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1.
Development ; 146(20)2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31558434

RESUMO

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFß pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.


Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Células Caliciformes/citologia , Mucosa Respiratória/citologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Células Epiteliais/metabolismo , Células Caliciformes/metabolismo , Humanos , Camundongos , RNA-Seq , Mucosa Respiratória/metabolismo , Suínos , Traqueia/citologia , Traqueia/metabolismo
2.
Blood ; 136(14): 1645-1656, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32559766

RESUMO

Light chain (LC) deposition disease (LCDD) is a rare disorder characterized by glomerular and peritubular amorphous deposits of a monoclonal immunoglobulin LC, leading to nodular glomerulosclerosis and nephrotic syndrome. We developed a transgenic model using site-directed insertion of the variable domain of a pathogenic human LC gene into the mouse immunoglobulin κ locus, ensuring its production by all plasma cells (PCs). High free LC levels were achieved after backcrossing with mice presenting increased PC differentiation and no immunoglobulin heavy chain production. Our mouse model recapitulates the characteristic features of LCDD, including progressive glomerulosclerosis, nephrotic-range proteinuria, and finally kidney failure. The variable domain of the LC bears alone the structural properties involved in its pathogenicity. RNA sequencing conducted on PCs demonstrated that LCDD LC induces endoplasmic reticulum stress, likely accounting for the high efficiency of proteasome inhibitor-based therapy. Accordingly, reduction of circulating pathogenic LC was efficiently achieved and not only preserved renal function but also partially reversed kidney lesions. Finally, transcriptome analysis of presclerotic glomeruli revealed that proliferation and extracellular matrix remodeling represented the first steps of glomerulosclerosis, paving the way for future therapeutic strategies in LCDD and other kidney diseases featuring diffuse glomerulosclerosis, particularly diabetic nephropathy.


Assuntos
Cadeias Leves de Imunoglobulina/metabolismo , Paraproteinemias/diagnóstico , Paraproteinemias/etiologia , Animais , Biomarcadores , Ciclo Celular/genética , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Matriz Extracelular , Citometria de Fluxo , Perfilação da Expressão Gênica , Ordem dos Genes , Marcação de Genes , Vetores Genéticos/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Camundongos , Camundongos Transgênicos , Paraproteinemias/complicações , Paraproteinemias/mortalidade , Agregados Proteicos , Agregação Patológica de Proteínas , Insuficiência Renal/diagnóstico , Insuficiência Renal/etiologia , Insuficiência Renal/metabolismo , Insuficiência Renal/mortalidade
3.
Am J Respir Crit Care Med ; 202(12): 1636-1645, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-32726565

RESUMO

Rationale: The respiratory tract constitutes an elaborate line of defense that is based on a unique cellular ecosystem.Objectives: We aimed to investigate cell population distributions and transcriptional changes along the airways by using single-cell RNA profiling.Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. A total of 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree.Measurements and Main Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%) but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type-specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuroendocrine cells, and brush cells and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse "hillock" cells and with squamous cells expressing SCEL and SPRR1A/B.Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.


Assuntos
Brônquios/citologia , Brônquios/crescimento & desenvolvimento , Diferenciação Celular/genética , Proliferação de Células/genética , Células Epiteliais/citologia , Mucosa Nasal/citologia , Mucosa Nasal/crescimento & desenvolvimento , Células Estromais/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação da Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade
4.
Brief Bioinform ; 19(6): 1203-1217, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-28575140

RESUMO

In therapeutic research, the safety and efficacy of pharmaceutical products are necessarily tested on humans via clinical trials after an extensive and expensive preclinical development period. Methodologies such as computer modeling and clinical trial simulation (CTS) might represent a valuable option to reduce animal and human assays. The relevance of these methods is well recognized in pharmacokinetics and pharmacodynamics from the preclinical phase to postmarketing. However, they are barely used and are poorly regarded for drug approval, despite Food and Drug Administration and European Medicines Agency recommendations. The generalization of CTS could be greatly facilitated by the availability of software for modeling biological systems, by clinical trial studies and hospital databases. Data sharing and data merging raise legal, policy and technical issues that will need to be addressed. Development of future molecules will have to use CTS for faster development and thus enable better patient management. Drug activity modeling coupled with disease modeling, optimal use of medical data and increased computing speed should allow this leap forward. The realization of CTS requires not only bioinformatics tools to allow interconnection and global integration of all clinical data but also a universal legal framework to protect the privacy of every patient. While recognizing that CTS can never replace 'real-life' trials, they should be implemented in future drug development schemes to provide quantitative support for decision-making. This in silico medicine opens the way to the P4 medicine: predictive, preventive, personalized and participatory.


