RESUMEN
Biological sex influences inflammatory response, as there is a greater incidence of acute inflammation in men and chronic inflammation in women. Here, we report that acute inflammation is attenuated by X-inactive specific transcript (Xist), a female cell-specific nuclear long noncoding RNA crucial for X-chromosome inactivation. Lipopolysaccharide-mediated acute inflammation increased Xist levels in the cytoplasm of female mouse J774A.1 macrophages and human AML193 monocytes. In both cell types, cytoplasmic Xist colocalizes with the p65 subunit of NF-κB. This interaction was associated with reduced NF-κB nuclear migration, suggesting a novel mechanism to suppress acute inflammation. Further supporting this hypothesis, expression of 5' XIST in male cells significantly reduced IL-6 and NF-κB activity. Adoptive transfer of male splenocytes expressing Xist reduced acute paw swelling in male mice indicating that Xist can have a protective anti-inflammatory effect. These findings show that XIST has functions beyond X chromosome inactivation and suggest that XIST can contribute to sex-specific differences underlying inflammatory response by attenuating acute inflammation in women.
Asunto(s)
Inflamación/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Células Cultivadas , Citoplasma/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/patología , Inflamación/prevención & control , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Monocitos/metabolismo , FN-kappa B/metabolismo , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Factores Sexuales , Factor de Transcripción ReIA/metabolismoRESUMEN
Mutations of the SPAST gene, which encodes the microtubule-severing protein spastin, are the most common cause of hereditary spastic paraplegia (HSP). Haploinsufficiency is the prevalent opinion as to the mechanism of the disease, but gain-of-function toxicity of the mutant proteins is another possibility. Here, we report a new transgenic mouse (termed SPASTC448Y mouse) that is not haploinsufficient but expresses human spastin bearing the HSP pathogenic C448Y mutation. Expression of the mutant spastin was documented from fetus to adult, but gait defects reminiscent of HSP (not observed in spastin knockout mice) were adult onset, as is typical of human patients. Results of histological and tracer studies on the mouse are consistent with progressive dying back of corticospinal axons, which is characteristic of the disease. The C448Y-mutated spastin alters microtubule stability in a manner that is opposite to the expectations of haploinsufficiency. Neurons cultured from the mouse display deficits in organelle transport typical of axonal degenerative diseases, and these deficits were worsened by depletion of endogenous mouse spastin. These results on the SPASTC448Y mouse are consistent with a gain-of-function mechanism underlying HSP, with spastin haploinsufficiency exacerbating the toxicity of the mutant spastin proteins. These findings reveal the need for a different therapeutic approach than indicated by haploinsufficiency alone.
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Paraplejía Espástica Hereditaria/genética , Espastina/genética , Animales , Transporte Axonal/fisiología , Axones/metabolismo , Modelos Animales de Enfermedad , Mutación con Ganancia de Función/genética , Haploinsuficiencia , Haplotipos , Ratones , Ratones Transgénicos , Microtúbulos/metabolismo , Proteínas Mutantes/genética , Mutación , Neuronas/metabolismo , Paraplejía Espástica Hereditaria/fisiopatología , Espastina/fisiologíaRESUMEN
BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE.
