RESUMEN
Glaucoma is a leading cause of blindness. Current glaucoma medications work by lowering intraocular pressure (IOP), a risk factor for glaucoma, but most treatments do not directly target the pathological changes leading to increased IOP, which can manifest as medication resistance as disease progresses. To identify physiological modulators of IOP, we performed genome- and exome-wide association analysis in >129,000 individuals with IOP measurements and extended these findings to an analysis of glaucoma risk. We report the identification and functional characterization of rare coding variants (including loss-of-function variants) in ANGPTL7 associated with reduction in IOP and glaucoma protection. We validated the human genetics findings in mice by establishing that Angptl7 knockout mice have lower (~2 mmHg) basal IOP compared to wild-type, with a trend towards lower IOP also in heterozygotes. Conversely, increasing murine Angptl7 levels via injection into mouse eyes increases the IOP. We also show that acute Angptl7 silencing in adult mice lowers the IOP (~2-4 mmHg), reproducing the observations in knockout mice. Collectively, our data suggest that ANGPTL7 is important for IOP homeostasis and is amenable to therapeutic modulation to help maintain a healthy IOP that can prevent onset or slow the progression of glaucoma.
Asunto(s)
Glaucoma , Presión Intraocular , Adulto , Proteína 7 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/genética , Animales , Ceguera , Glaucoma/tratamiento farmacológico , Glaucoma/genética , Humanos , Ratones , Ratones NoqueadosRESUMEN
The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Enfermedad de Alzheimer/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Tauopatías/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Bulk RNA sequencing provides the opportunity to understand biology at the whole transcriptome level without the prohibitive cost of single cell profiling. Advances in spatial transcriptomics enable to dissect tissue organization and function by genome-wide gene expressions. However, the readout of both technologies is the overall gene expression across potentially many cell types without directly providing the information of cell type constitution. Although several in-silico approaches have been proposed to deconvolute RNA-Seq data composed of multiple cell types, many suffer a deterioration of performance in complex tissues. Here we present AdRoit, an accurate and robust method to infer the cell composition from transcriptome data of mixed cell types. AdRoit uses gene expression profiles obtained from single cell RNA sequencing as a reference. It employs an adaptive learning approach to alleviate the sequencing technique difference between the single cell and the bulk (or spatial) transcriptome data, enhancing cross-platform readout comparability. Our systematic benchmarking and applications, which include deconvoluting complex mixtures that encompass 30 cell types, demonstrate its preferable sensitivity and specificity compared to many existing methods as well as its utilities. In addition, AdRoit is computationally efficient and runs orders of magnitude faster than most methods.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Genoma , Transcriptoma , Sensibilidad y EspecificidadRESUMEN
Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.
Asunto(s)
Córnea/citología , Adulto , Diferenciación Celular/fisiología , Conjuntiva/citología , Córnea/patología , Enfermedades de la Córnea/patología , Células Epiteliales/citología , Epitelio Corneal/citología , Humanos , Limbo de la Córnea/citología , Análisis de Secuencia de ARN/métodos , Nicho de Células Madre/fisiología , Células Madre/citología , Transcriptoma/fisiologíaRESUMEN
A standard pathway/gene-set enrichment analysis, the over-representation analysis, is based on four values: the size of two gene-sets, size of their overlap, and size of the gene universe from which the gene-sets are chosen. The standard result of such an analysis is based on the p-value of a statistical test. We supplement this standard pipeline by six cautions: (1) any p-value threshold to distinguish enriched gene-sets from not-enriched ones is to certain degree arbitrary; (2) genes in a gene-set may be correlated, which potentially overcount the gene-set size; (3) any attempt to impose multiple testing correction will increase the false negative rate; (4) gene-sets in a gene-set database may be correlated, potentially overcount the factor for multiple testing correction; (5) the discrete nature of the data make it possible that a minimum change in counts may lead to a quantum change in the p-value threshold-based conclusion; (6) the two gene-sets may not be chosen from the universe of all human genes, but in fact from a subset of that universe, or even two different subsets of all genes. Careful reconsideration of these issues can have an impact on an enrichment analysis conclusion. Part of our cautions mirror the call from statistician that reaching conclusion from data is not a simple matter of p-value smaller than 0.05, but a thoughtful process with due diligences.
