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1.
J Biol Chem ; 299(6): 104755, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37116708

RESUMO

The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia. However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in chronic neutrophilic leukemia and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues-the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at WT threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain. Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.


Assuntos
Leucemia Neutrofílica Crônica , Humanos , Leucemia Neutrofílica Crônica/diagnóstico , Leucemia Neutrofílica Crônica/genética , Leucemia Neutrofílica Crônica/metabolismo , Glicosilação , Ligantes , Mutação , Receptores de Fator Estimulador de Colônias/genética , Receptores de Fator Estimulador de Colônias/metabolismo , Treonina/metabolismo , Fatores Estimuladores de Colônias/genética , Fatores Estimuladores de Colônias/metabolismo
2.
Microbiol Spectr ; 11(3): e0402422, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37036344

RESUMO

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-ß, IFN-γ, interleukin-1ß (IL-1ß), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.


Assuntos
Infecções por Adenoviridae , Doença de Newcastle , Doenças das Aves Domésticas , Vacinas Virais , Humanos , Animais , Vírus da Doença de Newcastle/genética , Galinhas , Neuraminidase , Hemaglutininas , Doença de Newcastle/prevenção & controle , Adenoviridae , Antivirais , Monócitos , Vacinas Virais/genética , Vacinas Sintéticas , Genótipo , Anticorpos Antivirais , Fatores Estimuladores de Colônias/genética , Granulócitos
4.
Leukemia ; 36(10): 2499-2508, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35941213

RESUMO

Alternatively spliced colony stimulating factor 3 receptor (CSF3R) isoforms Class III and Class IV are observed in myelodysplastic syndromes (MDS), but their roles in disease remain unclear. We report that the MDS-associated splicing factor SRSF2 affects the expression of Class III and Class IV isoforms and perturbs granulopoiesis. Add-back of the Class IV isoform in Csf3r-null mouse progenitor cells increased granulocyte progenitors with impaired neutrophil differentiation, while add-back of the Class III produced dysmorphic neutrophils in fewer numbers. These CSF3R isoforms were elevated in patients with myeloid neoplasms harboring SRSF2 mutations. Using in vitro splicing assays, we confirmed increased Class III and Class IV transcripts when SRSF2 P95 mutations were co-expressed with the CSF3R minigene in K562 cells. Since SRSF2 regulates splicing partly by recognizing exonic splicing enhancer (ESE) sequences on pre-mRNA, deletion of either ESE motifs within CSF3R exon 17 decreased Class IV transcript levels without affecting Class III. CD34+ cells expressing SRSF2 P95H showed impaired neutrophil differentiation in response to G-CSF and was accompanied by increased levels of Class IV. Our findings suggest that SRSF2 P95H promotes Class IV splicing by binding to key ESE sequences in CSF3R exon 17, and that SRSF2, when mutated, contributes to dysgranulopoiesis.


Assuntos
Síndromes Mielodisplásicas , Transtornos Mieloproliferativos , Neoplasias , Animais , Fatores Estimuladores de Colônias/genética , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Camundongos , Mutação , Síndromes Mielodisplásicas/genética , Isoformas de Proteínas/genética , Precursores de RNA , Proteínas de Ligação a RNA/genética , Receptores de Fator Estimulador de Colônias , Fatores de Processamento de Serina-Arginina/genética
5.
Probl Radiac Med Radiobiol ; 25: 456-477, 2020 Dec.
Artigo em Inglês, Ucraniano | MEDLINE | ID: mdl-33361854

