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1.
Nat Immunol ; 14(11): 1183-1189, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24097111

RESUMO

The ability of activation-induced cytidine deaminase (AID) to efficiently mediate class-switch recombination (CSR) is dependent on its phosphorylation at Ser38; however, the trigger that induces AID phosphorylation and the mechanism by which phosphorylated AID drives CSR have not been elucidated. Here we found that phosphorylation of AID at Ser38 was induced by DNA breaks. Conversely, in the absence of AID phosphorylation, DNA breaks were not efficiently generated at switch (S) regions in the immunoglobulin heavy-chain locus (Igh), consistent with a failure of AID to interact with the endonuclease APE1. Additionally, deficiency in the DNA-damage sensor ATM impaired the phosphorylation of AID at Ser38 and the interaction of AID with APE1. Our results identify a positive feedback loop for the amplification of DNA breaks at S regions through the phosphorylation- and ATM-dependent interaction of AID with APE1.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/imunologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/imunologia , Retroalimentação Fisiológica , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/imunologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/imunologia , Linfócitos B/citologia , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Regulação da Expressão Gênica , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Fosforilação , Ligação Proteica , Serina/imunologia , Serina/metabolismo , Transdução de Sinais
2.
Nat Immunol ; 12(2): 160-6, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21186367

RESUMO

During immunoglobulin class-switch recombination (CSR), the cytidine deaminase AID induces double-strand breaks into transcribed, repetitive DNA elements called switch sequences. The mechanism that promotes the binding of AID specifically to switch regions remains to be elucidated. Here we used a proteomic screen with in vivo biotinylation of AID to identify the splicing regulator PTBP2 as a protein that interacts with AID. Knockdown of PTBP2 mediated by short hairpin RNA in B cells led to a decrease in binding of AID to transcribed switch regions, which resulted in considerable impairment of CSR. PTBP2 is thus an effector of CSR that promotes the binding of AID to switch-region DNA.


Assuntos
Citidina Desaminase/metabolismo , DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citidina Desaminase/genética , Citidina Desaminase/imunologia , DNA/genética , Switching de Imunoglobulina/genética , Região de Troca de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/imunologia , Ligação Proteica/genética , RNA Interferente Pequeno/genética , Ativação Transcricional/genética , Ativação Transcricional/imunologia , Transgenes/genética
3.
Int J Mol Sci ; 23(1)2021 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-35008598

RESUMO

BACKGROUND: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. METHODS: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D3 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). RESULTS: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC50 = 2.98 ± 1.10 nmol/L, in 14433 cells: EC50 = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC50 = n/d, in 14433 cells: EC50 = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. CONCLUSIONS: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs' structure might lead to new treatment options against ovarian cancer.


Assuntos
Sobrevivência Celular , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Calcitriol/genética , Vitamina D3 24-Hidroxilase/genética , Vitamina D/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Ergocalciferóis/metabolismo , Ergocalciferóis/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/metabolismo , Vitamina D/análogos & derivados
4.
Int J Mol Sci ; 18(6)2017 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-28635660

RESUMO

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development.


Assuntos
Células Sanguíneas/metabolismo , Regulação da Expressão Gênica , Receptores de Calcitriol/genética , Tretinoína/metabolismo , Vitamina D/análogos & derivados , Animais , Células Sanguíneas/citologia , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Células HL-60 , Hematopoese , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ácido Retinoico 4 Hidroxilase/genética , Vitamina D/metabolismo
5.
J Autoimmun ; 56: 66-80, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25457307

RESUMO

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.