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Síndromes de Dolor Regional Complejo/sangre , Síndromes de Dolor Regional Complejo/genética , Exosomas/genética , MicroARNs/genética , Intercambio Plasmático , Regiones no Traducidas 3'/genética , Adulto , Secuencia de Bases , Exosomas/ultraestructura , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Interleucina-6/sangre , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células THP-1 , Factor de Necrosis Tumoral alfa/sangreRESUMEN
BACKGROUND: Ketamine provides relief for a subset of patients with complex regional pain syndrome (CRPS). The poor responders had a lower body mass index (BMI) relative to responders. Regulation of proopiomelanocortin (POMC) expression is crucial in normal body weight homeostasis. The main objectives of this study were to investigate the mechanisms underlying lower BMI characterizing CRPS patients responding poorly to intravenous ketamine therapy and identify potential biomarkers for predicting response. METHODS: We investigated POMC transcript levels in blood from CRPS patients grouped as responders and poor responders to ketamine therapy. Plasma levels of ß-endorphin, ACTH and α-MSH were measured by ELISA. We previously identified differential expression of small noncoding microRNA hsa-miR-34a in blood between responders and poor responders. We investigated whether a 11-fold downregulation of hsa-miR-34a in poor responders relative to responders is contributing to the differences in POMC levels by targeting POMC regulator CRHR1. Binding of miR-34a to CRHR1 was assessed using reporter assay; changes in mRNA and protein levels of CRHR1 were used to determine the regulation of CRHR1 by miR-34a. RNA from blood of CRPS and control subjects were used for quantitative PCR for CRHR1. RESULTS: Though ketamine treatment did not alter POMC expression, poor responders had higher levels of POMC mRNA than responders, both before and after treatment. Corticotropin-releasing hormone (CRH) is a key regulator of POMC expression and the biological effects are mediated through its receptor corticotrophin releasing hormone receptor 1 (CRHR1). We show that hsa-miR-34a is a negative regulator of CRHR1; overexpression of hsa-miR-34a in Jurkat cells resulted in reduction of CRH-mediated POMC expression. Poor responders had higher expression of CRHR1 transcripts than responders, indicating a regulatory role for miR-34a. In addition, we found positive correlations between the pretreatment levels of miR-34a to BMI and response to ketamine therapy. CONCLUSIONS: Our findings indicate a mechanism by which hsa-miR-34a can regulate the CRH/CRHR1/POMC axis and may influence BMI. Studies in larger patient cohorts are required to confirm the biomarker utility of circulating hsa-miR-34a levels in predicting treatment response to ketamine therapy.
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Síndromes de Dolor Regional Complejo/genética , MicroARNs/metabolismo , Proopiomelanocortina/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Hormona Adrenocorticotrópica/sangre , Índice de Masa Corporal , Síndromes de Dolor Regional Complejo/sangre , Síndromes de Dolor Regional Complejo/tratamiento farmacológico , Regulación de la Expresión Génica , Células HEK293 , Humanos , Células Jurkat , Ketamina/uso terapéutico , MicroARNs/genética , Proopiomelanocortina/sangre , Receptores de Hormona Liberadora de Corticotropina/genética , Reproducibilidad de los Resultados , alfa-MSH/sangre , betaendorfina/sangreRESUMEN
OBJECTIVE: The objective of this study was to use a genome-wide association (GWAS) approach and pooled DNA strategy to search for new genomic loci associated with complex regional pain syndrome (CRPS). DESIGN: The study cohort consisted of 230 patients with established diagnosis of CRPS. The control group consisted of 230 age- and gender-matched subjects without chronic pain. We tested the association of common single nucleotide polymorphisms (SNPs), genotyped using a high-density microarray platform, with CRPS phenotype. This was followed by individual genotyping of the most significant SNPs identified in the microarray genomic scan, in both original discovery (N = 115) and independent verification (N = 115) groups of patients with CRPS, as well as in the appropriate matched control subjects. RESULTS: The results of our study provide no support for the initial hypothesis of the existence of an association between any investigated genomic targets (including GWAS for all genomic loci available on the microarray, and focused scan of the HLA locus on chromosome 6) and CRPS phenotype. CONCLUSIONS: Despite the fact that we interrogated about 83% of all of common SNPs in the human genome, we did not find evidence that any of the investigated common SNPs may be associated with CRPS phenotype.
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Síndromes de Dolor Regional Complejo/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Anciano , Estudios de Cohortes , ADN/análisis , ADN/genética , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Adulto JovenRESUMEN
Complex Regional Pain Syndrome (CRPS) is a serious and painful condition involving the peripheral and central nervous systems. Full comprehension of the disorder's pathophysiology remains incomplete, but research implicates the immune system as a contributor to chronic pain. Because of the impact gastrointestinal bacteria have in the development and behavior of the immune system, this study compares the GI microbial communities of 16 participants with CRPS (5 of whom have intestinal discomforts) and 16 healthy controls using 454 sequencing technology. CRPS subjects were found to have significantly less diversity than their healthy counterparts. Statistical analysis of the phylogenetic classifications revealed significantly increased levels of Proteobacteria and decreased levels of Firmicutes in CRPS subjects. Clustering analysis showed significant separation between healthy controls and CRPS subjects. These results support the hypothesis that the GI microbial communities of CRPS participants differ from those of their healthy counterparts. These variations may hold the key to understanding how CRPS develops and provide information that could yield a potential treatment.
