Navigating the New EU Medical Devices Regulation: Retrospective Post-Market Follow-Up of Hyaluronic Acid Injections for Knee Osteoarthritis.

Purpose: The entry into force of the new Medical Device Regulation (EU) 2017/745 highlights the need for post-market clinical follow-up to ensure the safety and efficacy throughout the life cycle of medical devices. This study evaluates the efficacy and safety of a single intra-articular hyaluronic acid injection in knee osteoarthritis in real-world conditions, over a six-month period, aligning with the summary of safety and clinical performance (SSCP) required by the new regulation. Patients and Methods: Patients over 18 years of age with knee osteoarthritis, treated with a single injection of HA (Adant® One, Meiji Pharma Spain, Spain) at a 3rd level hospital. Patients were treated and followed between January 1, 2020 and June 30, 2022. Demographic, clinical, and treatment-related data were collected, and efficacy regarding pain relief and/or function improvement was assessed using a Likert-type scale. Data were pseudo-anonymized and the comparison was performed using Fisher' or Mann Whitney' test. The study was approved by the Ethics Review Board of the Hospital Puerta de Hierro (Madrid, Spain). Results: We followed 20 patients with knee osteoarthritis, with a mean age of 61 years, 80% women, and with a high burden of comorbidities (90%). A total of 60% of patients presented Kellgren-Lawrence grade III-IV. Four patients (20%) returned before 6 months due to lack of efficacy. Of the other patients, 65% showed a clinical response that lasted more than 12 months in 38.5% of cases. Time until medical appointment and taking concomitant medication for knee osteoarthritis were associated with better clinical response (p < 0.05). Conclusion: The administration of a Adant® One single intra-articular hyaluronic acid injection in knee osteoarthritis is effective, safe, and maintains the improvement over a six-month period. Our findings also emphasize the need of using standardized tools for accurate efficacy assessment and optimal patient care.

EVOLUTION OF DIAGNOSTIC DELAY IN PEDIATRIC INFLAMMATORY BOWEL DISEASE AND THE IMPACT OF THE COVID-19 PANDEMIC.

BACKGROUND: Pediatric inflammatory bowel disease (IBD) is increasingly prevalent, but diagnosis can still be challenging. Diagnostic delay is particularly deleterious in this age group. OBJECTIVE: This study explores the evolution of diagnostic delay in pediatric IBD and the influence of the COVID-19 pandemic. METHODS: Retrospective study including all pediatric IBD patients diagnosed during 2014, 2019 and 2020 in a tertiary hospital. Diagnostic delay, time to first medical visit, time to pediatric gastroenterologist (PG) visit and time to diagnosis were calculated and compared within a gap of five years (2019 and 2014) and with the year of onset of the pandemic (2020 and 2019). RESULTS: A total of 93 participants were included (2014: 32, 2019: 30, 2020: 31). No significant differences were observed in diagnostic delay, time to first medical visit in Crohn's disease (CD), time to PG visit and time to diagnosis when comparing 2019-2014 and 2020-2019. Time to first visit in ulcerative colitis (UC) and Undetermined-IBD increased in 2019 (P=0.03), with new decrease in 2020 (P=0.04). Diagnostic delay was longer in DC compared to UC plus Undetermined-IBD. CONCLUSION: Diagnostic delay is still an important matter in pediatric IBD, with no significant change over the last years. The time to the first PG visit and the time for diagnosis seem to have the greatest impact on diagnostic delay. Thus, strategies to enhance recognition of IBD symptoms among first-line physicians and to improve communication, facilitating referral, are of utmost importance. Despite the restraints in the health care system caused by the pandemic, time to diagnosis in pediatric IBD was not impaired during 2020 in our center.