Assuntos
Interleucina-2/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Biomarcadores/metabolismo , Antígeno CTLA-4/metabolismo , Relação Dose-Resposta a Droga , Eosinofilia/induzido quimicamente , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interleucina-2/análogos & derivados , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/deficiência , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Contagem de Linfócitos , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos
6.
J Steroid Biochem Mol Biol ; 224: 106173, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36031072

RESUMO

Many malignancies are driven by mutations within the gene for fibroblast growth factor receptor 1 (FGFR1). Previously, we have shown that signal transduction from the FOP2-FGFR1 fusion protein in acute myeloid leukemia KG1 cells is responsible for a low level of expression of the vitamin D receptor gene. In this paper, we address whether other fibroblast growth factor receptors regulate the vitamin D receptor (VDR) gene. We used the human myeloid leukemia U937 and HL60 cells, the bone cancer cell line U2OS, and cell transfection methods to answer the question. For myeloid leukemia cells, overexpression of FGFRs 1-3 genes caused a shift towards monocytic differentiation; this was extracellular regulated kinase (Erk) 1,2-dependent. Overexpression of FGFRs 1-3 genes also upregulated expression of the VDR gene, further sensitizing these cells to 1,25-dihydroxyvitamin D-induced monocyte differentiation. When we increased expression in bone cells, fibroblast growth factor receptors did not upregulate VDR gene expression, nor influence the activity of VDR. Fibroblast growth factor receptors are overexpressed in many neoplasms. Therefore, it may be reasonable to use vitamin D analogs to treat these cancers, to activate VDR and drive cell differentiation.


Assuntos
Leucemia Mieloide Aguda , Receptores de Calcitriol , Humanos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Leucemia Mieloide Aguda/metabolismo , Diferenciação Celular , Células HL-60 , Di-Hidroxicolecalciferóis
7.
Cells ; 9(9)2020 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-32872475

RESUMO

(1) Background: Vitamin D receptor (VDR) is present in multiple types of blood cells, and its ligand, 1,25-dihydroxyvitamin D (1,25D), is important for the proper functioning of the immune system. Activity of VDR is higher in hematopoietic stem and progenitor cells than in fully differentiated blood cells of mice and humans. In some human acute myeloid leukemia (AML) blasts, the expression of the VDR gene is also high. The mechanism of silencing the VDR gene expression during differentiation of blood cells has been addressed in this work. (2) Methods: The cells have been obtained using fluorescence activated sorting from murine tissues and from human umbilical cord blood (UCB). Then, the expression of the VDR gene and transcriptional activity of the VDR protein has been tested in real-time polymerase chain reaction (PCR). Eventually, the methylation of VDR promoter regions was tested using bisulfite sequencing. (3) Results: The CpG islands in VDR promoters were not methylated in the cells studied both in mice and in humans. The use of hypomethylating agents had no effect toward expression of human VDR transcripts, but it increased expression of the VDR-target gene, CYP24A1. (4) Conclusions: The expression of the VDR gene and transcriptional activity of the VDR protein varies at successive stages of hematopoietic differentiation in humans and mice, and in blasts from AML patients. The experiments presented in this case indicate that methylation of the promoter region of the VDR gene is not the major mechanism responsible for these differences.


Assuntos
Metilação de DNA/genética , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Receptores de Calcitriol/genética , Adulto , Idoso , Diferenciação Celular , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
8.
Med Pr ; 57(6): 537-42, 2006.
Artigo em Polonês | MEDLINE | ID: mdl-17533991

RESUMO

Biological monitoring plays a significant part in the assessment of occupational exposure to lead. The method basically comprises determinations of blood lead concentration and of one of early toxic-effect biomarkers: the level of either zincprotoporphyrin (ZnPP) in blood or deltaaminolevulinic acid (ALA) in urine. However, biological monitoring is conducted only in 25% of all industrial plants, where the employed technological processes are the source of occupational lead exposure. The project that has been implemented by the Nofer Institute of Occupational Medicine, Lódz, Poland and Outpatient Clinic for Occupational Health, Poznan, Poland, since 1997 made it possible to assess the dynamics of lead exposure in one of the largest battery plants in Western Poland. Based on the analysis of blood lead determinations in lead-exposed workers, the authors could trace the effectiveness of the project aimed at reducing lead exposure in the plant. The findings revealed that the preventive measures applied over the years 1997-2005 resulted in a considerably decreased percentage of blood lead concentrations exceeding the relevant BEI (biological exposure index) value of 500 microg/l: from 19% in 1997 to 1% in 2005. Such a high effectiveness of the project can be mostly attributed to a successful cooperation between the occupational physician, in-plant occupational health services, and the employer. They were all actively involved in a wide-range of activities for workers' health protection and workplace health promotion.