Asunto(s)
Algoritmos , Biología Computacional , Bases de Datos Genéticas , Perfilación de la Expresión Génica , HumanosRESUMEN
OBJECTIVES: To characterize the safety, tolerability, and anti-tumor activity of cemiplimab as monotherapy or in combination with hypofractionated radiation therapy (hfRT) in patients with recurrent or metastatic cervical cancer. To determine the association between histology and programmed death-ligand 1 (PD-L1) expression. METHODS: In non-randomized phase I expansion cohorts, patients (squamous or non-squamous histology) received cemiplimab 3â¯mg/kg intravenously every 2â¯weeks for 48â¯weeks, either alone (monotherapy cohort) or with hfRT during week 2 (combination cohort). Due to insufficient tissue material, PD-L1 protein expression was evaluated in commercially purchased samples and mRNA expression levels were analyzed from The Cancer Genome Atlas (TCGA). RESULTS: Twenty patients enrolled in both cohorts in total; 10 had squamous histology. The most common adverse events of any grade were diarrhea, fatigue, and hypokalemia, occurring in 35%, 25%, and 25%, respectively. Objective response rate was 10% in each cohort; responders had squamous histology. Duration of response was 11.2â¯months and 6.4â¯months for the responder in the monotherapy and combination cohort, respectively. Irradiated lesions were not included in the response assessments. In separate archived specimens (Nâ¯=â¯155), PD-L1 protein expression in tumor and immune cells was negative (<1%) more commonly in adenocarcinoma than in squamous tumors. PD-L1 mRNA levels were lower in adenocarcinoma than squamous cell tumors (1.2 vs 5.0 mean transcripts per million, respectively) in TCGA. CONCLUSIONS: Cemiplimab has activity in cervical squamous cell carcinoma. The phase I results, combined with results from other anti-PD-1 trials in cervical cancer and our biomarker analyses have informed the design of the ongoing phase III trial, with the primary overall survival hierarchical analyses being done first in patients with squamous histology.
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Adenocarcinoma/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Administración Intravenosa , Adulto , Anciano , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Femenino , Humanos , Persona de Mediana EdadRESUMEN
BACKGROUND AND PURPOSE: Stroke is a major cause of chronic neurological disability. There is considerable interest in understanding how acute transcriptome changes evolve into subacute and chronic patterns that facilitate or limit spontaneous recovery. Here we mapped longitudinal changes in gene expression at multiple time points after stroke in mice out to 6 months. METHODS: Adult C57BL/6 mice were subjected to transient middle cerebral artery occlusion. Longitudinal transcriptome levels were measured at 10 time points after stroke from acute to recovery phases of ischemic stroke. Localization and the number of mononuclear phagocytes were determined in the postischemic brain. Whole-mount brain imaging was performed in asplenic mice receiving GFP+ (green fluorescent protein)-tagged splenocytes. RESULTS: Sustained stroke-induced mRNA abundance changes were observed in both hemispheres with 2989 ipsilateral and 822 contralateral genes significantly perturbed. In the hemisphere ipsilateral to the infarct, genes associated with immune functions were strongly affected, including temporally overlapping innate and adaptive immunity and macrophage M1 and M2 phenotype-related genes. The strong immune gene activation was accompanied by the sustained infiltration of peripheral immune cells at acute, subacute, and recovery stages of stroke. The infiltrated immune cells were found in the infarcted area but also in remote regions at 2 months after stroke. CONCLUSIONS: The study identifies that immune components are the predominant molecular signatures and they may propagate or continuously respond to brain injury in the subacute to chronic phase after central nervous system injury. The study suggests a potential immune-based strategy to modify injury progression and tissue remodeling in ischemic stroke, even months after the initiating event.
Asunto(s)
Isquemia Encefálica/diagnóstico por imagen , Isquemia Encefálica/inmunología , Movimiento Celular/fisiología , Inmunidad Celular/fisiología , Recuperación de la Función/fisiología , Transcripción Genética/fisiología , Animales , Isquemia Encefálica/genética , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The conventional outflow pathway is a complex tissue responsible for maintaining intraocular pressure (IOP) homeostasis. The coordinated effort of multiple cells with differing responsibilities ensures healthy outflow function and IOP maintenance. Dysfunction of one or more resident cell types results in ocular hypertension and risk for glaucoma, a leading cause of blindness. In this study, single-cell RNA sequencing was performed to generate a comprehensive cell atlas of human conventional outflow tissues. We obtained expression profiles of 17,757 genes from 8,758 cells from eight eyes of human donors representing the outflow cell transcriptome. Upon clustering analysis, 12 distinct cell types were identified, and region-specific expression of candidate genes was mapped in human tissues. Significantly, we identified two distinct expression patterns (myofibroblast- and fibroblast-like) from cells located in the trabecular meshwork (TM), the primary structural component of the conventional outflow pathway. We also located Schwann cell and macrophage signatures in the TM. The second primary component structure, Schlemm's canal, displayed a unique combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the utility of our atlas was demonstrated by mapping glaucoma-relevant genes to outflow cell clusters. Our study provides a comprehensive molecular and cellular classification of conventional and unconventional outflow pathway structures responsible for IOP homeostasis.
