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1.
Mol Carcinog ; 58(11): 2008-2016, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31385375

RESUMO

Treatment of acute myeloid leukemia (AML) is still a challenge because of common relapses or resistance to treatment. Therefore, the development of new therapeutic approaches is necessary. Various studies have shown that certain cancers, including some chemoresistant AML subsets, have upregulated oxidative phosphorylation. In this study, we aimed to assess treatment-resistant AML patients' cell modulation using oxidative phosphorylation inhibitors metformin and atovaquone alone and in various combinations with cytosine analog cytarabine and apoptosis inducer venetoclax. Metabolic activity analysis using Agilent Seahorse XF Extracellular Flux Analyzer revealed that peripheral blood mononuclear cells' metabolic state was different among treatment-resistant AML patients. We demonstrated that metformin decreased therapy-resistant-AML cell oxidative phosphorylation ex vivo, cotreatment with cytarabine and venetoclax slightly increased the effect. However, treatment with atovaquone did not have a marked effect in our experiment. Cell treatment had a slight effect on cell proliferation inhibition; combination of metformin, cytarabine, and venetoclax had the strongest effect. Moreover, a slightly higher effect on cell proliferation and cell cycle regulation was demonstrated in the cells with higher initial oxidative phosphorylation rate as demonstrated by gene expression analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Proteomic analysis by liquid chromatography-mass spectrometry demonstrated that chemoresistant AML cell treatment with metformin modulated metabolic pathways, while metformin combination with cytarabine and venetoclax boosted the effect. We suggest that oxidative phosphorylation inhibition is effective but not sufficient for chemoresistant AML treatment. Indeed, it causes anticancerous changes that might have an important additive role in combinatory treatment.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Metformina/farmacologia , Proteômica , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Proteínas de Neoplasias/genética , Fosforilação Oxidativa/efeitos dos fármacos
2.
Immunology ; 158(3): 206-218, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31393598

RESUMO

Behçet's disease (BD) is a chronic systemic inflammatory disease with unclear etiopathogenesis. Although gene variants of CC chemokine receptor type 1 (CCR1) have been reported, the protein expression of CCR1 in patients with BD remains unclear. The objective of this study was to analyze the frequencies of CCR1+ cells in a herpes simplex virus-induced mouse model of BD. The frequencies of CCR1+ cells on the surface and in the cytoplasm of peripheral blood mononuclear cells and lymph nodes were analyzed by flow cytometry. The CCR1+ cells were significantly down-regulated in BD mice compared with the normal control and symptom-free control mice. Colchicine and pentoxifylline treatment improved the symptoms of BD and increased the frequencies of CCR1+ cells in BD mice. Treatment with chemokine CC motif ligand 3 (CCL3), a ligand of CCR1, caused BD symptoms to deteriorate in 10 of 16 BD mice (62·5%) via down-regulation of CCR1+ cells. Anti-CCL3 antibody treatment ameliorated BD symptoms in 10 of 20 mice (50%) and significantly decreased the disease severity score compared with CCL3-treated BD mice (P = 0·01) via up-regulation of CCR1+ cell frequencies. In patients with BD, plasma levels of CCL3 in an active state were significantly higher than in healthy control individuals (P = 0·02). These results show that the up-regulation of CCR1+ cells was related to the control of systemic inflammation of BD in mouse models.


Assuntos
Anticorpos/farmacologia , Síndrome de Behçet/tratamento farmacológico , Quimiocina CCL3/antagonistas & inibidores , Herpes Simples/tratamento farmacológico , Leucócitos Mononucleares/imunologia , Receptores CCR1/imunologia , Simplexvirus/imunologia , Animais , Síndrome de Behçet/imunologia , Síndrome de Behçet/patologia , Síndrome de Behçet/virologia , Quimiocina CCL3/imunologia , Modelos Animais de Doenças , Herpes Simples/imunologia , Herpes Simples/patologia , Humanos , Leucócitos Mononucleares/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR
3.
Graefes Arch Clin Exp Ophthalmol ; 257(10): 2307-2314, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31338585

