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We report on a study of next-generation sequencing in 257 patients undergoing investigations for cytopenias. We sequenced bone marrow aspirates using a target enrichment panel comprising 82 genes and used T cells from paired blood as a control. One hundred and sixty patients had idiopathic cytopenias, 81 had myeloid malignancies and 16 had lymphoid malignancies or other diagnoses. Forty-seven of the 160 patients with idiopathic cytopenias had evidence of somatic pathogenic variants consistent with clonal cytopenias. Only 39 genes of the 82 tested were mutated in the 241 patients with either idiopathic cytopenias or myeloid neoplasms. We confirm that T cells can be used as a control to distinguish between germline and somatic variants. The use of paired analysis with a T-cell control significantly reduced the time molecular scientists spent reporting compared to unpaired analysis. We identified somatic variants of uncertain significance (VUS) in a higher proportion (24%) of patients with myeloid malignancies or clonal cytopenias compared to less than 2% of patients with non-clonal cytopenias. This suggests that somatic VUS are indicators of a clonal process. Lastly, we show that blood depleted of lymphocytes can be used in place of bone marrow as a source of material for sequencing.
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Citopenia , Síndromes Mielodisplásicas , Transtornos Mieloproliferativos , Neoplasias , Humanos , Síndromes Mielodisplásicas/genética , Mutação , Linfócitos T/patologia , Transtornos Mieloproliferativos/genéticaRESUMO
AIMS: Glioneuronal tumours (GNTs) are poorly distinguished by their histology and lack robust diagnostic indicators. Previously, we showed that common GNTs comprise two molecularly distinct groups, correlating poorly with histology. To refine diagnosis, we constructed a methylation-based model for GNT classification, subsequently evaluating standards for molecular stratification by methylation, histology and radiology. METHODS: We comprehensively analysed methylation, radiology and histology for 83 GNT samples: a training cohort of 49, previously classified into molecularly defined groups by genomic profiles, plus a validation cohort of 34. We identified histological and radiological correlates to molecular classification and constructed a methylation-based support vector machine (SVM) model for prediction. Subsequently, we contrasted methylation, radiological and histological classifications in validation GNTs. RESULTS: By methylation clustering, all training and 23/34 validation GNTs segregated into two groups, the remaining 11 clustering alongside control cortex. Histological review identified prominent astrocytic/oligodendrocyte-like components, dysplastic neurons and a specific glioneuronal element as discriminators between groups. However, these were present in only a subset of tumours. Radiological review identified location, margin definition, enhancement and T2 FLAIR-rim sign as discriminators. When validation GNTs were classified by SVM, 22/23 classified correctly, comparing favourably against histology and radiology that resolved 17/22 and 15/21, respectively, where data were available for comparison. CONCLUSIONS: Diagnostic criteria inadequately reflect glioneuronal tumour biology, leaving a proportion unresolvable. In the largest cohort of molecularly defined glioneuronal tumours, we develop molecular, histological and radiological approaches for biologically meaningful classification and demonstrate almost all cases are resolvable, emphasising the importance of an integrated diagnostic approach.
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Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central , Neoplasias Neuroepiteliomatosas , Radiologia , Humanos , Neoplasias Encefálicas/patologia , Metilação de DNA , Neoplasias Neuroepiteliomatosas/genética , Neoplasias do Sistema Nervoso Central/genéticaRESUMO
BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.
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Neuroblastoma , Medicina de Precisão , Quinase do Linfoma Anaplásico/genética , Linhagem Celular Tumoral , Criança , Humanos , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de SinaisRESUMO
BACKGROUND: Minimal residual disease (MRD) measured on end-of-induction bone marrow (BM) is the most important biomarker for guiding therapy in pediatric acute lymphoblastic leukemia (ALL). Due to limited sensitivity of current approaches, peripheral blood (PB) is not a reliable source for identifying patients needing treatment changes. We sought to determine if high-throughput sequencing (HTS) (next-generation sequencing) of rearranged immunoglobulin and T-cell receptor genes can overcome this and be used to measure MRD in PB. PROCEDURE: We employed a quantitative HTS approach to accurately measure MRD from one million cell equivalents of DNA from 17 PB samples collected at day 29 after induction therapy in patients with precursor B-cell ALL. We compared these results to the gold-standard real-time PCR result obtained from their paired BM samples, median follow-up 49 months. RESULTS: With the increased sensitivity, detecting up to one abnormal cell in a million normal cells, we were able to detect MRD in the PB by HTS in all those patients requiring treatment intensification (MRD ≥ 0.005% in BM). CONCLUSION: This is proof of principle that using the increased sensitivity of HTS, PB can be used to measure MRD and stratify children with ALL. The method is cost effective, rapid, accurate, and reproducible, with inherent advantages in children. Importantly, increasing the frequency testing by PB as opposed to intermittent BM sampling may allow extension of the dynamic range of MRD, giving a more complete picture of the kinetics of disease remission while improving relapse prediction and speed of detection.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Criança , Estudos de Viabilidade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Células Precursoras de Linfócitos B , Estudos ProspectivosRESUMO
Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We, therefore, investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of cell state accounts for a significant component of bottleneck selection during induction chemotherapy.