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Bacterias/genética , Síndromes de Dolor Regional Complejo/microbiología , Tracto Gastrointestinal/microbiología , Adulto , Análisis de Varianza , Bacterias/clasificación , ADN/genética , ADN/aislamiento & purificación , Femenino , Genes Bacterianos/genética , Humanos , Masculino , Persona de Mediana Edad , ARN Ribosómico 16S/biosíntesis , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disorder. Although most cases of ALS are sporadic, 5-10% of cases are familial, with mutations associated with over 40 genes. There is variation of ALS symptoms within families carrying the same mutation; the disease may develop in one sibling and not in another despite the presence of the mutation in both. Although the cause of this phenotypic variation is unknown, it is likely related to genetic modifiers of disease expression. The identification of ALS causing genes has led to the development of transgenic mouse models of motor neuron disease. Similar to families with familial ALS, there are background-dependent differences in disease phenotype in transgenic mouse models of ALS suggesting that, as in human ALS, differences in phenotype may be ascribed to genetic modifiers. These genetic modifiers may not cause ALS rather their expression either exacerbates or ameliorates the effect of the mutant ALS causing genes. We have reported that in both the G93A-hSOD1 and G59S-hDCTN1 mouse models, SJL mice demonstrated a more severe phenotype than C57BL6 mice. From reciprocal intercrosses between G93A-hSOD1 transgenic mice on SJL and C57BL6 strains, we identified a major quantitative trait locus (QTL) on mouse chromosome 17 that results in a significant shift in lifespan. In this study we generated reciprocal intercrosses between transgenic G59S-hDCTN1 mice on SJL and C57BL6 strains and identified survival QTLs on mouse chromosomes 17 and 18. The chromosome 17 survival QTL on G93A-hSOD1 and G59S-hDCTN1 mice partly overlap, suggesting that the genetic modifiers located in this region may be shared by these two ALS models despite the fact that motor neuron degeneration is caused by mutations in different proteins. The overlapping region contains eighty-seven genes with non-synonymous variations predicted to be deleterious and/or damaging. Two genes in this segment, NOTCH3 and Safb/SAFB1, have been associated with motor neuron disease. The identification of genetic modifiers of motor neuron disease, especially those modifiers that are shared by SOD1 and dynactin-1 transgenic mice, may result in the identification of novel targets for therapies that can alter the course of this devastating illness.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedad de la Neurona Motora , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Complejo Dinactina/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedad de la Neurona Motora/genética , Sitios de Carácter Cuantitativo/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
BACKGROUND: Aberrant expression of small noncoding RNAs called microRNAs (miRNAs) is a common feature of several human diseases. The objective of the study was to identify miRNA modulation in patients with complex regional pain syndrome (CRPS) a chronic pain condition resulting from dysfunction in the central and/or peripheral nervous systems. Due to a multitude of inciting pathologies, symptoms and treatment conditions, the CRPS patient population is very heterogeneous. Our goal was to identify differentially expressed miRNAs in blood and explore their utility in patient stratification. METHODS: We profiled miRNAs in whole blood from 41 patients with CRPS and 20 controls using TaqMan low density array cards. Since neurogenic inflammation is known to play a significant role in CRPS we measured inflammatory markers including chemokines, cytokines, and their soluble receptors in blood from the same individuals. Correlation analyses were performed for miRNAs, inflammatory markers and other parameters including disease symptoms, medication, and comorbid conditions. RESULTS: Three different groups emerged from miRNA profiling. One group was comprised of 60% of CRPS patients and contained no control subjects. miRNA profiles from the remaining patients were interspersed among control samples in the other two groups. We identified differential expression of 18 miRNAs in CRPS patients. Analysis of inflammatory markers showed that vascular endothelial growth factor (VEGF), interleukin1 receptor antagonist (IL1Ra) and monocyte chemotactic protein-1 (MCP1) were significantly elevated in CRPS patients. VEGF and IL1Ra showed significant correlation with the patients reported pain levels. Analysis of the patients who were clustered according to their miRNA profile revealed correlations that were not significant in the total patient population. Correlation analysis of miRNAs detected in blood with additional parameters identified miRNAs associated with comorbidities such as headache, thyroid disorder and use of narcotics and antiepileptic drugs. CONCLUSIONS: miRNA profiles can be useful in patient stratification and have utility as potential biomarkers for pain. Differentially expressed miRNAs can provide molecular insights into gene regulation and could lead to new therapeutic intervention strategies for CRPS.