Assuntos
Ensaios Clínicos como Assunto , Desenvolvimento de Medicamentos , Oncologia , Neoplasias/terapia , Pesquisa Biomédica , Biologia Computacional , Simulação por Computador , Aprovação de Drogas/legislação & jurisprudência , Europa (Continente) , Política de Saúde , Humanos , Vigilância de Produtos Comercializados , Estados Unidos
5.
Am J Respir Crit Care Med ; 200(2): 184-198, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30964696

RESUMO

Rationale: Given the paucity of effective treatments for idiopathic pulmonary fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. TGF-ß (transforming growth factor-ß) is the main profibrotic factor, but its inhibition is associated with severe side effects because of its pleiotropic role. Objectives: To determine if downstream noncoding effectors of TGF-ß in fibroblasts may represent new effective therapeutic targets whose modulation may be well tolerated. Methods: We investigated the whole noncoding fraction of TGF-ß-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblasts. Differential expression of the long noncoding RNA (lncRNA) DNM3OS (dynamin 3 opposite strand) and its associated microRNAs (miRNAs) was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis. Measurements and Main Results: We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-ß-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e., miR-199a-5p/3p and miR-214-3p), which influence SMAD and non-SMAD components of TGF-ß signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis. Conclusions: Pharmacological approaches aiming at interfering with the lncRNA DNM3OS may represent new effective therapeutic strategies in IPF.


Assuntos
Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , RNA Longo não Codificante/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Caveolina 1/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Camundongos , MicroRNAs/metabolismo , Miofibroblastos/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Via de Sinalização Wnt
6.
Nucleic Acids Res ; 46(12): 6344-6355, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29668986

RESUMO

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the functional deficiency of the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in translational regulation of many messenger RNAs, playing key roles in synaptic morphology and plasticity. To date, no effective treatment for FXS is available. We searched for FMRP targets by HITS-CLIP during early development of multiple mouse brain regions (hippocampus, cortex and cerebellum) at a time of brain development when FMRP is most highly expressed and synaptogenesis reaches a peak. We identified the largest dataset of mRNA targets of FMRP available in brain and we defined their cellular origin. We confirmed the G-quadruplex containing structure as an enriched motif in FMRP RNA targets. In addition to four less represented motifs, our study points out that, in the brain, CTGKA is the prominent motif bound by FMRP, which recognizes it when not engaged in Watson-Crick pairing. All of these motifs negatively modulated the expression level of a reporter protein. While the repertoire of FMRP RNA targets in cerebellum is quite divergent, the ones of cortex and hippocampus are vastly overlapping. In these two brain regions, the Phosphodiesterase 2a (Pde2a) mRNA is a prominent target of FMRP, which modulates its translation and intracellular transport. This enzyme regulates the homeostasis of cAMP and cGMP and represents a novel and attractive therapeutic target to treat FXS.


Assuntos
Encéfalo/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , RNA Mensageiro/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Cerebelo/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Hipocampo/metabolismo , Imunoprecipitação , Masculino , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , RNA Mensageiro/química , Análise de Sequência de RNA
7.
Neuroimmunomodulation ; 26(2): 59-66, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30703773

RESUMO

BACKGROUND: Others and we have shown that T cells have an important role in hippocampal synaptic plasticity, including neurogenesis in the dentate gyrus, spinogenesis, and glutamatergic synaptic function in the CA of the hippocampus. Hippocampus plasticity is particularly involved in the brain effects of the enriched environment (EE), and interestingly CD4+ and CD8+ T cells play essential and differential roles in these effects. However, the precise mechanisms by which they act on the brain remain elusive. OBJECTIVES: We searched for a putative mechanism of action by which CD4+ T cells could influence brain plasticity and hypothesized that they could regulate protein transport at the level of the blood-CSF barrier in the choroid plexus. METHOD: We compared mice housed in EE and deprived of CD4+ T cells using a depleting antibody with a control group injected with the control isotype. We analyzed in the hippocampus the gene expression profiles using the Agilent system, and the expression of target proteins in plasma, CSF, and the choroid plexus using ELISA. RESULTS: We show that CD4+ T cells may influence EE-induced hippocampus plasticity via thyroid hormone signaling by regulating in the choroid plexus the expression of transthyretin, the major transporter of thyroxine (T4) to the brain parenchyma. CONCLUSIONS: Our study highlights the contribution of close interactions between the immune and neuroendocrine systems in brain plasticity and function.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Plexo Corióideo/metabolismo , Plasticidade Neuronal/fisiologia , Pré-Albumina/metabolismo , Tiroxina/metabolismo , Animais , Feminino , Hipocampo/metabolismo , Abrigo para Animais , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/fisiologia , Hormônios Tireóideos/metabolismo
8.
Nucleic Acids Res ; 45(7): e48, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-27940562

