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1.
Aging Cell ; 20(10): e13470, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547174

RESUMO

Aging is paradoxically associated with a deteriorated immune defense (immunosenescence) and increased basal levels of tissue inflammation (inflammaging). The lung is particularly sensitive to the effects of aging. The immune cell mechanisms underlying physiological lung aging remain poorly understood. Here we reveal that aging leads to increased interferon signaling and elevated concentrations of chemokines in the lung, which is associated with infiltration of monocytes into the lung parenchyma. scRNA-seq identified a novel Type-1 interferon signaling dependent monocyte subset (MO-ifn) that upregulated IFNAR1 expression and exhibited greater transcriptomal changes with aging than the other monocytes. Blockade of type-1 interferon signaling by treatment with anti-IFNAR1 neutralizing antibodies rapidly ablated MO-ifn cells. Treatment with anti-IFNAR1 antibodies also reduced airway chemokine concentrations and repressed the accumulation of the overall monocyte population in the parenchyma of the aged lung. Together, our work suggests that physiological aging is associated with increased basal level of airway monocyte infiltration and inflammation in part due to elevated type-1 interferon signaling.


Assuntos
Interferon Tipo I/metabolismo , Pulmão/patologia , Monócitos/metabolismo , Transcriptoma/fisiologia , Envelhecimento , Animais , Humanos , Camundongos , Transdução de Sinais
2.
J Neuroinflammation ; 18(1): 152, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34229727

RESUMO

BACKGROUND: The immune pathways in Alzheimer's disease (AD) remain incompletely understood. Our recent study indicates that tissue-resident group 2 innate lymphoid cells (ILC2) accumulate in the brain barriers of aged mice and that their activation alleviates aging-associated cognitive decline. The regulation and function of ILC2 in AD, however, remain unknown. METHODS: In this study, we examined the numbers and functional capability of ILC2 from the triple transgenic AD mice (3xTg-AD) and control wild-type mice. We investigated the effects of treatment with IL-5, a cytokine produced by ILC2, on the cognitive function of 3xTg-AD mice. RESULTS: We demonstrate that brain-associated ILC2 are numerically and functionally defective in the triple transgenic AD mouse model (3xTg-AD). The numbers of brain-associated ILC2 were greatly reduced in 7-month-old 3xTg-AD mice of both sexes, compared to those in age- and sex-matched control wild-type mice. The remaining ILC2 in 3xTg-AD mice failed to efficiently produce the type 2 cytokine IL-5 but gained the capability to express a number of proinflammatory genes. Administration of IL-5, a cytokine produced by ILC2, transiently improved spatial recognition and learning in 3xTg-AD mice. CONCLUSION: Our results collectively indicate that numerical and functional deficiency of ILC2 might contribute to the cognitive impairment of 3xTg-AD mice.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Linfócitos/imunologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos
3.
J Biomol Tech ; 32(4)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-35837267

RESUMO

Single-cell RNA sequencing (scRNA-seq) offers great new opportunities for increasing our understanding of complex biological processes. In particular, development of an accurate Human Cell Atlas is largely dependent on the rapidly advancing technologies and molecular chemistries employed in scRNA-seq. These advances have already allowed an increase in throughput for scRNA-seq from 96 to 80,000 cells on a single instrument run by capturing cells within nanoliter droplets. Although this increase in throughput is critical for many experimental questions, a thorough comparison between microfluidic-based, plate-based, and droplet-based technologies or between multiple available platforms utilizing these technologies is largely lacking. Here, we report scRNA-seq data from SUM149PT cells treated with the histone deacetylase inhibitor trichostatin A versus untreated controls across several scRNA-seq platforms (Fluidigm C1, WaferGen iCell8, 10x Genomics Chromium Controller, and Illumina/BioRad ddSEQ). The primary goal of this project was to demonstrate RNA sequencing methods for profiling the ultra-low amounts of RNA present in individual cells, and this report discusses the results of the study, as well as technical challenges and lessons learned and present general guidelines for best practices in sample preparation and analysis.


Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA/genética , Análise de Sequência de RNA/métodos
4.
J Biomol Tech ; 32(3): 148-157, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-35027872

RESUMO

Here we present an inexpensive, rapid, and robust reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection method that is easily scalable, enabling point-of-care facilities and clinical labs to determine results from patients' saliva directly in 30 minutes for less than $2 per reaction. The method uses a novel combination of widely available reagents that can be prepared in bulk, plated, and frozen and remain stable until samples are received. This innovation dramatically reduces preparation time, enabling high-throughput automation and testing with time to results (including setup) in less than 1 hour for 96 patient samples simultaneously when using a 384-well format. By using a dual reporter (phenol red pH indicator for end-point detection and SYTO-9 fluorescent dye for real time), the assay also provides internal validation of results and redundancy in the event of an instrument malfunction.


Assuntos
COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral , Saliva , Sensibilidade e Especificidade
5.
J Biomol Tech ; 32(3): 199-205, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-35027877

RESUMO

Loop-mediated isothermal amplification (LAMP) is a power tool for the amplification of specific RNA and DNA targets. Much like PCR, LAMP requires primers that surround a target amplification region and generates exponential product through a unique highly specific daisy-chain single-temperature amplification reaction. However, until recently, attempts to amplify targets of greater than 200 base pairs (bp) have been mostly unsuccessful and limited to short amplicon targets of less than 150 bp. Although short amplicons have the benefit of a rapid detection (<40 min), they do not allow for the prediction of RNA integrity based on RNA length and possible intactness. In this study, 8 primer sets were developed using 2 LAMP primer-specific software packages against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid gene with insert lengths ranging from 262 to 945 bp in order to amplify and infer the integrity of viral RNA. Because these amplification lengths are greater than the current methods that use an insert length of 130 or less, they require a longer incubation, modified primer and temperature strategies, and the addition of specific adjuncts to prevent nonspecific amplification. This proof of concept study resulted in successful reverse transcription LAMP reactions for amplicon targets of 262, 687, 693, and 945 bp using a clinical nasopharyngeal NP sample, purified SARS-CoV-2 RNA, and crude lysate containing inactivated virus.


Assuntos
COVID-19 , Transcrição Reversa , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
6.
J Immunol ; 205(2): 502-510, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32503894

RESUMO

Despite mounting evidence suggesting the involvement of the immune system in regulating brain function, the specific role of immune and inflammatory cells in neurodegenerative diseases remain poorly understood. In this study, we report that depletion of NK cells, a type of innate lymphocytes, alleviates neuroinflammation, stimulates neurogenesis, and improves cognitive function in a triple-transgenic Alzheimer disease (AD) mouse model. NK cells in the brains of triple-transgenic AD mouse model (3xTg-AD) mice exhibited an enhanced proinflammatory profile. Depletion of NK cells by anti-NK1.1 Abs drastically improved cognitive function of 3xTg-AD mice. NK cell depletion did not affect amyloid ß concentrations but enhanced neurogenesis and reduced neuroinflammation. Notably, in 3xTg-AD mice depleted of NK cells, microglia demonstrated a homeostatic-like morphology, decreased proliferative response and reduced expression of neurodestructive proinflammatory cytokines. Together, our results suggest a proinflammatory role for NK cells in 3xTg-AD mice and indicate that targeting NK cells might unlock novel strategies to combat AD.


Assuntos
Doença de Alzheimer/imunologia , Células Matadoras Naturais/imunologia , Inflamação Neurogênica/imunologia , Doença de Alzheimer/terapia , Animais , Anticorpos/metabolismo , Antígenos Ly/metabolismo , Apoptose , Cognição , Modelos Animais de Doenças , Humanos , Depleção Linfocítica , Camundongos , Camundongos Transgênicos , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neurogênese , Inflamação Neurogênica/terapia , Recuperação de Função Fisiológica
7.
J Exp Med ; 217(4)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32022838

RESUMO

Increasing evidence has challenged the traditional view about the immune privilege of the brain, but the precise roles of immune cells in regulating brain physiology and function remain poorly understood. Here, we report that tissue-resident group 2 innate lymphoid cells (ILC2) accumulate in the choroid plexus of aged brains. ILC2 in the aged brain are long-lived, are relatively resistant to cellular senescence and exhaustion, and are capable of switching between cell cycle dormancy and proliferation. They are functionally quiescent at homeostasis but can be activated by IL-33 to produce large amounts of type 2 cytokines and other effector molecules in vitro and in vivo. Intracerebroventricular transfer of activated ILC2 revitalized the aged brain and enhanced the cognitive function of aged mice. Administration of IL-5, a major ILC2 product, was sufficient to repress aging-associated neuroinflammation and alleviate aging-associated cognitive decline. Targeting ILC2 in the aged brain may provide new avenues to combat aging-associated neurodegenerative disorders.