Assuntos
Monitoramento Ambiental , Poluição Ambiental/prevenção & controle , Chumbo/sangue , Exposição Ocupacional , Prevenção Primária , Animais , Humanos , Chumbo/efeitos adversos , Masculino , Polônia
9.
Polymers (Basel) ; 8(9)2016 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-30974593

RESUMO

Current regenerative strategies used for cartilage repair rely on biomaterial functionality as a scaffold for cells that may have potential in chondrogenic differentiation. The purpose of the research was to investigate the biocompatibility of enzymatically treated alginate/chitosan hydrosol sponges and their suitability to support chondrogenic differentiation of human adipose derived multipotent stromal cells (hASCs). The alginate/chitosan and enzyme/alginate/chitosan sponges were formed from hydrosols with various proportions and were used as a biomaterial in this study. Sponges were tested for porosity and wettability. The porosity of each sponge was higher than 80%. An equal dose of alginate and chitosan in the composition of sponges improved their swelling ability. It was found that equal concentrations of alginate and chitosan in hydrosols sponges assure high biocompatibility properties that may be further improved by enzymatic treatment. Importantly, the high biocompatibility of these biomaterials turned out to be crucial in the context of hydrosols' pro-chondrogenic function. After exposure to the chondrogenic conditions, the hASCs in N/A/C and L/A/C sponges formed well developed nodules and revealed increased expression of collagen type II, aggrecan and decreased expression of collagen type I. Moreover, in these cultures, the reactive oxygen species level was lowered while superoxide dismutase activity increased. Based on the obtained results, we conclude that N/A/C and L/A/C sponges may have prospective application as hASCs carriers for cartilage repair.

10.
Int Arch Allergy Immunol ; 138(2): 180-8, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16210858

RESUMO

BACKGROUND: Rheumatoid factors (RFs) are autoantibodies associated with rheumatoid arthritis. They can be detected in normal individuals, although transiently. This dichotomy has led to questions about the origins and types of RFs. Recently it has been shown that B cells that produce RFs only do so when activated by two signals, one from engagement of the B-cell receptor and the other from recognition of a pathogen-associated molecular pattern through a Toll-like receptor (TLR). These autoantibodies thus link the innate and acquired immune responses. OBJECTIVE: Through a review of the literature, an examination of the current knowledge of RF induction is presented. The focus is on a discussion of a beneficial or detrimental role for RFs in normal individuals and in those with chronic disease. RESULTS: What makes RF 'good' in some cases and 'bad' in others may reflect the type of RF produced. Low-affinity polyreactive IgM RFs are probably beneficial as they aid in the clearance of immune complexes that are more efficiently cleared, and the RF B cell can act as an antigen-presenting cell and stimulate host defense. However, large amounts of high-affinity RFs found in patients with chronic disease may be harmful by participation in a vicious cycle of autoantibody production by stimulation of self lymphocytes, and/or deposition in blood vessels thus causing vasculitis. CONCLUSIONS: Whether RFs are beneficial or detrimental depends on the context in which they are expressed, the type and amount of RF produced, whether the response is perpetuated by TLR ligation and whether other cells are stimulated either directly or indirectly by RF-positive B cells.


Assuntos
Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Fator Reumatoide/imunologia , Animais , Humanos , Tolerância Imunológica , Imunoglobulina G/imunologia , Camundongos , Fator Reumatoide/genética , Fator Reumatoide/metabolismo , Linfócitos T/imunologia
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