidative stress in the lungs. It also prevented airway remodeling. Myrtenol may be suggested as a potent herbal medicine for the treatment of allergic asthma


No disponible


Assuntos
Humanos , Animais , Masculino , Remodelação das Vias Aéreas/efeitos dos fármacos , Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Células Caliciformes/patologia , Leucócitos/imunologia , Pulmão/imunologia , Mucosa Respiratória/patologia , Monoterpenos/uso terapêutico , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Ratos Wistar , Mucosa Respiratória/efeitos dos fármacos
6.
Rev. osteoporos. metab. miner. (Internet) ; 11(1): 12-18, mar. 2019. graf
Artigo em Espanhol | IBECS | ID: ibc-184080

RESUMO

La fuerza mecánica es importante para el modelado, el remodelado y la regeneración ósea; estimula a los osteocitos provocando una alteración en la producción y secreción de moléculas de señalización que regulan la actividad de los osteoblastos y los osteoclastos. El objetivo del presente estudio fue evaluar el efecto del medio condicionado de células osteocíticas de ratón estimuladas mecánicamente sobre la capacidad proliferativa y migratoria de células mesenquimales y células óseas. Para ello, se analizó la proliferación y migración de las células preosteoblásticas de ratón, células mesenquimales preadiposas humanas y macrófagos de ratón en presencia del medio condicionado de las células osteocíticas, tras 6 y 24 horas después de ser sometidas a un estrés mecánico de 10 dinas/cm2 por flujo de fluido (FF) durante 10 minutos. Se encontró que la migración de células preosteoblásticas aumentó significativamente en presencia de medios condicionados de células osteocíticas con respecto al grupo control estático (SC) (SC=12,63±5,44; FF6h=23,03±11,57; FF24h=29,72±15,76; p<0,0001). De la misma manera, las células preadiposas también incrementaron significativamente su migración en presencia de dichos medios condicionados (SC=11,48±4,75; FF6h=18,43±9,94; FF24h=18,80±10,03; p≤0,0007). Sin embargo, la migración de los macrófagos disminuyó en presencia del medio condicionado recogido a las 24 horas con respecto al grupo control estático (SC=69±22,71; FF24h=26,57±5,47; p<0,0001). Estos efectos se asociaron con la disminución de la expresión proteica de ciertas quimioquinas, como la proteína quimiotáctica de monocitos de tipo I (SC=0,25±0,06; FF24h=0,09±0,005; p=0,0262), la proteína del grupo I de alta movilidad (SC=0,25±0,04; FF24h=0,15±0,05; p=0,0159) y la proteína reguladora de la activación de linfocitos T y monocitos (SC=3,29±0,88; FF6h=1,33±1,09; FF24h=0,97±0,66; p≤0,0314), por parte de los osteocitos en presencia de estímulo mecánico con respecto al grupo control estático. En conclusión, este estudio in vitro demuestra que la mecanotransducción de los osteocitos potencia el reclutamiento de osteoblastos y células mesenquimales preadiposas mientras que reduce la migración de los macrófagos


Mechanical force is important for modeling, remodeling and bone regeneration. It stimulates the osteocytes, causingan alteration in the production and secretion of signaling molecules that regulate osteoblast and osteoclast activity.The present study aims to evaluate the effect of the conditioned medium of mechanically stimulated mouse osteocyticcells on the proliferative and migratory capacity of mesenchymal cells and bone cells. For this, the proliferation andmigration of mouse pre‐osteoblastic cells, human pre‐adult mesenchymal cells and mouse macrophages in the pre‐sence of the conditioned medium of osteocytic cells were analyzed, after 6 and 24 hours after being subjected to amechanical stress of 10 dynes/cm2by fluid flow (FF) for 10 minutes. The migration of pre‐osteoblastic cells has beenfound to increase significantly in the presence of conditioned media of osteocytic cells compared to the static controlgroup (SC)(SC=12.63±5.44, FF6h=23.03±11.57, FF24h=29.72±15.76, p<0.0001). In the same way, the pre‐adipose cellsalso significantly increased their migration in the presence of this conditioned media (SC=11.48±4.75, FF6h=18.43±9.94,FF24h=18.80±10.03; p≤0.0007). However, macrophage migration decreased in the presence of the conditioned mediumcollected at 24 hours with respect to the static control group (SC=69±22.71, FF24h=26.57±5.47, p<0.0001). Theseeffects were associated with decreased protein expression of certain chemokines, such as the monocyte chemotacticprotein type I (SC=0.25±0.06, FF24h=0.09±0.005, p=0.0262), the protein of group I of high mobility (SC=0.25±0.04,FF24h=0.15±0.05, p=0.0159) and the regulatory protein of the activation of T lymphocytes and monocytes(SC=3.29±0.88, FF6h=1.33±1.09, FF24h=0.97±0.66, p≤0.0314), by the osteocytes in the presence of mechanical stimu‐lation with respect to the static control group. In conclusion, this in vitro study demonstrates that osteocyte mechano‐transduction enhances recruitment of osteoblasts and pre‐adipose mesenchymal cells while reducing the migration ofmacrophages


Assuntos
Humanos , Mecanotransdução Celular/fisiologia , Osteócitos/fisiologia , Proliferação de Células , Movimento Celular , Células-Tronco Mesenquimais/fisiologia , Células Cultivadas
7.
Allergol. immunopatol ; 47(1): 38-42, ene.-feb. 2019. graf
Artigo em Inglês | IBECS | ID: ibc-180769

