RESUMEN
BACKGROUND: Thymus mongolicus (family Lamiaceae) is a Thyme subshrub with strong aroma and remarkable environmental adaptability. Limited genomic information limits the use of this plant. RESULTS: Chromosome-level 605.2 Mb genome of T. mongolicus was generated, with 96.28% anchored to 12 pseudochromosomes. The repetitive sequences were dominant, accounting for 70.98%, and 32,593 protein-coding genes were predicted. Synteny analysis revealed that Lamiaceae species generally underwent two rounds of whole genome duplication; moreover, species-specific genome duplication was identified. A recent LTR retrotransposon burst and tandem duplication might play important roles in the formation of the Thymus genome. Using comparative genomic analysis, phylogenetic tree of seven Lamiaceae species was constructed, which revealed that Thyme plants evolved recently in the family. Under the phylogenetic framework, we performed functional enrichment analysis of the genes on nodes that contained the most gene duplication events (> 50% support) and of relevant significant expanded gene families. These genes were highly associated with environmental adaptation and biosynthesis of secondary metabolites. Combined transcriptome and metabolome analyses revealed that Peroxidases, Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferases, and 4-coumarate-CoA ligases genes were the essential regulators of the phenylpropanoid-flavonoid pathway. Their catalytic products (e.g., apigenin, naringenin chalcone, and several apigenin-related compounds) might be responsible for the environmental tolerance and aromatic properties of T. mongolicus. CONCLUSION: This study enhanced the understanding of the genomic evolution of T. mongolicus, enabling further exploration of its unique traits and applications, and contributed to the understanding of Lamiaceae genomics and evolutionary biology.
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Flavonoides , Thymus (Planta) , Filogenia , Apigenina , Cromosomas , Evolución MolecularRESUMEN
Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.
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Errores Innatos del Metabolismo de los Carbohidratos/genética , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas de Transporte de Monosacáridos/deficiencia , Proteína Quinasa C-alfa/fisiología , Secuencia de Aminoácidos , Animales , Transporte Biológico , Errores Innatos del Metabolismo de los Carbohidratos/enzimología , Línea Celular , Células Endoteliales/metabolismo , Eritrocitos/metabolismo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Mutación Missense , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Xenopus laevisRESUMEN
Over 60 mutations of SCN4A encoding the NaV1.4 sodium channel of skeletal muscle have been identified in patients with myotonia, periodic paralysis, myasthenia, or congenital myopathy. Most mutations are missense with gain-of-function defects that cause susceptibility to myotonia or periodic paralysis. Loss-of-function from enhanced inactivation or null alleles is rare and has been associated with myasthenia and congenital myopathy, while a mix of loss and gain of function changes has an uncertain relation to hypokalaemic periodic paralysis. To better define the functional consequences for a loss-of-function, we generated NaV1.4 null mice by deletion of exon 12. Heterozygous null mice have latent myasthenia and a right shift of the force-stimulus relation, without evidence of periodic paralysis. Sodium current density was half that of wild-type muscle and no compensation by retained expression of the foetal NaV1.5 isoform was detected. Mice null for NaV1.4 did not survive beyond the second postnatal day. This mouse model shows remarkable preservation of muscle function and viability for haploinsufficiency of NaV1.4, as has been reported in humans, with a propensity for pseudo-myasthenia caused by a marginal Na(+) current density to support sustained high-frequency action potentials in muscle.
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Debilidad Muscular/genética , Canal de Sodio Activado por Voltaje NAV1.4/genética , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Modelos Animales de Enfermedad , Heterocigoto , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Músculo Esquelético/fisiología , LinajeRESUMEN
Antigen-specific T cell tolerance holds great promise for the treatment of autoimmune diseases. However, strategies to induce durable tolerance using high doses of soluble antigen have to date been unsuccessful, due to lack of efficacy and the risk of hypersensitivity. In the current study we have overcome these limitations by developing a platform for tolerance induction based on engineering the immunoglobulin Fc region to modulate the dynamic properties of low doses (1 µg/mouse; â¼50 µg/kg) of Fc-antigen fusions. Using this approach, we demonstrate that antigen persistence is a dominant factor governing the elicitation of tolerance in the model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), induced by immunizing B10.PL mice with the N-terminal epitope of myelin basic protein. Unexpectedly, our analyses reveal a stringent threshold of antigen persistence for both prophylactic and therapeutic treatments, although distinct mechanisms lead to tolerance in these two settings. Importantly, the delivery of tolerogenic Fc-antigen fusions during ongoing disease results in the downregulation of T-bet and CD40L combined with amplification of Foxp3(+) T cell numbers. The generation of effective, low dose tolerogens using Fc engineering has potential for the regulation of autoreactive T cells.