Asunto(s)
Síndromes de Dolor Regional Complejo/genética , MicroARNs/genética , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Síndromes de Dolor Regional Complejo/sangre , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/sangre , Inflamación/genética , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Estadística como AsuntoRESUMEN
Complex regional pain syndrome (CRPS) is a chronic pain condition characterized by inflammation and debilitating pain. CRPS patients with pain refractory to more conventional analgesics can be treated with subanesthetic doses of ketamine. Our previous studies found that poor responders to ketamine had a 22-fold downregulation of the miRNA hsa-miR-605 in blood prior to ketamine treatment. Hence, we sought to investigate the functional significance of miR-605 downregulation and its impact on target gene expression, as investigating target mRNAs of differentially expressed miRNAs can provide important insights on aberrant gene expression that may contribute to disease etiology. Using a bioinformatics prediction, we identified that miR-605 can target the proinflammatory chemokine CXCL5, which plays a role in leukocyte recruitment and activation. We hypothesized that downregulation of miR-605 in poor responders to ketamine could increase CXCL5 expression and thereby contribute to inflammation in these patients. We confirmed that miR-605 regulates CXCL5 by using a miRNA mimic and inhibitor in human primary endothelial cells. Inhibition of miR-605 increased CXCL5 secretion and migration of human monocytic cells, thereby demonstrating a functional impact of miR-605 on chemotaxis. Additionally, CXCL5 mRNA was upregulated in whole blood from poor responders to ketamine, and CXCL5 protein was increased in plasma from CRPS patients. Thus, our studies suggest that miR-605 regulation of CXCL5 can regulate inflammation.
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Quimiocina CXCL5/inmunología , Síndromes de Dolor Regional Complejo/inmunología , MicroARNs/inmunología , Analgésicos/uso terapéutico , Movimiento Celular , Quimiocina CXCL5/sangre , Quimiocina CXCL5/genética , Síndromes de Dolor Regional Complejo/sangre , Síndromes de Dolor Regional Complejo/tratamiento farmacológico , Síndromes de Dolor Regional Complejo/genética , Regulación hacia Abajo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ketamina/uso terapéutico , MicroARNs/metabolismo , Monocitos/inmunología , Monocitos/fisiología , Células THP-1 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: To compare and contrast body mass indices calculated based on self-reported height and weight as compared with measured height and weight in migraine patients. BACKGROUND: Obesity is a risk factor for multiple neurological disorders including stroke, dementia, and migraine chronification. In addition, several cytokines and adipocytokines associated with migraine are modulated by body mass. The body mass index (BMI) is a commonly used anthropometric measure to estimate total body fat and is often calculated based on patient's self-reported height and weight. METHODS: This was a retrospective study evaluating consecutive migraine patients presenting to a headache clinic.Demographic characteristics and self-reported height and weight were obtained from a standardized questionnaire that each new patient completes upon presentation to the clinic. In addition, as depression has been shown to be associated with both migraine and obesity, information in regards to major depression utilizing the Patient Healthcare Questionnaire-9 was extracted as well. Following completion of the questionnaire, body mass indices are routinely measured, with height measured to the nearest 0.5 inch utilizing a mounted stadiometer, and weight measured with a standard scale to the nearest 0.5 lb. After this information was extracted from the charts, BMI was then calculated for both self-reported and measured body mass indices.Using the measured body mass indices as a standard, this was then compared and contrasted to the patient's self-reported body mass indices. RESULTS: A total of 110 patients were included in the study. Patients were predominantly female (91%) with a mean age of 38.6 +/- 11.6 years. Of the total patients included, no significant difference in self-reported height (mean 64.7 +/- 3.1 inches) as compared with measured height (mean 64.5 +/- 3.4 inches) was seen, P = .463. However, self-reported weight (169 +/- 41.3) was underestimated as compared with the measured weight (173.5 +/- 43.2), P = .001. And, the self-reported BMI (28.4 +/- 6.8) was significantly less than the measured BMI (29.4 +/- 7.5), P < .001. CONCLUSIONS: In our study, the self-reported mean weight and BMI for migraineurs was significantly less than the measured mean weight and BMI, and was of greater magnitude in the obese migraineurs. This suggests that conclusions drawn from studies evaluating obesity utilizing self-reported BMI in migraineurs may undercall the effect of total body obesity.