Asunto(s)
Humor Acuoso/metabolismo , Glaucoma/patología , Presión Intraocular/fisiología , Miofibroblastos/metabolismo , Malla Trabecular/metabolismo , Glaucoma/genética , Humanos , Macrófagos/metabolismo , RNA-Seq , Células de Schwann/metabolismo , Análisis de la Célula Individual , Malla Trabecular/citologíaRESUMEN
Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)-induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease.
Asunto(s)
Dermatitis/etiología , Dermatitis/metabolismo , Gastroenteritis/etiología , Gastroenteritis/metabolismo , Receptores de Interleucina-1/metabolismo , Transducción de Señal , Animales , Biomarcadores , Dermatitis/patología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Gastroenteritis/patología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Piel/metabolismo , Piel/patologíaRESUMEN
BACKGROUND: Severe inflammatory airway diseases are associated with inflammation that does not resolve, leading to structural changes and an overall environment primed for exacerbations. OBJECTIVE: We sought to identify and inhibit pathways that perpetuate this heightened inflammatory state because this could lead to therapies that allow for a more quiescent lung that is less predisposed to symptoms and exacerbations. METHODS: Using prolonged exposure to house dust mite in mice, we developed a mouse model of persistent and exacerbating airway disease characterized by a mixed inflammatory phenotype. RESULTS: We show that lung IL-33 drives inflammation and remodeling beyond the type 2 response classically associated with IL-33 signaling. IL-33 blockade with an IL-33 neutralizing antibody normalized established inflammation and improved remodeling of both the lung epithelium and lung parenchyma. Specifically, IL-33 blockade normalized persisting and exacerbating inflammatory end points, including eosinophilic, neutrophilic, and ST2+CD4+ T-cell infiltration. Importantly, we identified a key role for IL-33 in driving lung remodeling because anti-IL-33 also re-established the presence of ciliated cells over mucus-producing cells and decreased myofibroblast numbers, even in the context of continuous allergen exposure, resulting in improved lung function. CONCLUSION: Overall, this study shows that increased IL-33 levels drive a self-perpetuating amplification loop that maintains the lung in a state of lasting inflammation and remodeled tissue primed for exacerbations. Thus IL-33 blockade might ameliorate symptoms and prevent exacerbations by quelling persistent inflammation and airway remodeling.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Asma/inmunología , Interleucina-33/inmunología , Pulmón/inmunología , Pyroglyphidae/inmunología , Transducción de Señal/inmunología , Animales , Asma/inducido químicamente , Asma/patología , Asma/terapia , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Interleucina-33/antagonistas & inhibidores , Pulmón/patología , Ratones , Ratones Transgénicos , Células Th2/inmunología , Células Th2/patologíaRESUMEN
BACKGROUND: Critical illness myopathy (CIM) is associated with severe skeletal muscle wasting and impaired function in intensive care unit (ICU) patients. The mechanisms underlying CIM remain incompletely understood. To elucidate the biological activities occurring at the transcriptional level in the skeletal muscle of ICU patients with CIM, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using RNA sequencing (RNA-seq). We also compared the skeletal muscle gene signatures in ICU patients with CIM and genes perturbed by mechanical loading in one leg of the ICU patients, with an aim of reducing the loss of muscle function. METHODS: RNA-seq was used to assess gene expression changes in tibialis anterior skeletal muscle samples from seven critically ill, immobilized, and mechanically ventilated ICU patients with CIM and matched control subjects. We also examined skeletal muscle gene expression for both legs of six ICU patients with CIM, where one leg was mechanically loaded for 10 h/day for an average of 9 days. RESULTS: In total, 6257 of 17,221 detected genes were differentially expressed (84% upregulated; p < 0.05 and fold change ≥ 1.5) in skeletal muscle from ICU patients with CIM when compared to control subjects. The differentially expressed genes were highly associated with gene changes identified in patients with myopathy, sepsis, long-term inactivity, polymyositis, tumor, and repeat exercise resistance. Upstream regulator analysis revealed that the CIM signature could be a result of the activation of MYOD1, p38 MAPK, or treatment with dexamethasone. Passive mechanical loading only reversed expression of 0.74% of the affected genes (46 of 6257 genes). CONCLUSIONS: RNA-seq analysis revealed that the marked muscle atrophy and weakness observed in ICU patients with CIM were associated with the altered expression of genes involved in muscle contraction, newly identified E3 ligases, autophagy and calpain systems, apoptosis, and chaperone expression. In addition, MYOD1, p38 MAPK, and dexamethasone were identified as potential upstream regulators of skeletal muscle gene expression in ICU patients with CIM. Mechanical loading only marginally affected the skeletal muscle transcriptome profiling of ICU patients diagnosed with CIM.