RESUMO

Introduction: Disseminated BCG infections among other complications of Bacillus Calmette-Guérin (BCG) vaccine are rare and have occurred in children with immunodeficiency disorders such as mendelian susceptibility to mycobacterial disease (MSMD) which could be due to defects in some elements of IL-12/IFN-γ axis. MSMD-causing mutations have been identified in 10 genes during the last two decades. Among them, mutations in the IL12Rβ1 and IFN gamma R1 genes constitute about 80% of recorded cases of MSMD syndrome. The aim of this study was to investigate IL-12RBeta1 and IFN- gammaR1 deficiencies in patients with disseminated BCG infection. Methods: This study was performed on 31 children with disseminated BCG infections who referred to children's medical center. Whole blood cell culture was performed in presence of BCG, IL-12 and IFN- gamma stimulators. The supernatants were assayed for IFN-gamma and IL-12p70 by ELISA method. In order to evaluate IL12Rbeta1 and IFN- gammaR1 receptors expression, flow cytometry staining was performed on the patients’ T-cells stimulated with PHA. Results: Flow cytometry staining of 31 Iranian patients with disseminated BCG infections with the average age of 43 months showed lack of the expression of IL-12RBeta1 and IFN- gamma R1 genes in PHA-T-cells of the nine and one patients, respectively in whom the incomplete production of IFN- gamma and IL-12 was reported by ELISA. Among these 10 patients, eight cases had related parents (80%). Conclusion: It is recommended that to avoid BCG complications, screening be performed for MSMD before BCG inoculation in individuals with positive family history of primary immunodeficiency diseases and inhabitants of areas with high frequency of consanguinity


No disponible


Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Vacina BCG/imunologia , Síndromes de Imunodeficiência/epidemiologia , Mutação/genética , Infecções por Mycobacterium/epidemiologia , Receptores de Interferon/genética , Interleucina-12/genética , Linfócitos T/imunologia , Células Cultivadas , Predisposição Genética para Doença , Imunização , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Interferon gama/metabolismo , Interleucina-12/metabolismo , Irã (Geográfico)/epidemiologia , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia
8.
Av. odontoestomatol ; 34(6): 299-309, nov.-dic. 2018. ilus, graf
Artigo em Espanhol | IBECS | ID: ibc-182274

RESUMO

Introducción: Los fibroblastos gingivales (FGs) son células responsables del mantenimiento de la homeostasis, cicatrización y tolerancia inmunitaria del tejido conjuntivo gingival. Por sus características fisiológicas, los FGs pueden ser un candidato en la terapia celular. Sin embargo, la conservación de su fenotipo en cultivo celulares requiere de condiciones estrictamente definidas. Una de ellas es la concentración de Suero Fetal Bovino (SFB). Nuestro objetivo es evaluar el efecto concentración-dependiente de la suplementación de SFB en el medio de cultivo sobre el comportamiento, crecimiento, proliferación y supervivencia de los fibroblastos gingivales humanos. Materiales y métodos: FGs fueron cultivados con DMEM (Dulbecco's modification of Eagle médium) y concentraciones de 0%, 0.5% y 10% de SFB durante tres semanas. Análisis morfológico, de proliferación e inmunohistoquímicos fueron llevados a cabo en el presente trabajo. Se llevó a cabo un análisis factorial de varianza (ANOVA) por medio el software R Project. Los resultados fueron considerados significativos con un valor de p<0,05. Resultados y Discusión: Los FGs cultivados con 10% de SFB alcanzaron un desarrollo morfológico más notorio y en menor tiempo comparados con los FGs cultivados a 0.5% y 0% de SFB. La efectividad de la concentración del SFB 0,5% fue mucho más alta en relación a la concentración del SFB 10%. La inmunodetención de la actina, vimentina y fibronectina fueron más notorias en los FGs tratados con 10% y 0.5% de SFB. Este estudio concluye que los FGs humanos presentan una mejor capacidad de supervivencia, desarrollo y proliferación cuando son cultivados en presencia de SFB


Introduction: Gingival fibroblasts (FGs) are cells responsible for the maintenance of homeostasis, healing and immune tolerance of the gingival connective tissue. Due to their physiological characteristics, FGs can be a candidate in cell therapy. However, the conservation of their phenotype in cell culture requires strictly defined conditions. One of them is the concentration of Bovine Fetal Serum (FBS). Our aim is to evaluate the concentration-dependent effect of SFB supplementation in the culture medium on the behavior, growth, proliferation and survival of human gingival fibroblasts. Materials and methods: FGs were cultured with DMEM (Dulbecco's modification of Eagle medium) and concentrations of 0%, 0.5% and 10% of SFB for three weeks. Morphological, proliferation and immunohistochemical analyzes were carried out in the present work. A factorial analysis of variance (ANOVA) was carried out using the R Project software. The results were considered significant with a value of p <0.05. Results and discussion: The FGs grown with 10% FBS reached a more noticeable morphological development and in a shorter time compared to the FGs grown at 0.5% and 0% FBS. The effectiveness of the concentration of the SFB 0.5% was much higher in relation to the concentration of the SFB 10%. The immunodetention of actin, vimentin and fibronectin were more evident in the FGs treated with 10% and 0.5% FBS. This study concludes that human FGs have a better capacity for survival, development and proliferation when they are grown in the presence of FBS