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Índice de Masa Corporal , Peso Corporal/fisiología , Trastornos Migrañosos/complicaciones , Obesidad/complicaciones , Obesidad/diagnóstico , Autoevaluación (Psicología) , Adulto , Trastorno Depresivo/complicaciones , Trastorno Depresivo/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad/psicología , Relaciones Médico-Paciente , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Estudios Retrospectivos , Autocuidado/psicología , Autoimagen , Autorrevelación , Encuestas y CuestionariosRESUMEN
OBJECTIVE: Chronic regional pain syndrome (CRPS) is a severe pain condition that usually results from an injury or surgical procedure. The pain in CRPS often spreads from the site of injury, and with time becomes refractory to conventional therapy. The present study was undertaken to evaluate the effects of 5-day continuous intravenous lidocaine treatment in patients afflicted with CRPS. METHODS: Intravenous lidocaine was administered in an escalating dose schedule to 49 severely affected CRPS patients in a monitored setting over 5 days. Evaluation of pain parameters and other signs and symptoms of CRPS were obtained during the infusion and at 1, 3, and 6 months following therapy. RESULTS: The majority of patients demonstrated a significant decrease in pain parameters and other symptoms and signs of CRPS. The pain reduction lasted an average of 3 months. Lidocaine may be particularly effective for thermal and mechanical allodynia. Less clinically significant effects were documented on the motor aspects of the syndrome. DISCUSSION: Intravenous lidocaine administration titrated to 5 mg/L demonstrated: 1) a significant decrease in mechanical and thermal allodynia for three months, 2) lessened associated inflammatory components of CRPS, and 3) only minimal side effects and no severe complications.
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Anestésicos Locales/administración & dosificación , Síndromes de Dolor Regional Complejo/tratamiento farmacológico , Lidocaína/administración & dosificación , Adolescente , Adulto , Anestésicos Locales/efectos adversos , Niño , Femenino , Humanos , Infusiones Intravenosas , Lidocaína/efectos adversos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Extracellular RNA in circulation mediates intercellular communication in normal and pathological processes. One mode of circulating miRNA transport in bodily fluids is within 30-150 nm small extracellular vesicles (sEVs) or exosomes. Uptake of sEVs can regulate gene expression in recipient cells enabling circulating miRNAs to exert paracrine and systemic effects. Complex regional pain syndrome (CRPS) is a debilitating pain disorder characterized by chronic inflammation. Our previous investigations identified a significant decrease of hsa-miR-939 in whole blood from CRPS patients compared to control; we also observed that overexpression of miR-939 can negatively regulate several proinflammatory genes in vitro. Though downregulated in whole blood, miR-939 was significantly upregulated in sEVs isolated from patient serum. Here we investigated miR-939 packaging into sEVs in vitro under inflammation induced by monocyte chemoattractant protein-1 (MCP-1), a chemokine that is upregulated in CRPS patients. Stimulation of THP-1 monocytes by MCP-1 led to elevated levels of miR-939 in sEVs, which was abrogated using inhibitors of exosome secretion. miRNAs loaded into exosomes largely contain short miRNA sequence motifs called EXOmotifs. Mutation analysis of miR-939 showed that EXOmotif is one of the possible cellular mechanisms responsible for packaging miR-939 into sEVs. We confirmed gene expression changes in recipient cells following the uptake of sEVs enriched in miR-939 using RNA sequencing. Additionally, our data from primary immune cell-derived sEVs of CRPS patients and controls demonstrate that while the relative expression of miR-939 is higher in sEVs derived from B cells, T cells and NK cells relative to monocyte-derived sEVs in controls, only the B cell-derived sEVs showed a significantly higher level of miR-939 in CRPS patients. Differential miRNA sorting into exosomes and its functional impact on recipient cells may contribute to the underlying pathophysiology of CRPS.
RESUMEN
BACKGROUND: Evidence is overwhelming for sex differences in pain, with women representing the majority of the chronic pain patient population. There is a need to explore novel avenues to elucidate this sex bias in the development of chronic inflammatory pain conditions. Complex regional pain syndrome (CRPS) is a chronic neuropathic pain disorder, and the incidence of CRPS is greater in women than in men by ~4:1. Since neurogenic inflammation is a key feature of CRPS, dysregulation of inflammatory responses can be a factor in predisposing women to chronic pain. METHODS: Our studies investigating alterations in circulating microRNAs (miRNAs) in whole blood from female CRPS patients showed significant differential expression of miRNAs between responders and poor responders to ketamine treatment. Several of these miRNAs are predicted to target the long noncoding RNA, X-inactive-specific transcript (XIST). XIST mediates X-chromosome inactivation and is essential for equalizing the expression of X-linked genes between females and males. Based on the well-established role in inflammatory process, we focused on miR-34a, one of the miRNAs predicted to target XIST, and downregulated in CRPS patients responding poorly to ketamine. RESULTS: Our in vitro and in vivo models of acute inflammation and data from patients with CRPS showed that miR-34a can regulate XIST under inflammation directly, and through pro-inflammatory transcription factor Yin-Yang 1 (YY1). XIST was significantly upregulated in a subset of CRPS patients responding poorly to ketamine. CONCLUSION: Since dysregulation of XIST can result in genes escaping inactivation or reactivation in female cells, further investigations on the role of XIST in the predominance of chronic inflammatory and pain disorders in women is warranted.
