The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain.

Nerve injury-induced neuropathic pain is difficult to treat and mechanistically characterized by strong neuroimmune interactions, involving signaling lipids that act via specific G-protein coupled receptors. Here, we investigated the role of the signaling lipid receptor G2A (GPR132) in nerve injury-induced neuropathic pain using the robust spared nerve injury (SNI) mouse model. We found that the concentrations of the G2A agonist 9-HODE (9-Hydroxyoctadecadienoic acid) are strongly increased at the site of nerve injury during neuropathic pain. Moreover, G2A-deficient mice show a strong reduction of mechanical hypersensitivity after nerve injury. This phenotype is accompanied by a massive reduction of invading macrophages and neutrophils in G2A-deficient mice and a strongly reduced release of the proalgesic mediators TNFα, IL-6 and VEGF at the site of injury. Using a global proteome analysis to identify the underlying signaling pathways, we found that G2A activation in macrophages initiates MyD88-PI3K-AKT signaling and transient MMP9 release to trigger cytoskeleton remodeling and migration. We conclude that G2A-deficiency reduces inflammatory responses by decreasing the number of immune cells and the release of proinflammatory cytokines and growth factors at the site of nerve injury. Inhibiting the G2A receptor after nerve injury may reduce immune cell-mediated peripheral sensitization and may thus ameliorate neuropathic pain.

[Perception of following gluten-free diet and adherence to treatment in pediatric patients with celiac disease]. / Percepción de estar haciendo bien la dieta sin gluten y adherencia al tratamiento en pacientes pediátricos con enfermedad celíaca.

INTRODUCTION: Celiac disease (CD) is a chronic immune-mediated enteropathy present in ~1% of population. Gluten-free diet (GFD) is the only treatment for this condition and the main limitation of its efficacy is the lack of adherence. OBJECTIVE: To assess factors influencing adherence to GFD in pediatric patients and measure the concordance between serological results and a nutritional adhe rence questionnaire. PATIENTS AND METHODS: celiac patients younger than 18 years of age, diagnosed CD following ESPGHAN criteria, on GFD for at least 6 months and consulting at Hospital Roberto del Río, Santiago, in 2008-2016, were assessed. Clinical presentation, nutritional evaluation and fac tors related to adherence to treatment (diet) were registered. A subsample answered Biaggi's nutri tional questionnaire. RESULTS: Of 65 evaluated patients, 44% and 30,1% adhered to GFD according to blood autoantibodies (TTG and EMA) and the adherence questionnaire, respectively. "Age at debut" (p = 0.049), "perception of following GFD correctly" (p = 0.002) and "behavior in social events" (p = 0.005) were significantly associated with adherence to GFD. There was concordance between serological test and Biagi's questionnaire (p = 0.0001). DISCUSSION: Adherence to GFD was lower than reported in literature. Intervention of some of the identified variables associated with adherence may help improving follow-up of celiac patients, especially those that due to diverse situations cannot measure their antibodies periodically.

Percepción de estar haciendo bien la dieta sin gluten y adherencia al tratamiento en pacientes pediátricos con enfermedad celíaca / Perception of following gluten-free diet and adherence to treatment in pediatric patients with celiac disease