Assuntos
Humanos , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Gengiva/citologia , Proliferação de Células , Fibroblastos/efeitos dos fármacos , Células Cultivadas , Tecido Conjuntivo/fisiologia , Análise de Variância , Imuno-Histoquímica , Fotomicrografia , Citoesqueleto
9.
Rev. osteoporos. metab. miner. (Internet) ; 10(4): 119-124, nov.-dic. 2018. ilus, graf
Artigo em Espanhol | IBECS | ID: ibc-178637

RESUMO

Objetivo: En las últimas décadas se han identificado genes asociados a la masa ósea y al riesgo de fractura osteoporótica, varios de los cuales pertenecen a la vía de Wnt. En este proyecto se estudió la funcionalidad de 7 mutaciones de cambio de sentido del gen DKK1 -un inhibidor de la vía de Wnt- presentes en la población general. Material y métodos: Se realizaron estudios in vitro del gen reportero luciferasa para medir la actividad de la vía de Wnt en presencia o ausencia de DKK1 silvestre o mutada, y estudios de western blot, para evaluar si las distintas mutaciones afectan a su síntesis y/o a su estabilidad. Resultados: La proteína DKK1 con la variante p.Ala41Thr presenta menor actividad inhibidora de la vía en comparación con la proteína silvestre. También se observaron diferencias significativas entre los experimentos realizados en ausencia de DKK1 y los que incluyen DKK1 con la mutación p.Ala41Thr. Los western blots mostraron que la cantidad de proteína era similar para todas las variantes, tanto las mutadas como la silvestre, por lo que la pérdida de actividad de p.Ala41Thr no parecía deberse a falta de proteína. El resto de las mutaciones no presentaron un comportamiento diferente al de la proteína DKK1 silvestre. Conclusiones: La variante de cambio de sentido p.Ala41Thr de la proteína DKK1, con una frecuencia poblacional de 0,013%, presenta una pérdida parcial de su función inhibidora, que no es debida a la falta de expresión de ésta. Esta variante génica podría conllevar un aumento de la densidad mineral ósea en las personas de la población general portadoras de esta mutación


Objective: In recent decades, genes associated with bone mass and osteoporotic fracture risk have been identified, several of which belong to the Wnt pathway. In this project, the functionality of 7 missense mutations of the gene DKK1-an inhibitor of the Wnt pathway- present in the general population was studied. Material and methods: In vitro studies of the luciferase reporter gene were carried out to measure Wnt pathway activity in the presence or absence of wild-type or mutated DKK1, and western blot studies, to evaluate if the different mutations affect its synthesis and/or stability. Results: The DKK1 protein with the p.Ala41Thr variant shows lower pathway inhibitory activity compared to the wild-type protein. Significant differences were also observed between the experiments performed in the absence of DKK1 and those that include DKK1 with the p.Ala41Thr mutation. Western blots showed that the amount of protein was similar for all variants, both mutated and "wild-type, so the loss of p.Ala41Thr activity did not seem to be due to a lack of protein. The rest of the mutations did not show different behavior from that of the wild DKK1 protein. Conclusions: The missense variant p.Ala41Thr of the DKK1 protein, with a population frequency of 0.013%, shows a partial loss of its inhibitory function, which is not due to the lack of expression. This gene variant could lead to an increase in bone mineral density in those people in the general population who carry this mutation


Assuntos
Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Via de Sinalização Wnt/genética , Mutação/genética , Western Blotting , Células Cultivadas , Fenótipo
10.
Gastroenterol. hepatol. (Ed. impr.) ; 41(8): 490-497, oct. 2018. graf
Artigo em Inglês | IBECS | ID: ibc-178102

RESUMO

Background: The "secondary injury" theory of liver failure indicated that hyperammonaemia due to liver failure causes further deterioration of hepatocytes. Our previous studies have demonstrated that high blood ammonia levels may lead to hepatocyte apoptosis, as NH4Cl loading caused metabolic acidosis and an increase in sodium-hydrogen exchanger isoform 1 (NHE1). In this study, we established a hyperammonia hepatocyte model to determine the role of NHE1 in the regulation of hepatocyte apoptosis induced by NH4Cl. Materials and methods: In current studies, intracellular pH (pHi) and NHE1 activity were analyzed using the pHi-sensitive dye BCECF-AM. The results showed that intracellular pH dropped and NHE1 activity increased in hepatocytes under NH4Cl treatment. As expected, decreased pHi induced by NH4Cl was associated with increased apoptosis, low cell proliferation and ATP depletion, which was exacerbated by exposure to the NHE1 inhibitor cariporide. We also found that NH4Cl treatment stimulated PI3K and Akt phosphorylation and this effect was considerably reduced by NHE1 inhibition. Conclusion: This study highlighted the significant role of NHE1 in the regulation of cell apoptosis induced by hyperammonaemia