RESUMO

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Células Cultivadas , DNA Complementar , Perfilação da Expressão Gênica/economia , Células HEK293 , Humanos , Análise de Sequência de RNA/economia , Análise de Célula Única
9.
Eur Respir J ; 52(4)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30190271

RESUMO

In line with the pathophysiological continuum described between nose and bronchus in allergic respiratory diseases, we assessed whether nasal epithelium could mirror the Type 2 T-helper cell (Th2) status of bronchial epithelium.Nasal and bronchial cells were collected by brushing from healthy controls (C, n=13), patients with allergic rhinitis and asthma (AR, n=12), and patients with isolated allergic rhinitis (R, n=14). Cellular composition was assessed by flow cytometry, gene expression was analysed by RNA sequencing and Th2, Type 17 T-helper cell (Th17) and interferon (IFN) signatures were derived from the literature.Infiltration by polymorphonuclear neutrophils (PMN) in the nose excluded 30% of the initial cohort. All bronchial samples from the AR group were Th2-high. The gene expression profile of nasal samples from the AR group correctly predicted the paired bronchial sample Th2 status in 71% of cases. Nevertheless, nasal cells did not appear to be a reliable surrogate for the Th2 response, in particular due to a more robust influence of the IFN response in 14 out of 26 nasal samples. The Th2 scores in the nose and bronchi correlated with mast cell count (both p<0.001) and number of sensitisations (p=0.006 and 0.002), while the Th17 scores correlated with PMN count (p=0.006 and 0.003).The large variability in nasal cell composition and type of inflammation restricts its use as a surrogate for assessing bronchial Th2 inflammation in AR patients.


Assuntos
Asma/imunologia , Rinite Alérgica/imunologia , Células Th17/citologia , Células Th2/citologia , Adulto , Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Casos e Controles , Feminino , Expressão Gênica , Humanos , Inflamação/imunologia , Interferons/metabolismo , Masculino , Líquido da Lavagem Nasal/citologia , Mucosa Respiratória/metabolismo , Rinite Alérgica/fisiopatologia , Análise de Sequência de RNA , Células Th17/imunologia , Células Th2/imunologia , Adulto Jovem
10.
Brain Behav Immun ; 69: 235-254, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29175168

RESUMO

Enriched environment (EE) induces plasticity changes in the brain. Recently, CD4+ T cells have been shown to be involved in brain plasticity processes. Here, we show that CD8+ T cells are required for EE-induced brain plasticity in mice, as revealed by measurements of hippocampal volume, neurogenesis in the DG of the hippocampus, spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. As a consequence, EE-induced behavioral benefits depend, at least in part, on CD8+ T cells. In addition, we show that spleen CD8+ T cells from mice housed in standard environment (SE) and EE have different properties in terms of 1) TNFα release after in vitro CD3/CD28 or PMA/Iono stimulation 2) in vitro proliferation properties 3) CD8+ CD44+ CD62Llow and CD62Lhi T cells repartition 4) transcriptomic signature as revealed by RNA sequencing. CD8+ T cells purified from the choroid plexus of SE and EE mice also exhibit different transcriptomic profiles as highlighted by single-cell mRNA sequencing. We show that CD8+ T cells are essential mediators of beneficial EE effects on brain plasticity and cognition. Additionally, we propose that EE differentially primes CD8+ T cells leading to behavioral improvement.