Evolução do atraso diagnóstico na doença inflamatória intestinal em idade pediátrica e o impacto da pandemia COVID-19 / Evolution of diagnostic delay in pediatric inflammatory bowel disease and the impact of the covid-19 pandemic

ABSTRACT Background: Pediatric inflammatory bowel disease (IBD) is increasingly prevalent, but diagnosis can still be challenging. Diagnostic delay is particularly deleterious in this age group. Objective This study explores the evolution of diagnostic delay in pediatric IBD and the influence of the COVID-19 pandemic. Methods Retrospective study including all pediatric IBD patients diagnosed during 2014, 2019 and 2020 in a tertiary hospital. Diagnostic delay, time to first medical visit, time to pediatric gastroenterologist (PG) visit and time to diagnosis were calculated and compared within a gap of five years (2019 and 2014) and with the year of onset of the pandemic (2020 and 2019). Results A total of 93 participants were included (2014: 32, 2019: 30, 2020: 31). No significant differences were observed in diagnostic delay, time to first medical visit in Crohn's disease (CD), time to PG visit and time to diagnosis when comparing 2019-2014 and 2020-2019. Time to first visit in ulcerative colitis (UC) and Undetermined-IBD increased in 2019 (P=0.03), with new decrease in 2020 (P=0.04). Diagnostic delay was longer in DC compared to UC plus Undetermined-IBD. Conclusion Diagnostic delay is still an important matter in pediatric IBD, with no significant change over the last years. The time to the first PG visit and the time for diagnosis seem to have the greatest impact on diagnostic delay. Thus, strategies to enhance recognition of IBD symptoms among first-line physicians and to improve communication, facilitating referral, are of utmost importance. Despite the restraints in the health care system caused by the pandemic, time to diagnosis in pediatric IBD was not impaired during 2020 in our center.

RESUMO Contexto Apesar da prevalência crescente da doença inflamatória intestinal (DII) em idade pediátrica, o seu diagnóstico pode ser desafiante. Um atraso no diagnóstico é particularmente deletério nesta faixa etária. Objetivo Este estudo investiga a evolução do atraso diagnóstico na DII pediátrica e o impacto da pandemia COVID-19 no mesmo. Métodos Estudo retrospetivo que incluiu todos os doentes em idade pediátrica diagnosticados com DII durante 2014, 2019 e 2020 num hospital terciário. O atraso diagnóstico, o tempo para a primeira visita médica, o tempo para a primeira visita ao gastroenterologista pediátrico (GP) e o tempo para o diagnóstico foram calculados e comparados num intervalo de cinco anos (2019 e 2014) e com o ano marcado pelo surgimento da pandemia COVID-19 (2020 e 2019). Resultados Foram incluídos 93 participantes (2014: 32, 2019: 30, 2020: 31). Não se observou diferença significativa no atraso diagnóstico, no tempo para a primeira visita médica na doença de Crohn (DC), no tempo para a primeira visita ao GP e no tempo para o diagnóstico após comparação entre 2019-2014 e 2020-2019. Na colite ulcerosa e colite indeterminada, o tempo para a primeira visita médica aumentou em 2019 (P=0,03), com nova diminuição em 2020 (P=0,04). O atraso diagnóstico foi superior na DC comparativamente com a colite ulcerosa e colite indeterminada. Conclusão O atraso diagnóstico na DII pediátrica continua a ser um tema importante, que não sofreu alteração significativa ao longo dos últimos anos. O tempo para a primeira visita ao GP e o tempo para o diagnóstico parecem ter maior impacto no atraso diagnóstico, pelo que são necessárias estratégias para aumentar o reconhecimento dos sintomas da DII entre os médicos de primeira linha, bem como melhorar a comunicação e a referenciação. Apesar das restrições causadas pela pandemia no sistema de saúde, o tempo para o diagnóstico na DII pediátrica não foi comprometido no nosso centro em 2020.

COVID-19 pandemic lockdown effect on neonatal hospital admissions from the community.