RESUMO

OBJECTIVE: to establish the connection of radiation-induced changes in gene expression with the realized pathology of the broncho-pulmonary and cardiovascular systems in Chornobyl clean-up workers. MATERIALS AND METHODS: We examined 314 male Chornobyl clean-up workers (main group; age (58.94 ± 6.82) years(M ± SD); min 33, max 79 years; radiation dose (411.82 ± 625.41) mSv (M ± SD); min 1.74, max 3600 mSv) with various nosological forms of cardiovascular and broncho-pulmonary pathology (BPP) and 50 subjects of the controlgroup: age (50.50 ± 5.73) years (M ± SD); min 41, max 67 years. The relative level of BCL2, CDKN2A, CLSTN2, GSTM1,IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2, TERT, TNF, TP53, CCND1, CSF2, VEGFA genes expression was determined inperipheral blood leukocytes by real-time PCR (7900 HT Fast Real-Time PCR System (Applied Biosystems, USA)). The«gene-disease¼ association was determined on statistical models stratified separately for each disease and gene.Logistic regression was used to calculate the odds ratio. RESULTS: Increased GSTM1 gene expression and no changes in angiogenesis-related VEGFA gene expression werefound in the main group of patients with coronary heart disease (CHD). It was established overexpression of TP53,VEGF and IFNG genes in the group of patients with arterial hypertension (AH). At combination of these diseases anincrease of expression of СSF2, TERF1, TERF2 genes was established. The detected changes demonstrate an activationof the antioxidative defense system in patients with CHD, while AH is associated with the expression of genes ofangiogenesis and immune inflammation. It was shown an increase in the expression of genes associated with apoptosis and kinase activity (BCL2, CLSTN2, CDKN2), immune inflammation (CSF2, IL1B, TNF) in Chornobyl clean-upworkers with BPP. Expression of TP53 and GSTM1 (gene, associated with the glutathione system) was significantlyupregulated in the group of individuals with chronic bronchitis, whereas in patients with chronic obstructive pulmonary disease, no increase was detected; the expression of SERPINB9 and MCF2L genes was downregulated. CONCLUSIONS: Changes in the expression of genes, associated with the development of somatic pathology in theremote period after irradiation, in particular the genes of the immune response and inflammatory reactions CSF2,IFNG, IL1B, TNF; expression of genes that regulate cell proliferation, aging and apoptosis TP53, BCL2, MCF2L, CDKN2A,SERPINB9, TERF1, TERF2, TERT; genes that regulate cell adhesion and angiogenesis CLSTN2, VEGF.


Assuntos
Doenças Cardiovasculares/genética , Acidente Nuclear de Chernobyl , Expressão Gênica/efeitos da radiação , Pneumopatias/genética , Exposição à Radiação/efeitos adversos , Lesões por Radiação/genética , Idoso , Poluentes Radioativos do Ar/efeitos adversos , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fatores Estimuladores de Colônias/genética , Fatores Estimuladores de Colônias/metabolismo , Socorristas , Contaminação Radioativa de Alimentos , Humanos , Interferons/genética , Interferons/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/efeitos da radiação , Pneumopatias/etiologia , Pneumopatias/metabolismo , Pneumopatias/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Lesões por Radiação/etiologia , Lesões por Radiação/metabolismo , Lesões por Radiação/patologia , Radioisótopos , Poluentes Radioativos do Solo/efeitos adversos , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ucrânia
6.
Toxicol In Vitro ; 69: 104994, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32891722

RESUMO

In this study, we aimed to identify signaling alteration caused by exposure to diesel exhaust particles (DEPs) using primary human nasal epithelial cells (PHNECs). Global gene expression profiles in PHNECs following 50 and 200 µg/ml of DEP exposure were identified using microarray analysis. To cover the limitation of array-based mRNA expression analysis, text-mining-based software was used to analyze the integrative biological networks and relevant disease-focused functions among identified DEP-responsive genes. The confidence was valued based on the connectivity between the analyzed pathway and marker candidates. Through a literature-based pathway analysis, the stimulation of inflammation- and immune response-related processes mediated by TNF were predicted as major signaling alterations in PHNECs caused by DEP exposure. CSF3, CXCL8, MMP1, and VEGFA were identified as key hub genes in the predicted pathway. Significant expression level changes in the five key genes following DEP exposure were validated in terms of protein and mRNA expression. Although further studies are required, our toxicogenomic investigation provides key clues to the exact mechanism underlying DEP-induced nasal inflammatory damage. It also suggests an efficient approach for other research on adverse effects occurring in the upper respiratory tract following DEP exposure.