Assuntos
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfoma de Burkitt/tratamento farmacológico , Ciclo Celular , Humanos , Quimioterapia de Indução , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RecidivaRESUMO
PURPOSE: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. EXPERIMENTAL DESIGN: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. RESULTS: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. CONCLUSIONS: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ciclina D1/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Neurofibromina 1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Feminino , Fulvestranto/administração & dosagem , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Piperazinas/administração & dosagem , Estudos Prospectivos , Piridinas/administração & dosagem , Resultado do TratamentoRESUMO
Glioneuronal tumours are an important cause of treatment-resistant epilepsy. Subtypes of tumour are often poorly discriminated by histological features and may be difficult to diagnose due to a lack of robust diagnostic tools. This is illustrated by marked variability in the reported frequencies across different epilepsy surgical series. To address this, we used DNA methylation arrays and RNA sequencing to assay the methylation and expression profiles within a large cohort of glioneuronal tumours. By adopting a class discovery approach, we were able to identify two distinct groups of glioneuronal tumour, which only partially corresponded to the existing histological classification. Furthermore, by additional molecular analyses, we were able to identify pathogenic mutations in BRAF and FGFR1, specific to each group, in a high proportion of cases. Finally, by interrogating our expression data, we were able to show that each molecular group possessed expression phenotypes suggesting different cellular differentiation: astrocytic in one group and oligodendroglial in the second. Informed by this, we were able to identify CCND1, CSPG4, and PDGFRA as immunohistochemical targets which could distinguish between molecular groups. Our data suggest that the current histological classification of glioneuronal tumours does not adequately represent their underlying biology. Instead, we show that there are two molecular groups within glioneuronal tumours. The first of these displays astrocytic differentiation and is driven by BRAF mutations, while the second displays oligodendroglial differentiation and is driven by FGFR1 mutations.
Assuntos
Neoplasias Encefálicas/metabolismo , Epilepsia/metabolismo , Ganglioglioma/metabolismo , Neoplasias Neuroepiteliomatosas/metabolismo , Adolescente , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Criança , Pré-Escolar , Estudos de Coortes , Metilação de DNA , Epilepsia/genética , Epilepsia/patologia , Epilepsia/cirurgia , Feminino , Ganglioglioma/genética , Ganglioglioma/patologia , Ganglioglioma/cirurgia , Expressão Gênica , Humanos , Lactente , Masculino , Mutação , Neoplasias Neuroepiteliomatosas/genética , Neoplasias Neuroepiteliomatosas/patologia , Neoplasias Neuroepiteliomatosas/cirurgia , Fenótipo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoAssuntos
Medula Óssea/patologia , Sistema Nervoso Central/patologia , Células Clonais/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pré-Escolar , Humanos , Lactente , Invasividade Neoplásica , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , RecidivaRESUMO
Loss of cone photoreceptors, crucial for daylight vision, has the greatest impact on sight in retinal degeneration. Transplantation of stem cell-derived L/M-opsin cones, which form 90% of the human cone population, could provide a feasible therapy to restore vision. However, transcriptomic similarities between fetal and stem cell-derived cones remain to be defined, in addition to development of cone cell purification strategies. Here, we report an analysis of the human L/M-opsin cone photoreceptor transcriptome using an AAV2/9.pR2.1:GFP reporter. This led to the identification of a cone-enriched gene signature, which we used to demonstrate similar gene expression between fetal and stem cell-derived cones. We then defined a cluster of differentiation marker combination that, when used for cell sorting, significantly enriches for cone photoreceptors from the fetal retina and stem cell-derived retinal organoids, respectively. These data may facilitate more efficient isolation of human stem cell-derived cones for use in clinical transplantation studies.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Opsinas de Bastonetes/genética , Transcriptoma/genética , Diferenciação Celular/genética , Feto/citologia , Feto/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Retina/crescimento & desenvolvimento , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/transplante , Degeneração Retiniana/patologiaRESUMO
