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1.
Cell ; 185(11): 1905-1923.e25, 2022 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-35523183

RESUMO

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.


Assuntos
Neoplasias , Animais , Genes ras , Camundongos , Neoplasias/genética , Filogenia , Sequenciamento do Exoma
2.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
3.
Genes Dev ; 30(2): 191-207, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26773003

RESUMO

Many long noncoding RNAs (lncRNAs) can regulate chromatin states, but the evolutionary origin and dynamics driving lncRNA-genome interactions are unclear. We adapted an integrative strategy that identifies lncRNA orthologs in different species despite limited sequence similarity, which is applicable to mammalian and insect lncRNAs. Analysis of the roX lncRNAs, which are essential for dosage compensation of the single X chromosome in Drosophila males, revealed 47 new roX orthologs in diverse Drosophilid species across ∼40 million years of evolution. Genetic rescue by roX orthologs and engineered synthetic lncRNAs showed that altering the number of focal, repetitive RNA structures determines roX ortholog function. Genomic occupancy maps of roX RNAs in four species revealed conserved targeting of X chromosome neighborhoods but rapid turnover of individual binding sites. Many new roX-binding sites evolved from DNA encoding a pre-existing RNA splicing signal, effectively linking dosage compensation to transcribed genes. Thus, dynamic change in lncRNAs and their genomic targets underlies conserved and essential lncRNA-genome interactions.


Assuntos
Evolução Biológica , Drosophila melanogaster/fisiologia , Genoma de Inseto/genética , RNA Longo não Codificante/metabolismo , Animais , Sítios de Ligação , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Mecanismo Genético de Compensação de Dose/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Masculino , Ligação Proteica
4.
J Infect Dis ; 228(3): 311-320, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-36722133

RESUMO

BACKGROUND: Mathematical models explain how antivirals control viral infections. Hepatitis C virus (HCV) treatment results in at least 2 phases of decline in viremia. The first phase reflects clearance of rapidly produced virions. The second phase is hypothesized to derive from loss of infected cells but has been challenging to prove. METHODS: Using single-cell methods, we quantified the number of hepatitis C virus (HCV)-infected hepatocytes in liver biopsies taken before and within 7 days of initiating direct-acting antivirals (DAAs) in a double-blinded randomized controlled trial testing 2 (sofosbuvir-velpatasvir) versus 3 (sofosbuvir-velpatasvir-voxilaprevir) DAAs. RESULTS: We employed thousands of intrahepatic measurements in 10 persons with chronic genotype 1a HCV infection: median proportion of infected hepatocytes declined from 11.3% (range, 1.3%-59%) to 0.6% (range, <0.3%-5.8%), a loss of 75%-95% infected hepatocytes. Plasma viremia correlated with numbers of HCV-infected hepatocytes (r = 0.77; P < .0001). Second-phase plasma dynamics and changes in infected hepatocytes were indistinct (P = .16), demonstrating that second-phase viral dynamics derive from loss of infected cells. DAAs led to a decline in intracellular HCV RNA and interferon-stimulated gene expression (P < .05 for both). CONCLUSIONS: We proved that second-phase viral dynamics reflect decay of intrahepatic burden of HCV, partly due to clearance of HCV RNA from hepatocytes. CLINICAL TRIALS REGISTRATION: NCT02938013.


Assuntos
Hepatite C Crônica , Hepatite C , Humanos , Sofosbuvir/uso terapêutico , Antivirais/uso terapêutico , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Viremia/tratamento farmacológico , Cinética , Lactamas Macrocíclicas/uso terapêutico , Hepatite C/tratamento farmacológico , RNA Viral , Genótipo
5.
Nat Rev Genet ; 17(1): 47-62, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26666209

RESUMO

Long non-coding RNAs (lncRNAs) are a diverse class of RNAs that engage in numerous biological processes across every branch of life. Although initially discovered as mRNA-like transcripts that do not encode proteins, recent studies have revealed features of lncRNAs that further distinguish them from mRNAs. In this Review, we describe special events in the lifetimes of lncRNAs - before, during and after transcription - and discuss how these events ultimately shape the unique characteristics and functional roles of lncRNAs.