Assuntos
Células Epiteliais/efeitos dos fármacos , Mucosa Nasal/citologia , Transcriptoma/efeitos dos fármacos , Emissões de Veículos/toxicidade , Células Cultivadas , Fatores Estimuladores de Colônias/genética , Células Epiteliais/metabolismo , Humanos , Inflamação/genética , Interleucina-8/genética , Metaloproteinase 1 da Matriz/genética , Transdução de Sinais/efeitos dos fármacos , Toxicogenética , Fator A de Crescimento do Endotélio Vascular/genética
7.
Dev Comp Immunol ; 108: 103671, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32147469

RESUMO

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are related cytokines that signal through receptors possessing the ß common (ßc) chain. As a family, these cytokines combine rather non-specific hematopoietic growth factor properties with a special importance for eosinophils, basophils, and mast cells. In fish the cytokines of this family are called IL-5fam, and the present study, using carp, constitutes their first functional analysis. Carp il-5fam expression was enhanced by stimulation with phytohemagglutinin and killed bacteria. Reminiscent of mammalian IL-3/IL-5/GM-CSF family members, recombinant carp IL-5fam (rcIL-5fam) induced activation of transcription factor STAT5 and efficiently promoted proliferation and colony-formation of eosinophil/basophil/mast-cell type (EBM) granulocytes. Upon addition of recombinant carp ßc the growth effect of rcIL-5fam was reduced, suggesting ßc participation in the signaling route. In summary, despite differences in individual cytokines and cell populations, fish and mammalian IL-3/IL-5/GM-CSF family members share growth factor functions for non-neutrophil granulocytes.


Assuntos
Carpas/imunologia , Fatores Estimuladores de Colônias/metabolismo , Proteínas de Peixes/metabolismo , Granulócitos/imunologia , Interleucinas/metabolismo , Animais , Carpas/genética , Carpas/metabolismo , Carpas/microbiologia , Proliferação de Células , Fatores Estimuladores de Colônias/genética , Fatores Estimuladores de Colônias/isolamento & purificação , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/isolamento & purificação , Granulócitos/metabolismo , Interleucinas/genética , Interleucinas/isolamento & purificação , Fito-Hemaglutininas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT5/metabolismo
8.
Int J Biol Macromol ; 156: 204-216, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32156537

RESUMO

Calf thymus polypeptide (CTP) is prepared from calf thymus. It has a molecular mass of <10 kilodalton (kDa) and contains 17 types of amino acids. This study investigated the hematopoietic function-improvement effect of CTP in CHRF, K562, and bone marrow mononuclear cells; mice with immunosuppression; and with hematopoietic dysfunction. In mice with immunosuppression, CTP enhanced the cytotoxic activity of natural killer cells and the proliferation of lymphocytes and regulated the levels of immunoglobulins. It also enhanced the proliferation and differentiation of CHRF and K562 cells by upregulating the expression of proliferation- and differentiation-related proteins. In mice with hematopoietic dysfunction, CTP restored white blood cell, neutrophil, and hemoglobin proportions in the peripheral blood and enhanced the levels of B lymphocytes and hematopoietic stem cells and progenitor cells in the bone marrow. CTP effectively regulated the levels of hematopoiesis-related cytokines, such as granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin 2, and interferons-γ, and enhanced the expression of hematopoiesis-related proteins in both primary bone marrow cells and mice with hematopoietic dysfunction. These results indicate that CTP has hematopoietic function-improvement effect and this effect may be related to the modulation of colony-stimulating factors (CSFs) and related signaling pathways.