BACKGROUND: The lockdowns imposed by countries due to COVID-19 pandemic had enormous impact on healthcare. Our goal is to determine consequences of the COVID-19 pandemic lockdown on neonatal hospitalizations and disease incidence in our hospital. METHODS: Observational retrospective study comparing newborns admitted to the neonatal care unit (NCU) from emergency department (ED). Newborns were distributed in two groups according to the date of the lockdown (22/3/2020): pre-lockdown group (12 month before) and post-lockdown group (12 month after). Categorical variables were compared according to chi square test and continuous variables with Mann-Whitney test. A Bonferroni corrected p-value < 0.006 was considered statistically significant. The monthly hospitalization rate between the two groups was analyzed with Generalized Method of Moments - System. RESULTS: We included 99 patients, 65/99 (65.7%) in the pre-lockdown group and 34/99 (34.3%) in the post-lockdown group. Pre-lockdown group: median age at hospitalization 19 (10-26) days, duration of symptoms 12 (5.5-36) hours. Post-lockdown group: median age at hospitalization 16 (6-24) days, duration of symptoms 14 (6-72) hours. The incidence of contagious disease was higher in the pre-lockdown group: 27/65 (41.5%) versus 3/34 (8.8%) in post-lockdown group (p = 0.001). No statistically significant difference in gestational age, gender, age at hospitalization, duration of symptoms and length of stay. CONCLUSION: Lockdown decreased admissions in NCU due to the decrease in contagious infections. The similar duration of symptoms before age at hospitalization in the two groups might indicate that health care accessibility has been maintained.

Impact of COVID-19 in Pediatric Patients and Young Adults with Inflammatory Bowel Disease.

Introduction: Acute COVID-19 in pediatric and young adult patients tends to be milder in severity compared to adult infection. Recent studies seem to show that inflammatory bowel disease (IBD) patients are at no greater risk than the general population. We aim to describe our experience in the follow-up of pediatric and young adult patients with IBD followed in our center and determine possible risk factors of said population for severe COVID-19. Methods: We performed a retrospective study of all patients aged under 25 years followed for IBD at the Unit of Pediatric Gastroenterology in a tertiary center between December 2019 and April 2021 evaluating the incidence of COVID-19 and characterization of positive cases. Results: Of the 268 participants, 24 had COVID-19: the mean age was 19 years old and gender had an equal distribution; 75% (n = 18) had Crohn's disease, whereas only 25% (n = 6) had ulcerative colitis. Most patients were in clinical remission (n = 21). The majority of patients were under treatment with a tumor necrosis factor (TNF) antagonist (58%, n = 14), mainly infliximab, and most had no comorbidities other than IBD (83%). Regarding COVID-19, 17% of the patients were asymptomatic while the rest had only mild symptoms. There were no reported gastrointestinal complaints, no complications nor hospitalizations. Most patients did not require interruption of their IBD treatment. Conclusions: Our data suggest that pediatric and young adult IBD patients have a low risk for complications and hospitalization, regardless of IBD treatment. We believe that this experience is encouraging and allows for safe counseling regarding treatment options and school attendance in pediatric and young adult IBD patients.

Introdução: Na população pediátrica e de jovens adultos a gravidade da COVID-19 tende a ser moderada quando comparada com os doentes adultos. Os estudos mais recentes sugerem que os doentes com doença inflamatória intestinal (DII) não têm risco acrescido em relação à população geral. O objetivo do presente estudo é a descrição da nossa experiência no follow-up de crianças e jovens adultos com DII a COVID-19 e determinar a existência de possíveis fatores de risco para doença grave na referida população. Métodos: Foi realizado um estudo retrospetivo de todos os doentes com idade inferior a 25 anos, seguidos na Unidade de Gastrenterologia Pediátrico de um centro terciário por DII, com avaliação da incidência de COVID-19 entre dezembro de 2019 e abril de 2021, e caracterização dos casos postivos. Resultados: Entre os 268 participantes, 24 tiveram COVID-19. A idade média foi de 19 anos com uma distribuição por género equiparável. Destes, 75% (n = 18) tinham doença de Crohn, enquanto 25% (6) tinham colite ulcerosa. A maior parte dos doentes apresentavam-se em remissão clínica (n = 21) e, à data da doença COVID-19. A sua maioria, os doentes encontravam-se sob tratamento com antagonistas do fator de necrose tumoral (58%, n = 14), predominantemente o infliximab, e a generalidade dos doentes (83%) não apresentava outras comorbilidades além da DII. Relativamente à COVID-19, 17% eram assintomáticos enquanto os restantes apresentavam apenas sintomas ligeiros. Não houve relato de queixas gastrointestinais, complicações ou necessidade de hospitalização. Na maioria dos casos, não houve necessidade de interromper o tratamento da DII. Conclusão: Os nossos dados sugerem que doentes pediátricos e jovens adultos com DII apresentam um risco baixo de complicações ou hospitalização associados à COVID-19, independentemente do tratamento em curso para a DII. Este estudo apresenta resultados encorajadores e contribui para o aconselhamento adequado e fundamentado aos doentes e respetivos cuidadores, no que diz respeito às opções terapêuticas e frequência escolar dos doentes pediátricos e jovens adultos com DII.