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Antígenos/inmunología , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Tolerancia Inmunológica/inmunología , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Epítopos/inmunología , Femenino , Citometría de Flujo , Humanos , Inmunización , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Masculino , Ratones Transgénicos , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/prevención & control , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Transient attacks of weakness in hypokalaemic periodic paralysis are caused by reduced fibre excitability from paradoxical depolarization of the resting potential in low potassium. Mutations of calcium channel and sodium channel genes have been identified as the underlying molecular defects that cause instability of the resting potential. Despite these scientific advances, therapeutic options remain limited. In a mouse model of hypokalaemic periodic paralysis from a sodium channel mutation (NaV1.4-R669H), we recently showed that inhibition of chloride influx with bumetanide reduced the susceptibility to attacks of weakness, in vitro. The R528H mutation in the calcium channel gene (CACNA1S encoding CaV1.1) is the most common cause of hypokalaemic periodic paralysis. We developed a CaV1.1-R528H knock-in mouse model of hypokalaemic periodic paralysis and show herein that bumetanide protects against both muscle weakness from low K+ challenge in vitro and loss of muscle excitability in vivo from a glucose plus insulin infusion. This work demonstrates the critical role of the chloride gradient in modulating the susceptibility to ictal weakness and establishes bumetanide as a potential therapy for hypokalaemic periodic paralysis arising from either NaV1.4 or CaV1.1 mutations.
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Bumetanida/uso terapéutico , Canales de Calcio Tipo L/genética , Parálisis Periódica Hipopotasémica/tratamiento farmacológico , Parálisis Periódica Hipopotasémica/genética , Mutación/genética , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Acetazolamida/farmacología , Animales , Arginina/genética , Inhibidores de Anhidrasa Carbónica/farmacología , Modelos Animales de Enfermedad , Potenciales Evocados Motores/efectos de los fármacos , Potenciales Evocados Motores/genética , Furosemida/farmacología , Glucosa/metabolismo , Histidina/genética , Parálisis Periódica Hipopotasémica/patología , Parálisis Periódica Hipopotasémica/fisiopatología , Técnicas In Vitro , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/genética , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/fisiopatologíaRESUMEN
ß-Catenin, a key component of the Wnt signaling pathway, has been implicated in the development of the neuromuscular junction (NMJ) in mice, but its precise role in this process remains unclear. Here we use a ß-catenin gain-of-function mouse model to stabilize ß-catenin selectively in either skeletal muscles or motor neurons. We found that ß-catenin stabilization in skeletal muscles resulted in increased motor axon number and excessive intramuscular nerve defasciculation and branching. In contrast, ß-catenin stabilization in motor neurons had no adverse effect on motor innervation pattern. Furthermore, stabilization of ß-catenin, either in skeletal muscles or in motor neurons, had no adverse effect on the formation and function of the NMJ. Our findings demonstrate that ß-catenin levels in developing muscles in mice are crucial for proper muscle innervation, rather than specifically affecting synapse formation at the NMJ, and that the regulation of muscle innervation by ß-catenin is mediated by a non-cell autonomous mechanism.
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Músculo Esquelético/inervación , Unión Neuromuscular/fisiología , beta Catenina/fisiología , Animales , Ratones , Neuronas Motoras/fisiología , Músculo Esquelético/embriología , Unión Neuromuscular/embriología , Especificidad de Órganos , Sinapsis/fisiologíaRESUMEN
Experimental autoimmune encephalomyelitis is induced in B10.PL (H-2(u)) mice by immunization with the immunodominant N-terminal epitope of myelin basic protein, Ac1-9. In the present study, we show that the site of immunization impacts disease incidence and severity. This effect is more marked in female mice than in males. Although immunization in the flanks is effective in eliciting disease, delivery of Ag in the footpad and tailbase results in poor induction. Analyses of the immune responses in female mice following different immunization regimens indicates that resistance to disease is accompanied by higher levels of IFN-gamma and CD11b(+)Gr-1(int) myeloid cells. Such myeloid cells are known to have a suppressive function, and consistent with this knowledge, blockade of IFN-gamma results in increased disease activity and decreased levels of splenic CD11b(+)Gr-1(int) cells. Conversely, injection of adjuvants (CFA or Pam(3)CSK(4)) in the footpad decreases experimental autoimmune encephalomyelitis incidence and severity. Our study indicates that the site of immunization can impact the magnitude of the ensuing inflammatory response, and that at a certain threshold a protective, regulatory circuit can be elicited.