Assuntos
Fatores Estimuladores de Colônias/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Bovinos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fatores Estimuladores de Colônias/metabolismo , Citocinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hospedeiro Imunocomprometido , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Ativação Linfocitária , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Especificidade de Órgãos/imunologia , Peptídeos/química , Timo/metabolismo
9.
Invest Ophthalmol Vis Sci ; 59(12): 4986-4997, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30326070

RESUMO

Purpose: Lens epithelial cell (LEC) conversion to myofibroblast is responsible for fibrotic cataract surgery complications including posterior capsular opacification. While transforming growth factor beta (TGFß) signaling is important, the mechanisms by which the TGFß pathway is activated post cataract surgery (PCS) are not well understood. Methods: RNA-seq was performed on LECs obtained from a mouse cataract surgery model at the time of surgery and 24 hours later. Bioinformatic analysis was performed with iPathwayGuide. Expression dynamics were determined by immunofluorescence. Results: The LEC transcriptome is massively altered by 24 hours PCS. The differentially expressed genes included those important for lens biology, and fibrotic markers. However, the most dramatic changes were in the expression of genes regulating the innate immune response, with the top three altered genes exhibiting greater than 1000-fold upregulation. Immunolocalization revealed that CXCL1, S100a9, CSF3, COX-2, CCL2, LCN2, and HMOX1 protein levels upregulate in LECs between 1 hour and 6 hours PCS and peak at 24 hours PCS, while their levels sharply attenuate by 3 days PCS. This massive upregulation of known inflammatory mediators precedes the infiltration of neutrophils into the eye at 18 hours PCS, the upregulation of canonical TGFß signaling at 48 hours PCS, and the infiltration of macrophages at 3 days PCS. Conclusions: These data demonstrate that LECs produce proinflammatory cytokines immediately following lens injury that could drive postsurgical flare, and suggest that inflammation may be a major player in the onset of lens-associated fibrotic disease PCS.


Assuntos
Opacificação da Cápsula/metabolismo , Extração de Catarata , Citocinas/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Cristalino/metabolismo , Animais , Calgranulina B/genética , Quimiocina CCL2/genética , Quimiocina CXCL1/genética , Fatores Estimuladores de Colônias/genética , Ciclo-Oxigenase 2/genética , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Heme Oxigenase-1/genética , Lipocalina-2/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Infiltração de Neutrófilos/fisiologia , Análise de Sequência de RNA , Transcriptoma/genética
10.
Free Radic Biol Med ; 104: 10-19, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28063941

RESUMO

Many diseases accompanied by chronic inflammation are connected with dysregulated activation of macrophage subpopulations. Recently, we reported that nitro-fatty acids (NO2-FAs), products of metabolic and inflammatory reactions of nitric oxide and nitrite, modulate macrophage and other immune cell functions. Bone marrow cell suspensions were isolated from mice and supplemented with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with NO2-OA for different times. RAW 264.7 macrophages were used for short-term (1-5min) experiments. We discovered that NO2-OA reduces cell numbers, cell colony formation, and proliferation of macrophages differentiated with colony-stimulating factors (CSFs), all in the absence of toxicity. In a case of GM-CSF-induced bone marrow-derived macrophages (BMMs), NO2-OA acts via downregulation of signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) activation. In the case of M-CSF-induced BMMs, NO2-OA decreases activation of M-CSFR and activation of related PI3K and ERK. Additionally, NO2-OA also attenuates activation of BMMs. In aggregate, we demonstrate that NO2-OA regulates the process of macrophage differentiation and that NO2-FAs represent a promising therapeutic tool in the treatment of inflammatory pathologies linked with increased accumulation of macrophages in inflamed tissues.


Assuntos
Fatores Estimuladores de Colônias/genética , Inflamação/tratamento farmacológico , Óxido Nítrico/administração & dosagem , Ácido Oleico/administração & dosagem , Animais , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/química , Ácido Oleico/química , Fosfatidilinositol 3-Quinases/genética , Células RAW 264.7 , Fator de Transcrição STAT5/genética
11.
Immunol Lett ; 176: 18-27, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27208804