Assuntos
Comportamento Animal/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Meio Ambiente , Hipocampo/fisiologia , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Proliferação de Células/fisiologia , Comportamento Alimentar/fisiologia , Feminino , Camundongos , Atividade Motora/fisiologia
11.
Arch Toxicol ; 92(4): 1539-1550, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29362864

RESUMO

Although Tacrolimus is an immunosuppressive drug widely used in renal transplantation, its chronic use paradoxically induces nephrotoxic effects, in particular renal fibrosis, which is responsible for chronic allograft dysfunction and represents a major prognostic factor of allograft survival. As molecular pathways and mechanisms involved in Tacrolimus-induced fibrogenic response are poorly elucidated, we assessed whether miRNAs are involved in the nephrotoxic effects mediated by Tacrolimus. Treatment of CD-1 mice with Tacrolimus (1 mg/kg/d for 28 days) resulted in kidney injury and was associated with alteration of a gene expression signature associated with cellular stress, fibrosis and inflammation. Tacrolimus also affected renal miRNA expression, including miRNAs previously involved in fibrotic and inflammatory processes as "fibromirs" such as miR-21-5p, miR-199a-5p and miR-214-3p. In agreement with in vivo data, Renal Proximal Tubular Epithelial cells exposed to Tacrolimus (25 and 50 µM) showed upregulation of miR-21-5p and the concomitant induction of epithelial phenotypic changes, inflammation and oxidative stress. In conclusion, this study suggests for the first time that miRNAs, especially fibromiRs, participate to Tacrolimus-induced nephrotoxic effects. Therefore, targeting miRNAs may be a new therapeutic option to counteract Tacrolimus deleterious effects on kidney.


Assuntos
Imunossupressores/toxicidade , Rim/efeitos dos fármacos , MicroRNAs/metabolismo , Tacrolimo/toxicidade , Animais , Células Cultivadas , Fibrose , Humanos , Rim/metabolismo , Rim/patologia , Camundongos , Transcriptoma/efeitos dos fármacos , Regulação para Cima
12.
Breast Cancer Res ; 17: 41, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25886996

RESUMO

INTRODUCTION: Accurate assessment of HER2 status is critical in determining appropriate therapy for breast cancer patients but the best HER2 testing methodology has yet to be defined. In this study, we compared quantitative HER2 expression by the HERmark™ Breast Cancer Assay (HERmark) with routine HER2 testing by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and correlated HER2 results with overall survival (OS) of breast cancer patients in a multicenter Collaborative Biomarker Study (CBS). METHODS: Two hundred and thirty-two formalin-fixed, paraffin-embedded breast cancer tissues and local laboratory HER2 testing results were provided by 11 CBS sites. HERmark assay and central laboratory HER2 IHC retesting were retrospectively performed in a blinded fashion. HER2 results by all testing methods were obtained in 192 cases. RESULTS: HERmark yielded a continuum of total HER2 expression (H2T) ranging from 0.3 to 403 RF/mm2 (approximately 3 logs). The distribution of H2T levels correlated significantly (P<0.0001) with all routine HER2 testing results. The concordance of positive and negative values (equivocal cases excluded) between HERmark and routine HER2 testing was 84% for local IHC, 96% for central IHC, 85% for local FISH, and 84% for local HER2 status. OS analysis revealed a significant correlation of shorter OS with HER2 positivity by local IHC (HR=2.6, P=0.016), central IHC (HR=3.2, P=0.015), and HERmark (HR=5.1, P<0.0001) in this cohort of patients most of whom received no HER2-targeted therapy. The OS curve of discordant low (HER2 positive but H2T low, 10% of all cases) was aligned with concordant negative (HER2 negative and H2T low, HR=1.9, P=0.444), but showed a significantly longer OS than concordant positive (HER2 positive and H2T high, HR=0.31, P=0.024). Conversely, the OS curve of discordant high (HER2 negative but H2T high, 9% of all cases) was aligned with concordant positive (HR=0.41, P=0.105), but showed a significantly shorter OS than concordant negative (HR=41, P<0.0001). CONCLUSIONS: Quantitative HER2 measurement by HERmark is highly sensitive, accurately quantifies HER2 protein expression and correlates well with routine HER2 testing. When HERmark and local HER2 results were discordant, HERmark more accurately predicted overall survival.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Feminino , Seguimentos , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Carga Tumoral , Adulto Jovem
13.
J Infect Dis ; 209(1): 66-73, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23922373