Assuntos
RNA Longo não Codificante/biossíntese , Animais , Cromatina/genética , Cromatina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Impressão Genômica , Humanos , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Transporte de RNA , RNA Longo não Codificante/genética
6.
J Viral Hepat ; 28(2): 334-344, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33128322

RESUMO

HCV and HIV coinfection is common and HIV leads to increased HCV viraemia and accelerated disease progression. However, the biological basis of this interaction remains poorly understood and little is known about the impact of HIV on HCV replication at the cellular level. We analysed HCV RNA, based on single-cell laser-capture microdissection, in liver biopsies from monoinfected (n = 4) and HCV/HIV-coinfected (n = 5) participants. HCV RNA was assayed in 3200 hepatocytes with information of spatial position. We compared HCV RNA levels and clustering properties of infection between mono- and coinfected participants, and developed a mathematical model of infection. Although the median plasma HCV RNA level and the fraction of infected cells were comparable in monoinfected (7.0 log10 IU/mL and ~ 30%) and coinfected (7.3 log10 IU/mL and ~ 40%) participants, the median HCV RNA per infected hepatocyte in monoinfected (2.8IU) was significantly lower than in coinfected (8.2IU) participants (p = .03). Clustering of infected cells was more prominent in monoinfected participants (91% of samples) than in coinfected participants (~48%), p = .0045, suggesting that spatial spread may be influenced by HIV coinfection. Interestingly, when clustering does occur, the size of clusters is similar in both types of infection. A mathematical model of infection suggested that HIV allows higher intracellular accumulation of HCV RNA by impeding the export of HCV RNA. Our observations show that HIV coinfection impacts intracellular accumulation of HCV RNA and the clustering of HCV-infected cells, but to a less extent the fraction of HCV-infected cells.


Assuntos
Coinfecção , Infecções por HIV , Hepatite C Crônica , Hepatite C , Análise por Conglomerados , Infecções por HIV/complicações , Hepacivirus , Hepatite C/complicações , Humanos
7.
Mol Cell ; 51(2): 156-73, 2013 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-23870142

RESUMO

Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. Here, by using UV crosslinking followed by deep sequencing, we show that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem-loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, we show that different roX RNA domains have overlapping function, since only combinatorial mutations in the tandem stem-loops result in severe loss of dosage compensation and consequently male-specific lethality. We propose that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes.


Assuntos
Mecanismo Genético de Compensação de Dose/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Fatores de Transcrição/genética , Cromossomo X/genética , Animais , Animais Geneticamente Modificados , Pareamento de Bases , Western Blotting , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Imunoprecipitação , Masculino , Mutação/genética , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequências de Repetição em Tandem/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Cromossomo X/metabolismo
8.
J Infect Dis ; 221(9): 1462-1469, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-31740931

RESUMO

BACKGROUND: Hepatitis B virus (HBV) is a leading cause of liver failure and hepatocellular carcinoma. Approximately 10% of people with HIV also have HBV and are at higher risk of liver disease progression than in HBV monoinfection. Antivirals, common to HIV and HBV, suppress HBV DNA levels but do not eradicate virus because the transcriptional template, covalently closed circular DNA (cccDNA), is long lived in infected hepatocytes. METHODS: Using single-cell laser capture microdissection, we isolated >1100 hepatocytes from 5 HIV/HBV coinfected persons with increasing exposure to HBV antivirals (HB1-HB5; no exposure to >7 years exposure), quantifying cccDNA and pregenomic RNA (pgRNA) in each cell using droplet digital polymerase chain reaction. RESULTS: The proportion of infected hepatocytes decreased with antiviral exposure from 96.4% (HB1) to 29.8% (HB5). Upper cccDNA range and median pgRNA decreased from HB1 to HB5 (P < .05 for both). The amount of pgRNA transcribed per cccDNA also decreased from HB1 to HB5 (P < .05). Cells with inactive pgRNA transcription were enriched from 0% (HB1) to 14.3% (HB5) of infected hepatocytes. CONCLUSIONS: cccDNA transcription is reduced in HIV/HBV coinfected people with longer antiviral duration. Understanding HBV transcriptional regulation may be critical to develop a functional cure.