INTRODUCCIÓN: La enfermedad celíaca (EC) es una enteropatía crónica mediada inmunológicamente que afecta ~1% de la población. La dieta libre de gluten (DLG) es su único tratamiento y la principal limitante de su eficacia es la falta de adherencia. OBJETIVOS: Evaluar factores que influyen en la adherencia a la DLG de pacientes celiacos pediátricos. Medir la concordancia entre la serología y un cuestionario nutricional de adherencia. PACIENTES Y MÉTODO: Estudio transversal en celiacos menores de 18 años, en DLG por más de 6 meses. Se aplicó un cuestionario con 5 grupos de factores (OMS). Se registraron características clínicas, dieta de los últimos 3 meses, percepción (de padres/cuidadores, y del paciente adolescente) de la DLG; el conocimiento de los alimentos permitidos y disponibles en el país de la simbología "libre de gluten", y si lee/no lee ingredientes de un alimento antes de comprarlo. Se aplicó un score dando un punto a cada respuesta correcta (0-4). A un subgrupo se le aplicó el cuestionario de adherencia a la DLG de Biagi. Se midió EMA y TTG dentro de las 2 semanas posteriores a la entrevista. Se usó índice Kappa para evaluar la concordancia entre TTG y encuesta nutricional de adherencia; Chi cuadrado para la asociación entre los factores evaluados y los resultados de EMA y TTG, y Odds ratio como medida de asociación. Se aplicó un modelo de regresión logística a los factores asociados a los resultados de los exámenes de anticuerpos EMA y TTG (positivo-negativo). Se definió "buena adherencia a la DLG" cuando EMA y TTG fueron negativos. Resultados: De 65 pacientes; 44% y 30,1% adherían correctamente a la DLG según medición de anticuerpos (TTG y EMA) y el cuestionario, respectivamente. La "edad de inicio de la enfermedad" (p = 0,049), "percepción de estar realizando bien la DLG" (p = 0,002) y la "conducta del paciente frente a alimentos en reuniones sociales" (p = 0,005), se asociaron significativamente con adherencia a DLG. Hubo concordancia entre los exámenes serológicos y el cuestionario de adherencia (p = 0,0001). DISCUSIÓN: La adherencia fue menor que la reportada en la literatura. La intervención de variables asociadas a adherencia identificadas, podría ayudar al mejor seguimiento de los pacientes, especialmente en aquellos quienes por diversos motivos no puedan realizarse exámenes serológicos con la frecuencia adecuada.

INTRODUCTION: Celiac disease (CD) is a chronic immune-mediated enteropathy present in ~1% of population. Gluten-free diet (GFD) is the only treatment for this condition and the main limitation of its efficacy is the lack of adherence. OBJECTIVE: To assess factors influencing adherence to GFD in pediatric patients and measure the concordance between serological results and a nutritional adhe rence questionnaire. PATIENTS AND METHODS: celiac patients younger than 18 years of age, diagnosed CD following ESPGHAN criteria, on GFD for at least 6 months and consulting at Hospital Roberto del Río, Santiago, in 2008-2016, were assessed. Clinical presentation, nutritional evaluation and fac tors related to adherence to treatment (diet) were registered. A subsample answered Biaggi's nutri tional questionnaire. RESULTS: Of 65 evaluated patients, 44% and 30,1% adhered to GFD according to blood autoantibodies (TTG and EMA) and the adherence questionnaire, respectively. "Age at debut" (p = 0.049), "perception of following GFD correctly" (p = 0.002) and "behavior in social events" (p = 0.005) were significantly associated with adherence to GFD. There was concordance between serological test and Biagi's questionnaire (p = 0.0001). DISCUSSION: Adherence to GFD was lower than reported in literature. Intervention of some of the identified variables associated with adherence may help improving follow-up of celiac patients, especially those that due to diverse situations cannot measure their antibodies periodically.

Induction of apoptosis by R-flurbiprofen in human colon carcinoma cells: involvement of p53.

R-flurbiprofen, a non cyclooxygenase inhibiting non-steroidal anti-inflammatory drug (NSAID), has been found to inhibit tumor growth in various animal models. In vitro experiments have shown that this effect is based on the induction of a cell cycle block and apoptosis. Cell cycle inhibition has been explained by activation of the c-Jun-N-terminal kinase (JNK) and downregulation of cyclin D1 expression. However, the molecular mechanism leading to apoptosis is unknown. Here, we show that treatment of the human colon carcinoma cell line HCT116 with different concentrations of R-flurbiprofen leads to an accumulation of p53 protein which is accompanied by an increase in phosphorylated p53 at serine 15. Mutation of serine 15 to alanine by site directed mutagenesis and overexpression of the mutated p53 gene in HCT116 cells, revealed that these cells are significantly less sensitive to apoptosis induced by R-flurbiprofen than pcDNA control cells, as measured by PARP-cleavage and flow cytometry. By contrast, no difference was detected between HCT116p53ser15ala cells and HCT116 pcDNA cells with respect to induction of a cell cycle block after R-flurbiprofen treatment. Moreover, in nude mice HCT116p53ser15ala overexpressing xenografts were significantly less sensitive to R-flurbiprofen than HCT116 pcDNA control xenografts. In conclusion, we were able to show that induction of apoptosis in HCT116 cells after R-flurbiprofen treatment is at least partly dependent on the tumor suppressor gene p53 and that mutation of p53 at serine 15 impairs the apoptotic potency of R-flurbiprofen.