Asunto(s)
Apoptosis , Autofagia , Chaperonas Moleculares/metabolismo , Contracción Muscular , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad Crítica , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Debilidad Muscular/genética , Debilidad Muscular/metabolismo , Atrofia Muscular/etiología , Atrofia Muscular/metabolismo , Enfermedades Musculares/etiología , Análisis de Secuencia de ARN , Transcriptoma , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct.
Asunto(s)
Proteínas ADAM/metabolismo , Infertilidad Masculina/genética , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Proteínas ADAM/genética , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Oviductos , Motilidad Espermática/genéticaRESUMEN
Profiling T cell receptor (TCR) repertoire via short read transcriptome sequencing (RNA-Seq) has a unique advantage of probing simultaneously TCRs and the genome-wide RNA expression of other genes. However, compared to targeted amplicon approaches, the shorter read length is more prone to mapping error. In addition, only a small percentage of the genome-wide reads may cover the TCR loci and thus the repertoire could be significantly under-sampled. Although this approach has been applied in a few studies, the utility of transcriptome sequencing in probing TCR repertoires has not been evaluated extensively. Here we present a systematic assessment of RNA-Seq in TCR profiling. We evaluate the power of both Fluidigm C1 full-length single cell RNA-Seq and bulk RNA-Seq in characterizing the repertoires of different diversities under either naïve conditions or after immunogenic challenges. Standard read length and sequencing coverage were employed so that the evaluation was conducted in accord with the current RNA-Seq practices. Despite high sequencing depth in bulk RNA-Seq, we encountered difficulty quantifying TCRs with low transcript abundance (<1%). Nevertheless, top enriched TCRs with an abundance of 1-3% or higher can be faithfully detected and quantified. When top TCR sequences are of interest and transcriptome sequencing is available, it is worthwhile to conduct a TCR profiling using the RNA-Seq data.
Asunto(s)
ARN/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Sitios Genéticos , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones , Ratones Endogámicos C57BL , ARN/química , ARN/aislamiento & purificación , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Bazo/citología , Bazo/inmunología , Bazo/virología , TranscriptomaRESUMEN
Inferring HLA types from genome-wide sequencing data has gained growing attention with the development of new cost-efficient sequencing technologies and the increasing need to integrate HLA types with transcriptomic or other genomic information for insights into immune-mediated diseases, vaccination, and cancer immunotherapy. PHLAT is a computational tool designed for high-resolution (4-digit) typing of the major class I and class II HLA genes using RNAseq or exome sequencing data as input. We illustrate here how PHLAT can be installed, configured, and executed. This document also provides guidance for how to read and interpret the output results. Finally, the best practices of using PHLAT are also discussed.
Asunto(s)
Algoritmos , Secuenciación del Exoma/métodos , Antígenos HLA/genética , Análisis de Secuencia de ARN/métodos , Alelos , Prueba de Histocompatibilidad , HumanosRESUMEN
Growth and differentiation factor 8 (GDF8) is a TGF-ß superfamily member, and negative regulator of skeletal muscle mass. GDF8 inhibition results in prominent muscle growth in mice, but less impressive hypertrophy in primates, including man. Broad TGF-ß inhibition suggests another family member negatively regulates muscle mass, and its blockade enhances muscle growth seen with GDF8-specific inhibition. Here we show that activin A is the long-sought second negative muscle regulator. Activin A specific inhibition, on top of GDF8 inhibition, leads to pronounced muscle hypertrophy and force production in mice and monkeys. Inhibition of these two ligands mimics the hypertrophy seen with broad TGF-ß blockers, while avoiding the adverse effects due to inhibition of multiple family members. Altogether, we identify activin A as a second negative regulator of muscle mass, and suggest that inhibition of both ligands provides a preferred therapeutic approach, which maximizes the benefit:risk ratio for muscle diseases in man.