Leaf economics and slow-fast adaptation across the geographic range of Arabidopsis thaliana.

Life history strategies of most organisms are constrained by resource allocation patterns that follow a 'slow-fast continuum'. It opposes slow growing and long-lived organisms with late investment in reproduction to those that grow faster, have earlier and larger reproductive effort and a short longevity. In plants, the Leaf Economics Spectrum (LES) depicts a leaf-level trade-off between the rate of carbon assimilation and leaf lifespan, as stressed in functional ecology from interspecific comparative studies. However, it is still unclear how the LES is connected to the slow-fast syndrome. Interspecific comparisons also impede a deep exploration of the linkage between LES variation and adaptation to climate. Here, we measured growth, morpho-physiological and life-history traits, at both the leaf and whole-plant levels, in 378 natural accessions of Arabidopsis thaliana. We found that the LES is tightly linked to variation in whole-plant functioning, and aligns with the slow-fast continuum. A genetic analysis further suggested that phenotypic differentiation results from the selection of different slow-fast strategies in contrasted climates. Slow growing and long-lived plants were preferentially found in cold and arid habitats while fast growing and short-lived ones in more favorable habitats. Our findings shed light on the role of the slow-fast continuum for plant adaptation to climate. More broadly, they encourage future studies to bridge functional ecology, genetics and evolutionary biology to improve our understanding of plant adaptation to environmental changes.

Prevalence of erectile dysfunction in Spanish primary care setting and its association with cardiovascular risk factors and cardiovascular diseases. SIMETAP-ED study / Prevalencia de la disfunción eréctil en el ámbito de la atención primaria española y su asociación con factores de riesgo cardiovasculares y enfermedades cardiovasculares. Estudio SIMETAP-ED

Introduction: Few studies conducted in primary care setting report about age-adjusted prevalence rates of erectile dysfunction (ED). Aims of SIMETAP-ED study were to determine crude and age-adjusted prevalence rates of ED diagnosis, to compare these rates with other similar studies, and to compare prevalence rates of cardiovascular risk factors (CVRF), cardiovascular diseases (CVD), metabolic diseases and chronic kidney disease (CKD) between populations with and without ED. Methods: Cross-sectional observational study conducted in primary care setting. Population-based random sample: 2934 adult men. Response rate: 66%. A clinical interview was conducted to diagnose ED using a question derived from ED definition. The medical records of patients were reviewed to identify their CVRF and diseases associated with ED. The age-adjustments were standardized to Spanish population. Results: The prevalence rates of metabolic diseases, CVD, CVRF, and CKD in population with ED were higher than population without ED, highlighting the CVD. The crude prevalence of ED was 17.2% (95% confidence interval: 15.8-18.6). The age-adjusted prevalence rates of ED were 0.71% in men under 40 years, 12.4% in men over 18 years, 10.8% in men aged 40-69 years, 18.9% in men over 40 years, and 48.6% in men over 70 years. Conclusions: SIMETAP-ED study showed association of ED with metabolic diseases, CKD, CVRF, and highlighting CVD. The age-adjusted prevalence of ED was 12.4% in adult men, 19% in men over 40 years, and almost 50% in men over 70 years