Cyclooxygenase-2 (COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anticarcinogenic effects of celecoxib is still not fully understood. To investigate the extent to which the anticarcinogenic effect of celecoxib depends on COX-2 expression, we transfected human colon carcinoma cells (Caco-2) with the human COX-2 cDNA, in both sense and in antisense orientation, to generate cells which either overexpress COX-2 (human COX-2-sense, hCOX-2-s), express no COX-2 (human COX-2-antisense, hCOX-2-as) or express only very small amounts of COX-2 (control cells). Treatment of these cells with celecoxib dose-dependently (0-100microM) reduced cell survival which was accompanied by an induction of a G(0)/G(1) phase block and apoptosis. The effect of celecoxib treatment on both, cell survival and induction of apoptosis in hCOX-2-as cells was less marked than in the COX-2-expressing cells. Apoptosis was accompanied by an activation of caspase-3 and caspase-9 and cytochrome c release. In contrast, we observed no difference in sensitivity with regard to the induction of a cell cycle block between the different cell clones. The G(0)/G(1) phase block caused by celecoxib correlated with a decrease in expression levels of cyclin A and cyclin B1 and an increase in the expression of the cell cycle inhibitory proteins p21(Waf1) and p27(Kip1) irrespective of the type of cell used. These data indicate that apoptosis-inducing effects of celecoxib partly depend on COX-2 expression of the cells, whereas induction of a cell cycle block occurred COX-2 independently. Thus, the anticarinogenic effects of celecoxib can be explained by both COX-2-dependent and -independent mechanisms.

Activation of c-Jun-N-terminal-kinase is crucial for the induction of a cell cycle arrest in human colon carcinoma cells caused by flurbiprofen enantiomers.

The unselective cyclooxygenase (COX) inhibitor S-flurbiprofen and its-in terms of COX-inhibition-"inactive" enantiomer R-flurbiprofen have been previously found to inhibit tumor development and growth in various animal models. The underlying mechanisms are unknown. Here, we show that both R- and S-flurbiprofen reduce survival of three colon cancer cell lines, which differ in the expression of COX-2 (HCT-15, no COX-2; Caco-2, inducible COX-2; and HT-29, constitutive COX-2). The IC50 for S- and R-flurbiprofen ranged from 250 to 450 microM. Both flurbiprofen enantiomers induced apoptosis in all three cell lines as indicated by DNA- and PARP-cleavage. In addition, R- and S-flurbiprofen caused a G1-cell cycle block. The latter was associated with an activation of c-Jun N-terminal kinase (JNK), an increase of the DNA binding activity of the transcription factor AP-1 and down-regulation of cyclin D1 expression. Western blot analysis, as well as supershift experiments, revealed that the AP-1 activation was associated with a change of AP-1 composition toward an increase of JunB. The JNK inhibitor SP600125 antagonized R- and S-flurbiprofen-induced AP-1 DNA binding, suppression of cyclin D1 expression, and the G1-cell cycle block. However, JNK inhibition had no effect on flurbiprofen-induced apoptosis. Hence, the cell cycle arrest is obviously mediated, at least in part, through JNK-activation, whereas R- and S-flurbiprofen-induced apoptosis is largely independent of JNK. Although in vitro effects of R- and S-flurbiprofen were indistinguishable, only R-flurbiprofen inhibited HCT-15 tumor growth in nude mice, suggesting the involvement of additional in vivo targets, which are differently affected by R- and S-flurbiprofen.