RESUMO

BACKGROUND: Determinants of intersubtype differences in human immunodeficiency virus type 1 (HIV-1) clinical disease progression remain unknown. METHODS: HIV-1 subtype was independently determined for 5 separate genomic regions in 396 HIV-1 seroconverters from Rakai, Uganda, using a multiregion hybridization assay. Replication capacities (RC) in samples from a subset of 145 of these subjects were determined. HIV-1 genomic regions and pol RC were examined for association with disease progression. Amino acid polymorphisms were examined for association with pol RC. RESULTS: In multivariate analyses, the hazard for progression to the composite end point (defined as a CD4(+) T-cell count <250 cells/mm(3), antiretroviral therapy initiation, or death) among patients with subtype D pol infection was 2.4 times the hazard for those infected with subtype A pol infection (P = .001). Compared with subtype A pol (the reference group), the hazard for progression to the composite end point for subtype D pol infection with a pol RC >67% (ie, the median pol RC) was significantly greater (HR, 4.6; 95% confidence interval [CI], 1.9-11.0; P = .001), whereas the hazard for progression to the composite end point for subtype D pol infection with a pol RC ≤67% was not significantly different (HR, 2.2; 95% CI, 1.0-4.9; P = .051). Amino acid substitutions at protease positions 62 and 64 and at reverse transcriptase position 272 were associated with significant differences in pol RC. CONCLUSIONS: HIV-1 pol gene intersubtype and RC differences are associated with disease progression and may be influenced by amino acid polymorphisms.


Assuntos
Genes pol , Infecções por HIV/virologia , HIV-1/fisiologia , Replicação Viral/genética , Adolescente , Adulto , Substituição de Aminoácidos , Progressão da Doença , Feminino , HIV-1/enzimologia , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Uganda , Carga Viral
14.
BMC Bioinformatics ; 15: 77, 2014 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-24646213

RESUMO

BACKGROUND: Recent efforts in HIV-1 vaccine design have focused on immunogens that evoke potent neutralizing antibody responses to a broad spectrum of viruses circulating worldwide. However, the development of effective vaccines will depend on the identification and characterization of the neutralizing antibodies and their epitopes. We developed bioinformatics methods to predict epitope networks and antigenic determinants using structural information, as well as corresponding genotypes and phenotypes generated by a highly sensitive and reproducible neutralization assay.282 clonal envelope sequences from a multiclade panel of HIV-1 viruses were tested in viral neutralization assays with an array of broadly neutralizing monoclonal antibodies (mAbs: b12, PG9,16, PGT121 - 128, PGT130 - 131, PGT135 - 137, PGT141 - 145, and PGV04). We correlated IC50 titers with the envelope sequences, and used this information to predict antibody epitope networks. Structural patches were defined as amino acid groups based on solvent-accessibility, radius, atomic depth, and interaction networks within 3D envelope models. We applied a boosted algorithm consisting of multiple machine-learning and statistical models to evaluate these patches as possible antibody epitope regions, evidenced by strong correlations with the neutralization response for each antibody. RESULTS: We identified patch clusters with significant correlation to IC50 titers as sites that impact neutralization sensitivity and therefore are potentially part of the antibody binding sites. Predicted epitope networks were mostly located within the variable loops of the envelope glycoprotein (gp120), particularly in V1/V2. Site-directed mutagenesis experiments involving residues identified as epitope networks across multiple mAbs confirmed association of these residues with loss or gain of neutralization sensitivity. CONCLUSIONS: Computational methods were implemented to rapidly survey protein structures and predict epitope networks associated with response to individual monoclonal antibodies, which resulted in the identification and deeper understanding of immunological hotspots targeted by broadly neutralizing HIV-1 antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Biologia Computacional/métodos , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Epitopos/genética , Epitopos/metabolismo , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Testes de Neutralização
15.
Breast Cancer Res Treat ; 141(1): 43-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23959396

RESUMO

Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT­mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan­Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/secundário , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica , Genes erbB-2 , Proteínas de Neoplasias/biossíntese , Receptor ErbB-2/análise , Receptor ErbB-3/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/classificação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Estudos de Coortes , Árvores de Decisões , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Estimativa de Kaplan-Meier , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologia , Prognóstico , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptor ErbB-3/genética , Receptor ErbB-3/imunologia , Estudos Retrospectivos , Método Simples-Cego , Trastuzumab , Resultado do Tratamento
16.
Oncologist ; 17(1): 26-35, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22234627