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Activinas/metabolismo , Hipertrofia/patología , Hipotonía Muscular/patología , Músculo Esquelético/crecimiento & desarrollo , Miostatina/metabolismo , Receptores de Activinas Tipo II/metabolismo , Activinas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Índice de Masa Corporal , Dexametasona/farmacología , Humanos , Contracción Isométrica/fisiología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Músculo Esquelético/fisiología , Miostatina/antagonistas & inhibidores , RatasRESUMEN
OBJECTIVE: Kawasaki disease (KD) is the most common cause of acquired cardiac disease in US children. In addition to coronary artery abnormalities and aneurysms, it can be associated with systemic arterial aneurysms. We evaluated the development of systemic arterial dilatation and aneurysms, including abdominal aortic aneurysm (AAA) in the Lactobacillus casei cell-wall extract (LCWE)-induced KD vasculitis mouse model. METHODS AND RESULTS: We discovered that in addition to aortitis, coronary arteritis and myocarditis, the LCWE-induced KD mouse model is also associated with abdominal aorta dilatation and AAA, as well as renal and iliac artery aneurysms. AAA induced in KD mice was exclusively infrarenal, both fusiform and saccular, with intimal proliferation, myofibroblastic proliferation, break in the elastin layer, vascular smooth muscle cell loss, and inflammatory cell accumulation in the media and adventitia. Il1r(-/-), Il1a(-/-), and Il1b(-/-) mice were protected from KD associated AAA. Infiltrating CD11c(+) macrophages produced active caspase-1, and caspase-1 or NLRP3 deficiency inhibited AAA formation. Treatment with interleukin (IL)-1R antagonist (Anakinra), anti-IL-1α, or anti-IL-1ß mAb blocked LCWE-induced AAA formation. CONCLUSIONS: Similar to clinical KD, the LCWE-induced KD vasculitis mouse model can also be accompanied by AAA formation. Both IL-1α and IL-1ß play a key role, and use of an IL-1R blocking agent that inhibits both pathways may be a promising therapeutic target not only for KD coronary arteritis, but also for the other systemic arterial aneurysms including AAA that maybe seen in severe cases of KD. The LCWE-induced vasculitis model may also represent an alternative model for AAA disease.
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Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Síndrome Mucocutáneo Linfonodular/complicaciones , Receptores Tipo I de Interleucina-1/metabolismo , Transducción de Señal , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/prevención & control , Aortitis/genética , Aortitis/metabolismo , Aortitis/patología , Caspasa 1/deficiencia , Caspasa 1/genética , Proliferación Celular , Pared Celular , Dilatación Patológica , Modelos Animales de Enfermedad , Elastina/metabolismo , Femenino , Perfilación de la Expresión Génica , Genotipo , Humanos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1alfa/deficiencia , Interleucina-1alfa/genética , Interleucina-1beta/deficiencia , Interleucina-1beta/genética , Lacticaseibacillus casei , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome Mucocutáneo Linfonodular/inducido químicamente , Síndrome Mucocutáneo Linfonodular/tratamiento farmacológico , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fenotipo , Receptores Tipo I de Interleucina-1/deficiencia , Receptores Tipo I de Interleucina-1/genética , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.
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Autoanticuerpos/biosíntesis , Autoinmunidad , Glomerulonefritis Membranoproliferativa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Animales , Autoanticuerpos/sangre , Proteína C9orf72 , Citocinas/sangre , Femenino , Glomerulonefritis Membranoproliferativa/sangre , Glomerulonefritis Membranoproliferativa/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Tejido Linfoide/patología , Macrófagos/inmunología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/inmunología , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
KEY POINTS: Using an experimental rat intensive care unit (ICU) model, not limited by early mortality, we have previously shown that passive mechanical loading attenuates the loss of muscle mass and force-generation capacity associated with the ICU intervention. Mitochondrial dynamics have recently been shown to play a more important role in muscle atrophy than previously recognized. In this study we demonstrate that mitochondrial dynamics, as well as mitophagy, is affected by mechanosensing at the transcriptional level, and muscle changes induced by unloading are counteracted by passive mechanical loading. The recently discovered ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that similarly is prevented by passive mechanical loading. ABSTRACT: The complete loss of mechanical stimuli of skeletal muscles, i.e. loss of external strain related to weight bearing and internal strain related to activation of contractile proteins, in mechanically ventilated, deeply sedated and/or pharmacologically paralysed intensive care unit (ICU) patients is an important factor triggering the critical illness myopathy (CIM). Using a unique experimental ICU rat model, mimicking basic ICU conditions, we have recently shown that mechanical silencing is a dominant factor triggering the preferential loss of myosin, muscle atrophy and decreased specific force in fast- and slow-twitch muscles and muscle fibres. The aim of this study is to gain improved understanding of the gene signature and molecular pathways regulating the process of mechanical activation of skeletal muscle that are affected by the ICU condition. We have focused on pathways controlling myofibrillar protein synthesis and degradation, mitochondrial homeostasis and apoptosis. We demonstrate that genes regulating mitochondrial dynamics, as well as mitophagy are induced by mechanical silencing and that these effects are counteracted by passive mechanical loading. In addition, the recently identified ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that is reversed by passive mechanical loading. Thus, mechano-cell signalling events are identified which may play an important role for the improved clinical outcomes reported in response to the early mobilization and physical therapy in immobilized ICU patients.