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Autoantígenos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Inmunidad Innata , Mediadores de Inflamación/fisiología , Interferón gamma/fisiología , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Vacunación/métodos , Animales , Autoantígenos/administración & dosificación , Antígeno CD11b/biosíntesis , Células Cultivadas , Encefalomielitis Autoinmune Experimental/epidemiología , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Incidencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/administración & dosificación , Células Mieloides/inmunología , Células Mieloides/metabolismo , Fragmentos de Péptidos/administración & dosificación , Receptores de Quimiocina/biosíntesis , Índice de Severidad de la EnfermedadRESUMEN
Psychological stress is an important factor in susceptibility to many diseases. Our laboratory has been investigating the impact of stress on the susceptibility to Theiler's virus-induced demyelination (TVID), a mouse model of multiple sclerosis. Using immunodominant viral peptides specific for identification of either CD4(+) or CD8(+) T cells, stress reduced IFN-gamma-producing virus-specific CD4(+) and CD8(+) T cells in the spleen and CD8(+) T cells in the CNS. Expression of mRNA for the Th1 transcription factor T-bet and the Th2 transcription factor GATA-3 were decreased in spleen cells isolated from stressed mice. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus indicated that stress decreased both type 1 and type 2 responses. The adverse effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone, indicating a major role for glucocorticoids. Global stress-induced immunosuppression resulted in higher levels of virus replication and dissemination. The higher viral load subsequently led to an earlier disease onset and more severe clinical and histological signs of demyelinating disease. Our results have important implications for understanding the pathogenesis of MS, and suggest that stressful events during early infection with an agent capable of inducing demyelination may result in immunosuppression and failure to eliminate the pathogen, which in turn may lead to the development of MS.
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Autoinmunidad/fisiología , Infecciones por Cardiovirus/inmunología , Esclerosis Múltiple/inmunología , Estrés Psicológico/inmunología , Theilovirus/inmunología , Animales , Infecciones por Cardiovirus/fisiopatología , Citocinas/inmunología , Citocinas/metabolismo , Glucocorticoides/metabolismo , Humanos , Tolerancia Inmunológica/inmunología , Ratones , Esclerosis Múltiple/fisiopatología , Estrés Psicológico/complicaciones , Estrés Psicológico/fisiopatología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
The development of approaches for Ag delivery to the appropriate subcellular compartments of APCs and the optimization of Ag persistence are both of central relevance for the induction of protective immunity or tolerance. The expression of the neonatal Fc receptor, FcRn, in APCs and its localization to the endosomal system suggest that it might serve as a target for Ag delivery using engineered Fc fragment-epitope fusions. The impact of FcRn binding characteristics of an Fc fragment on in vivo persistence allows this property to also be modulated. We have therefore generated recombinant Fc (mouse IgG1-derived) fusions containing the N-terminal epitope of myelin basic protein that is associated with experimental autoimmune encephalomyelitis in H-2(u) mice. The Fc fragments have distinct binding properties for FcRn that result in differences in intracellular trafficking and in vivo half-lives, allowing the impact of these characteristics on CD4(+) T cell responses to be evaluated. To dissect the relative roles of FcRn and the "classical" FcgammaRs in Ag delivery, analogous aglycosylated Fc-MBP fusions have been generated. We show that engineered Fc fragments with increased affinities for FcRn at pH 6.0-7.4 are more effective in delivering Ag to FcRn-expressing APCs in vitro relative to their lower affinity counterparts. However, higher affinity of the FcRn-Fc interaction at near neutral pH results in decreased in vivo persistence. The trade-off between improved FcRn targeting efficiency and lower half-life becomes apparent during analyses of T cell proliferative responses in mice, particularly when Fc-MBP fusions with both FcRn and FcgammaR binding activity are used.