RESUMO

BACKGROUND: Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. METHODS: The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. RESULTS: A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). CONCLUSIONS: These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Receptor ErbB-2/biossíntese , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias Encefálicas/induzido quimicamente , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Metástase Neoplásica , Receptor ErbB-2/genética , Fatores de Risco , Trastuzumab
17.
STAR Protoc ; 3(3): 101600, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36042886

RESUMO

Cell response variability is a starting point in cancer drug resistance that has been difficult to analyze because the tolerant cell states are short lived. Here, we present fate-seq, an approach to isolate single cells in their transient states of drug sensitivity or tolerance before profiling. The drug response is predicted in live cells, which are laser-captured by microdissection before any drug-induced change can alter their states. This framework enables the identification of the cell-state signatures causing differential cell decisions upon treatment. For complete details on the use and execution of this protocol, please refer to Meyer et al. (2020).


Assuntos
Diagnóstico por Imagem , Microdissecção , Lasers , Microdissecção/métodos
18.
J Biol Chem ; 285(7): 4278-90, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-19996317

RESUMO

Exogenous or endogenous beta(2)-adrenergic receptor agonists enhance alveolar epithelial fluid transport via a cAMP-dependent mechanism that protects the lungs from alveolar flooding in acute lung injury. However, impaired alveolar fluid clearance is present in most of the patients with acute lung injury and is associated with increased mortality, although the mechanisms responsible for this inhibition of the alveolar epithelial fluid transport are not completely understood. Here, we found that transforming growth factor beta1 (TGF-beta1), a critical mediator of acute lung injury, inhibits beta(2)-adrenergic receptor agonist-stimulated vectorial fluid and Cl(-) transport across primary rat and human alveolar epithelial type II cell monolayers. This inhibition is due to a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis mediated by a phosphatidylinositol 3-kinase (PI3K)-dependent heterologous desensitization and down-regulation of the beta(2)-adrenergic receptors. Consistent with these in vitro results, inhibition of the PI3K pathway or pretreatment with soluble chimeric TGF-beta type II receptor restored beta(2)-adrenergic receptor agonist-stimulated alveolar epithelial fluid transport in an in vivo model of acute lung injury induced by hemorrhagic shock in rats. The results demonstrate a novel role for TGF-beta1 in impairing the beta- adrenergic agonist-stimulated alveolar fluid clearance in acute lung injury, an effect that could be corrected by using PI3K inhibitors that are safe to use in humans.


Assuntos
AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Alvéolos Pulmonares/citologia , Fator de Crescimento Transformador beta1/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2 , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cloretos/metabolismo , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Hemorrágico/metabolismo
19.
J Cyst Fibros ; 20(1): 173-182, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32978064

RESUMO

BACKGROUND: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process. METHODS: Newborn CFTR+/+ and CFTR-/- were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina. Tracheal mucosa and the bronchoalveolar lavage (BAL) fluid were collected to determine the level of mucin O-glycosylation, bacteria binding to mucins and the airways transcriptome. Disturbances in mucociliary transport were determined by ex-vivo imaging of luminescent Pseudomonas aeruginosa. RESULTS: We provide evidence of an increased sialylation of CF airway mucins and impaired mucociliary transport that occur before the onset of inflammation. Hypersialylation of mucins was reproduced on tracheal explants from non CF animals treated with GlyH101, an inhibitor of CFTR channel activity, indicating a causal relationship between the absence of CFTR expression and the sialylation of mucins. This increased sialylation was correlated to an increased adherence of P. aeruginosa to mucins. In vivo infection of newborn CF piglets by live luminescent P. aeruginosa demonstrated an impairment of mucociliary transport of this bacterium, with no evidence of pre-existing inflammation. CONCLUSIONS: Our results document for the first time in a well-defined CF animal model modifications that affect the O-glycan chains of mucins. These alterations precede infection and inflammation of airway tissues, and provide a favorable context for microbial development in CF lung that hallmarks this disease.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Mucinas/metabolismo , Depuração Mucociliar , Mucosa Respiratória/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Glicosilação , Masculino , Pseudomonas aeruginosa , Mucosa Respiratória/microbiologia , Suínos , Traqueia
20.
Oncogene ; 40(14): 2621, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33686243

RESUMO

Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, Nuclear LUCAT1 (NLUCAT1), which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-κB and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs.

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