Exome sequencing is becoming widely popular and affordable, making it one of the most desirable methods for the identification of rare genetic variants for clinical diagnosis. Here, we report the clinical application of whole exome sequencing for the ultimate diagnosis of a ciliary chondrodysplasia case presented with an initial clinical diagnosis of Asphyxiating Thoracic Dystrophy (ATD, Jeune Syndrome). We have identified a novel homozygous missense mutation in WDR35 (c.206G > A), a gene previously associated with Sensenbrenner Syndrome, Ellis-van Creveld syndrome and Short-rib polydactyly syndrome type V. The genetic findings in this family led to the re-evaluation of the initial diagnosis and a differential diagnosis of Sensenbrenner Syndrome was made after cautious re-examination of the patient. Cell culture studies revealed normal subcellular localization of the mutant WDR35 protein in comparison to wildtype protein, pointing towards impaired protein-protein interaction and/or altered cell signaling pathways as a consequence of the mutated allele. This research study highlights the importance of including pathogenic variant identification in the diagnosis pipeline of ciliary chondrodysplasias, especially for clinically not fully defined phenotypes.
Assuntos
Osso e Ossos/anormalidades , Ciliopatias/genética , Craniossinostoses/genética , Displasia Ectodérmica/genética , Síndrome de Ellis-Van Creveld/genética , Mutação de Sentido Incorreto , Proteínas/genética , Adulto , Células Cultivadas , Criança , Ciliopatias/diagnóstico , Craniossinostoses/diagnóstico , Proteínas do Citoesqueleto , Diagnóstico Diferencial , Displasia Ectodérmica/diagnóstico , Síndrome de Ellis-Van Creveld/diagnóstico , Feminino , Proteínas Hedgehog , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Linhagem , Ligação Proteica , Transporte Proteico , Proteínas/metabolismo , Sequenciamento do ExomaRESUMO
BACKGROUND: In humans, the adrenal glands and gonads undergo distinct biological events between 6-10 weeks post conception (wpc), such as testis determination, the onset of steroidogenesis and primordial germ cell development. However, relatively little is currently known about the genetic mechanisms underlying these processes. We therefore aimed to generate a detailed genomic atlas of adrenal and gonad development across these critical stages of human embryonic and fetal development. METHODS: RNA was extracted from 53 tissue samples between 6-10 wpc (adrenal, testis, ovary and control). Affymetrix array analysis was performed and differential gene expression was analysed using Bioconductor. A mathematical model was constructed to investigate time-series changes across the dataset. Pathway analysis was performed using ClueGo and cellular localisation of novel factors confirmed using immunohistochemistry. RESULTS: Using this approach, we have identified novel components of adrenal development (e.g. ASB4, NPR3) and confirmed the role of SRY as the main human testis-determining gene. By mathematical modelling time-series data we have found new genes up-regulated with SOX9 in the testis (e.g. CITED1), which may represent components of the testis development pathway. We have shown that testicular steroidogenesis has a distinct onset at around 8 wpc and identified potential novel components in adrenal and testicular steroidogenesis (e.g. MGARP, FOXO4, MAP3K15, GRAMD1B, RMND2), as well as testis biomarkers (e.g. SCUBE1). We have also shown that the developing human ovary expresses distinct subsets of genes (e.g. OR10G9, OR4D5), but enrichment for established biological pathways is limited. CONCLUSION: This genomic atlas is revealing important novel aspects of human development and new candidate genes for adrenal and reproductive disorders.
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Routine childhood vaccination against measles, mumps and rubella has virtually abolished virus-related morbidity and mortality. Notwithstanding this, we describe here devastating neurological complications associated with the detection of live-attenuated mumps virus Jeryl Lynn (MuVJL5) in the brain of a child who had undergone successful allogeneic transplantation for severe combined immunodeficiency (SCID). This is the first confirmed report of MuVJL5 associated with chronic encephalitis and highlights the need to exclude immunodeficient individuals from immunisation with live-attenuated vaccines. The diagnosis was only possible by deep sequencing of the brain biopsy. Sequence comparison of the vaccine batch to the MuVJL5 isolated from brain identified biased hypermutation, particularly in the matrix gene, similar to those found in measles from cases of SSPE. The findings provide unique insights into the pathogenesis of paramyxovirus brain infections.