RESUMO

PURPOSE: To compare IL-6, sIL-6R, and IL-17 secretion in peripheral blood mononuclear cells (PBMCs) cultured with tocilizumab (a humanized monoclonal antibody against the interleukin-6 receptor), dexamethasone, and placebo, obtained from patients with thyroid eye disease (TED) and healthy controls. METHODS: The study was a prospective proof of concept test. We cultured peripheral blood mononuclear cells from TED patients and healthy controls with tocilizumab, dexamethasone, and placebo. IL-6, sIL-6R, and IL-17 levels in supernatants obtained from PBMCs cultures were analyzed by ELISA. RESULTS: We included seventeen patients with thyroid eye disease (12 females and five males). The mean age was 49 years. Both dexamethasone and tocilizumab influenced IL-6 and IL-6Rs levels in patients' group. Supernatants obtained from PBMCs treated with dexamethasone showed 77.2% and 82.8% lower IL-6 levels compared with those cultured with placebo and tocilizumab, respectively. Furthermore, overnight culture of PBMCs with dexamethasone showed significantly lower sIL-6R secretion compared with untreated (33.71%, p = 0.04) and tocilizumab treated (58.21%, p = 0.01) PBMCs. Neither dexamethasone nor tocilizumab affected IL-17 concentrations in PBMCs cultures. CONCLUSIONS: Both dexamethasone and tocilizumab affect the IL-6/sIL-6R system. Specifically, dexamethasone reduces and tocilizumab increases the levels of these cytokines in PBMCs cultures. These results strengthen the molecular rationale for interrogating the efficacy of tocilizumab in steroid-resistant TED, as IL-6 seems to be a common target for both anti-IL-6R antibody and steroids.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Citocinas/biossíntese , Dexametasona/uso terapêutico , Oftalmopatia de Graves/tratamento farmacológico , Adulto , Idoso , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Glucocorticoides/uso terapêutico , Oftalmopatia de Graves/diagnóstico , Oftalmopatia de Graves/metabolismo , Humanos , Interleucina-6/antagonistas & inibidores , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tomografia Computadorizada por Raios X , Resultado do Tratamento
4.
Zhonghua Gan Zang Bing Za Zhi ; 27(4): 286-290, 2019 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-31082340

RESUMO

Objective: To investigate TLR2 and TLR4 expressional situation on the surface of peripheral blood mononuclear cells (PBMC) in patients with hepatocellular carcinoma (HCC) and their relationship with small intestinal bacterial overgrowth (SIBO). Methods: Flow cytometry was used to detect TLR2 and TLR4 expressional situation on the surface of PBMC in 78 cases with HCC, 56 cases with cirrhosis and 33 healthy controls. Furthermore, lactose hydrogen breath test (LHBT) was used to detect small intestinal bacterial overgrowth. Results: Of the 78 cases with HCC, 56 cases (71.8%) were SIBO-positive, 23 cases (41.1%) were SIBO- positive in 56 cases with cirrhosis, and 1 (3.0%) was SIBO-positive in 33 healthy controls. The incidence of SIBO in HCC patients was higher than cirrhosis patients (χ(2) = 12.72, P < 0.05) and healthy controls (χ(2) = 41.18, P < 0.05). The expression levels of TLR2 and TLR4 in HCC patients (100.55 ± 24.22, 42.76 ± 15.96) were significantly higher than cirrhosis (67.42 ± 18.36, 24.38 ± 8.68)and healthy control group (33.06 ± 11.72, 12.52 ± 4.46) (P < 0.05). Furthermore, the expression levels of TLR2 and TLR4 in SIBO-positive patients (108.75 ± 20.40, 48.1 ± 14.98) were higher than SIBO-negative patients (79.67 ± 20.60, 28.62 ± 7.36) (P < 0.05). Conclusion: The expression of TLR2 and TLR4 and the incidence of SIBO in HCC patients are significantly higher than cirrhosis and healthy control group. Moreover, the high expressions of TLR2 and TLR4 in SIBO-positive HCC patients may promote the development of HCC.


