Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment.

The pH-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1; GPR68) is expressed in the gut. Inflammatory bowel disease is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated, pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration, and proliferation were measured using electric cell-substrate impedance-sensing technology. Localization of the tight junction proteins zonula occludens protein 1 and occludin and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability and protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant upregulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. The deconstruction of OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms.

The proton-activated G protein coupled receptor OGR1 acutely regulates the activity of epithelial proton transport proteins.

The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H(+)-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl(2) and CuCl(2) that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.

Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling.

Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide-binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell-specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116-/- mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia.

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.

Fluid balance and length of mechanical ventilation in children admitted to a single Pediatric Intensive Care Unit. / Balance de fluidos y duración de la ventilación mecánica en niños internados en una Unidad de Terapia Intensiva Pediátrica.

INTRODUCTION: Associations between cumulative fluid balance and a prolonged duration of assisted mechanical ventilation have been described in adults. The aim of this study was to evaluate whether fluid balance in the first 48 hours of assisted mechanical ventilation initiation was associated with a prolonged duration of this process among children in the Pediatric Intensive Care Unit (PICU). METHODS: Retrospective cohort of patients in the PICU o, Hospital Italiano de Buenos Aires, between 1/1/2010 and 6/30/2012. Balance was calculated in percentage of body weight; prolonged mechanical ventilation was defined as >7 days, and confounders were registered. Univariate and multivariate analyses were performed. RESULTS: Two hundred and forty-nine patients were mechanically ventilated for over 48 hours; 163 were included in the study. Balance during the first 48 hours of mechanical ventilation was 5.7% ± 5.86; 82 patients (50.3%) were on mechanical ventilation for more than 7 days. Age 〈 4 years old (OR 3.21, 95% CI 1.38-7.48, p 0.007), respiratory disease (OR 4.94, 95% CI 1.51-16.10, p 0.008), septic shock (OR 4.66, 95% CI 1.10-19.65, p 0.036), Pediatric Logistic Organ Dysfunction (PELOD) 〉 10 (OR 2.44, 95% CI 1.234.85, p 0.011), and positive balance 〉 13% (OR 4.02, 95% CI 1.08-15.02, p 0.038) were associated with prolonged mechanical ventilation. The multivariate model resulted in an OR 2.58, 95% CI: 1.17-5.58, p= 0.018 for PELOD 〉 10, and an OR 3.7, 95% CI: 0.91-14.94, p= 0.066 for positive balance 〉 13%. CONCLUSIONS: Regarding prolonged mechanical ventilation, the multivariate model showed an independent association with organ dysfunction (PELOD 〉 10) and a trend towards an association with positive balance 〉 13%.

INTRODUCCIÓN: Se han descrito asociaciones entre balance de fluido acumulado y mayor estadía en asistencia respiratoria mecánica en adultos. El objetivo fue evaluar si el balance de las primeras 48 horas de iniciada la asistencia respiratoria mecánica se asociaba a su prolongación en niños internados en Terapia Intensiva Pediátrica (UCIP). MÉTODOS: Cohorte retrospectiva de pacientes de la UCIP de, Hospital Italiano de Buenos Aires, entre el 1/1/2010 y el 30/6/2012. El balance se calculó en porcentaje del peso corporal; ventilación mecánica prolongada se definió como 〉 7 días y se registraron confundidores. Se realizó un análisis univariado y multivariado. RESULTADOS: 249 pacientes permanecieron ventilados más de 48 horas; se incluyeron 163. El balance de las primeras 48 horas en ventilación mecánica fue 5,7%±5,86; 82 pacientes (50,3%) permanecieron más de 7 días con respirador. La edad 〈 4 años (OR 3,21; IC 95% 1,38-7,48; p 0,007), enfermedad respiratoria (OR 4,94; IC 95% 1,51-16,10; p 0,008), shock séptico (OR 4,66; IC 95% 1,10-19,65; p 0,036), puntaje de disfunción orgánica (PELOD) 〉 10 (OR 2,44; IC 95% 1,23-4,85; p 0,011) y balance positivo 〉 13% (OR 4,02; IC 95% 1,08-15,02; p 0,038) se asociaron a ventilación mecánica prolongada. El modelo multivariado mostró para PELOD 〉 10 un OR 2,58; IC 95%: 1,17-5,58; p 0,018, y para balance positivo 〉 13% un OR 3,7; IC 95%: 0,91-14,94; p 0,066. CONCLUSIONES: En relación a ventilación mecánica prolongada, el modelo multivariado mostró una asociación independiente con disfunción de órganos (PELOD 〉 10) y una tendencia hacia la asociación con balance positivo 〉 13%.