Assuntos
Encéfalo/virologia , Encefalite Viral/virologia , Vacina contra Caxumba/efeitos adversos , Vírus da Caxumba/isolamento & purificação , Biópsia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Doença Crônica , Encefalite Viral/complicações , Encefalite Viral/diagnóstico por imagem , Encefalite Viral/terapia , Evolução Fatal , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Vírus da Caxumba/genética , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/diagnóstico por imagem , Imunodeficiência Combinada Severa/terapiaRESUMO
Omission of in vivo T-cell depletion promotes rapid, thymic-independent CD4+-biased T-cell recovery after cord blood transplant. This enhanced T-cell reconstitution differs from that seen after stem cell transplant from other stem cell sources, but the mechanism is not known. Here, we demonstrate that the transcription profile of naive CD4+ T cells from cord blood and that of lymphocytes reconstituting after cord blood transplantation is similar to the transcription profile of fetal CD4+ T cells. This profile is distinct to that of naive CD4+ T cells from peripheral blood and that of lymphocytes reconstituting after T-replete bone marrow transplantation. The transcription profile of reconstituting naive CD4+ T cells from cord blood transplant recipients was upregulated in the T-cell receptor (TCR) signaling pathway and its transcription factor activator protein-1 (AP-1). Furthermore, a small molecule inhibitor of AP-1 proportionally inhibited cord blood CD4+ T-cell proliferation (P < .05). Together, these findings suggest that reconstituting cord blood CD4+ T cells reflect the properties of fetal ontogenesis, and enhanced TCR signaling is responsible for the rapid restoration of the unique CD4+ T-cell biased adaptive immunity after cord blood transplantation.
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High-throughput sequencing (HTS) (next-generation sequencing) of the rearranged Ig and T-cell receptor genes promises to be less expensive and more sensitive than current methods of monitoring minimal residual disease (MRD) in patients with acute lymphoblastic leukemia. However, the adoption of new approaches by clinical laboratories requires careful evaluation of all potential sources of error and the development of strategies to ensure the highest accuracy. Timely and efficient clinical use of HTS platforms will depend on combining multiple samples (multiplexing) in each sequencing run. Here we examine the Ig heavy-chain gene HTS on the Illumina MiSeq platform for MRD. We identify errors associated with multiplexing that could potentially impact the accuracy of MRD analysis. We optimize a strategy that combines high-purity, sequence-optimized oligonucleotides, dual indexing, and an error-aware demultiplexing approach to minimize errors and maximize sensitivity. We present a probability-based, demultiplexing pipeline Error-Aware Demultiplexer that is suitable for all MiSeq strategies and accurately assigns samples to the correct identifier without excessive loss of data. Finally, using controls quantified by digital PCR, we show that HTS-MRD can accurately detect as few as 1 in 10(6) copies of specific leukemic MRD.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Reação em Cadeia da Polimerase Multiplex , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Biologia Computacional/métodos , Humanos , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SoftwareRESUMO
Using immunohistochemistry and flow cytometry to define phases of the cell cycle, this study shows that a high proportion of acute myeloid leukaemia (AML) blasts obtained from trephine biopsies are cycling, whereas >95% of peripheral blood-derived blasts are arrested in G1 . Results obtained from bone marrow aspirates are more similar to those from blood rather than from trephine biopsies. These differences were confirmed by gene expression profiling in a patient with high count AML. This has implications for cell cycle and other biological studies using aspirates rather than trephine biopsies and for the use of cell mobilising agents before chemotherapy.