RESUMEN
Circulating microRNAs are beneficial biomarkers because of their stability and dysregulation in diseases. Here we sought to determine the role of miR-939, a miRNA downregulated in patients with complex regional pain syndrome (CRPS). Hsa-miR-939 is predicted to target several proinflammatory genes, including IL-6, VEGFA, TNFα, NFκB2, and nitric oxide synthase 2 (NOS2A). Binding of miR-939 to the 3' untranslated region of these genes was confirmed by reporter assay. Overexpression of miR-939 in vitro resulted in reduction of IL-6, NOS2A and NFκB2 mRNAs, IL-6, VEGFA, and NOS2 proteins and NFκB activation. We observed a significant decrease in the NOS substrate l-arginine in plasma from CRPS patients, suggesting reduced miR-939 levels may contribute to an increase in endogenous NOS2A levels and NO, and thereby to pain and inflammation. Pathway analysis showed that miR-939 represents a critical regulatory node in a network of inflammatory mediators. Collectively, our data suggest that miR-939 may regulate multiple proinflammatory genes and that downregulation of miR-939 in CRPS patients may increase expression of these genes, resulting in amplification of the inflammatory pain signal transduction cascade. Circulating miRNAs may function as crucial signaling nodes, and small changes in miRNA levels may influence target gene expression and thus disease.
Asunto(s)
MicroARN Circulante/metabolismo , Síndromes de Dolor Regional Complejo/patología , Regulación de la Expresión Génica , Factores Inmunológicos/biosíntesis , Inflamación/patología , MicroARNs/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , HumanosRESUMEN
Complex Regional Pain Syndrome (CRPS) Types I and II are characterized by various combinations of sensory, autonomic and motor abnormalities. Pain disproportionate to the severity and duration of the inciting event is the most devastating symptom. In animal studies, conditions resulting in exaggerated pain states demonstrate elevated pro-inflammatory cytokines. In addition, pro-inflammatory cytokines have been shown to induce or increase neuropathic and inflammatory pain. Utilizing high sensitivity enzyme linked immunosorbent assay (ELISA), we compared the levels of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-alpha) in the cerebrospinal fluid (CSF) of patients afflicted with CRPS to CSF levels found in other patients with and without painful conditions. The results from this study demonstrated significant increases in IL-1beta and IL-6, but not TNF-alpha in the CSF of individuals afflicted with CRPS as compared to controls. CSF cytokine levels in controls with painful conditions did not differ from levels in controls without pain. These increases showed no correlation with the patient's gender or weight. These results are consistent with studies that suggest that the pathogenesis of CRPS is due in part to central neuroimmune activation.