Introducción: Existen pocos estudios realizados en atención primaria sobre prevalencias ajustadas por edad de la disfunción eréctil (ED, por sus siglas en inglés). Los objetivos del estudio SIMETAP-ED fueron determinar las prevalencias crudas y ajustadas por edad del diagnóstico de la ED, comparar estas tasas con otros estudios similares, y comparar las prevalencias de factores de riesgo cardiovasculares (FRCV), enfermedades cardiovasculares (ECV), enfermedades metabólicas y enfermedad renal crónica (ERC) entre las poblaciones con y sin ED. Métodos: Estudio observacional transversal realizado en atención primaria. Muestra aleatoria base poblacional: 2.934 varones adultos. Tasa de respuesta: 66%. Se realizó una entrevista clínica para diagnosticar ED mediante una pregunta derivada de la definición de ED. Se revisaron las historias clínicas de los pacientes para identificar sus FRCV y enfermedades asociadas con la ED. Los ajustes de tasas se estandarizaron con respecto a la población española. Resultados: Las prevalencias de enfermedades metabólicas, ECV, FRCV y ERC en la población con ED fueron más altas que en la población sin ED, destacando las ECV. La prevalencia cruda de la ED fue del 17,21% (intervalo de confianza del 95%: 15,86-18,63). Las tasas de prevalencia ajustadas por edad de la ED fueron del 0,71% en menores de 40 años, del 12,4% en mayores de 18 años, del 10,8% en varones entre 40 y 69 años, del 18,9% en mayores de 40 años y del 48,6% en mayores de 70 años. Conclusiones: El estudio SIMETAP-ED mostró asociación de la ED con las enfermedades metabólicas, ERC, FRCV y, sobre todo, con ECV. La prevalencia ajustada por edad de la ED fue del 12,4% en varones adultos, del 19% en mayores de 40 años y casi del 50% en mayores de 70 años

Prevalence of erectile dysfunction in Spanish primary care setting and its association with cardiovascular risk factors and cardiovascular diseases. SIMETAP-ED study.

INTRODUCTION: Few studies conducted in primary care setting report about age-adjusted prevalence rates of erectile dysfunction (ED). Aims of SIMETAP-ED study were to determine crude and age-adjusted prevalence rates of ED diagnosis, to compare these rates with other similar studies, and to compare prevalence rates of cardiovascular risk factors (CVRF), cardiovascular diseases (CVD), metabolic diseases and chronic kidney disease (CKD) between populations with and without ED. METHODS: Cross-sectional observational study conducted in primary care setting. Population-based random sample: 2934 adult men. Response rate: 66%. A clinical interview was conducted to diagnose ED using a question derived from ED definition. The medical records of patients were reviewed to identify their CVRF and diseases associated with ED. The age-adjustments were standardized to Spanish population. RESULTS: The prevalence rates of metabolic diseases, CVD, CVRF, and CKD in population with ED were higher than population without ED, highlighting the CVD. The crude prevalence of ED was 17.2% (95% confidence interval: 15.8-18.6). The age-adjusted prevalence rates of ED were 0.71% in men under 40 years, 12.4% in men over 18 years, 10.8% in men aged 40-69 years, 18.9% in men over 40 years, and 48.6% in men over 70 years. CONCLUSIONS: SIMETAP-ED study showed association of ED with metabolic diseases, CKD, CVRF, and highlighting CVD. The age-adjusted prevalence of ED was 12.4% in adult men, 19% in men over 40 years, and almost 50% in men over 70 years.

Traits determining the digestibility-decomposability relationships in species from Mediterranean rangelands.