Assuntos
Bactérias/isolamento & purificação , Carcinoma Hepatocelular/metabolismo , Leucócitos Mononucleares/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Infecções Bacterianas/sangue , Infecções Bacterianas/epidemiologia , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , China/epidemiologia , Humanos , Incidência , Leucócitos Mononucleares/patologia , Cirrose Hepática/sangue , Cirrose Hepática/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/patologia , Receptor 2 Toll-Like/sangue , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/sangue , Receptor 4 Toll-Like/genética
5.
Nucleic Acids Res ; 47(15): e87, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31127310

RESUMO

Detection of cancer-associated somatic mutations has broad applications for oncology and precision medicine. However, this becomes challenging when cancer-derived DNA is in low abundance, such as in impure tissue specimens or in circulating cell-free DNA. Next-generation sequencing (NGS) is particularly prone to technical artefacts that can limit the accuracy for calling low-allele-frequency mutations. State-of-the-art methods to improve detection of low-frequency mutations often employ unique molecular identifiers (UMIs) for error suppression; however, these methods are highly inefficient as they depend on redundant sequencing to assemble consensus sequences. Here, we present a novel strategy to enhance the efficiency of UMI-based error suppression by retaining single reads (singletons) that can participate in consensus assembly. This 'Singleton Correction' methodology outperformed other UMI-based strategies in efficiency, leading to greater sensitivity with high specificity in a cell line dilution series. Significant benefits were seen with Singleton Correction at sequencing depths ≤16 000×. We validated the utility and generalizability of this approach in a cohort of >300 individuals whose peripheral blood DNA was subjected to hybrid capture sequencing at ∼5000× depth. Singleton Correction can be incorporated into existing UMI-based error suppression workflows to boost mutation detection accuracy, thus improving the cost-effectiveness and clinical impact of NGS.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Leucemia Mieloide Aguda/genética , Mutação , Proteínas de Neoplasias/genética , Análise de Sequência de DNA/métodos , Alelos , Linhagem Celular Tumoral , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Frequência do Gene , Células HCT116 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Medicina de Precisão/métodos , Erro Experimental
6.
Nat Methods ; 16(5): 409-412, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011186

RESUMO

Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Camundongos , Células NIH 3T3 , RNA Guia/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas
7.
Life Sci ; 224: 241-248, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30867120

RESUMO

AIMS: DNA methylation and hydroxymethylation are significantly related to the occurrence and development of coronary heart disease (CHD) and atherosclerosis (AS). 5-Methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) are used to assess DNA methylation and hydroxymethylation levels, respectively. However, 5-mC and 5-hmC levels associated with CHD remain controversial. In the present study, we aimed to investigate the association of the peripheral blood levels of 5-mC and 5-hmC and the degree of coronary atherosclerosis in elderly CHD patients. MAIN METHODS: 5-mC and 5-hmC levels in peripheral blood mononuclear cells (PBMCs) were measured in 44 CHD patients and 42 matched control subjects by ELISA and dot blot analysis. Immunohistochemical staining was used to observe 5-mC, 5-hmC and TET2 expression in human aortic tissue. Gensini score was used to evaluate the degree of coronary atherosclerosis. KEY FINDINGS: 5-mC and 5-hmC levels in PBMCs from CHD patients and in human aortic atherosclerosis plaque were both higher than those in control subjects and in tissue samples. TET2 expression was significantly upregulated in CHD patients compared with control subjects, while only an increasing trend in the expression of DNMT1, DNMT3A and all the other TET genes were found. Spearman correlation analysis demonstrated that 5-mC and 5-hmC levels were positively correlated with Gensini score. 5-mC and 5-hmC were considered as the risk factors for CHD after adjustment. SIGNIFICANCE: DNA methylation and hydroxymethylation levels in PBMCs from elderly CHD patients were significantly increased, showing a positive correlation with the degree of coronary atherosclerosis.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Aterosclerose/patologia , Doença das Coronárias/patologia , Metilação de DNA , Regulação da Expressão Gênica , Leucócitos Mononucleares/patologia , Idoso , Aterosclerose/genética , Estudos de Casos e Controles , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima
8.
Immunopharmacol Immunotoxicol ; 41(1): 123-129, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30721634