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Síndromes de Dolor Regional Complejo/líquido cefalorraquídeo , Citocinas/líquido cefalorraquídeo , Adolescente , Adulto , Síndromes de Dolor Regional Complejo/clasificación , Síndromes de Dolor Regional Complejo/complicaciones , Citocinas/clasificación , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Interleucina-1/líquido cefalorraquídeo , Interleucina-6/líquido cefalorraquídeo , Masculino , Persona de Mediana Edad , Neuralgia/líquido cefalorraquídeo , Neuralgia/etiología , Dimensión del Dolor/métodos , Análisis de Regresión , Factor de Necrosis Tumoral alfa/líquido cefalorraquídeoRESUMEN
BACKGROUND: Complex regional pain syndrome (CRPS) is a severe chronic pain condition that most often develops following trauma. Some investigators have postulated CRPS to be a post-traumatic neuralgia associated with distal degeneration of small-diameter peripheral axons. Intravenous immunoglobulin treatment (IVIG) has been shown to be efficacious in the treatment of painful polyneuropathies. Some CRPS patients have been reported to respond to IVIG. Based on a recent hypothesis proposing an autoimmune etiology for CRPS, we decided to offer plasma exchange therapy (PE) to CRPS patients with a clinical presentation suggestive of a small fiber neuropathy. OBJECTIVES: To evaluate the efficacy of PE in a group of CRPS patients with a clinical presentation suggestive of a small fiber neuropathy that were either non-responders or poor responders to their current treatment. STUDY DESIGN: This is a retrospective case series study of CRPS patients that met the Budapest diagnostic criteria for CRPS and received PE as treatment for their illness between September 2012 and June 2014. Approval for this review was granted by the Drexel University Institutional Review Board. SETTING: Drexel University College of Medicine pain clinic METHODS: Thirty-three CRPS patients that received PE treatment were retrospectively studied. The workup for these patients consisted of a complete medical and pain evaluation, the completion of the short-form McGill questionnaire, quantitative sensory testing (QST), and skin punch biopsy. The PE protocol was as follows: all patients had a series of PE therapies (range 5 to 11 with a mean of 7.2) performed over a 2 to 3 week period. Following the PE series, the patients had a pain evaluation and completed the short-form McGill questionnaire. Patients that responded to PE were offered maintenance therapy consisting of either weekly PE or other immune modulating agents. In these patients, their pain was evaluated during the maintenance phase. RESULTS: Thirty of the 33 patients demonstrated significant (P < 0.01) median pain reduction of 64% following the initial series of PE. Three patients demonstrated no improvement. Twenty-four patients are receiving maintenance therapy, the pain reduction in these patients following the initial PE series has been maintained with either weekly PE (n = 15), oral immune modulating agents (n = 8), or IVIG (n = 1). The remaining 6 patients are not receiving maintenance therapy and their pain has returned to pre-treatment levels. In addition, this study suggests that patients with the greatest loss of small fibers and the greatest temperature sensory deficits are most likey to benefit from PE therapy. LIMITATIONS: The major limitation of this study is its retrospective nature which includes non-randomization, non-blinding, and an uncontrolled design. CONCLUSIONS: This study shows that PE is effective in a subset of patients with severe long-standing CRPS and that the reduction in pain following the initial series of PE treatments can be maintained on a weekly PE schedule, IVIG, or with other immune modulating drugs. Large, randomized, placebo controlled studies may be required to confirm and expand these results. Such studies may lead to new therapies for this severe life-altering condition.
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Síndromes de Dolor Regional Complejo/diagnóstico , Síndromes de Dolor Regional Complejo/terapia , Manejo del Dolor/métodos , Intercambio Plasmático/métodos , Adulto , Anciano , Analgésicos/uso terapéutico , Método Doble Ciego , Femenino , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Dimensión del Dolor/métodos , Estudios Retrospectivos , Adulto JovenRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease primarily affecting motor neurons in the central nervous system. Although most cases of ALS are sporadic, about 5-10% of cases are familial (FALS) with approximately 20% of FALS caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. We have reported that hSOD1-G93A transgenic mice modeling this disease show a more severe phenotype when the transgene is bred on a pure SJL background and a milder phenotype when bred on a pure B6 background and that these phenotype differences link to a region on mouse Chromosome 17.To examine whether other models of motor neuron degeneration are affected by genetic background, we bred the mutant human dynactin p150Glued (G59S-hDCTN1) transgene onto inbred SJL and B6 congenic lines. This model is based on an autosomal dominant lower motor neuron disease in humans linked to a mutation in the p150Glued subunit of the dynactin complex. As seen in hSOD1-G93A mice, we observed a more severe phenotype with earlier disease onset (p<0.001) and decreased survival (p<0.00001) when the G59S-hDCTN1 transgene was bred onto the SJL background and delayed onset (p<0.0001) with increased survival (p<0.00001) when bred onto the B6 background. Furthermore, B6 mice with an SJL derived chromosome 17 interval previously shown to delay disease onset in hSOD1-G93A mice also showed delays onset in G59S-hDCTN1 mice suggesting that at least some genetic modifiers are shared. We have shown that genetic background influences phenotype in G59S-hDCTN1 mice, in part through a region of chromosome 17 similar to the G93-hSOD1 ALS mouse model. These results support the presence of genetic modifiers in both these models some of which may be shared. Identification of these modifiers will highlight intracellular pathways involved in motor neuron disease and provide new therapeutic targets that may be applicable to motor neuron degeneration.