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Células Presentadoras de Antígenos/inmunología , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Proteína Básica de Mielina/inmunología , Receptores Fc/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Antígenos/genética , Antígenos/inmunología , Antígenos/farmacología , Linfocitos T CD4-Positivos/inmunología , Células CHO , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Epítopos/genética , Epítopos/farmacología , Antígenos de Histocompatibilidad Clase I/genética , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Ratones , Ratones Transgénicos , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/inmunología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Receptores Fc/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The first randomized blinded study of thymectomy in nonthymomatous myasthenia gravis was designed to answer 3 questions: does the combination of prednisone and removal of the thymus gland via extended transsternal thymectomy after 3 years compared with an identical dosing protocol of prednisone alone (1) lead to better disease status for generalized MG patients with antiacetylcholine receptor antibodies, (2) reduce their prednisone requirements, and/or (3) reduce the side-effect burden from medications used to treat the disease? The study demonstrated that thymectomy confers these benefits for patients and sets the stage for inquiries into the benefits of less-invasive approaches to thymic resection.
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Miastenia Gravis/cirugía , Timectomía , Ensayos Clínicos como Asunto , HumanosRESUMEN
Periodic paralysis is an ion channelopathy of skeletal muscle in which recurrent episodes of weakness or paralysis are caused by sustained depolarization of the resting potential and thus reduction of fiber excitability. Episodes are often triggered by environmental stresses, such as changes in extracellular K+, cooling, or exercise. Rest after vigorous exercise is the most common trigger for weakness in periodic paralysis, but the mechanism is unknown. Here, we use knock-in mutant mouse models of hypokalemic periodic paralysis (HypoKPP; NaV1.4-R669H or CaV1.1-R528H) and hyperkalemic periodic paralysis (HyperKPP; NaV1.4-M1592V) to investigate whether the coupling between pH and susceptibility to loss of muscle force is a possible contributor to exercise-induced weakness. In both mouse models, acidosis (pH 6.7 in 25% CO2) is mildly protective, but a return to pH 7.4 (5% CO2) unexpectedly elicits a robust loss of force in HypoKPP but not HyperKPP muscle. Prolonged exposure to low pH (tens of minutes) is required to cause susceptibility to post-acidosis loss of force, and the force decrement can be prevented by maneuvers that impede Cl- entry. Based on these data, we propose a mechanism for post-acidosis loss of force wherein the reduced Cl- conductance in acidosis leads to a slow accumulation of myoplasmic Cl- A rapid recovery of both pH and Cl- conductance, in the context of increased [Cl]in/[Cl]out, favors the anomalously depolarized state of the bistable resting potential in HypoKPP muscle, which reduces fiber excitability. This mechanism is consistent with the delayed onset of exercise-induced weakness that occurs with rest after vigorous activity.
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Parálisis Periódica Hipopotasémica/fisiopatología , Contracción Muscular , Acidosis , Animales , Técnicas de Sustitución del Gen , Concentración de Iones de Hidrógeno , Ratones , Músculo Esquelético/fisiopatología , Mutación , PotasioRESUMEN
Objective. Systemic sclerosis (SSc) is a systemic connective tissue disease of unknown etiology. Aberrant gene expression and epigenetic modifications in circulating immune cells have been implicated in the pathogenesis of SSc. This study is to delineate the interaction network between gene transcription and DNA methylation in PBMC of SSc patients and to identify methylation-regulated genes which are involved in the pathogenesis of SSc. Methods. Genome-wide mRNA transcription and global DNA methylation analysis were performed on PBMC from 18 SSc patients and 19 matched normal controls (NC) using Illumina BeadChips. Differentially expressed genes (DEGs) and differentially methylated positions (DMPs) were integrative analyzed to identify methylation-regulated genes and associated molecular pathways. Results. Transcriptome analysis distinguished 453 DEGs (269 up- and 184 downregulated) in SSc from NC. Global DNA methylation analysis identified 925 DMPs located on 618 genes. Integration of the two lists revealed only 20 DEGs which harbor inversely correlated DMPs, including 12 upregulated (ELANE, CTSG, LTBR, C3AR1, CSTA, SPI1, ODF3B, SAMD4A, PLAUR, NFE2, ZYX, and CTSZ) and eight downregulated genes (RUNX3, PRF1, PRKCH, PAG1, RASSF5, FYN, CXCR6, and F2R). These potential methylation-regulated DEGs (MeDEGs) are enriched in the pathways related to immune cell migration, proliferation, activation, and inflammation activities. Using a machine learning algorism, we identified six out of the 20 MeDEGs, including F2R, CXCR6, FYN, LTBR, CTSG, and ELANE, which distinguished SSc from NC with 100% accuracy. Four genes (F2R, FYN, PAG1, and PRKCH) differentially expressed in SSc with interstitial lung disease (ILD) compared to SSc without ILD. Conclusion. The identified MeDEGs may represent novel candidate factors which lead to the abnormal activation of immune regulatory pathways in the pathogenesis of SSc. They may also be used as diagnostic biomarkers for SSc and clinical complications.