Assuntos
DNA Circular/genética , DNA Viral/genética , Infecções por HIV/virologia , Hepatite B Crônica/virologia , Hepatócitos/virologia , Adulto , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Replicação Viral/genética
9.
J Infect Dis ; 222(4): 601-610, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32201883

RESUMO

BACKGROUND: Direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) have revolutionized outcomes in human immunodeficiency virus (HIV) coinfection. METHODS: We examined early events in liver and plasma through A5335S, a substudy of trial A5329 (paritaprevir/ritonavir, ombitasvir, dasabuvir, with ribavirin) that enrolled chronic genotype 1a HCV-infected persons coinfected with suppressed HIV: 5 of 6 treatment-naive enrollees completed A5335S. RESULTS: Mean baseline plasma HCV ribonucleic acid (RNA) = 6.7 log10 IU/mL and changed by -4.1 log10 IU/mL by Day 7. In liver, laser capture microdissection was used to quantify HCV. At liver biopsy 1, mean %HCV-infected cells = 25.2% (95% confidence interval [CI], 7.4%-42.9%), correlating with plasma HCV RNA (Spearman rank correlation r = 0.9); at biopsy 2 (Day 7 in 4 of 5 participants), mean %HCV-infected cells = 1.0% (95% CI, 0.2%-1.7%) (P < .05 for change), and DAAs were detectable in liver. Plasma C-X-C motif chemokine 10 (CXCL10) concentrations changed by mean = -160 pg/mL per day at 24 hours, but no further after Day 4. CONCLUSIONS: We conclude that HCV infection is rapidly cleared from liver with DAA leaving <2% HCV-infected hepatocytes at Day 7. We extrapolate that HCV eradication could occur in these participants by 63 days, although immune activation might persist. Single-cell longitudinal estimates of HCV clearance from liver have never been reported previously and could be applied to estimating the minimum treatment duration required for HCV infection.


Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Antivirais/uso terapêutico , Coinfecção/tratamento farmacológico , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , 2-Naftilamina , Adulto , Anilidas , Antivirais/farmacocinética , Carbamatos , Ciclopropanos , Feminino , Humanos , Cinética , Lactamas Macrocíclicas , Masculino , Pessoa de Meia-Idade , Prolina/análogos & derivados , Ribavirina , Ritonavir/uso terapêutico , Sulfonamidas , Resultado do Tratamento , Estados Unidos , Uracila/análogos & derivados , Valina , Carga Viral
10.
J Hepatol ; 73(2): 294-302, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32240715

RESUMO

BACKGROUND & AIMS: Accurate HCV incidence estimates are critical for monitoring progress towards HCV elimination goals, including an 80% reduction in HCV incidence by 2030. Moreover, incidence estimates can help guide prevention and treatment programming, particularly in the context of the US opioid epidemic. METHODS: An inexpensive, Genedia-based HCV IgG antibody avidity assay was evaluated as a platform to estimate cross-sectional, population-level primary HCV incidence using 1,840 HCV antibody and RNA-positive samples from 875 individuals enrolled in 5 cohort studies in the US and India. Using samples collected <2 years following HCV seroconversion, the mean duration of recent infection (MDRI) was calculated by fitting a maximum likelihood binomial regression model to the probability of appearing recent. Among samples collected ≥2 years post-HCV seroconversion, an individual-level false recent ratio (FRR) was calculated by estimating the probability of appearing recent using an exact binomial test. Factors associated with falsely appearing recent among samples collected ≥2 years post seroconversion were determined by Poisson regression with generalized estimating equations and robust variance estimators. RESULTS: An avidity index cut-off of <40% resulted in an MDRI of 113 days (95% CI 84-146), and FRRs of 0.4% (95% CI 0.0-1.2), 4.6% (95% CI 2.2-8.3), and 9.5% (95% CI 3.6-19.6) among individuals who were HIV-uninfected, HIV-infected, and HIV-infected with a CD4 count <200/µl, respectively. No variation was seen between HCV genotypes 1 and 3. In hypothetical scenarios of high-risk settings, a sample size of <1,000 individuals could reliably estimate primary HCV incidence. CONCLUSIONS: This cross-sectional approach can estimate primary HCV incidence for the most common genotypes. This tool can serve as a valuable resource for program and policy planners seeking to monitor and reduce HCV burden. LAY SUMMARY: Determining the rate of new hepatitis C virus (HCV) infections in a population is critical to monitoring progress toward HCV elimination and to appropriately guide control efforts. However, since HCV infections are most often initially asymptomatic, it is difficult to estimate the rate of new HCV infections without following HCV-uninfected people over time and repeatedly testing them for HCV infection. Here, we present a novel, resource-efficient method to estimate the rate of new HCV infections in a population using data from a single timepoint.