Antecedentes: La teoría de la «lesión secundaria» de la insuficiencia hepática mostró que la hiperamoniaquemia provocada por la insuficiencia hepática causa mayor deterioro de los hepatocitos. Nuestros anteriores estudios previos han demostrado que los niveles altos de amoníaco en sangre pueden conducir a la apoptosis de los hepatocitos. Como la carga de NH4Cl provocó acidosis metabólica y un aumento de la isoforma 1 del intercambiador de sodio/hidrógeno (NHE1). En este estudio, establecimos un modelo de hepatocitos de hiperamonia para establecer el papel de NHE1 en la regulación de la apoptosis de hepatocitos inducida por NH4Cl. Materiales y métodos: En los estudios actuales, el pH intracelular (pHi) y la actividad del NHE1 se analizaron con el colorante BCECF-AM, sensible al pHi. Los resultados mostraron que el pH intracelular disminuyó y la actividad del NHE1 aumentó en hepatocitos con tratamiento del NH4Cl. Como se esperaba, la disminución del pHi inducido por NH4Cl se relacionó con un aumento de la apoptosis, baja proliferación celular y reducción del ATP, que se exacerbó por la exposición a cariporide, inhibidor del NHE1. También encontramos que el tratamiento del NH4Cl estimuló la fosforilación de PI3K y Akt, y este efecto se redujo considerablemente por la inhibición del NHE1. Conclusión: Este trabajo ha destacado el importante papel del NHE1 en la regulación de la apoptosis celular inducida por hiperamoniaquemia


Assuntos
Humanos , Cloreto de Amônio/farmacologia , Apoptose , Hepatócitos/metabolismo , Hiperamonemia/metabolismo , /fisiologia , Trifosfato de Adenosina/biossíntese , Células Cultivadas , Guanidinas/farmacologia , Hepatócitos/citologia , Hepatócitos , Líquido Intracelular , Concentração de Íons de Hidrogênio , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sulfonas/farmacologia , /antagonistas & inibidores
11.
Neurología (Barc., Ed. impr.) ; 33(4): 211-223, mayo 2018. tab, graf
Artigo em Espanhol | IBECS | ID: ibc-175837

RESUMO

INTRODUCCIÓN: La exposición de líquido cefalorraquídeo (LCR) de pacientes con esclerosis lateral amiotrófica (ELA) induce efectos citotóxicos en cultivos celulares de neuronas motoras in vitro. MATERIAL Y MÉTODOS: Se seleccionó LCR de 32 pacientes con ELA que previamente habían demostrado efectos citotóxicos. Se implantaron con minibombas osmóticas intracerebroventriculares (ICV) en 28 ratas macho adultas y se dividieron en 3 grupos: 9 ratas de LCR de pacientes no-ELA, 15 ratas de ELA-LCR citotóxico y 4 ratas de una solución salina fisiológica. El LCR se administró por vía ICV de forma continua durante periodos de 20 o 43 días. Se realizó la evaluación clínica, electromiográfica y análisis de tejidos después de sacrificio a los 20, 45 y 82 días tras la cirugía. RESULTADOS: Los estudios inmunohistoquímicos muestran daño en los tejidos con características similares a las encontradas en formas esporádicas de ELA, tales como sobre expresión de cistatina C, transferrina y la proteína en el TDP-43 citoplasmática. Los primeros cambios observados parecían jugar un papel protector por la sobreexpresión de periferina, panAKT, fosfoAKT y metalotioneínas; esta expresión habría disminuido al momento de analizar las ratas que se sacrificaron al día 82, en el que hay un aumento de apoptosis. Los primeros cambios celulares identificados fueron la constatación de activación de la microglía seguido por astrogliosis con sobreexpresión de GFAP y proteína S100B. CONCLUSIONES: Nuestros datos parecen indicar que la ELA podría propagarse a través del LCR, y que la administración ICV de ELA-LCR citotóxico produce cambios similares a los encontrados en las formas esporádicas de la enfermedad


INTRODUCTION: Cerebrospinal fluid (CSF) from amyotrophic lateral sclerosis (ALS) patients induces cytotoxic effects in in vitro cultured motor neurons. MATERIAL AND METHODS: We selected CSF with previously reported cytotoxic effects from 32 ALS patients. Twenty-eight adult male rats were intracerebroventricularly implanted with osmotic mini-pumps and divided into 3 groups: 9 rats injected with CSF from non-ALS patients, 15 rats injected with cytotoxic ALS-CSF, and 4 rats injected with a physiological saline solution. CSF was intracerebroventricularly and continuously infused for periods of 20 or 43days after implantation. We conducted clinical assessments and electromyographic examinations, and histological analyses were conducted in rats euthanised 20, 45, and 82days after surgery. RESULTS: Immunohistochemical studies revealed tissue damage with similar characteristics to those found in the sporadic forms of ALS, such as overexpression of cystatin C, transferrin, and TDP-43 protein in the cytoplasm. The earliest changes observed seemed to play a protective role due to the overexpression of peripherin, AKTpan, AKTphospho, and metallothioneins; this expression had diminished by the time we analysed rats euthanised on day 82, when an increase in apoptosis was observed. The first cellular changes identified were activated microglia followed by astrogliosis and overexpression of GFAP and S100B proteins. CONCLUSION: Our data suggest that ALS could spread through CSF and that intracerebroventricular administration of cytotoxic ALS-CSF provokes changes similar to those found in sporadic forms of the disease


Assuntos
Humanos , Animais , Masculino , Adulto , Ratos , Esclerose Amiotrófica Lateral/líquido cefalorraquidiano , Cérebro/patologia , Líquido Cefalorraquidiano/metabolismo , Infusões Intraventriculares , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/patologia , Líquido Cefalorraquidiano/química , Citotoxinas/farmacologia , Modelos Animais , Neurônios Motores/citologia , Neurônios Motores , Neurônios Motores/metabolismo , Células Cultivadas
12.
J. physiol. biochem ; 74(2): 207-221, mayo 2018. graf, tab
Artigo em Inglês | IBECS | ID: ibc-178978

