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BACKGROUND: Flow and mass cytometry are important modern immunology tools for measuring expression levels of multiple proteins on single cells. The goal is to better understand the mechanisms of responses on a single cell basis by studying differential expression of proteins. Most current data analysis tools compare expressions across many computationally discovered cell types. Our goal is to focus on just one cell type. Our narrower field of application allows us to define a more specific statistical model with easier to control statistical guarantees. RESULTS: Differential analysis of marker expressions can be difficult due to marker correlations and inter-subject heterogeneity, particularly for studies of human immunology. We address these challenges with two multiple regression strategies: a bootstrapped generalized linear model and a generalized linear mixed model. On simulated datasets, we compare the robustness towards marker correlations and heterogeneity of both strategies. For paired experiments, we find that both strategies maintain the target false discovery rate under medium correlations and that mixed models are statistically more powerful under the correct model specification. For unpaired experiments, our results indicate that much larger patient sample sizes are required to detect differences. We illustrate the CytoGLMM R package and workflow for both strategies on a pregnancy dataset. CONCLUSION: Our approach to finding differential proteins in flow and mass cytometry data reduces biases arising from marker correlations and safeguards against false discoveries induced by patient heterogeneity.
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Citometria de Fluxo , Modelos Estatísticos , Humanos , Modelos Lineares , Tamanho da AmostraRESUMO
The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation. Specifically, we generate primary data, create bioinformatics tools and provide analysis to support the work of expert manual gene annotators and automated gene annotation pipelines. In addition, manual and computational annotation workflows use any and all publicly available data and analysis, along with the research literature to identify and characterise gene loci to the highest standard. GENCODE gene annotations are accessible via the Ensembl and UCSC Genome Browsers, the Ensembl FTP site, Ensembl Biomart, Ensembl Perl and REST APIs as well as https://www.gencodegenes.org.
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Bases de Dados Genéticas , Genoma Humano/genética , Genômica , Pseudogenes/genética , Animais , Biologia Computacional , Humanos , Internet , Camundongos , Anotação de Sequência Molecular , SoftwareRESUMO
IMPORTANCE: The association of copy number variations (CNVs), differing numbers of copies of genetic sequence at locations in the genome, with phenotypes such as intellectual disability has been almost exclusively evaluated using clinically ascertained cohorts. The contribution of these genetic variants to cognitive phenotypes in the general population remains unclear. OBJECTIVE: To investigate the clinical features conferred by CNVs associated with known syndromes in adult carriers without clinical preselection and to assess the genome-wide consequences of rare CNVs (frequency ≤0.05%; size ≥250 kilobase pairs [kb]) on carriers' educational attainment and intellectual disability prevalence in the general population. DESIGN, SETTING, AND PARTICIPANTS: The population biobank of Estonia contains 52,000 participants enrolled from 2002 through 2010. General practitioners examined participants and filled out a questionnaire of health- and lifestyle-related questions, as well as reported diagnoses. Copy number variant analysis was conducted on a random sample of 7877 individuals and genotype-phenotype associations with education and disease traits were evaluated. Our results were replicated on a high-functioning group of 993 Estonians and 3 geographically distinct populations in the United Kingdom, the United States, and Italy. MAIN OUTCOMES AND MEASURES: Phenotypes of genomic disorders in the general population, prevalence of autosomal CNVs, and association of these variants with educational attainment (from less than primary school through scientific degree) and prevalence of intellectual disability. RESULTS: Of the 7877 in the Estonian cohort, we identified 56 carriers of CNVs associated with known syndromes. Their phenotypes, including cognitive and psychiatric problems, epilepsy, neuropathies, obesity, and congenital malformations are similar to those described for carriers of identical rearrangements ascertained in clinical cohorts. A genome-wide evaluation of rare autosomal CNVs (frequency, ≤0.05%; ≥250 kb) identified 831 carriers (10.5%) of the screened general population. Eleven of 216 (5.1%) carriers of a deletion of at least 250 kb (odds ratio [OR], 3.16; 95% CI, 1.51-5.98; P = 1.5e-03) and 6 of 102 (5.9%) carriers of a duplication of at least 1 Mb (OR, 3.67; 95% CI, 1.29-8.54; P = .008) had an intellectual disability compared with 114 of 6819 (1.7%) in the Estonian cohort. The mean education attainment was 3.81 (P = 1.06e-04) among 248 (≥250 kb) deletion carriers and 3.69 (P = 5.024e-05) among 115 duplication carriers (≥1 Mb). Of the deletion carriers, 33.5% did not graduate from high school (OR, 1.48; 95% CI, 1.12-1.95; P = .005) and 39.1% of duplication carriers did not graduate high school (OR, 1.89; 95% CI, 1.27-2.8; P = 1.6e-03). Evidence for an association between rare CNVs and lower educational attainment was supported by analyses of cohorts of adults from Italy and the United States and adolescents from the United Kingdom. CONCLUSIONS AND RELEVANCE: Known pathogenic CNVs in unselected, but assumed to be healthy, adult populations may be associated with unrecognized clinical sequelae. Additionally, individually rare but collectively common intermediate-size CNVs may be negatively associated with educational attainment. Replication of these findings in additional population groups is warranted given the potential implications of this observation for genomics research, clinical care, and public health.