Assuntos
Infecções por HIV , Hepacivirus , Anticorpos Anti-Hepatite C/isolamento & purificação , Hepatite C , Imunoglobulina G/isolamento & purificação , Afinidade de Anticorpos , Contagem de Linfócito CD4/métodos , Contagem de Linfócito CD4/estatística & dados numéricos , Estudos de Coortes , Monitoramento Epidemiológico , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Infecções por HIV/epidemiologia , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Hepatite C/epidemiologia , Hepatite C/imunologia , Humanos , Incidência , Índia , Soroconversão , Testes Sorológicos/métodos , Testes Sorológicos/estatística & dados numéricos , Estados Unidos/epidemiologia , Carga Viral/métodos , Carga Viral/estatística & dados numéricos
11.
J Stroke Cerebrovasc Dis ; 28(6): e66-e67, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30930242

RESUMO

Fibrocartilaginous embolism (FCE) is an uncommon cause of spinal cord infarction often misdiagnosed as transverse myelitis. The mechanism of ischemia is suspected to be due to retrograde embolization of nucleus pulposus material originating from Schmorl's nodes to the spinal vessels following acute disk herniation. We describe the clinical and imaging findings of FCE in 3 healthy young women with history of trivial spinal cord trauma, and recommend that FCE should be considered in the differential diagnosis of acute myelopathy.


Assuntos
Doenças das Cartilagens/complicações , Embolia/complicações , Infarto/etiologia , Extremidade Inferior/inervação , Isquemia do Cordão Espinal/etiologia , Medula Espinal/irrigação sanguínea , Extremidade Superior/inervação , Adolescente , Doenças das Cartilagens/diagnóstico por imagem , Doenças das Cartilagens/terapia , Diagnóstico Diferencial , Imagem de Difusão por Ressonância Magnética , Embolia/diagnóstico por imagem , Embolia/terapia , Feminino , Humanos , Infarto/diagnóstico por imagem , Infarto/fisiopatologia , Infarto/terapia , Valor Preditivo dos Testes , Recuperação de Função Fisiológica , Isquemia do Cordão Espinal/diagnóstico por imagem , Isquemia do Cordão Espinal/fisiopatologia , Isquemia do Cordão Espinal/terapia , Resultado do Tratamento , Adulto Jovem
12.
PLoS Genet ; 11(12): e1005680, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26633036

RESUMO

Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), LNCRNA-HIT in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, LncRNA-HIT was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of LncRNA-HIT-p100/CBP complexes by ChIRP-seq revealed LncRNA-HIT associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by LncRNA-HIT-p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in LncRNA-HIT or p100 transcripts revealed a significant decrease in expression of many of the LncRNA-HIT-associated loci. LncRNA-HIT siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the LncRNA-HIT siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that LncRNA-HIT is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using LncRNA-HIT and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.


Assuntos
Diferenciação Celular/genética , Condrogênese/genética , RNA Longo não Codificante/genética , Proteína p120 Ativadora de GTPase/genética , Animais , Epigênese Genética/genética , Extremidades/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/crescimento & desenvolvimento , Mesoderma/crescimento & desenvolvimento , Camundongos , RNA Longo não Codificante/biossíntese , Proteína p120 Ativadora de GTPase/biossíntese
13.
J Infect Dis ; 214(3): 344-52, 2016 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26768250

RESUMO

BACKGROUND: Sensitive methods are needed to estimate the population-level incidence of hepatitis C virus (HCV) infection. METHODS: We developed an HCV immunoglobulin G (IgG) antibody avidity assay by modifying the Ortho 3.0 HCV enzyme-linked immunoassay and tested 997 serum or plasma samples from 568 people who inject drugs enrolled in prospective cohort studies. Avidity-based testing algorithms were evaluated by their (1) mean duration of recent infection (MDRI), defined as the average time an individual is identified as having been recently infected, according to a given algorithm; (2) false-recent rate, defined as the proportion of samples collected >2 years after HCV seroconversion that were misclassified as recent; (3) sample sizes needed to estimate incidence; and (4) power to detect a reduction in incidence between serial cross-sectional surveys. RESULTS: A multiassay algorithm (defined as an avidity index of <30%, followed by HCV viremia detection) had an MDRI of 147 days (95% confidence interval [CI], 125-195 days), and the false-recent rates were 0.7% (95% CI, .2%-1.8%) and 7.6% (95% CI, 4.2%-12.3%) among human immunodeficiency virus (HIV)-negative and HIV-positive persons, respectively. In various simulated high-risk populations, this algorithm required <1000 individuals to estimate incidence (relative standard error, 30%) and had >80% power to detect a 50% reduction in incidence. CONCLUSIONS: Avidity-based algorithms have the capacity to accurately estimate HCV infection incidence and rapidly assess the impact of public health efforts among high-risk populations. Efforts to optimize this method should be prioritized.