Asunto(s)
Enfermedad Crítica , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Animales , Femenino , Perfilación de la Expresión Génica , Inmunoglobulina G/metabolismo , Unidades de Cuidados Intensivos , Atrofia Muscular/metabolismo , Ventilación Pulmonar , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
BACKGROUND: Accurate HLA typing at amino acid level (four-digit resolution) is critical in hematopoietic and organ transplantations, pathogenesis studies of autoimmune and infectious diseases, as well as the development of immunoncology therapies. With the rapid adoption of genome-wide sequencing in biomedical research, HLA typing based on transcriptome and whole exome/genome sequencing data becomes increasingly attractive due to its high throughput and convenience. However, unlike targeted amplicon sequencing, genome-wide sequencing often employs a reduced read length and coverage that impose great challenges in resolving the highly homologous HLA alleles. Though several algorithms exist and have been applied to four-digit typing, some deliver low to moderate accuracies, some output ambiguous predictions. Moreover, few methods suit diverse read lengths and depths, and both RNA and DNA sequencing inputs. New algorithms are therefore needed to leverage the accuracy and flexibility of HLA typing at high resolution using genome-wide sequencing data. RESULTS: We have developed a new algorithm named PHLAT to discover the most probable pair of HLA alleles at four-digit resolution or higher, via a unique integration of a candidate allele selection and a likelihood scoring. Over a comprehensive set of benchmarking data (a total of 768 HLA alleles) from both RNA and DNA sequencing and with a broad range of read lengths and coverage, PHLAT consistently achieves a high accuracy at four-digit (92%-95%) and two-digit resolutions (96%-99%), outcompeting most of the existing methods. It also supports targeted amplicon sequencing data from Illumina Miseq. CONCLUSIONS: PHLAT significantly leverages the accuracy and flexibility of high resolution HLA typing based on genome-wide sequencing data. It may benefit both basic and applied research in immunology and related fields as well as numerous clinical applications.
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ADN/genética , Antígenos HLA/genética , ARN/genética , Algoritmos , Alelos , Genoma Humano , HumanosRESUMEN
BACKGROUND: Transforming growth factor (TGF)-ß is a multifunctional peptide that is important in T-cell activation and cardiovascular remodeling, both of which are important features of Kawasaki disease (KD). We postulated that variation in TGF-ß signaling might be important in KD susceptibility and disease outcome. METHODS AND RESULTS: We investigated genetic variation in 15 genes belonging to the TGF-ß pathway in a total of 771 KD subjects of mainly European descent from the United States, the United Kingdom, Australia, and the Netherlands. We analyzed transcript abundance patterns using microarray and reverse transcriptase-polymerase chain reaction for these same genes, and measured TGF-ß2 protein levels in plasma. Genetic variants in TGFB2, TGFBR2, and SMAD3 and their haplotypes were consistently and reproducibly associated with KD susceptibility, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. A SMAD3 haplotype associated with KD susceptibility replicated in 2 independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (A/G, rs4776338) (P=0.000022; odds ratio, 1.50; 95% confidence interval, 1.25 to 1.81). Pathway analysis using all 15 genes further confirmed the importance of the TGF-ß pathway in KD pathogenesis. Whole-blood transcript abundance for these genes and TGF-ß2 plasma protein levels changed dynamically over the course of the illness. CONCLUSIONS: These studies suggest that genetic variation in the TGF-ß pathway influences KD susceptibility, disease outcome, and response to therapy, and that aortic root and coronary artery Z scores can be used for phenotype/genotype analyses. Analysis of transcript abundance and protein levels further support the importance of this pathway in KD pathogenesis.