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2.
Front Immunol ; 10: 2367, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31681275

RESUMO

Cytometry by Time-Of-Flight (CyTOF) uses antibodies conjugated to isotopically pure metals to identify and quantify a large number of cellular features with single-cell resolution. A barcoding approach allows for 20 unique samples to be pooled and processed together in one tube, reducing the intra-barcode technical variability. However, with only 20 samples per barcode, multiple barcode sets (batches) are required to address questions in robustly powered study designs. A batch adjustment procedure is required to reduce variability across batches and to facilitate direct comparison of runs performed across multiple barcodes run over weeks, months, or years. We describe a method using technical replicates that are included in each run to determine and apply an appropriate adjustment per batch without manual intervention. The use of technical replicate samples (i.e., anchors or reference samples) avoids assumptions of sample homogeneity among batches, and allows direct estimation of batch effects and appropriate adjustment parameters applicable to all samples within a batch. Quantification of cell subpopulations and mean signal intensity pre- and post-adjustment using both manual gating and unsupervised clustering demonstrate substantial mitigation of batch effects in the anchor samples used for this adjustment calculation, and in a second validation set of technical replicates.

3.
Front Immunol ; 10: 998, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156616

RESUMO

CTLA-4 is essential for immune tolerance. Heterozygous CTLA4 mutations cause immune dysregulation evident in defective regulatory T cells with low levels of CTLA-4 expression. Biallelic mutations in LRBA also result in immune dysregulation with low levels of CTLA-4 and clinical presentation indistinguishable from CTLA-4 haploinsufficiency. CTLA-4 has become an immunotherapy target whereby its blockade with a monoclonal antibody has resulted in improved survival in advanced melanoma patients, amongst other malignancies. However, this therapeutic manipulation can result in autoimmune/inflammatory complications reminiscent of those seen in genetic defects affecting the CTLA-4 pathway. Despite efforts made to understand and establish disease genotype/phenotype correlations in CTLA-4-haploinsufficiency and LRBA-deficiency, such relationships remain elusive. There is currently no specific immunological marker to assess the degree of CTLA-4 pathway disruption or its relationship with clinical manifestations. Here we compare three different patient groups with disturbances in the CTLA-4 pathway-CTLA-4-haploinsufficiency, LRBA-deficiency, and ipilimumab-treated melanoma patients. Assessment of CTLA4 mRNA expression in these patient groups demonstrated an inverse correlation between the CTLA4 message and degree of CTLA-4 pathway disruption. CTLA4 mRNA levels from melanoma patients under therapeutic CTLA-4 blockade (ipilimumab) were increased compared to patients with either CTLA4 or LRBA mutations that were clinically stable with abatacept treatment. In summary, we show that increased CTLA4 mRNA levels correlate with the degree of CTLA-4 pathway disruption, suggesting that CTLA4 mRNA levels may be a quantifiable surrogate for altered CTLA-4 expression.

4.
J Exp Med ; 216(6): 1255-1267, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31040184

RESUMO

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rß, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rß, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rß surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rß-/- animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rß expression and signaling in T and NK cells.

6.
J Vis Exp ; (136)2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-30010641

RESUMO

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of "intrinsic" patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native "intrinsic pathogenic" disease state, or in the presence of therapeutic agents (in vivo or ex vivo).


Assuntos
Citometria de Fluxo/métodos , Sistema Imunitário/irrigação sanguínea , Imunofenotipagem/mortalidade , Análise de Célula Única/métodos , Citocinas/imunologia , Humanos
7.
J Clin Immunol ; 38(4): 540-541, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29781065

RESUMO

The original version of this article unfortunately contained mistakes in some of the author names and affiliations. The correct list of author names and affiliations is below, with the corrections in bold.

8.
J Clin Immunol ; 38(3): 320-329, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29675737

RESUMO

Genetic testing plays a critical role in diagnosis for many primary immunodeficiency diseases. The goals of this report are to outline some of the challenges that clinical immunologists face routinely in the use of genetic testing for patient care. In addition, we provide a review of the types of genetic testing used in the diagnosis of PID, including their strengths and limitations. We describe the strengths and limitations of different genetic testing approaches for specific clinical contexts that raise concern for specific PID disorders in light of the challenges reported by the clinical immunologist members of the CIS in a recent membership survey. Finally, we delineate the CIS's recommendations for the use of genetic testing in light of these issues.