RESUMO

Context: Fluconazole (FNZ) is a drug used in antifungal therapy. However, the minimum FNZ dose to interfering with immune responses or inducing DNA damage is still unknown. Objective: This study investigated the toxicological profile of FNZ on cultured human peripheral blood mononuclear cells (PBMCs) treated with different concentrations of this azole. Materials and methods: Cultured PBMCs were exposed to FNZ (6, 12, 30, 60 and 120 µg/mL) and the toxicological profile was assessed by the following parameters: cytotoxic and nuclear division index (necrotic, apoptotic and viable cells), DNA damage (alkaline comet test), mutagenic potential (micronucleus test), cytokine modulation (IL-1, IL-6, IL-10, TNF-α, IFN-γ), and predictive toxicity (Osiris® and LAZAR® programs). Results: Our results demonstrated that FNZ induced cellular DNA damage and mutagenicity at concentrations above the plasma peak (>30 µg/mL) and 6 µg/mL, respectively, which was associated with increased TNF-α, and decrease IL-6 and IL-10 concentrations. These effects may be related to increased apoptosis and cytotoxic nuclear division index in the cultured PBMCs. In silico results indicated potential mutagenic, tumorigenic, irritant, and carcinogenic effects, which were partially confirmed by the above assays. Discussion and conclusions: Together, these findings suggest the need to rationalize the use of FNZ, especially if it is used for long periods or with concomitant pathologies requiring azole therapy that may increase FNZ's plasma concentration.


Assuntos
Antifúngicos/toxicidade , Citocinas/imunologia , Dano ao DNA , Fluconazol/toxicidade , Leucócitos Mononucleares/efeitos dos fármacos , Mutagênicos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interleucina-10/imunologia , Interleucina-6/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Fator de Necrose Tumoral alfa/imunologia
9.
Hematology ; 24(1): 331-336, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30727851

RESUMO

OBJECT: To explore the critical role of growth differentiation factor 11 (GDF11) in the pathobiology of aplastic anemia (AA). METHODS: We have examined the serum GDF11 levels for 79 AA patients and 30 healthy controls. A total of 79 AA patients, which included 29 new diagnosed (untreated) cases, 14 cases with no response, 21 partial remission (PR) cases and 15 complete remission (CR) cases after immunosuppressive therapy (IST). GDF11 serum levels were assessed by an enzyme-linked immunosorbent assay. GDF11 mRNA expression in peripheral blood mononuclear (PBMNC) was detected through real time polymerase chain reaction. The correlation between GDF11 expression and erythropoietic function was evaluated. RESULTS: The serum GDF11 levels in untreated AA patients were higher than that of the control group. The serum GDF11 levels of PR patients or CR patients after IST was decreased, compared with untreated patients, but did not recover back to the normal levels. GDF11 levels had a negative correlation with hemoglobin (Hb) levels and reticulocyte counts in AA patients. GDF11 levels did not correlate with age, sex and severity of in AA patients. CONCLUSION: Serum GDF11 levels were increased and negatively correlated with Hb levels and reticulocyte counts in AA patients. This suggests an impaired GDF11 response contributing to anemia in AA patients.