RESUMO

The dissociated dorsal root ganglion (DRG) neurons with or without culture were widely used for investigation of their electrophysiological properties. The culture procedures, however, may alter the properties of these neurons and the effects are not clear. In the present study, we recorded the action potentials (AP) and the voltage-gated Na+, K+, and Ca2+ currents with patch clamp technique and measured the mRNA of Nav1.6-1.9 and Cav2.1-2.2 with real-time PCR technique from acutely dissociated and 1-day (1-d) cultured DRG neurons. The effects of the nerve growth factor (NGF) on the expression of Nav1.6-1.9 and Cav2.1-2.2 were evaluated. The neurons were classified as small (DRG-S), medium (DRG-M), and large (DRG-L), according to their size frequency distribution pattern. We found 1-d culture increased the AP size but reduced the excitability, and reduced the voltage-gated Na+ and Ca2+ currents and their corresponding mRNA expression in all types of neurons. The lack of NGF in the culture medium may contribute to the reduced Na+ and Ca2+ current, as the application of NGF recovered some of the reduced transcripts (Nav1.9, Cav2.1, and Cav2.2). 1-d culture showed neuron-type specific effects on some of the AP properties: it increased the maximum AP depolarizing rate (MDR) and hyperpolarized the resting membrane potential (RP) in DRG-M and DRG-L neurons, but slowed the maximum AP repolarizing rate (MRR) in DRG-S neurons. In conclusion, the 1-d cultured neurons had different properties with those of the acutely dissociated neurons, and lack of NGF may contribute to some of these differences


No disponible


Assuntos
Animais , Feminino , Ratos , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Neurônios/fisiologia , Potenciais de Ação , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Células Cultivadas , Fator de Crescimento Neural , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , RNA Mensageiro/genética , Ratos Sprague-Dawley
13.
Endocrinol. diabetes nutr. (Ed. impr.) ; 65(4): 200-205, abr. 2018. graf
Artigo em Inglês | IBECS | ID: ibc-172150

RESUMO

Introduction: Vascular endothelial growth factor (VEGF) plays an essential role in development of diabetic macular edema (DME). While there is evidence suggesting that silymarin, a flavonoid extracted from Silybum marianum, could be useful for prevention and treatment of diabetic nephropathy, no studies have been conducted in diabetic retinopathy (DR). The aim of this study was to assess the effect of silymarin on disruption of inner blood retinal barrier (BRB), the primary cause of DME. Materials and methods: Human retinal endothelial cells (HRECs) were cultured under standard (5.5mM D-glucose) and diabetogenic conditions (25mM D-glucose and 25mM D-glucose + recombinant vascular endothelial growth factor [rVEGF, 25mg/mL]). To assess cell viability, three concentrations of silymarin were tested (2, 4 and 10μg/mL). The effect of silymarin on HREC disruption was determined using a dextran (70kD) permeability asssay. Results: No differences were found in the viability of HRECs treated with 2 or 4μg/mL of silymarin as compared to untreated cells, but viability significantly decreased after using 10 μg/mL. The concentration of 4 μg/mL was therefore selected. Silymarin (4μg/mL) caused a significant decrease in VEGF-induced permeability in both media with 5.5nM (422±58 vs. 600±72 ng/mL/cm2; p<0.03) and 25nM of D-glucose (354 ± 28 vs. 567 ± 102 ng/mL/cm2; p<0.04). Discussion: Our results show that silymarin is effective for preventing hyperpermeability induced by diabetic conditions in HRECs. Further studies are needed to assess whether silymarin could be useful to treat DME (AU)


Introducción: El Vascular endothelial growth factor (VEGF) juega un papel esencial en el desarrollo del edema macular diabético (EMD). Existe evidencia que indica que el uso de la silimarina, extracto flavonoide del Silybum marianum, podría ser útil en la prevención y el tratamiento de la nefropatía diabética pero no se dispone de datos en retinopatía diabética (RD). El objetivo del estudio es evaluar el efecto de la silimarina sobre la disrupción de la barrera hematorretininana, que es la causa primaria del EMD. Material y métodos: Células endoteliales de retina humana (HRECs) se cultivaron en condiciones estándar (5.5mM de D-glucosa) y en condiciones suprafisiológicas de glucosa (25mM de D-glucosa y 25mM de D-glucosa + VEGF 25mg/dl). Para evaluar la viabilidad de las células se probaron 3 concentraciones de silimarina (2, 4 y 10μg/ml). El efecto de la silimarina sobre la disrupción de las HRECs se determinó mediante análisis de permeabilidad a dextrano (70kD). Resultados: No se observaron diferencias en la viabilidad de las HRECs tratadas con 2 o 4μg/ml de silimarina en comparación con las células no tratadas, pero se observó una reducción de la viabilidad con la concentración de 10μg/ml. Por consiguiente, se seleccionó la concentración de 4μg/ml de silimarina. La silimarina (4μg/ml) produjo un descenso significativo de la permeabilidad inducida por VEGF tanto en medio con 5.5mM de D-glucosa (422 ±58 vs. 600 ±72 ng/ml/cm2; p<0.03) como en medio con 25mM de D-glucosa (354±28 vs. 567±102 ng/ml/cm2; p<0.04). Discusión: Nuestros resultados demuestran que la silimarina es efectiva para prevenir la hiperpermeabilidad inducida por condiciones suprafisiológicas de glucosa en HRECs. Son necesarios más estudios para evaluar si la silimarina podría ser útil para el tratamiento del EMD (AU)