Assuntos
Envelhecimento/imunologia , Disfunção Cognitiva/imunologia , Imunidade Inata/imunologia , Linfócitos/imunologia , Idoso , Animais , Ciclo Celular/imunologia , Células Cultivadas , Senescência Celular/imunologia , Feminino , Homeostase/imunologia , Humanos , Inflamação/imunologia , Interleucina-33/imunologia , Interleucina-5/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/imunologia
8.
Aging Cell ; 18(6): e13019, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31429526

RESUMO

The effects of aging on innate immunity and the resulting impacts on immunosenescence remain poorly understood. Here, we report that aging induces compartmentalized changes to the development and function of group 2 innate lymphoid cells (ILC2), an ILC subset implicated in pulmonary homeostasis and tissue repair. Aging enhances bone marrow early ILC2 development through Notch signaling, but the newly generated circulating ILC2 are unable to settle in the lungs to replenish the concomitantly declining mature lung ILC2 pool in aged mice. Aged lung ILC2 are transcriptomically heterogeneous and functionally compromised, failing to produce cytokines at homeostasis and during influenza infection. They have reduced expression of Cyp2e1, a cytochrome P450 oxidase required for optimal ILC2 function. Transfer of lung ILC2 from young mice enhances resistance to influenza infection in old mice. These data highlight compartmentalized effects of aging on ILC and indicate that targeting tissue-resident ILCs might unlock therapies to enhance resistance to infections and diseases in the elderly.


Assuntos
Envelhecimento/imunologia , Imunidade Inata/imunologia , Linfócitos/imunologia , Animais , Diferenciação Celular/imunologia , Senescência Celular/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Methods Mol Biol ; 1507: 245-259, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27832545

RESUMO

Understanding differential isoform expression is critical to mechanistically illuminate the biology underlying both normal development and disease states. High-throughput expression profiling analysis of splice variants has thus far been limited by sample requirements and an appropriate platform for quantitation and analysis. Here we describe Affymetrix GeneChip Human Transcriptome Array 2.0, which is employed for comprehensive examination of all known transcript isoforms.


Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , RNA Mensageiro/genética , Transcriptoma , Sequência de Bases , Linhagem Celular Tumoral , Humanos , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Interface Usuário-Computador
10.
Methods Mol Biol ; 632: 63-72, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20217571

RESUMO

Alternative splicing plays an important role in regulation of normal cellular function. Alternative splicing of pre-mRNA leads to the diversity of downstream protein products in the cell. The Affymetrix Exon arrays allow for a high throughput evaluation of the differences in spliced mRNA expressed in a biological system. In this study, we describe a method using this technology to study the generation of alternative mRNA transcripts in breast cancer cells that differ in the levels of a particular integrin, alpha3beta1.


Assuntos
Processamento Alternativo , Éxons/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Separação Celular , DNA Complementar/química , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Hibridização de Ácido Nucleico , RNA/química , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Ribossomos/metabolismo , Coloração e Rotulagem
11.
Methods Mol Biol ; 632: 159-71, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20217577

RESUMO

Microarrays are extensively used to evaluate the effects of compounds on gene expression in the cells. Most of the studies so far have analyzed the transcriptome of the cell. The basic assumption of this approach is that the changes in gene expression occur at the level of transcription of a gene. However, changes often occur at the posttranscriptional level and are not reflected in the analysis of whole transcriptome. We have pioneered the development of "ribonomic profiling" as a high-throughput method to study posttranscriptional regulation of gene expression in the cell. This method is also often referred to as RIP-CHIP. In this chapter, we describe how to use the RIP-CHIP technology to assess the posttranscriptional changes occurring in the cell in response to treatment with a drug.


Assuntos
Imunoprecipitação da Cromatina/métodos , Descoberta de Drogas/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas de Ligação a RNA/metabolismo , Soluções Tampão , Linhagem Celular , Ensaios de Triagem em Larga Escala/métodos , Humanos , Hibridização de Ácido Nucleico , RNA Complementar/biossíntese , RNA Complementar/química , RNA Complementar/genética , RNA Complementar/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Coloração e Rotulagem
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