Assuntos
Anemia Aplástica/sangue , Proteínas Morfogenéticas Ósseas/sangue , Regulação da Expressão Gênica , Fatores de Diferenciação de Crescimento/sangue , Leucócitos Mononucleares/metabolismo , Adolescente , Adulto , Idoso , Anemia Aplástica/patologia , Anemia Aplástica/terapia , Criança , Feminino , Humanos , Imunossupressão , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Contagem de Reticulócitos
10.
J Virol ; 93(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30760566

RESUMO

Simian-human immunodeficiency virus (SHIV) infection in rhesus macaques (RMs) resembles human immunodeficiency virus type 1 (HIV-1) infection in humans and serves as a tool to evaluate candidate AIDS vaccines. HIV-1 clade A (HIV-A) predominates in parts of Africa. We constructed an R5 clade A SHIV (SHIV-A; strain SHIV-KNH1144) carrying env from a Kenyan HIV-A. SHIV-A underwent rapid serial passage through six RMs. To allow unbridled replication without adaptive immunity, we simultaneously ablated CD8+ and B cells with cytotoxic monoclonal antibodies in the next RM, resulting in extremely high viremia and CD4+ T-cell loss. Infected blood was then transferred into two non-immune-depleted RMs, where progeny SHIV-A showed increased replicative capacity and caused AIDS. We reisolated SHIV-KNH1144p4, which was replication competent in peripheral blood mononuclear cells (PBMC) of all RMs tested. Next-generation sequencing of early- and late-passage SHIV-A strains identified mutations that arose due to "fitness" virus optimization in the former and mutations exhibiting signatures typical for adaptive host immunity in the latter. "Fitness" mutations are best described as mutations that allow for better fit of the HIV-A Env with SIV-derived virion building blocks or host proteins and mutations in noncoding regions that accelerate virus replication, all of which result in the outgrowth of virus variants in the absence of adaptive T-cell and antibody-mediated host immunity.IMPORTANCE In this study, we constructed a simian-human immunodeficiency virus carrying an R5 Kenyan HIV-1 clade A env (SHIV-A). To bypass host immunity, SHIV-A was rapidly passaged in naive macaques or animals depleted of both CD8+ and B cells. Next-generation sequencing identified different mutations that resulted from optimization of viral replicative fitness either in the absence of adaptive immunity or due to pressure from adaptive immune responses.


Assuntos
Imunidade Adaptativa , Infecções por HIV/imunologia , HIV-1/fisiologia , Mutação , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Infecções por HIV/genética , Infecções por HIV/patologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Replicação Viral/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
11.
Stem Cell Res ; 35: 101367, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30763735

RESUMO

Autosomal recessive osteopetrosis (ARO) is a genetic bone disease that can be caused by mutations in the CLCN7 gene preventing osteoclast-mediated bone resorption. We generated a human induced pluripotent stem cell (hiPSC) line, BIHi002-A, from peripheral blood mononuclear cells of an ARO patient carrying the CLCN7 mutations c.875G>A and c.1208G>A using Sendai viral vectors. The pluripotent identity of the BIHi002-A line was confirmed by their expression of typical markers for undifferentiated hiPSCs, their capacity to differentiate into cells of the three germ layers and by PluriTest analysis. The BIHi002-A line provides a tool for disease modelling and therapy development.


Assuntos
Linhagem Celular , Canais de Cloreto , Células-Tronco Pluripotentes Induzidas , Leucócitos Mononucleares , Mutação , Osteopetrose , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Lactente , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Osteopetrose/genética , Osteopetrose/metabolismo , Osteopetrose/patologia
12.
Stem Cell Res ; 35: 101393, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30711802

RESUMO

The Cri du Chat Syndrome (CdCS) is a genetic disease resulting from variable size deletion occurring on the short arm of chromosome 5. The main clinical features are a high-pitched monochromatic cry, microcephaly, severe psychomotor and mental retardation with characteristics of autism spectrum disorders such as hand flapping, obsessive attachments to objects, twirling objects, repetitive movements, and rocking. We reprogrammed to pluripotency peripheral blood mononuclear cells derived from a patient carrying large deletion on the short arm of chromosome 5, using a commercially available non-integrating expression system. The iPSCs expressed pluripotency markers and differentiated in the three embryonic germ layers.