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BACKGROUND: Recent achievement in genetics and epigenetics has led to the exploration of the pathogenesis of systemic lupus erythematosus (SLE). Identification of differentially expressed genes and their regulatory mechanism(s) at whole-genome level will provide a comprehensive understanding of the development of SLE and its devastating complications, lupus nephritis (LN). METHODS: We performed whole-genome transcription and DNA methylation analysis in PBMC of 30 SLE patients, including 15 with LN (SLE LN(+)) and 15 without LN (SLE LN(-)), and 25 normal controls (NC) using HumanHT-12 Beadchips and Illumina Human Methy450 chips. The serum proinflammatory cytokines were quantified using Bio-plex Human Cytokine 27-plex assay. Differentially expressed genes and differentially methylated CpG were analyzed with GenomeStudio, R, and SAM software. The association between DNA methylation and gene expression were tested. Gene interaction pathways of the differentially expressed genes were analyzed by IPA software. RESULTS: We identified 552 upregulated genes and 550 downregulated genes in PBMC of SLE. Integration of DNA methylation and gene expression profiling showed that 334 upregulated genes were hypomethylated, and 479 downregulated genes were hypermethylated. Pathway analysis on the differential genes in SLE revealed significant enrichment in interferon (IFN) signaling and toll-like receptor (TLR) signaling pathways. Nine IFN- and seven TLR-related genes were identified and displayed step-wise increase in SLE LN(-) and SLE LN(+). Hypomethylated CpG sites were detected on these genes. The gene expressions for MX1, GPR84, and E2F2 were increased in SLE LN(+) as compared to SLE LN(-) patients. The serum levels of inflammatory cytokines, including IL17A, IP-10, bFGF, TNF-α, IL-6, IL-15, GM-CSF, IL-1RA, IL-5, and IL-12p70, were significantly elevated in SLE compared with NC. The levels of IL-15 and IL1RA correlated with their mRNA expression. The upregulation of IL-15 may be regulated by hypomethylated CpG sites in the promotor region of the gene. CONCLUSIONS: Our study has demonstrated that significant number of differential genes in SLE were involved in IFN, TLR signaling pathways, and inflammatory cytokines. The enrichment of differential genes has been associated with aberrant DNA methylation, which may be relevant to the pathogenesis of SLE. Our observations have laid the groundwork for further diagnostic and mechanistic studies of SLE and LN.
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Metilación de ADN/genética , Regulación de la Expresión Génica/inmunología , Leucocitos Mononucleares , Lupus Eritematoso Sistémico/genética , Adulto , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Lupus Eritematoso Sistémico/inmunología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Missense mutations at arginine residues in the S4 voltage-sensor domains of NaV1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K(+). Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K(+) during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K(+), yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na(+) current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type NaV1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na(+) was 0.03%, which implies a specific conductance in muscle from heterozygous patients of â¼ 10 µS/cm(2) or 1% of the total resting conductance. Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na(+) current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na(+) current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of NaV, CaV, and KV channels associated with human disease.
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Parálisis Periódica Hipopotasémica/genética , Parálisis Periódica Hipopotasémica/metabolismo , Canales de Sodio/genética , Canales de Sodio/metabolismo , Potenciales de Acción , Animales , Técnicas de Sustitución del Gen , Humanos , Parálisis Periódica Hipopotasémica/fisiopatología , Activación del Canal Iónico/genética , Ratones , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
OBJECTIVE: To test the hypothesis that inhibition of the Na-K-2Cl transporter with bumetanide will reduce the susceptibility to decreases in muscle force in a mouse model of hypokalemic periodic paralysis (HypoPP). METHODS: In vitro contraction tests were performed on soleus muscle isolated from mice with knock-in missense mutations that result in HypoPP (sodium channel NaV1.4-R669H) or hyperkalemic periodic paralysis (HyperPP; sodium channel NaV1.4-M1592V). RESULTS: Bumetanide prevented the development of weakness in 2 mM K(+) and also restored force during an established attack of HypoPP. Stimulation of the Na-K-2Cl transporter via induction of hyperosmolality exacerbated the weakness seen in low K(+) and was also prevented by bumetanide. Bumetanide was more efficacious than acetazolamide for preventing weakness in low K(+) conditions. Decreases in force in HyperPP muscle exposed to 10 mM K(+) were not prevented by treatment with bumetanide. CONCLUSIONS: The Na-K-2Cl inhibitor bumetanide was highly effective in preventing attacks of weakness in the NaV1.4-R669H mouse model of HypoPP and should be considered for management of patients with HypoPP due to sodium channel mutations. Dehydration may aggravate HypoPP by stimulating the Na-K-2Cl transporter.
