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Stem Cell Reports ; 16(12): 2861-2870, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34861163


Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Progenitoras de Megacariócitos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Plaquetas/metabolismo , Linhagem Celular , Proliferação de Células , Células Clonais , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação para Cima , Proteína bcl-X/metabolismo
Toxicol Appl Pharmacol ; 420: 115532, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33845054


Limited information is available regarding the effects of arsenic exposure on immune function. We have recently reported that chronic exposure to As was associated asthma, as determined by spirometry and respiratory symptoms. Because T helper 2 (Th2)-driven immune responses are implicated in the pathogenesis of allergic diseases, including asthma, we studied the associations of serum Th1 and Th2 mediators with the As exposure markers and the features of asthma among individuals exposed to As. A total of 553 blood samples were selected from the same study subjects recruited in our previous asthma study. Serum levels of Th1 and Th2 cytokines were analyzed by immunoassay. Subjects' arsenic exposure levels (drinking water, hair and nail arsenic concentrations) were determined by inductively coupled plasma mass spectroscopy. Arsenic exposure levels of the subjects showed significant positive associations with serum Th2-mediators- interleukin (IL)-4, IL-5, IL-13, and eotaxin without any significant changes in Th1 mediators- interferon-γ and tumor necrosis factor-α. The ratios of Th2 to Th1 mediators were significantly increased with increasing exposure to As. Notably, most of the Th2 mediators were positively associated with serum levels of total immunoglobulin E and eotaxin. The serum levels of Th2 mediators were significantly higher in the subjects with asthma than those without asthma. The results of our study suggest that the exacerbated Th2-driven immune responses are involved in the increased susceptibility to allergic asthma among individuals chronically exposed to As.

Arsênio/efeitos adversos , Asma/induzido quimicamente , Citocinas/sangue , Células Th1/efeitos dos fármacos , Equilíbrio Th1-Th2/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Poluentes Químicos da Água/efeitos adversos , Adolescente , Adulto , Asma/diagnóstico , Asma/imunologia , Asma/metabolismo , Bangladesh , Carga Corporal (Radioterapia) , Estudos Transversais , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Medição de Risco , Fatores de Risco , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Adulto Jovem
Sci Total Environ ; 668: 1004-1012, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31018442


Arsenic (As) toxicity and diabetes mellitus (DM) are emerging public health concerns worldwide. Although exposure to high levels of As has been associated with DM, whether there is also an association between low and moderate As exposure and DM remains unclear. We explored the dose-dependent association between As exposure levels and hyperglycemia, with special consideration of the impact of demographic variables, in 641 subjects from rural Bangladesh. The total study participants were divided into three groups depending on their levels of exposure to As in drinking water (low, moderate and high exposure groups). Prevalence of hyperglycemia, including impaired glucose tolerance (IGT) and DM was significantly associated with the subjects' drinking water arsenic levels. Almost all exposure metrics (As levels in the subjects' drinking water, hair and nails) showed dose-dependent associations with the risk of hyperglycemia, IGT and DM. Among the variables considered, sex, age, and BMI were found to be associated with higher risk of hyperglycemia, IGT and DM. In sex-stratified analyses, As exposure showed a clearer pattern of dose-dependent risk for hyperglycemia in females than males. Finally, drinking water containing low-to-moderate levels of As (50.01-150 µg/L) was found to confer a greater risk of hyperglycemia than safe drinking water (As ≤10 µg/L). Thus the results suggested that As exposure was dose-dependently associated with hyperglycemia, especially in females.

Intoxicação por Arsênico/complicações , Diabetes Mellitus/fisiopatologia , Exposição Ambiental , Hiperglicemia/fisiopatologia , Poluentes Químicos da Água/análise , Adulto , Arsênio/análise , Intoxicação por Arsênico/epidemiologia , Bangladesh/epidemiologia , Índice de Massa Corporal , Diabetes Mellitus/epidemiologia , Suscetibilidade a Doenças , Água Potável/química , Feminino , Cabelo/química , Humanos , Hiperglicemia/epidemiologia , Masculino , Pessoa de Meia-Idade , Unhas/química , Prevalência , Abastecimento de Água
Int Immunol ; 30(4): 141-154, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29617862


Immunotherapies have led to the successful development of novel therapies for cancer. However, there is increasing concern regarding the adverse effects caused by non-tumor-specific immune responses. Here, we report an effective strategy to generate high-avidity tumor-antigen-specific CTLs, using Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) delivery. As a proof-of-principle demonstration, we selected the gp100 melanoma-associated tumor antigen, and cloned the gp100-specific high-avidity TCR from gp100-immunized mice. To enable rapid structural dissection of the TCR, we developed a 3D protein structure modeling system for the TCR/antigen-major histocompatibility complex (pMHC) interaction. Combining these technologies, we efficiently generated gp100-specific PD-1(-) CD8+ T cells, and demonstrated that the genetically engineered CD8+ T cells have high avidity against melanoma cells both in vitro and in vivo. Our methodology offers computational prediction of the TCR response, and enables efficient generation of tumor antigen-specific CD8+ T cells that can neutralize tumor-induced immune suppression leading to a potentially powerful cancer therapeutic.

Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/genética , Neoplasias/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/química , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Genes Reporter , Melanoma Experimental , Camundongos , Modelos Moleculares , Complexos Multiproteicos , Neoplasias/metabolismo , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Antígeno gp100 de Melanoma/química , Antígeno gp100 de Melanoma/imunologia , Antígeno gp100 de Melanoma/metabolismo
Environ Health ; 16(1): 20, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28270149


BACKGROUND: Chronic exposure to arsenic is associated with cancer and hypertension. Growing evidence suggests that altered methylation in long interspersed nuclear element-1 (LINE-1) is involved in many types of disorders, including cardiovascular disease. Here we evaluated the association between arsenic exposure and LINE-1 methylation levels, especially in relation to blood pressure (BP). METHODS: A total of 236 subjects (175 from arsenic-endemic areas and 61 from a non-endemic area) in rural Bangladesh were recruited. The subjects' arsenic exposure levels (i.e., drinking water, hair and nail arsenic concentrations) were measured by inductively coupled plasma mass spectroscopy. The subjects' LINE-1 methylation levels were determined by pyrosequencing. RESULTS: The average LINE-1 methylation levels of the subjects living in the arsenic-endemic areas were significantly (p < 0.01) lower than those of the subjects living in the non-endemic area. In a sex-stratified analysis, the arsenic exposure levels in female but not male subjects showed a significant inverse association with LINE-1 methylation levels before (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) and after (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) adjustment for age, body mass index and smoking. Analyses examining interactions among arsenic levels, BP and LINE-1 methylation showed that arsenic-related elevated levels of BP were associated with LINE-1 hypomethylation. CONCLUSIONS: Our findings demonstrated that chronic exposure to arsenic was inversely associated with LINE-1 methylation levels in blood leukocyte DNA and this was more pronounced in females than males; in addition, the decreased levels of LINE-1 methylation might be involved in the arsenic-induced elevation of BP.

Arsênio/efeitos adversos , Pressão Sanguínea/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Exposição Ambiental/efeitos adversos , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Poluentes Químicos da Água/efeitos adversos , Adulto , Arsênio/análise , Bangladesh , Estudos Transversais , Água Potável/análise , Exposição Ambiental/análise , Feminino , Cabelo/química , Humanos , Masculino , Pessoa de Meia-Idade , Unhas/química , Poluentes Químicos da Água/análise