Assuntos
Afinidade de Anticorpos , Biomarcadores/sangue , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C/diagnóstico , Hepatite C/epidemiologia , Imunoglobulina G/imunologia , Adolescente , Adulto , Idoso , Estudos Transversais , Feminino , Hepatite C/imunologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
14.
PLoS Pathog ; 10(5): e1004082, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24788318

RESUMO

Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-ß, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1ß. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.


Assuntos
HIV-1/imunologia , Hepacivirus/imunologia , Inflamassomos/metabolismo , Interferon Tipo I/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Receptores Toll-Like/fisiologia , Células Cultivadas , Citocinas/metabolismo , Endocitose/imunologia , Endossomos/imunologia , Endossomos/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/fisiologia , Células Hep G2 , Hepacivirus/fisiologia , Hepatite C/imunologia , Hepatite C/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/virologia , Monócitos/imunologia , Monócitos/virologia , Internalização do Vírus
15.
Hum Mol Genet ; 22(14): 2960-72, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23575222

RESUMO

To date, genome-wide single nucleotide polymorphism (SNP) and copy number variant (CNV) association studies of autism spectrum disorders (ASDs) have led to promising signals but not to easily interpretable or translatable results. Our own genome-wide association study (GWAS) showed significant association to an intergenic SNP near Semaphorin 5A (SEMA5A) and provided evidence for reduced expression of the same gene. In a novel GWAS follow-up approach, we map an expression regulatory pathway for a GWAS candidate gene, SEMA5A, in silico by using population expression and genotype data sets. We find that the SEMA5A regulatory network significantly overlaps rare autism-specific CNVs. The SEMA5A regulatory network includes previous autism candidate genes and regions, including MACROD2, A2BP1, MCPH1, MAST4, CDH8, CADM1, FOXP1, AUTS2, MBD5, 7q21, 20p, USH2A, KIRREL3, DBF4B and RELN, among others. Our results provide: (i) a novel data-derived network implicated in autism, (ii) evidence that the same pathway seeded by an initial SNP association shows association with rare genetic variation in ASDs, (iii) a potential mechanism of action and interpretation for the previous autism candidate genes and genetic variants that fall in this network, and (iv) a novel approach that can be applied to other candidate genes for complex genetic disorders. We take a step towards better understanding of the significance of SEMA5A pathways in autism that can guide interpretation of many other genetic results in ASDs.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Redes Reguladoras de Genes , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Locos de Características Quantitativas , Mapeamento Cromossômico , Variações do Número de Cópias de DNA , Predisposição Genética para Doença , Humanos , Polimorfismo de Nucleotídeo Único , Proteína Reelina , Fatores de Risco , Semaforinas
16.
Hepatology ; 60(2): 477-86, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24706559