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Bumetanida/uso terapéutico , Parálisis Periódica Hipopotasémica/prevención & control , Parálisis Periódica Hipopotasémica/fisiopatología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Animales , Bumetanida/farmacología , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Mutación Missense/fisiología , Técnicas de Cultivo de ÓrganosRESUMEN
Hypokalemic periodic paralysis (HypoPP) is a familial skeletal muscle disorder that presents with recurrent episodes of severe weakness lasting hours to days associated with reduced serum potassium (K+). HypoPP is genetically heterogeneous, with missense mutations of a calcium channel (Ca(V)1.1) or a sodium channel (Na(V)1.4) accounting for 60% and 20% of cases, respectively. The mechanistic link between Ca(V)1.1 mutations and the ictal loss of muscle excitability during an attack of weakness in HypoPP is unknown. To address this question, we developed a mouse model for HypoPP with a targeted Ca(V)1.1 R528H mutation. The Ca(V)1.1 R528H mice had a HypoPP phenotype for which low K+ challenge produced a paradoxical depolarization of the resting potential, loss of muscle excitability, and weakness. A vacuolar myopathy with dilated transverse tubules and disruption of the triad junctions impaired Ca2+ release and likely contributed to the mild permanent weakness. Fibers from the Ca(V)1.1 R528H mouse had a small anomalous inward current at the resting potential, similar to our observations in the Na(V)1.4 R669H HypoPP mouse model. This "gating pore current" may be a common mechanism for paradoxical depolarization and susceptibility to HypoPP arising from missense mutations in the S4 voltage sensor of either calcium or sodium channels.
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Canales de Calcio Tipo L/genética , Parálisis Periódica Hipopotasémica/genética , Fibras Musculares Esqueléticas/metabolismo , Mutación Missense , Potenciales de Acción , Análisis de Varianza , Animales , Canales de Calcio Tipo L/metabolismo , Modelos Animales de Enfermedad , Estimulación Eléctrica , Acoplamiento Excitación-Contracción , Femenino , Glucosa , Humanos , Parálisis Periódica Hipopotasémica/inducido químicamente , Parálisis Periódica Hipopotasémica/patología , Técnicas In Vitro , Insulina , Enfermedades por Almacenamiento Lisosomal/genética , Masculino , Ratones , Ratones de la Cepa 129 , Contracción Muscular , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/fisiología , Debilidad Muscular/genética , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Enfermedades Musculares/genética , FenotipoRESUMEN
Hypokalemic periodic paralysis (HypoPP) is an ion channelopathy of skeletal muscle characterized by attacks of muscle weakness associated with low serum K+. HypoPP results from a transient failure of muscle fiber excitability. Mutations in the genes encoding a calcium channel (CaV1.1) and a sodium channel (NaV1.4) have been identified in HypoPP families. Mutations of NaV1.4 give rise to a heterogeneous group of muscle disorders, with gain-of-function defects causing myotonia or hyperkalemic periodic paralysis. To address the question of specificity for the allele encoding the NaV1.4-R669H variant as a cause of HypoPP and to produce a model system in which to characterize functional defects of the mutant channel and susceptibility to paralysis, we generated knockin mice carrying the ortholog of the gene encoding the NaV1.4-R669H variant (referred to herein as R669H mice). Homozygous R669H mice had a robust HypoPP phenotype, with transient loss of muscle excitability and weakness in low-K+ challenge, insensitivity to high-K+ challenge, dominant inheritance, and absence of myotonia. Recovery was sensitive to the Na+/K+-ATPase pump inhibitor ouabain. Affected fibers had an anomalous inward current at hyperpolarized potentials, consistent with the proposal that a leaky gating pore in R669H channels triggers attacks, whereas a reduction in the amplitude of action potentials implies additional loss-of-function changes for the mutant NaV1.4 channels.