Assuntos
Crise Blástica/patologia , Ciclo Celular , Leucemia Mieloide Aguda/patologia , Adulto , Idoso , Biópsia , Células da Medula Óssea/patologia , Pontos de Checagem do Ciclo Celular , Feminino , Fase G1 , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/patologia , TrepanaçãoRESUMO
BACKGROUND: Various pathways involved in the pathogenesis of sJIA have been identified through gene expression profiling in peripheral blood mononuclear cells (PBMC), but not in neutrophils. Since neutrophils are important in tissue damage during inflammation, and are elevated as part of the acute phase response, we hypothesised that neutrophil pathways could also be important in the pathogenesis of sJIA. We therefore studied the gene profile in both PBMC and neutrophils of sJIA patients treated with tocilizumab. METHODS: We studied the transcriptomes of peripheral blood mononuclear cells (PBMC) and neutrophils from eight paired samples obtained from 4 sJIA patients taken before and after treatment, selected on the basis that they achieved ACR90 responses within 12 weeks of therapy initiation with tocilizumab. RNA was extracted and gene expression profiling was performed using Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray platform. A longitudinal analysis using paired t-test (p < 0.05 and FC ≥ 1.5) was applied to identify differentially expressed genes (DEGs) between the two time points followed by ingenuity pathway analysis. Gene Set Enrichment Analysis (GSEA) and quantitative real-time PCR were then performed to verify the microarray results. RESULTS: Gene ontology analysis in neutrophils revealed that response to tocilizumab significantly altered genes regulating mitochondrial dysfunction and oxidative stress (p = 4.6E-05). This was independently verified with GSEA, by identifying a set of oxidative genes whose expression correlated with response to tocilizumab. In PBMC, treatment of sJIA with tocilizumab appeared to affect genes in Oncostatin M signalling and B cell pathways. CONCLUSIONS: For the first time we demonstrate that neutrophils from sJIA patients responding to tocilizumab showed significantly different changes in gene expression. These data could highlight the importance of mitochondrial genes that modulate oxidative stress in the pathogenesis of sJIA.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite Juvenil/genética , Citocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Análise em Microsséries/métodos , Neutrófilos/metabolismo , Estresse Oxidativo/genética , RNA/análise , Adolescente , Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/metabolismo , Criança , Pré-Escolar , Citocinas/biossíntese , Feminino , Humanos , Masculino , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body. IMPORTANCE: Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.
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Antibacterianos/administração & dosagem , Fezes/microbiologia , Microbiota/efeitos dos fármacos , Saliva/microbiologia , Antibacterianos/farmacologia , DNA Ribossômico/química , DNA Ribossômico/genética , Voluntários Saudáveis , Humanos , Placebos/administração & dosagem , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Suécia , Fatores de Tempo , Reino UnidoRESUMO
Type I interferon (IFN-α/ß) is a fundamental antiviral defense mechanism. Mouse models have been pivotal to understanding the role of IFN-α/ß in immunity, although validation of these findings in humans has been limited. We investigated a previously healthy child with fatal encephalitis after inoculation of the live attenuated measles, mumps, and rubella (MMR) vaccine. By targeted resequencing, we identified a homozygous mutation in the high-affinity IFN-α/ß receptor (IFNAR2) in the proband, as well as a newborn sibling, that rendered cells unresponsive to IFN-α/ß. Reconstitution of the proband's cells with wild-type IFNAR2 restored IFN-α/ß responsiveness and control of IFN-attenuated viruses. Despite the severe outcome of systemic live vaccine challenge, the proband had previously shown no evidence of heightened susceptibility to respiratory viral pathogens. The phenotype of IFNAR2 deficiency, together with similar findings in STAT2-deficient patients, supports an essential but narrow role for IFN-α/ß in human antiviral immunity.
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Antivirais/metabolismo , Imunidade , Receptor de Interferon alfa e beta/deficiência , Evolução Fatal , Genes Recessivos , Teste de Complementação Genética , Humanos , Lactente , Interferons/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Transdução de SinaisRESUMO
Developing thymocytes require pre-TCR signalling to differentiate from CD4-CD8- double negative to CD4+CD8+ double positive cell. Here we followed the transcriptional response to pre-TCR signalling in a synchronised population of differentiating double negative thymocytes. This time series analysis revealed a complex transcriptional response, in which thousands of genes were up and down-regulated before changes in cell surface phenotype were detected. Genome-wide measurement of RNA degradation of individual genes showed great heterogeneity in the rate of degradation between different genes. We therefore used time course expression and degradation data and a genome wide transcriptional modelling (GWTM) strategy to model the transcriptional response of genes up-regulated on pre-TCR signal transduction. This analysis revealed five major temporally distinct transcriptional activities that up regulate transcription through time, whereas down-regulation of expression occurred in three waves. Our model thus placed known regulators in a temporal perspective, and in addition identified novel candidate regulators of thymocyte differentiation.