RESUMO

UNLABELLED: Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause substantial mortality, especially in persons chronically infected with both viruses. HIV infection raises plasma HCV RNA levels and diminishes the response to exogenous alpha interferon (IFN). The degree to which antiretroviral therapy (ART) control of infection overcomes these HIV effects is unknown. Participants with HIV-HCV coinfection were enrolled in a trial to measure HCV viral kinetics after IFN administration (ΔHCVIFN ) twice: initially before (pre-ART) and then after (post-ART) HIV RNA suppression. Liver tissue was obtained 2-4 hours before each IFN injection to measure interferon stimulated genes (ISGs). Following ART, the ΔHCVIFN at 72 hours (ΔHCVIFN,72 ) increased in 15/19 (78.9%) participants by a median (interquartile range [IQR]) of 0.11 log10 IU/mL (0.00-0.40; P < 0.05). Increases in ΔHCVIFN,72 post-ART were associated with decreased hepatic expression of several ISGs (r = -0.68; P = 0.001); a 2-fold reduction in a four-gene ISG signature predicted an increase in ΔHCVIFN,72 of 0.78 log10 IU/mL (95% confidence interval [CI] 0.36,1.20). Pre- and post-ART ΔHCVIFN,72 were closely associated (r = 0.87; P < 0.001). HCV virologic setpoint also changed after ART (ΔHCVART ): transient median increases of 0.28 log10 IU/mL were followed by eventual median decreases from baseline of 0.21 log10 IU/mL (P = 0.002). A bivariate model of HIV RNA control (P < 0.05) and increased expression of a nine-gene ISG signature (P < 0.001) predicted the eventual decreased ΔHCVART . CONCLUSION: ART is associated with lower post-IFN HCV RNA levels and that change is linked to reduced hepatic ISG expression. These data support recommendations to provide ART prior to IFN-based treatment of HCV and may provide insights into the pathogenesis of HIV-HCV coinfection.


Assuntos
Coinfecção/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Interferon-alfa/administração & dosagem , Adulto , Antirretrovirais/administração & dosagem , Antirretrovirais/efeitos adversos , Antirretrovirais/farmacocinética , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Antivirais/farmacocinética , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/genética , Hepacivirus/genética , Humanos , Interferon alfa-2 , Interferon-alfa/efeitos adversos , Interferon-alfa/farmacocinética , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Viral/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/farmacocinética , Resultado do Tratamento , Adulto Jovem
17.
Gastroenterology ; 145(6): 1404-13.e1-10, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23973767

RESUMO

BACKGROUND & AIMS: Hepatitis C virus (HCV) predominantly infects hepatocytes, but many hepatocytes are not infected; studies have shown that HCV antigens cluster within the liver. We investigated spatial distribution and determinants of HCV replication in human liver samples. METHODS: We analyzed liver samples from 4 patients with chronic HCV infection (genotype 1, Metavir scores 0-1) to estimate the proportion of infected hepatocytes and the amount of HCV viral RNA (vRNA) per cell. Single-cell laser capture microdissection was used to capture more than 1000 hepatocytes in grids, to preserve geometric relationships. HCV vRNA and interferon-induced transmembrane protein 3 (IFITM3) messenger RNA (the transcript of an interferon-stimulated gene) were measured in the same hepatocytes by quantitative polymerase chain reaction and assembled in maps to identify areas of high and low HCV replication. RESULTS: Patients' serum levels of HCV RNA ranged from 6.87 to 7.40 log10 IU/mL; the proportion of HCV-infected hepatocytes per person ranged from 21% to 45%, and the level of vRNA ranged from 1 to 50 IU/hepatocyte. Infection was not random; we identified clustering of HCV-positive hepatocytes using infected-neighbor analysis (P < .0005) and distance to the kth nearest neighbor compared with random distributions, obtained by bootstrap simulations (P < .02). Hepatocytes that expressed IFITM3 did not appear to cluster and were largely HCV negative. CONCLUSIONS: We used single-cell laser capture and high-resolution analysis to show that in human liver HCV infects hepatocytes in nonrandom clusters, whereas expression of antiviral molecules is scattered among hepatocytes. These findings show that quantitative single-cell RNA measurements can be used to estimate the abundance of HCV vRNA per infected human hepatocyte and are consistent with cell-cell propagation of infection in the absence of clustered IFITM3.


Assuntos
Hepacivirus/isolamento & purificação , Hepatite C/patologia , Hepatócitos/patologia , Hepatócitos/virologia , Microdissecção e Captura a Laser/métodos , Fígado/patologia , Adulto , Biópsia , Estudos Transversais , Feminino , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Humanos , Imunidade Inata/fisiologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estudos Retrospectivos , Transdução de Sinais/fisiologia , Replicação Viral
18.
Clin Infect Dis ; 57(12): 1747-50, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24051866

RESUMO

The prevalence of hepatitis C virus (HCV) infection in sub-Saharan Africa remains unclear. We tested 1000 individuals from Rakai, Uganda, with the Ortho version 3.0 HCV enzyme-linked immunosorbent assay. All serologically positive samples were tested for HCV RNA. Seventy-six of the 1000 (7.6%) participants were HCV antibody positive; none were confirmed by detection of HCV RNA.