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Parálisis Periódica Hipopotasémica/genética , Canales de Sodio/genética , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Glucosa/farmacología , Homocigoto , Humanos , Parálisis Periódica Hipopotasémica/fisiopatología , Insulina/farmacología , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/genética , Contracción Isométrica/fisiología , Masculino , Ratones , Ratones Mutantes , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Canal de Sodio Activado por Voltaje NAV1.4 , Ouabaína/farmacología , Fenotipo , Potasio/farmacología , Canales de Sodio/química , Canales de Sodio/fisiologíaRESUMEN
Psychological stress has been implicated in both the onset and exacerbation of multiple sclerosis (MS). Our research has focused on the role of stress at the onset of MS, using the mouse model Theiler's murine encephalomyelitis virus-induced demyelination. Theiler's virus is a natural pathogen of mice that causes a persistent infection of the central nervous system (CNS) and inflammatory immune-mediated demyelination that is very similar to MS. Our research has shown that restraint stress sufficiently increases corticosterone secretion to cause immunosuppression. Stressed mice develop decreased innate and adaptive immune responses, including decreased chemokine and cytokine responses, to virus, which leads to increased viral replication within the CNS. Higher levels of virus then cause increased later demyelinating disease. These findings may have important implications in our understanding of the interactions between stress and the development of autoimmune diseases induced by infectious agents.
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Sistema Inmunológico/patología , Esclerosis Múltiple/inmunología , Sistemas Neurosecretores/patología , Animales , Modelos Animales de Enfermedad , Humanos , Esclerosis Múltiple/complicaciones , Estrés Psicológico/complicaciones , Estrés Psicológico/inmunología , Theilovirus/inmunologíaRESUMEN
There is a paucity of knowledge concerning the immunologic sequelae that culminate in overt autoimmunity. In the present study, we have analyzed the factors that lead to disease in the model of autoimmunity, murine experimental autoimmune encephalomyelitis (EAE). EAE in H-2(u) mice involves autoreactive CD4(+) T cells that are induced by immunization with the immunodominant N-terminal epitope of myelin basic protein. The affinity of this epitope for I-A(u) can be increased by substituting lysine at position 4 with tyrosine, and this can be used to increase the effective Ag dose. Paradoxically, high doses of Ag are poorly encephalitogenic. We have used quantitative analyses to study autoreactive CD4(+) T cell responses following immunization of mice with Ag doses that are at the extremes of encephalitogenicity. A dose of autoantigen that is poorly encephalitogenic results in T cell hyperresponsiveness, triggering an anti-inflammatory feedback loop in which IFN-gamma plays a pivotal role. Our studies define a regulatory mechanism that serves to limit overly robust T cell responses. This feedback regulation has broad relevance to understanding the factors that determine T cell responsiveness.
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Autoinmunidad , Encefalomielitis Autoinmune Experimental/inmunología , Retroalimentación Fisiológica , Interferón gamma/metabolismo , Linfocitos T/inmunología , Animales , Anticuerpos/farmacología , Autoantígenos/inmunología , Autoinmunidad/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/patología , Epítopos/genética , Epítopos/inmunología , Antígenos H-2/genética , Inmunización , Epítopos Inmunodominantes/inmunología , Interferón gamma/antagonistas & inhibidores , Masculino , Ratones , Ratones Mutantes , Proteína Básica de Mielina/inmunologíaRESUMEN
Theiler's murine encephalomyelitis virus (TMEV), a Picornavirus used as a viral model for multiple sclerosis (MS), causes an acute encephalomyelitis and chronic demyelination. The failure to clear the virus, which can result from stress, is a prerequisite for development of the later disease. Similarly, stressful life events have been associated with the development of MS. In the present study, a restraint stress (RS) model was used to investigate the effect of stress on the systemic dissemination of TMEV during the early stage of disease. Experimental data demonstrated that repeated RS remarkably facilitated the spread of virus from the CNS to such systemic organs as the spleen, lymph nodes, thymus, lungs and heart and compromised the ability of viral clearance within those tissues. RS also altered the pathogenecity of TMEV, enabling it to become cardiotropic, resulting in higher myocardial infectivity. These results demonstrate the profound impact that RS has upon both the tissue and organ dissemination of the virus, and the organ tropism of TMEV. An additional finding associated with stress was hepatic necrosis in the restrained animals, regardless of whether or not they were infected.