Background and Aims: Forage quality for herbivores and litter quality for decomposers are two key plant properties affecting ecosystem carbon and nutrient cycling. Although there is a positive relationship between palatability and decomposition, very few studies have focused on larger vertebrate herbivores while considering links between the digestibility of living leaves and stems and the decomposability of litter and associated traits. The hypothesis tested is that some defences of living organs would reduce their digestibility and, as a consequence, their litter decomposability, through 'afterlife' effects. Additionally in high-fertility conditions the presence of intense herbivory would select for communities dominated by fast-growing plants, which are able to compensate for tissue loss by herbivory, producing both highly digestible organs and easily decomposable litter. Methods: Relationships between dry matter digestibility and decomposability were quantified in 16 dominant species from Mediterranean rangelands, which are subject to management regimes that differ in grazing intensity and fertilization. The digestibility and decomposability of leaves and stems were estimated at peak standing biomass, in plots that were either fertilized and intensively grazed or unfertilized and moderately grazed. Several traits were measured on living and senesced organs: fibre content, dry matter content and nitrogen, phosphorus and tannin concentrations. Key results: Digestibility was positively related to decomposability, both properties being influenced in the same direction by management regime, organ and growth forms. Digestibility of leaves and stems was negatively related to their fibre concentrations, and positively related to their nitrogen concentration. Decomposability was more strongly related to traits measured on living organs than on litter. Digestibility and decomposition were governed by similar structural traits, in particular fibre concentration, affecting both herbivores and micro-organisms through the afterlife effects. Conclusions: This study contributes to a better understanding of the interspecific relationships between forage quality and litter decomposition in leaves and stems and demonstrates the key role these traits play in the link between plant and soil via herbivory and decomposition. Fibre concentration and dry matter content can be considered as good predictors of both digestibility and decomposability.

Benefits of different postoperative treatments in patients undergoing knee arthroscopic debridement.

PURPOSE: To assess the effectiveness of viscosupplementation or platelet-rich plasma (PRP), compared to standard care, for pain relief after knee arthroscopic debridement in patients with meniscal pathology and osteoarthritis (OA), under normal clinical practice conditions. PATIENTS AND METHODS: We conducted a prospective, randomized, evaluator-blind, pilot study. After arthroscopy, patients were randomized to receive 1) five injections of HA1 (Suprahyal®/Adant®); 2) four injections of HA2 (Orthovisc®); 3) three injections of HA3 (Synvisc®); 4) a single injection of PRP (GPS™ II); or 5) standard care (control). Patients were followed up for 18 months. Clinical outcomes were evaluated using the Western Ontario and McMaster Universities Arthritis Index (WOMAC) at 3, 6, 12, and 18 months. Minimally Clinical Important Improvement (MCII), as relative improvement ≥20 for pain and function, was also calculated. RESULTS: Fifty patients were included. At early follow-up (3 months), total WOMAC scores improved in all groups compared to baseline with reductions of 44.79% (HA1), 24.02% (HA2), 40.38% (HA3), 39.77% (PRP), and 27.64% (control) (p=0.002 HA1 compared to HA2). At 18 months, the higher improvement in total WOMAC was in HA1 with a 65.20% reduction, followed by PRP (55.01%), HA3 (49.57%), and HA2 (29.82%), whereas the control group had a 14.55% increase over baseline (p=0.001 control compared to HA1 and HA3). The percentage of patients achieving the MCII for both pain and function at 18 months was 100% (HA1), 80% (HA3), 60% (HA2), and 60% (PRP), whereas, in the control group, all patients returned to pre-arthroscopy levels. There were no adverse events attributable to surgery or to intraarticular administration. CONCLUSION: Viscosupplementation following arthroscopy is more effective than PRP in adequately selected patients with meniscal lesions occurring concomitantly with OA. Further controlled studies with a larger sample size and/or alternative regimens would be of interest for the scientific community.

Assortative mating by colored ornaments in blue tits: space and time matter.

Assortative mating is a potential outcome of sexual selection, and estimating its level is important to better understand local adaptation and underlying trait evolution. However, assortative mating studies frequently base their conclusions on small numbers of individuals sampled over short periods of time and limited spatial scales even though spatiotemporal variation is common. Here, we characterized assortative mating patterns over 10 years in four populations of the blue tit (Cyanistes caeruleus), a passerine bird. We focused on two plumage ornaments-the blue crown and the yellow breast patch. Based on data for 1,657 pairs of birds, we found large interannual variation: assortative mating varied from positive to negative. To determine whether there was nonetheless a general trend in the data, we ran a within-study meta-analysis. It revealed that assortative mating was moderately positive for both ornaments. It also showed that mating patterns differed among populations and especially between two neighboring populations that displayed phenotypic divergence. Our results therefore underscore that long-term studies are needed to draw broad conclusions about mating patterns in natural populations. They also call for studying the potential role of assortative mating in local adaptation and evolution of ornaments in both sexes.