Assuntos
Humanos , Masculino , Feminino , Silimarina/uso terapêutico , Retinopatia Diabética/complicações , Retinopatia Diabética/dietoterapia , Degeneração Macular/dietoterapia , Edema Macular/complicações , Células Endoteliais , Dextranos/análise , Células Cultivadas , Proliferação de Células , Sobrevivência Celular , Análise de Variância
14.
J. physiol. biochem ; 73(1): 59-65, feb. 2017. graf, ilus
Artigo em Inglês | IBECS | ID: ibc-168393

RESUMO

Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16INK4a pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 μM of palm γ-tocotrienol for 24 hours. Silencing of p16INK4a was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16INK4a were determined by western blot technique. The finding of this study showed that p16INK4a mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (AU)


No disponible


Assuntos
Humanos , Masculino , Criança , Senescência Celular , Ciclina D1/antagonistas & inibidores , Regulação para Baixo , Cromanos/metabolismo , Fibroblastos/metabolismo , Vitamina E/análogos & derivados , Fenômenos Fisiológicos Celulares , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/antagonistas & inibidores , Proteínas de Ligação a Retinoblastoma/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores
15.
J. physiol. biochem ; 72(3): 421-434, sept. 2016. ilus, tab, graf
Artigo em Inglês | IBECS | ID: ibc-168285

RESUMO

Acute exercise induces changes in peripheral mononuclear cells’ (PBMCs) capabilities to produce cytokines. The aim was to investigate the effect of docosahexaenoic acid (DHA) diet supplementation on cytokine production, by lipopolysaccharide (LPS)-stimulated PBMCs after exercise, and the in vitro influence of temperature. Fifteen male soccer players were randomly assigned to a placebo or an experimental group. The experimental group consumed an almond-based beverage enriched with DHA (1.16 g DHA/day) for 8 weeks, whereas the placebo group consumed a similar non-enriched beverage. Blood samples were taken before and after the nutritional intervention in basal conditions and 2 h after acute exercise. Nutritional intervention significantly increased the DHA content in erythrocytes only in experimental group (from 34 ± 3.6 to 43 ± 3.6 nmols DHA/109 erythrocytes). Exercise significantly increased Toll-like receptor 4 (TLR4) in PBMCs but only in the placebo group (203 %). Exercise also significantly increased IL6, IL8, VEGF, INFγ, TNFα, IL1α, IL1β, MCP1, and EGG production rates by LPS-stimulated PBMCs, and this response was attenuated by DHA supplementation. Temperature but not DHA also affected the pattern of cytokine production increasing IL6, IL8, IL1β, and MCP1 synthesis. The higher change was evidenced in IL1β increasing the production rate at 39.5 °C from 3.19 ± 0.77 to 22.4 ± 6.1 pg/h 106 PBMC in placebo and from 2.36 ± 0.11 to 10.6 ± 0.38 pg/h 106 PBMC in the supplemented group. The profile of affected cytokines differs between temperature and exercise, suggesting a different PBMC activation pathway. DHA diet supplementation only attenuated cytokine production after exercise and not that induced by temperatura (AU)


No disponible


Assuntos
Humanos , Masculino , Atletas , Exercício Físico , Citocinas/antagonistas & inibidores , Alimentos Fortificados , Leucócitos Mononucleares/metabolismo , Ácidos Docosa-Hexaenoicos/administração & dosagem , Fenômenos Fisiológicos da Nutrição Esportiva , Estresse Oxidativo , Células Cultivadas , Eritrócitos/metabolismo , Temperatura Alta/efeitos adversos , Lipopolissacarídeos/toxicidade , Perda de Seguimento , Espanha , Futebol , Ativação Linfocitária
16.
J. physiol. biochem ; 72(2): 255-268, jun. 2016. ilus, tab, graf
Artigo em Inglês | IBECS | ID: ibc-168270

RESUMO

The interstitial myocardial matrix is a complex and dynamic structure that adapts to local fluctuations in pressure and actively contributes to the heart's fluid exchange and hydration. However, classical physiologic models tend to treat it as a passive conduit for water and solute, perhaps because local interstitial regulatory mechanisms are not easily accessible to experiment in vivo. Here, we examined the interstitial contribution to the fluid-driving pressure ex vivo. Interstitial hydration potentials were determined from influx/efflux rates measured in explants from healthy and ischemia-reperfusion-injured pigs during colloid osmotic pressure titrations. Adaptive responses were further explored by isolating myocardial fibroblasts and measuring their contractile responses to water activity changes in vitro. Results show hydration potentials between 5 and 60 mmHg in healthy myocardia and shifts in excess of 200 mmHg in edematous myocardia after ischemia-reperfusion injury. Further, rates of fluid transfer were temperature-dependent, and in collagen gel contraction assays, myocardial fibroblasts tended to preserve the micro-environment's hydration volume by slowing fluid efflux rates at pressures above 40 mmHg. Our studies quantify components of the fluid-driving forces in the heart interstitium that the classical Starling's equation does not explicitly consider. Measured hydration potentials in healthy myocardia and shifts with edema are larger than predicted from the known values of hydrostatic and colloid osmotic interstitial fluid pressures. Together with fibroblast responses in vitro, they are consistent with regulatory mechanisms that add local biological controls to classic fluid-balance models (AU)