Assuntos
Técnicas de Reprogramação Celular , Síndrome do Miado do Gato , Células-Tronco Pluripotentes Induzidas , Leucócitos Mononucleares , Adulto , Síndrome do Miado do Gato/genética , Síndrome do Miado do Gato/metabolismo , Síndrome do Miado do Gato/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino
13.
Stem Cell Res ; 34: 101380, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605840

RESUMO

We describe the generation and characterization of 5 human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/patologia , Leucócitos Mononucleares/patologia , Adulto , Linhagem Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
14.
Clin Transl Oncol ; 21(9): 1280-1285, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30680609

RESUMO

PURPOSE: Autophagy has lately emerged as an important biological process with implications in several hematological pathologies. Recently, a growing body of evidence supports a putative role of autophagy in chronic lymphocytic leukemia; however, no definitive clue has been established so far. To elucidate this issue, we have developed a pilot study to measure autophagic flux in peripheral blood mononuclear cells from chronic lymphocytic leukemia patients, and explored its correlation with classical clinical/analytical parameters. METHODS/PATIENTS: Thirty-three chronic lymphocytic leukemia patients participated in the study. Autophagic flux in peripheral blood mononuclear cells was determined by western blot measuring the levels of the proteins p62 and lipidated LC3. Moreover, p62 mRNA levels were analyzed by RT-qPCR. RESULTS: Lymphocytosis and the percentage of tumoral lymphocytes in chronic lymphocytic leukemia patients statistically correlate with a blocked autophagic flux. CONCLUSION: Alterations in autophagic flux could play an important role in the physiopathology of chronic lymphocytic leukemia.


Assuntos
Autofagia , Biomarcadores Tumorais/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Leucócitos Mononucleares/patologia , Linfocitose/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucócitos Mononucleares/metabolismo , Linfocitose/metabolismo , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Prognóstico
15.
Stem Cell Res ; 34: 101382, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30658253

RESUMO

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by mutation in the HTT gene encoding HTT protein. The mutant protein leads to the neuronal death through dysregulation of multiple cellular processes. HD human induced pluripotent stem cells (iPSCs) represent a useful and valid model for the disease study. iPSC line from HD patient with 47 CAG repeats in HTT was generated from blood mononuclear cells by non-integrating episomal vectors. The iPSC line retained the mutation, expressed pluripotency markers, had a normal karyotype and displayed in vitro differentiation to the three germ layers. Resource table.


Assuntos
Técnicas de Cultura de Células/métodos , Reprogramação Celular , Doença de Huntington/sangue , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Leucócitos Mononucleares/patologia , Adulto , Linhagem Celular , Feminino , Humanos
16.
Biomed Pharmacother ; 111: 714-723, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30611996

RESUMO

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in which the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (PKB or Akt) pathway is deregulated in response to phosphatase and tensin homolog (PTEN) overexpression. Lactoferrin (LF), a multifunctional iron-binding glycoprotein, is involved in AD pathology; however, direct evidence of its impact upon AD remains unclear. To elucidate LF's role in AD, the possible protective mechanism post-LF administration for 3 months was investigated in AD patients by observing changes in the p-Akt/PTEN pathway. AD patients showed decreased serum acetylcholine (ACh), serotonin (5-HT), antioxidant and anti-inflammatory markers, and decreased expression of Akt in peripheral blood lymphocytes (PBL), as well as PI3K, and p-Akt levels in PBL lysate; all these parameters were significantly improved after daily LF administration for 3 months. Similarly, elevated serum amyloid ß (Aß) 42, cholesterol, oxidative stress markers, IL-6, heat shock protein (HSP) 90, caspase-3, and p-tau, as well as increased expression of tau, MAPK1 and PTEN in AD patients, were significantly reduced upon LF intake. Improvement in the aforementioned AD surrogate markers post-LF treatment was reflected in enhanced cognitive function assessed by the Mini-Mental State Examination (MMSE) and Alzheimer's Disease Assessment Scale-Cognitive Subscale 11-item (ADAS-COG 11) questionnaires as clinical endpoints. These results provide a basis for a possible protective mechanism of LF in AD through its ability to alleviate the AD pathological cascade and cognitive decline via modulation of the p-Akt/PTEN pathway, which affects the key players of inflammation and oxidative stress that are involved in AD pathology.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/tratamento farmacológico , Lactoferrina/uso terapêutico , PTEN Fosfo-Hidrolase/sangue , Proteínas Proto-Oncogênicas c-akt/sangue , Transdução de Sinais/efeitos dos fármacos , Idoso , Doença de Alzheimer/patologia , Feminino , Humanos , Lactoferrina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Projetos Piloto , Proteínas Proto-Oncogênicas c-akt/agonistas , Transdução de Sinais/fisiologia , Resultado do Tratamento
17.
Glia ; 67(1): 193-211, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30597659