G Protein-coupled pH-sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation.

BACKGROUND: A novel family of proton-sensing G protein-coupled receptors, including OGR1, GPR4, and TDAG8, was identified to be important for physiological pH homeostasis and inflammation. Thus, we determined the function of proton-sensing OGR1 in the intestinal mucosa. MTEHODS: OGR1 expression in colonic tissues was investigated in controls and patients with IBD. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied for more than 200 days. Microarray profiling was performed using Ogr1 and Ogr1 (WT) residential peritoneal macrophages. RESULTS: Patients with IBD expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant upregulation of OGR1 expression, which could be reversed by the presence of NF-κB inhibitors. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1/Il-10 mice. These mice displayed significantly less rectal prolapses. Upregulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell-adhesion gene pathways. CONCLUSIONS: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF-κB inhibition reverses the induction of OGR1 expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 knockout model. Our data indicate a pathophysiological role for pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation.

Neuroplasticidad y ácidos grasos omega 3 en adultos mayores / Neuroplasticity and omega-3 fatty acids in older adults

El aumento en la expectativa de vida ha llevado a que las enfermedades neurodegenerativas relacionadas con la vejez sean más investigadas. Las diversas intervenciones no farmacológicas en el campo de las demencias tienen su punto de partida en el concepto de neuroplasticidad o capacidad plástica del cerebro. Se conoce como neuroplasticidad a la capacidad cerebral para minimizar los efectos de las lesiones a través de cambios estructurales y funcionales, lo que permite al cerebro reaccionar o ajustarse a cambios ambientales internos y externos bajo condiciones fisiológicas y patológicas, a través modificaciones morfológicas extensas, como las que se observan en la regeneración de axones, formación de nuevas sinapsis, promoción de la neurogénesis, hasta sutiles cambios moleculares que alteran la respuesta celular a los neurotransmisores. Se ha propuesto como una de las estrategias en el tratamiento de la Enfermedad de Alzheimer, el deterioro cognitivo leve y las intervenciones post ACV la suplementación con ácidos grasos poliinsaturados (AGPI). Los AGPI omega 3 (AGPI ω-3) poseen múltiples mecanismos de acción en el cerebro y en el sistema vascular que podrían generar cierta protección contra el declive cognitivo y la demencia. Los estudios encontrados que fueron realizados en humanos corresponden a pacientes con deterioro cognitivo leve y enfermedad de Alzheimer (EA) leve a moderada y en un solo trabajo se evaluó la suplementación con omega 3 en pacientes post ACV. Aunque la evidencia clínica es algo contradictoria, probablemente en gran parte debido a cuestiones metodológicas, diversos estudios han demostrado que los AGPI ω-3 pueden mejorar la función cognitiva en los individuos adultos sanos y atenuar el deterioro cognitivo en el envejecimiento y EA leve. En los pacientes con EA moderada no se observaron cambios significativos. Hasta hoy no existen resultados concluyentes para incluir a los AGPI omega 3 como parte de un protocolo de tratamiento en enfermedades neurodegenerativas. Se necesitan más estudios aleatorizados controlados para definir el tiempo, dosis y momento adecuado para la prescripción de estos ácidos grasos.

The increase in life expectancy has led to the fact that the neurodegenerative diseases related to old age are being more and more researched. The various non-pharmacological interventions in the field of dementias have their starting point in the concept of neuroplasticity or plastic capacity of the brain. Neuroplasticity is known as the brain capacity to minimize the effects of injuries through structural and functional changes, allowing the brain to react or adjust to internal and external environmental changes under physiological and pathological conditions, through extensive morphological modifications, as the ones observed in the regeneration of axons, formation of new synapses, promotion of neurogenesis, to subtle molecular changes that alter the cellular response to neurotransmitters. It has been proposed as one of the strategies in the treatment of Alzheimer's Disease (AD), mild cognitive impairment (MCI) and poststroke interventions with polyunsaturated fatty acid (PUFA) supplementation. The omega-3 PUFAs (ω-3PUFA) have multiple mechanisms of action in the brain and vascular system that could provide some protection against cognitive decline and dementia. The studies found that were performed in humans correspond to patients with mild cognitive impairment and mild to moderate AD and in only one study, supplementation with omega-3 in poststroke patients was evaluated. Although clinical evidence is somehow contradictory, probably largely due to methodological issues, several studies have shown that ω-3 PUFAs may improve cognitive function in healthy adult individuals and attenuate cognitive impairment in aging and mild AD. No significant changes were observed in patients with moderate AD. Until today, there are no conclusive results to include omega-3 PUFAs as part of a treatment protocol in neurodegenerative diseases. Further randomized controlled studies are needed to define the time, dose and appropriate timing for the prescription of these fatty acids.