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Cruzamiento/métodos , Cromosomas de los Mamíferos/genética , Proteínas Asociadas a Microtúbulos/genética , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/patología , Superóxido Dismutasa/genética , Animales , Modelos Animales de Enfermedad , Complejo Dinactina , Femenino , Humanos , Masculino , Ratones , Ratones Congénicos , Ratones Transgénicos , Mutación , Superóxido Dismutasa-1RESUMEN
Although ketamine is beneficial in treating complex regional pain syndrome (CRPS), a subset of patients respond poorly to therapy. We investigated treatment-induced microRNA (miRNA) changes and their predictive validity in determining treatment outcome by assessing miRNA changes in whole blood from patients with CRPS. Blood samples from female patients were collected before and after 5 days of intravenous ketamine administration. Seven patients were responders and 6 were poor responders. Differential miRNA expression was observed in whole blood before and after treatment. In addition, 33 miRNAs differed between responders and poor responders before therapy, suggesting the predictive utility of miRNAs as biomarkers. Investigation of the mechanistic significance of hsa-miR-548d-5p downregulation in poor responders showed that this miRNA can downregulate UDP-glucuronosyltransferase UGT1A1 mRNA. Poor responders had a higher conjugated/unconjugated bilirubin ratio, indicating increased UGT1A1 activity. We propose that lower pretreatment levels of miR-548d-5p may result in higher UDP-GT activity, leading to higher levels of inactive glucuronide conjugates, thereby minimizing the therapeutic efficacy of ketamine in poor responders. Differences in miRNA signatures can provide molecular insights distinguishing responders from poor responders. Extending this approach to other treatment and outcome assessments might permit stratification of patients for maximal therapeutic outcome. Perspective: This study suggests the usefulness of circulating miRNAs as potential biomarkers. Assessing miRNA signatures before and after treatment demonstrated miRNA alterations from therapy; differences in miRNA signature in responders and poor responders before therapy indicate prognostic value. Mechanistic studies on altered miRNAs can provide new insights into disease.
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Analgésicos/uso terapéutico , Síndromes de Dolor Regional Complejo/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Ketamina/uso terapéutico , MicroARNs/sangre , Administración Intravenosa , Adulto , Anciano , Síndromes de Dolor Regional Complejo/sangre , Femenino , Glucuronosiltransferasa/sangre , Glucuronosiltransferasa/genética , Células Hep G2 , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , ARN Mensajero/metabolismo , Estadística como Asunto , TransfecciónRESUMEN
Transgenic mice expressing multiple copies of the G93A mutant form of SOD1 develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis (ALS). The phenotype of these mice is dependent on the number of transgene copies in their genome. Changes in transgene copy number, although rare, can sometimes occur while mating due to intra locus recombination events during meiosis. The objective of this study was to develop a real time quantitative PCR method to determine changes in transgene copy number in these mice and to evaluate the effect of transgene copy number on the phenotype of the G93A SOD1 mouse model of ALS.
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Esclerosis Amiotrófica Lateral/genética , Modelos Animales de Enfermedad , Dosificación de Gen , Superóxido Dismutasa/genética , Transgenes/fisiología , Animales , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , ARN Mensajero/metabolismo , Recombinación Genética/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Superóxido Dismutasa/metabolismo , Sobrevida/fisiologíaRESUMEN
A decrease in expression of the glutamate transporter GLT-1 is thought to be responsible for the increase in extracellular glutamate observed in patients with amyotrophic lateral sclerosis (ALS) and in a transgenic mouse model of ALS. We examined protein levels of the glutamate transporters GLT-1, GLAST and EAAC1 in the G93A (SOD1) transgenic mouse model of ALS. GLT-1 was detected in two bands (72 and 150 kD). Semi-quantitative analysis of Western blots showed that GLT-1 levels in sensorimotor cortex, brain stem, and cervical and lumbar spinal cord of G93A mice did not differ significantly from controls, either at end stage or at 60- or 90-days old. Nevertheless, other differences were found in GLT-1 at end stage. The percentage of total GLT-1 in the 150 kD band increased significantly (p<0.05) in the spinal cord and was elevated in the brain stem and cortex. Furthermore, brain stem and spinal cord GLT-1 from G93A mice showed retarded mobility on gels compared to controls (M(r) approximately equal to 77.3+/-2.3 and 164.3+/-3.1 vs. 72.2+/-2.4 and 153.6+/-4.7, respectively). GLAST and EAAC1 were unchanged in both amount and mobility. These results show that a loss of GLT-1 protein is not necessary for ALS-like neurodegeneration in G93A mice. However, the changes in GLT-1 mobility and distribution indicate that GLT-1 is altered in mice with the SOD1 mutation.