Assuntos
Testes Genéticos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/genética , Biomarcadores , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Testes Genéticos/normas , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndromes de Imunodeficiência/terapia , Diagnóstico Pré-Natal , Análise de Sequência de DNA
9.
Curr Opin Allergy Clin Immunol ; 16(6): 549-556, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27749361

RESUMO

PURPOSE OF REVIEW: This review gives an overview of the systems-immunology single-cell proteomic and transcriptomic approaches that can be applied to study primary immunodeficiency. It also introduces recent advances in multiparameter tissue imaging, which allows extensive immune phenotyping in disease-affected tissue. RECENT FINDINGS: Mass cytometry is a variation of flow cytometry that uses rare earth metal isotopes instead of fluorophores as tags bound to antibodies, allowing simultaneous measurement of over 40 parameters per single-cell. Mass cytomety enables comprehensive single-cell immunophenotyping and functional assessments, capturing the complexity of the immune system, and the molecularly heterogeneous consequences of primary immunodeficiency defects. Protein epitopes and transcripts can be simultaneously detected allowing immunophenotype and gene expression evaluation in mixed cell populations. Multiplexed epitope imaging has the potential to provide extensive phenotypic characterization at the subcellular level, in the context of 3D tissue microenvironment. SUMMARY: Mass cytometry and multiplexed epitope imaging can complement genetic methods in diagnosis and study of the pathogenesis of primary immunodeficiencies. The ability to understand the effect of a specific defect across multiple immune cell types and pathways, and in affected tissues, may provide new insight into tissue-specific disease pathogenesis and evaluate effects of therapeutic interventions.


Assuntos
Perfilação da Expressão Gênica/métodos , Síndromes de Imunodeficiência/diagnóstico , Espectrometria de Massas , Proteogenômica/métodos , Animais , Humanos , Imagem Tridimensional , Imunofenotipagem , Metais , Análise de Célula Única/métodos
10.
Nat Methods ; 13(3): 269-75, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26808670

RESUMO

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.


Assuntos
Citometria de Fluxo/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/métodos , Análise Serial de Proteínas/métodos , Proteínas/metabolismo , RNA/metabolismo , Humanos , Células Jurkat , Proteínas/análise , RNA/análise
11.
J Allergy Clin Immunol ; 136(5): 1326-36, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26037552

RESUMO

BACKGROUND: Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described. OBJECTIVE: We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes. METHODS: Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE. RESULTS: Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity. CONCLUSION: Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.


Assuntos
Células Matadoras Naturais/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Receptores Toll-Like/metabolismo , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Lúpus Eritematoso Sistêmico/genética , Ativação Linfocitária , NF-kappa B/metabolismo , Especificidade de Órgãos , Transdução de Sinais , Análise de Célula Única/métodos , Transcriptoma
12.
Am J Med Genet A ; 146A(18): 2337-45, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18697196

RESUMO

Opitz G/BBB syndrome is characterized by midline abnormalities such as hypertelorism, cleft palate, and hypospadias. This syndrome is heterogeneous with an X-linked recessive form caused by mutations in the MID1 gene at band Xp22.3. However, mutations in MID1 have only been identified in 47% of familial cases of X-linked Opitz G/BBB syndrome, and 13% of sporadic cases. We performed a phenotype-genotype analysis of a group of nine new patients with clinical characteristics commonly seen in Opitz G/BBB syndrome, and of previously reported patients. We identified a novel mutation in exon 9 of the MID1 gene, c.1941insTGAGTCATCATCC, leading to a premature termination codon at amino acid 514 in a patient with hypertelorism, apparently low-set ears, a short philtrum, bilateral cleft of lip and palate and hypospadias. This mutation affects the PRY domain of the C-terminus of the MID1 protein.


Assuntos
Anormalidades Múltiplas/genética , Fenda Labial/genética , Fissura Palatina/genética , Hipertelorismo/genética , Hipospadia/genética , Proteínas dos Microtúbulos/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequência de Bases , Criança , Pré-Escolar , Códon sem Sentido/genética , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Lactente , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Síndrome
13.
Genomics ; 86(3): 259-70, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16039824

RESUMO

In the analysis of complex traits, congenic strains are powerful tools because they allow characterization of a single locus in the absence of genetic variation throughout the remainder of the genome. Here, we report the construction and initial characterization of a genome-wide panel of congenic strains derived from the donor strain DBA/2J on the background strain C57BL/6J. For many strains, we have carried out high-density SNP genotyping to precisely map the congenic interval and to identify any contaminating regions. Certain strains exhibit striking variation in litter size and in the ratio of females to males. We illustrate the utility of the set by "Mendelizing" the complex trait of myocardial calcification. These 65 strains cover more than 95% of the autosomal genome and should facilitate the analysis of the many genetic trait differences that have been reported between these parental strains.


Assuntos
Genômica , Camundongos Congênicos/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Animais , Feminino , Genoma , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA
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