RESUMO

Cellular metabolic state and individual metabolites have been reported to regulate the functional phenotype of immune cells. Cytokine production by regulatory and inflammatory macrophages is thought to mainly involve fatty acid oxidation and glycolysis, respectively, which fuel mitochondrial oxidative phosphorylation. However, the association between metabolic pathways and the acquisition of specific macrophage phenotypes remains unclear. This study assessed the relationship between glycolysis and the differentiation of regulatory macrophages. Human monocytes derived from peripheral blood were cultured in vitro in the presence of macrophage colony-stimulating factor to yield regulatory macrophages (M-Mϕs). M-Mϕs had a regulatory macrophage phenotype and produced substantial IL-10 following stimulation with lipopolysaccharide. To analyze the role of glycolysis, glycolysis inhibitors (2-deoxy-d-glucose or dichloroacetate) were added during M-Mϕ differentiation. These cells cultured with glycolysis inhibitors produced significantly lower amounts of IL-10, but produced significantly higher amounts of IL-6 compared to M-Mϕs differentiated without glycolysis inhibitors. Such phenotypic change of M-Mϕs differentiated with glycolysis inhibitors was associated with the alteration of the gene expression pattern related to macrophage differentiation, such as CSF1, MMP9 and VEGFA. M-Mϕs differentiated with glycolysis inhibitors seemed to retain plasticity to become IL-10 producing cells. Furthermore, increased level of pyruvate in culture medium was found to partially reverse the effects of glycolysis inhibitors on cytokine production of M-Mϕs. These results indicate the importance of glycolytic pathway in macrophage differentiation to a regulatory phenotype, and pyruvate may be one of the key metabolites in this process.


Assuntos
Macrófagos/imunologia , Monócitos/imunologia , Ácido Pirúvico/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fatores Estimuladores de Colônias/genética , Fatores Estimuladores de Colônias/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/antagonistas & inibidores , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Imunomodulação , Interleucina-10/metabolismo , Metaloproteinase 9 da Matriz/genética , Monócitos/efeitos dos fármacos , Transcriptoma , Fator A de Crescimento do Endotélio Vascular/genética
12.
Dev Comp Immunol ; 63: 136-43, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27234705

RESUMO

Myeloid progenitors reside within specific hematopoietic organs and commit to progenitor lineages bearing megakaryocyte/erythrocyte (MEP) or granulocyte/macrophage potentials (GMP) within these sites. Unlike other vertebrates, the amphibian Xenopus laevis committed macrophage precursors are absent from the hematopoietic subcapsular liver and instead reside within their bone marrow. Presently, we demonstrate that while these frogs' liver-derived cells are unresponsive to recombinant forms of principal X. laevis macrophage (colony-stimulating factor-1; CSF-1) and granulocyte (CSF-3) growth factors, bone marrow cells cultured with CSF-1 and CSF-3 exhibit respectively archetypal macrophage and granulocyte morphology, gene expression and functionalities. Moreover, we demonstrate that liver, but not bone marrow cells possess erythropoietic capacities when stimulated with a X. laevis erythropoietin. Together, our findings indicate that X. laevis retain their MEP within the hematopoietic liver while sequestering their GMP to the bone marrow, thus marking a very novel myelopoietic strategy as compared to those seen in other jawed vertebrate species.


Assuntos
Proteínas de Anfíbios/metabolismo , Fatores Estimuladores de Colônias/metabolismo , Granulócitos/fisiologia , Fígado/fisiologia , Macrófagos/fisiologia , Mielopoese , Xenopus laevis/imunologia , Proteínas de Anfíbios/genética , Animais , Fatores Estimuladores de Colônias/genética , Células-Tronco Hematopoéticas/fisiologia
14.
Clin Exp Nephrol ; 19(5): 968-73, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25543187

RESUMO

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disease, is a progressive disease characterized by a bilateral proliferation and enlargement of renal cysts. Recent reports have shown that tolvaptan, a vasopressin V2 receptor antagonist, has been effective in inhibiting renal cyst proliferation and enlargement in ADPKD patients, although no biomarker has identified to predict the effects of tolvaptan. We explored the effective urinary biomarkers in ADPKD in human and in an animal model. METHODS: We measured 28 biomarkers in urine taken from ADPKD patients to compare with that of healthy subjects. Next, a gene expression analysis of the kidney from DBA/2FG-pcy mice (ADPKD model animals) was performed to identify prospective biomarkers. Additionally, we investigated the DBA/2FG-pcy mouse urine samples to determine the biomarkers' efficacy. RESULTS: There were statistically significant differences in 12 of the 28 prospective urinary biomarkers between urine from ADPKD patients and that from healthy subjects. Six of these matched with highly expressed gene products of DBA/sFG-pcy mouse kidneys. Among those 6 biomarkers, NGAL, M-CSF, and MCP-1 showed significantly higher values in the urine of DBA/2FG-pcy mice than that of wild type. CONCLUSIONS: This study suggests that NGAL, M-CSF, MCP-1 are potential candidates of urinary biomarkers in ADPKD.