Balance de fluidos y duración de la ventilación mecánica en niños internados en una Unidad de Terapia Intensiva Pediátrica / Fluid balance and length of mechanical ventilation in children admitted to a single Pediatric Intensive Care Unit

Introducción. Se han descrito asociaciones entre balance de fluido acumulado y mayor estadía en asistencia respiratoria mecánica en adultos. El objetivo fue evaluar si el balance de las primeras 48 horas de iniciada la asistencia respiratoria mecánica se asociaba a su prolongación en niños internados en Terapia Intensiva Pediátrica (UCIP). Métodos. Cohorte retrospectiva de pacientes de la UCIP del Hospital Italiano de Buenos Aires, entre el 1/1/2010 y el 30/6/2012. El balance se calculó en porcentaje del peso corporal; ventilación mecánica prolongada se definió como > 7 días y se registraron confundidores. Se realizó un análisis univariado y multivariado. Resultados. 249 pacientes permanecieron ventilados más de 48 horas; se incluyeron 163. El balance de las primeras 48 horas en ventilación mecánica fue 5,7%±5,86; 82 pacientes (50,3%) permanecieron más de 7 días con respirador. La edad < 4 años (OR 3,21; IC 95% 1,38-7,48; p 0,007), enfermedad respiratoria (OR 4,94; IC 95% 1,51-16,10; p 0,008), shock séptico (OR 4,66; IC 95% 1,10-19,65; p 0,036), puntaje de disfunción orgánica (PELOD) > 10 (OR 2,44; IC 95% 1,23-4,85; p 0,011) y balance positivo > 13% (OR 4,02; IC 95% 1,08-15,02; p 0,038) se asociaron a ventilación mecánica prolongada. El modelo multivariado mostró para PELOD > 10 un OR 2,58; IC 95%: 1,17-5,58; p 0,018, y para balance positivo > 13% un OR 3,7; IC 95%: 0,91-14,94; p 0,066. Conclusiones. En relación a ventilación mecánica prolongada, el modelo multivariado mostró una asociación independiente con disfunción de órganos (PELOD > 10) y una tendencia hacia la asociación con balance positivo > 13%.

Introduction. Associations between cumulative fluid balance and a prolonged duration of assisted mechanical ventilation have been described in adults. The aim of this study was to evaluate whether fluid balance in the first 48 hours of assisted mechanical ventilation initiation was associated with a prolonged duration of this process among children in the Pediatric Intensive Care Unit (PICU). Methods. Retrospective cohort of patients in the PICU of Hospital Italiano de Buenos Aires, between 1/1/2010 and 6/30/2012. Balance was calculated in percentage of body weight; prolonged mechanical ventilation was defined as >7 days, and confounders were registered. Univariate and multivariate analyses were performed. Results. Two hundred and forty-nine patients were mechanically ventilated for over 48 hours; 163 were included in the study. Balance during the first 48 hours of mechanical ventilation was 5.7% ± 5.86; 82 patients (50.3%) were on mechanical ventilation for more than 7 days. Age < 4 years old (OR 3.21, 95% CI 1.38-7.48, p 0.007), respiratory disease (OR 4.94, 95% CI 1.51-16.10, p 0.008), septic shock (OR 4.66, 95% CI 1.10-19.65, p 0.036), Pediatric Logistic Organ Dysfunction (PELOD) > 10 (OR 2.44, 95% CI 1.234.85, p 0.011), and positive balance > 13% (OR 4.02, 95% CI 1.08-15.02, p 0.038) were associated with prolonged mechanical ventilation. The multivariate model resulted in an OR 2.58, 95% CI: 1.17-5.58, p= 0.018 for PELOD > 10, and an OR 3.7, 95% CI: 0.91-14.94, p= 0.066 for positive balance > 13%. Conclusions. Regarding prolonged mechanical ventilation, the multivariate model showed an independent association with organ dysfunction (PELOD > 10) and a trend towards an association with positive balance > 13%.