No disponible


Assuntos
Animais , Feminino , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Coração/fisiopatologia , Deslocamentos de Líquidos Corporais , Matriz Extracelular , Líquido Extracelular/diagnóstico por imagem , Edema Cardíaco/etiologia , Modelos Animais de Doenças , Equilíbrio Hidroeletrolítico , Técnicas de Cultura de Tecidos , Sus scrofa , Pressão Osmótica , Miofibroblastos/patologia , Forma Celular , Células Cultivadas , Rastreamento de Células , Imagem por Ressonância Magnética
17.
Allergol. immunopatol ; 43(6): 584-592, nov.-dic. 2015. tab, ilus
Artigo em Inglês | IBECS | ID: ibc-147076

RESUMO

Background: Asthma is a chronic inflammatory airway disease, the incidence of which has increased recently. In order to identify the potential biomarkers for allergic asthma therapy, microarray data were analysed to find meaningful information. Methods: Microarray data GSE18965 were downloaded from Gene Expression Ominibus (GEO), including seven asthmatic epithelium samples from children with allergic asthma and nine healthy controls. Limma package was used to detect differentially expressed genes (DEGs) and the criteria were (log fold change) > 0.5 and p value < 0.05. We used Database for Annotation, Visualization and Integrated Discovery (DAVID) tool to perform GO function and KEGG pathway analysis. STRING database was used to construct protein - protein interaction (PPI) network. MicroRNA (miRNA) regulation network was constructed according to miRecords database. Results: We identified 274 DEGs in asthma epithelium samples comparing with healthy controls. There were 123 up-regulated DEGs and 151 down-regulated DEGs. PPI network analysis showed that TSPO, G6PD and TXN had higher degree. miRNA regulation network demonstrated that miR-16 and miR-15a had higher degree. The target genes of miRNAs were significantly enriched in the apoptosis function. Conclusions: TSPO, G6PD and TXN, miR-16, miR-15a and apoptosis may be used as the targets for children’s allergic asthma therapy (AU)


No disponible


Assuntos
Humanos , Masculino , Feminino , Criança , Animais , MicroRNAs/genética , Células Epiteliais/fisiologia , Glucosefosfato Desidrogenase/metabolismo , Receptores de GABA/metabolismo , Mapas de Interação de Proteínas , Análise em Microsséries , Regulação da Expressão Gênica , Células Cultivadas , Biomarcadores/metabolismo , Apoptose/genética
18.
Allergol. immunopatol ; 43(6): 553-561, nov-dic. 2015. tab, graf
Artigo em Inglês | IBECS | ID: ibc-145500

RESUMO

Background: Autosomal dominant hyper-IgE syndrome (AD-HIES) is a primary immunodeficiency mainly caused by mutations in STAT3, a signalling molecule implicated in the development of appropriate immune responses. We aimed to characterise the innate immune response in AD-HIES. Methods: The frequency of innate immune cells in peripheral blood (PB) from seven AD-HIES patients and healthy controls were determined. CD80/CD86 surface expression and cytokine levels in supernatants from PBMC after stimulation with TLR-2, -4 and -9 agonists were also measured by flow cytometry. In addition, several SNPs within these TLR genes in genomic DNA samples from patients and controls were examined. Results: A significantly reduced number of PB iNKT cells was observed in the AD-HIES group. CpG-stimulated pDC and mDC from patients exhibited a lower increase in the expression of the costimulatory molecule CD80. We also observed an increase in the secretion of IL-12p70, TNF-alpha and IL-10 in PBMC from HIES patients after LTA or LPS stimuli. No association was found between the different SNPs detected and the HIES phenotype. Conclusions: These findings demonstrate that important mediators of the innate immunity responses are affected in AD-HIES. More studies are necessary to investigate how the STAT3 function interferes with development of iNKT cells and TLR-mediated responses (AU)


No disponible


Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Criança , Síndrome de Job/imunologia , Células Dendríticas/fisiologia , Lipopolissacarídeos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Células T Matadoras Naturais/fisiologia , Ácidos Teicoicos/farmacologia , Análise Mutacional de DNA , Hipersensibilidade/genética , Células Cultivadas , Citocinas/metabolismo , Predisposição Genética para Doença , Imunidade Inata/genética
19.
J. investig. allergol. clin. immunol ; 24(5): 338-345, ago. 2014. tab, ilus
Artigo em Inglês | IBECS | ID: ibc-128321