RESUMO

Neurotrophins mediate neuronal growth, differentiation, and survival via tropomyosin receptor kinase (Trk) or p75 neurotrophin receptor (p75NTR ) signaling. The p75NTR is not exclusively expressed by neurons but also by certain immune cells, implying a role for neurotrophin signaling in the immune system. In this study, we investigated the effect of p75NTR on innate immune cell behavior and on neuronal morphology upon chronic Toxoplasma gondii (T. gondii) infection-induced neuroinflammation. Characterization of the immune cells in the periphery and central nervous system (CNS) revealed that innate immune cell subsets in the brain upregulated p75NTR upon infection in wild-type mice. Although cell recruitment and phagocytic capacity of p75NTRexonIV knockout (p75-/- ) mice were not impaired, the activation status of resident microglia and recruited myeloid cell subsets was altered. Importantly, recruited mononuclear cells in brains of infected p75-/- mice upregulated the production of the cytokines interleukin (IL)-10, IL-6 as well as IL-1α. Protein levels of proBDNF, known to negatively influence neuronal morphology by binding p75NTR , were highly increased upon chronic infection in the brain of wild-type and p75-/- mice. Moreover, upon infection the activated immune cells contributed to the proBDNF release. Notably, the neuroinflammation-induced changes in spine density were rescued in the p75-/- mice. In conclusion, these findings indicate that neurotrophin signaling via the p75NTR affects innate immune cell behavior, thus, influencing the structural plasticity of neurons under inflammatory conditions.


Assuntos
Leucócitos Mononucleares/fisiologia , Neurônios/fisiologia , Receptor de Fator de Crescimento Neural/fisiologia , Toxoplasma , Toxoplasmose/imunologia , Animais , Feminino , Imunidade Inata/fisiologia , Inflamação/imunologia , Inflamação/patologia , Leucócitos Mononucleares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Toxoplasmose/patologia
18.
J Diabetes ; 11(6): 440-448, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30318734

RESUMO

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease resulting from an attack by autoreactive T lymphocytes against pancreatic islet ß- cells. In recent studies, regulatory T cells (Tregs) have been implicated in the process of T1D. Furthermore, cluster of differentiation 39 (CD39), which is involved in the suppression of inflammation, has been shown to be expressed on Tregs. However, the pathological importance of CD39 to the memory Treg population remains unclear. METHODS: This study investigated Treg subsets, focusing on resting, effector, and memory Tregs, and determined CD39 expression on Tregs. In addition, changes in Treg subsets and Treg-associated cytokine secretion after CD3/CD28 stimulation of peripheral blood mononuclear cells were evaluated in diabetic patients and healthy controls. The suppressive function of Tregs was measured using the mixed lymphocyte reaction (MLR) test. RESULTS: There was a higher percentage of memory Tregs in T1D patients than healthy controls. However, Tregs in T1D patients showed impaired suppression, with low forkhead box P3 (Foxp3) expression and low serum interleukin (IL)-10 levels. Furthermore, CD39 expression on Tregs, and on memory Tregs in particular, was lower in T1D patients than healthy controls. After stimulation, the percentage of resting Tregs was decreased and that of effector/memory Tregs was increased in both healthy controls and T1D patients, but CD39 expression on effector/memory Tregs was still lower and there was no increase in IL-10 secretion in T1D patients. CONCLUSIONS: The defective suppressive function of Tregs in T1D patients is due to lower expression of CD39 on memory Tregs.