Assuntos
Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Hepatite C/diagnóstico , Adulto , Análise de Variância , Reações Falso-Positivas , Feminino , Infecções por HIV/sangue , Infecções por HIV/etiologia , Infecções por HIV/virologia , Hepacivirus/imunologia , Hepacivirus/isolamento & purificação , Hepatite C/epidemiologia , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , RNA Viral/sangue , Estudos Soroepidemiológicos , Uganda/epidemiologia , Adulto Jovem
19.
Hepatology ; 56(2): 544-54, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22331678

RESUMO

UNLABELLED: Chronic hepatitis C virus (HCV) infection is complicated by hepatic fibrosis. Hypothesizing that early fibrogenic signals may originate in cells susceptible to HCV infection, hepatocyte gene expression was analyzed from persons with chronic HCV at different stages of liver fibrosis. Four HCV-infected subjects with precirrhosis liver fibrosis (Ishak fibrosis 3-5) were matched for age, race, and gender to five HCV-infected subjects with no evidence of fibrosis (Ishak fibrosis 0). Hepatocytes from each subject were isolated from liver biopsies using laser capture microdissection. Transcriptome profiling was performed on hepatocyte RNA using hybridization arrays. We found that hepatocytes in precirrhosis fibrosis were depleted for genes involved in small molecule and drug metabolism, especially butyrylcholinesterase (BCHE), a gene involved in the metabolism of drugs of abuse. Differential expression of BCHE was validated in the same tissues and cross-sectionally in an expanded cohort of 143 HCV-infected individuals. In a longitudinal study, serum BCHE activity was already decreased at study inception in 19 fibrosis progressors compared with 20 fibrosis nonprogressors (P < 0.05). Nonprogressors also had decreased BCHE activity over time compared with initial values, but these evolved a median (range) 8.6 (7.8-11.4) years after the study period inception (P < 0.05). Laser captured portal tracts were enriched for immune related genes when compared with hepatocytes but precirrhosis livers lost this enrichment. CONCLUSION: Chronic HCV is associated with hepatocyte BCHE loss years before hepatic synthetic function is impaired. These results indicate that BCHE may be involved in the pathogenesis of HCV-related fibrosis among injection drug users.


Assuntos
Butirilcolinesterase/genética , Hepatite C Crônica/genética , Hepatócitos/fisiologia , Cirrose Hepática/genética , Erros Inatos do Metabolismo/genética , Transtornos Relacionados ao Uso de Substâncias/genética , Adulto , Apneia , Butirilcolinesterase/deficiência , Butirilcolinesterase/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Genótipo , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Hepatócitos/virologia , Humanos , Microdissecção e Captura a Laser/métodos , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Estudos Longitudinais , Masculino , Erros Inatos do Metabolismo/patologia , Erros Inatos do Metabolismo/virologia , Pessoa de Meia-Idade , Sistema Porta/citologia , Sistema Porta/fisiologia , Sistema Porta/virologia , Transtornos Relacionados ao Uso de Substâncias/patologia , Transtornos Relacionados ao Uso de Substâncias/virologia
20.
Cell Syst ; 14(7): 605-619.e7, 2023 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-37473731

RESUMO

Spatial variation in cellular phenotypes underlies heterogeneity in immune recognition and response to therapy in cancer and many other diseases. Spatial transcriptomics holds the potential to quantify such variation, but existing analysis methods are limited by their focus on individual tasks such as spot deconvolution. We present BayesTME, an end-to-end Bayesian method for analyzing spatial transcriptomics data. BayesTME unifies several previously distinct analysis goals under a single, holistic generative model. This unified approach enables BayesTME to deconvolve spots into cell phenotypes without any need for paired single-cell RNA-seq. BayesTME then goes beyond spot deconvolution to uncover spatial expression patterns among coordinated subsets of genes within phenotypes, which we term spatial transcriptional programs. BayesTME achieves state-of-the-art performance across myriad benchmarks. On human and zebrafish melanoma tissues, BayesTME identifies spatial transcriptional programs that capture fundamental biological phenomena such as bilateral symmetry and tumor-associated fibroblast and macrophage reprogramming. BayesTME is open source.


Assuntos
Benchmarking , Peixe-Zebra , Humanos , Animais , Teorema de Bayes , Peixe-Zebra/genética , Perfilação da Expressão Gênica , Macrófagos
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