The intensity threshold of colour vision in a passerine bird, the blue tit (Cyanistes caeruleus).

Many vertebrates use colour vision for vital behaviour but their visual performance in dim light is largely unknown. The light intensity threshold of colour vision is known only for humans, horses and two parrot species. Here, we first explore this threshold in a passerine bird, the blue tit (Cyanistes caeruleus). Using classic conditioning of colour cues to food rewards in three individuals, we find a threshold ranging from 0.05 to 0.2 cd m(-2). Results are comparable to the two previously tested bird species. For tits, nest light conditions probably exceed that threshold, at least after sunrise. These results shed new light on the lively debate questioning the visual performance of cavity nesters and the evolutionary significance of egg and chick coloration. Although this needs further investigation, it is possible that blue tits exploit both colour and brightness cues when viewing their eggs, chicks or conspecifics in their nests.

New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase.

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)2D3 as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [³H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)2D3 and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)2D3 receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)2D3 supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)2D3 and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.

Activation of mTOR and RhoA is a major mechanism by which Ceramide 1-phosphate stimulates macrophage proliferation.

This study tested the hypothesis that Ceramide 1-phosphate (C1P) stimulates macrophage proliferation through activation of the mammalian target of rapamycin (mTOR). We first reported that C1P is mitogenic for fibroblasts and macrophages, but the mechanisms whereby it stimulates cell proliferation are incompletely understood. Here we demonstrate that C1P causes phosphorylation of mTOR in primary (bone marrow-derived) macrophages. Activation of this kinase was tested my measuring the phosphorylation state of its downstream target p70S6K after treatment with C1P. These actions were dependent upon prior activation of phosphoinositide 3 kinase (PI3-K), as selective inhibition of this kinase blocked mTOR phosphorylation and activation. In addition, C1P caused phosphorylation of PRAS40, a component of the mTOR complex 1 (mTORC1) that is absent in mTORC2. Furthermore, inhibition of the small G protein Ras homolog enriched in brain (Rheb), which is also a specific component of mTORC1, with FTI277, completely blocked C1P-stimulated mTOR phosphorylation, DNA synthesis and macrophage growth. In addition, C1P caused phosphorylation of another Ras homolog gene family member, RhoA, which is also involved in cell proliferation. Interestingly, inhibition of the RhoA downstream effector RhoA-associated kinase (ROCK) also blocked C1P-stimulated mTOR and cell proliferation. It can be concluded that mTORC1, and RhoA/ROCK are essential components of the mechanism whereby C1P stimulates macrophage proliferation.

Ceramide-1-phosphate in cell survival and inflammatory signaling.

An important metabolite of ceramide is ceramide-1-phosphate (C1P). This lipid second messenger was first demonstrated to be mitogenic for fibroblasts and macrophages and later shown to have antiapoptotic properties. C1P is also an important mediator of the inflammatory response, by stimulating the release of arachidonic acid through activation of group IVA cytosolic phospholipase A2, the initial rate-limiting step of eicosanoid biosynthesis. C1P is formed from ceramide by the action of a specific ceramide kinase (CerK), which is distinct from the sphingosine kinases that synthesize sphingosine-1-phosphate. CerK is specific for natural ceramides with the erythro configuration in the base component and esterified to long-chain fatty acids. CerK can be activated by different agonists, including interleukin 1-beta, macrophage colony stimulating factor, or calcium ions. Most of the effects of C1P so far described seem to take place in intracellular compartments; however, the recent observation that C1P stimulates cell migration implicates a specific plasma membrane receptor that is coupled to a G(i) protein. Therefore, C1P has a dual regulatory capacity acting as an intracellular second messenger to regulate cell survival, or as extracellular receptor ligand to stimulate chemotaxis.

Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease.