Assuntos
Apirase/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Memória Imunológica/imunologia , Leucócitos Mononucleares/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Biomarcadores/análise , Glicemia/análise , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Seguimentos , Fatores de Transcrição Forkhead/metabolismo , Hemoglobina A Glicada/análise , Humanos , Interleucina-10/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia
19.
Methods Mol Biol ; 1881: 267-276, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30350212

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that target specific mRNAs through interaction with complementary sequences usually found in the 3'-untranslated regions (UTRs) of target mRNAs. miRNAs have been shown to play a fundamental role in the management of chronic lymphocytic leukemia (CLL) by modulating gene expression patterns and cellular signaling pathways. In recent years, several studies have focused on the role of regulatory miRNAs in the pathogenesis of CLL. Aberrant expression of CLL-specific miRNAs has emerged as therapeutic and diagnostic biomarkers in patients with CLL. Here, we describe a method for the quantification of miRNAs in malignant B cells from the mononuclear cell compartment, isolated from peripheral blood. We focus on the isolation of human blood monocytes by Ficoll-Paque gradient centrifugation, total RNA extraction from human peripheral blood mononuclear cells, and quantitative reverse transcription (qRT)-PCR, which is useful for the measurement of miRNAs in monocytes isolated from blood samples.


Assuntos
Linfócitos B/metabolismo , Biomarcadores Tumorais/sangue , Leucemia Linfocítica Crônica de Células B/sangue , Leucócitos Mononucleares/metabolismo , MicroRNAs/sangue , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linfócitos B/patologia , Biomarcadores Tumorais/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucócitos Mononucleares/patologia , MicroRNAs/genética
20.
Biochem Biophys Res Commun ; 508(1): 225-229, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30473214

RESUMO

Pravastatin sodium on triggering receptor expressed on myeloid cell-1 (TREM-1)-mediated inflammation in human peripheral blood mononuclear cells (PBMCs) has been poorly investigated. In this study, we isolated PBMCs from the peripheral blood samples of patients with chronic obstructive pulmonary disease, treated the cells with pravastatin sodium, and determined a concentration at which more than 90% cells could survive. Then we treated cells with 10 ng/ml of lipopolysaccharide, added with 10, 50, 100 µM of pravastatin sodium combined with or without LR-12, a known TREM-1 inhibitor. The expression of TREM-1 was determined by quantitative RT-PCR. The levels of TREM-1, IL-6, and TNF-α in cell culture supernatant were measured with ELISA. Simultaneously, NF-κB signaling pathway-related protein p-p65 and p-IκBα were detected by Western blot assay. Results demonstrated that pravastatin sodium significantly mitigated lipopolysaccharide-stimulated TREM-1 over-expression at mRNA and protein levels dose-dependently. Elevated IL-6 and TNF-α levels changed synchronously. LR-12 inhibited the TREM-1 over-expression and inflammatory factor production but did not show extra synergistic effect to pravastatin. Lipopolysaccharide induced phospho-p65 and -IκBα over-expression was weakened significantly when cells were treated with pravastatin sodium. In conclusion, pravastatin could inhibit TREM-1-medieted inflammation and NF-κB signaling pathway was involved.


Assuntos
Inflamação/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Pravastatina/farmacologia , Receptor Gatilho 1 Expresso em Células Mieloides/antagonistas & inibidores , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Humanos , Inflamação/metabolismo , Inflamação/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo
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