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Variações do Número de Cópias de DNA , Heterozigoto , Deficiência Intelectual/genética , Transtornos Mentais/genética , Adolescente , Adulto , Cognição , Escolaridade , Epilepsia/genética , Estônia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Itália , Masculino , Obesidade/genética , Fenótipo , Reino Unido , Estados UnidosRESUMO
Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employ a combination of the viral vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.
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Cardiomiopatia Dilatada , Camundongos , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/terapia , Cardiomiopatia Dilatada/metabolismo , Edição de Genes , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Miocárdio/metabolismo , Mutação , Miócitos Cardíacos/metabolismoRESUMO
Natural killer (NK) cells are the predominant antiviral cells of the innate immune system, and may play an important role in acquisition and disease progression of HIV. While untreated HIV infection is associated with distinct alterations in the peripheral blood NK cell repertoire, less is known about how NK phenotype is altered in the setting of long-term viral suppression with antiretroviral therapy (ART), as well as how NK memory can impact functional responses. As such, we sought to identify changes in NK cell phenotype and function using high-dimensional mass cytometry to simultaneously analyze both surface and functional marker expression of peripheral blood NK cells in a cohort of ART-suppressed, HIV+ patients and HIV- healthy controls. We found that the NK cell repertoire following IL-2 treatment was altered in individuals with treated HIV infection compared to healthy controls, with increased expression of markers including NKG2C and CD2, and decreased expression of CD244 and NKp30. Using co-culture assays with autologous, in vitro HIV-infected CD4 T cells, we identified a subset of NK cells with enhanced responsiveness to HIV-1-infected cells, but no differences in the magnitude of anti-HIV NK cell responses between the HIV+ and HIV- groups. In addition, by profiling of NK cell receptors on responding cells, we found similar phenotypes of HIV-responsive NK cell subsets in both groups. Lastly, we identified clusters of NK cells that are altered in individuals with treated HIV infection compared to healthy controls, but found that these clusters are distinct from those that respond to HIV in vitro. As such, we conclude that while chronic, treated HIV infection induces a reshaping of the IL-2-stimulated peripheral blood NK cell repertoire, it does so in a way that does not make the repertoire more HIV-specific.
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Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Fenótipo , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Técnicas de Cocultura , Estudos de Coortes , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Interleucina-2/genética , Interleucina-2/uso terapêutico , Masculino , Pessoa de Meia-Idade , Receptores de Células Matadoras Naturais/metabolismo , Proteínas Recombinantes/uso terapêutico , Resultado do TratamentoRESUMO
Dengue virus (DENV) is a significant cause of morbidity in many regions of the world, with children at the greatest risk of developing severe dengue. NK cells, characterized by their ability to rapidly recognize and kill virally infected cells, are activated during acute DENV infection. However, their role in viral clearance versus pathogenesis has not been fully elucidated. Our goal was to profile the NK cell receptor-ligand repertoire to provide further insight into the function of NK cells during pediatric and adult DENV infection. We used mass cytometry to phenotype isolate NK cells and PBMCs from a cohort of DENV-infected children and adults. Using unsupervised clustering, we found that pediatric DENV infection leads to a decrease in total NK cell frequency with a reduction in the percentage of CD56dimCD38bright NK cells and an increase in the percentage of CD56dimperforinbright NK cells. No such changes were observed in adults. Next, we identified markers predictive of DENV infection using a differential state test. In adults, NK cell expression of activation markers, including CD69, perforin, and Fas-L, and myeloid cell expression of activating NK cell ligands, namely Fas, were predictive of infection. In contrast, increased NK cell expression of the maturation marker CD57 and myeloid cell expression of inhibitory ligands, such as HLA class I molecules, were predictive of pediatric DENV infection. These findings suggest that acute pediatric DENV infection may result in diminished NK cell activation, which could contribute to enhanced pathogenesis and disease severity.