RESUMO

Background and objective: Allergic airway diseases are not only a TH2-mediated chronic airway inflammation, but also a condition of epithelial barrier defects and dysfunction. Allergens with protease activities are known factors that initiate respiratory epithelial damage. Cockroach allergy is the second leading cause of allergic respiratory airway diseases in Taiwan, and cockroach allergens have strong serine protease activity. This study aimed to determine the protective effect of the direct local administration of gabexate mesilate (GM) on American cockroach allergen (CraA)-induced human bronchial epithelial cell inflammation. Methods: BEAS-2B cells, from the human bronchial epithelial cell line, were stimulated with CraA or co-cultured with different doses of GM. Cellular morphologic changes were observed by microscopy and changes in chemokine mRNA expression and protein levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and ELISA. Effects of specific inhibitors of ERK1/2 (U0126), JNK (SP600125), and p38 MAPK (SB203580) on CraA-induced chemokine mRNA expression were also tested by RT-PCR. Results: GM prevented CraA-induced bronchial epithelial cell detachment and morphological changes. It had superior and more extensive suppression effects than specific target MAPK inhibitors in CraA-induced mRNA expression of IL-8, monocyte chemotactic protein (MCP) 1, chemokine (C-C motif) ligand 20, and granulocyte-macrophage colony-stimulating factor from the cells in a dose-dependent manner. CraA-induced IL-8 and MCP-1 protein production from BEAS-2B cells was also attenuated by GM. Conclusions: The serine protease inhibitor GM has local protective effects against CraA-induced bronchial epithelial inflammation. The development of an inhaled or intranasal protease inhibitor may be a potential strategy for the treatment of allergic airway diseases induced by allergens with protease activities (AU)


Antecedentes y objetivo: Las enfermedades alergicas de las vias respiratorias no son solo fruto de una inflamacion cronica de las vias respiratorias mediada por Th2, sino que tambien se encuentran defectos fisicos y funcionales de la barrera epitelial. En este sentido, es conocido el papel de los alergenos con actividad proteasa que son los factores conocidos de inicio del dano epitelial respiratorio. La alergia a las cucarachas es la segunda causa principal de enfermedades alergicas de las vias respiratorias en Taiwan y estos alergenos poseen una potente actividad serin-proteasa. Este estudio tuvo como objetivo determinar el efecto protector del mesilato de gabexate (GM) contra la inflamacion inducida por la administracion local directa de alergenos de la cucaracha americana (CRAA), sobre las celulas epiteliales bronquiales humanas. Metodos: Se empleo la linea de celulas epiteliales bronquiales, celulas BEAS-2B, las cuales se estimularon con CRAA o fueron co-cultivadas con diferentes dosis de GM. Se analizaron los cambios morfologicos celulares por microscopia y los cambios en la expresion de ARNm de diferentes quimiocinas, asi como los niveles de proteina. Se utilizaron metodos semi-cuantitativos de RT-PCR y ELISA. Los efectos de inhibidores especificos de ERK1/2 (U0126), JNK (SP600125), y p38 MAPK (SB203580) en las expresion de ARNm de quimiocinas inducidas por CRAA, tambien se ensayaron por RT-PCR. Resultados: El mesilato de gabexate impidio el desprendimiento de las celulas epiteliales y los cambios morfologicos inducidos con CRAA a nivel bronquial. Sus efectos supresores fueron mas potentes y prolongados que los obtenidos con inhibidores especificos de MAPK sobre la expresion del ARNm de IL-8, MCP-1, CCL20 y GMSF en una forma dosis-dependiente. La produccion proteica de IL-8 y MCP-1 de las celulas BEAS-2B tambien fue menor cuando se anadio GM. Conclusiones: El mesilato de gabexate, inhibidor serin-proteasa , tiene efectos protectores locales contra la inflamacion bronquial epitelial inducida por la cucaracha americana. El desarrollo de un inhibidor de la proteasa, por via inhalada o intranasal, puede ser una estrategia potencial para el tratamiento de enfermedades de las vias respiratorias alergicas inducidas por alergenos con actividad proteasa (AU)


Assuntos
Humanos , Animais , Masculino , Feminino , Brônquios/patologia , Quimiocina CCL2/biossíntese , Inibidores de Serino Proteinase/farmacologia , Células Cultivadas , Quimiocinas/genética , Células Epiteliais/patologia , Interleucina-8/biossíntese , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação
20.
Clin. transl. oncol. (Print) ; 15(3): 211-218, mar. 2013. tab, ilus
Artigo em Inglês | IBECS | ID: ibc-127080

RESUMO

INTRODUCTION: Real time RT-PCR is a widely used technique to evaluate and confirm gene expression data obtained in different cell systems and experimental conditions. However, there are many conflicting reports about the same gene or sets of gene expression. A common method is to report the interest gene expression relative to an internal control, usually a housekeeping gene (HKG), which should be constant in cells independently of experimental conditions. MATERIALS AND METHODS: In this study, the expression stability of ten HKGs was considered in parallel in two cell systems (endothelial and osteosarcoma cells): beta actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (TBP), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Cyclophilin A (PPIA), beta-2-microglobulin (B2M), glucuronidase beta (GUSB), eukaryotic translation elongation factor 1 alpha1 (EEF1A1), transferrin receptor (TFRC), ribosomal protein S18 (RPS18). In order to study the stability of candidate reference genes, data have been also analyzed by several algorithms (geNorm, NormFinder, BestKeeper and delta-Ct method). RESULTS AND CONCLUSIONS: The overall analysis obtained by the comprehensive ranking showed that RPS18 and PPIA are appropriate internal reference genes for tumor neovascularization studies where it is necessary to analyze both systems at the same time (AU)


Assuntos
Humanos , Neoplasias Ósseas/genética , Endotélio Vascular/metabolismo , Genes Essenciais/genética , Neovascularização Patológica/genética , Osteoblastos/metabolismo , Osteossarcoma/genética , Algoritmos , Neoplasias Ósseas/patologia , Células Cultivadas , Endotélio Vascular/citologia , Perfilação da Expressão Gênica , Osteoblastos/citologia , Osteossarcoma/patologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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