Simple bioactive sphingolipids include ceramide, sphingosine and their phosphorylated forms sphingosine 1-phosphate and ceramide 1-phosphate. These molecules are crucial regulators of cell functions. In particular, they play important roles in the regulation of angiogenesis, apoptosis, cell proliferation, differentiation, migration, and inflammation. Decoding the mechanisms by which these cellular functions are regulated requires detailed understanding of the signaling pathways that are implicated in these processes. Most importantly, the development of inhibitors of the enzymes involved in their metabolism may be crucial for establishing new therapeutic strategies for treatment of disease.

Activation of protein kinase C-alpha is essential for stimulation of cell proliferation by ceramide 1-phosphate.

We previously demonstrated that ceramide-1-phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described. Here we demonstrate that C1P induces translocation of protein kinase C-alpha (PKC-alpha) from the soluble to the membrane fraction of bone marrow-derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC-alpha was independent of prior stimulation of phosphatidylinositol-dependent or phosphatidylcholine-dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC-alpha activation also blocked C1P-stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.

Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages.

We previously reported that incubation of bone-marrow derived macrophages in the absence of macrophage-colony stimulating factor (M-CSF), a cytokine that is essential for their growth and survival, resulted in stimulation of acid sphingomyelinase, accumulation of ceramides, and induction of apoptosis [A. Gomez-Munoz et al. 2004. Ceramide 1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res 45: 99-105]. Here, we show that alveolar NR8383 macrophages, which are not dependent on M-CSF for viability, undergo apoptosis when they are incubated in the absence of serum. NR8383 cells showed increased levels of ceramides under apoptotic conditions, but in contrast to bone marrow macrophage acid and neutral sphingomyelinases were only slightly activated. We found that the major mechanism for ceramide generation in NR8383 macrophages was stimulation of their synthesis de novo. This action involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of this pathway. A relevant finding was that ceramide 1-phosphate (C1P) inhibited SPT activity and ceramide accumulation leading to inhibition of apoptosis. Furthermore, C1P enhanced the activity of antiapoptotic protein kinase B and its downstream effector nuclear factor kappa B. These observations add a new dimension to the understanding of the pro-survival actions of C1P in mammalian cells.

Ceramide 1-phosphate (C1P) promotes cell migration Involvement of a specific C1P receptor.

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid that is implicated in the regulation of cell homeostasis and the control of inflammation. It is mitogenic for fibroblasts and macrophages, and has been described as potent inhibitor of apoptosis. Using RAW 264.7 macrophages we have now discovered a new biological activity of C1P: stimulation of cell migration. This novel action can only be observed when C1P is applied exogenously to the cells in culture, and not by increasing the intracellular levels of C1P. This fact led to identify a specific receptor through which C1P stimulates cell migration. The receptor is coupled to G(i) proteins and causes phosphorylation of extracellularly regulated kinases 1 and 2, and protein kinase B (also known as Akt) upon ligation with C1P. Inhibition of either of these pathways completely abolished C1P-stimulated macrophage migration. In addition, C1P stimulated the DNA binding activity of nuclear factor kappa B, and blockade of this transcription factor resulted in complete inhibition of macrophage migration. This newly identified receptor could be an important drug target for treatment of illnesses that are associated to inflammatory processes, or to diseases in which cell migration is a major cause of pathology, as it occurs in metastatic tumors.

Chain length specificity for activation of cPLA2alpha by C1P: use of the dodecane delivery system to determine lipid-specific effects.

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of >or=6 carbons efficiently activated cPLA(2)alpha in vitro, whereas C(2)-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA(2)alpha, AA release, and PGE(2) synthesis (EC(50) = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specific increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more efficiently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C(2)-C1P was shown to be ineffective in the induction of AA release as compared with C(6)-C1P, C(16)-C1P, and C(18:1) C1P. Here, we demonstrate that C1P requires >or=6 carbon acyl-chain to activate cPLA(2)alpha. Thus, published reports on the biological activity of C(2)-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently deliver exogenous phospholipids to cells for the examination of specific biological effects.