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Antígenos CD57/imunologia , Dengue/imunologia , Citometria de Fluxo/métodos , Células Matadoras Naturais/imunologia , Receptores de Células Matadoras Naturais/imunologia , Adolescente , Adulto , Anticorpos Monoclonais/imunologia , Biomarcadores , Criança , Pré-Escolar , Dengue/sangue , Proteína Ligante Fas/metabolismo , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Perforina/metabolismo , Coloração e Rotulagem , Adulto JovemRESUMO
Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56bright subpopulation of NK cells that is correlated with the drug's therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions.
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Daclizumabe/administração & dosagem , Imunossupressores/administração & dosagem , Células Matadoras Naturais/efeitos dos fármacos , Espectrometria de Massas/métodos , Esclerose Múltipla/sangue , Esclerose Múltipla/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/imunologia , Antígeno CD56/metabolismo , Estudos de Coortes , Feminino , Humanos , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Células Matadoras Naturais/metabolismo , Adulto JovemRESUMO
Highly exposed seronegative (HESN) individuals present a unique setting to study mechanisms of protection against HIV acquisition. As natural killer (NK) cell activation and function have been implicated as a correlate of protection in HESN individuals, we sought to better understand the features of NK cells that may confer protection. We used mass cytometry to phenotypically profile NK cells from a cohort of Beninese sex workers and healthy controls. We found that NK cells from HESN women had increased expression of NKG2A, NKp30 and LILRB1, as well as the Fc receptor CD16, and decreased expression of DNAM-1, CD94, Siglec-7, and NKp44. Using functional assessments of NK cells from healthy donors against autologous HIV-infected CD4+ T cells, we observed that NKp30+ and Siglec-7+ cells had improved functional activity. Further, we found that NK cells from HESN women trended towards increased antibody-dependent cellular cytotoxicity (ADCC) activity; this activity correlated with increased CD16 expression. Overall, we identify features of NK cells in HESN women that may contribute to protection from HIV infection. Follow up studies with larger cohorts are warranted to confirm these findings.
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Infecções por HIV/patologia , Células Matadoras Naturais/metabolismo , Adulto , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Genótipo , Infecções por HIV/imunologia , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Lectinas/metabolismo , Modelos Lineares , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Fenótipo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Profissionais do SexoRESUMO
BACKGROUNDElevated levels of inflammatory cytokines have been associated with poor outcomes among COVID-19 patients. It is unknown, however, how these levels compare with those observed in critically ill patients with acute respiratory distress syndrome (ARDS) or sepsis due to other causes.METHODSWe used a Luminex assay to determine expression of 76 cytokines from plasma of hospitalized COVID-19 patients and banked plasma samples from ARDS and sepsis patients. Our analysis focused on detecting statistical differences in levels of 6 cytokines associated with cytokine storm (IL-1ß, IL-1RA, IL-6, IL-8, IL-18, and TNF-α) between patients with moderate COVID-19, severe COVID-19, and ARDS or sepsis.RESULTSFifteen hospitalized COVID-19 patients, 9 of whom were critically ill, were compared with critically ill patients with ARDS (n = 12) or sepsis (n = 16). There were no statistically significant differences in baseline levels of IL-1ß, IL-1RA, IL-6, IL-8, IL-18, and TNF-α between patients with COVID-19 and critically ill controls with ARDS or sepsis.CONCLUSIONLevels of inflammatory cytokines were not higher in severe COVID-19 patients than in moderate COVID-19 or critically ill patients with ARDS or sepsis in this small cohort. Broad use of immunosuppressive therapies in ARDS has failed in numerous Phase 3 studies; use of these therapies in unselected patients with COVID-19 may be unwarranted.FUNDINGFunding was received from NHLBI K23 HL125663 (AJR); The Bill and Melinda Gates Foundation OPP1113682 (AJR and CAB); Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687 NIH/NIAID U19AI057229-16; Stanford Maternal Child Health Research Institute; and Chan Zuckerberg Biohub (CAB).