Assuntos
Rim Policístico Autossômico Dominante/urina , Proteínas de Fase Aguda/genética , Adulto , Animais , Biomarcadores/urina , Quimiocina CCL2/genética , Fatores Estimuladores de Colônias/genética , Feminino , Expressão Gênica/genética , Humanos , Lipocalina-2 , Lipocalinas/genética , Masculino , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Proteínas Oncogênicas/genética , Rim Policístico Autossômico Dominante/genética , Proteínas Proto-Oncogênicas/genética , Reprodutibilidade dos Testes , Adulto Jovem
15.
J Virol ; 87(7): 3678-86, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23325678

RESUMO

Adenovirus serotype 5 (Ad5) naturally infects the liver after intravenous injection, making it a candidate for hepatocyte-directed gene transfer. While Ad5 can be efficient, most of the dose is destroyed by liver Kupffer cells before it can reach hepatocytes. In contrast, Ad5 bearing the hexon from Ad6 (Ad5/6) evades Kupffer cells. While Ad5/6 dramatically increases hepatocyte transduction in BALB/c mice, it has surprisingly little effect on C57BL/6 mice. To determine the source of this strain-specific difference, the roles of Kupffer cells, liver sinusoidal endothelial cells (LSECs), hepatocytes, scavenger receptors, clotting factors, and immunoglobulins were analyzed. The numbers of Kupffer cells and LSECs, the level of clotting factor X, and hepatocyte infectibility did not differ between different strains of mice. In contrast, high levels of immunoglobulins correlated negatively with Ad5 liver transduction in different mouse strains. Removal of immunoglobulins by use of Rag-deficient mice restored Ad5 transduction to maximal levels. Removal of Kupffer cells by predosing or by testing in colony-stimulating factor knockout mice restored Ad5 transduction in the presence of immunoglobulins. Partial reconstitution of IgM in Rag mice resulted in significant reductions in liver transduction by Ad5 but not by Ad5/6. These data suggest a role for IgM-mediated clearance of Ad5 via Kupffer cells and may explain the mechanism by which Ad5/6 evades these cells. These mechanisms may play a vital role in Ad pharmacology in animals and in humans.


Assuntos
Adenoviridae/imunologia , Anticorpos Antivirais/imunologia , Capsídeo/imunologia , Terapia Genética/métodos , Hepatócitos/virologia , Células de Kupffer/imunologia , Macrófagos/imunologia , Animais , Anticorpos Antivirais/sangue , Fatores Estimuladores de Colônias/genética , Células Endoteliais/virologia , Imunoglobulina M/imunologia , Células de Kupffer/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade da Espécie , Transdução Genética/métodos
16.
J Neuroinflammation ; 9: 83, 2012 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-22546005

RESUMO

BACKGROUND: Maternal viral infection during pregnancy is associated with an increase in the incidence of psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The enhanced risk for developing mental illness appears to be caused by deleterious effects of innate immune response-associated factors on the development of the central nervous system, which predispose the offspring to pathological behaviors in adolescence and adulthood. To identify the immune response-associated soluble factors that may affect central nervous system development, we examined the effect of innate immune response activation by polyriboinosinic-polyribocytidylic acid (poly(I:C)), a synthetic analogue of viral double-stranded RNA, on the expression levels of pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors in fetal and postnatal mouse brain 6 h and 24 h after treatment. METHODS: C57BL/6J pregnant mice (gestational day 16) or newborn mice (postnatal day 4) received a single intraperitoneal injection of the synthetic analogue of viral double-stranded RNA poly(I:C) (20 mg/kg). Thirty-two immune response-associated soluble factors, including pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors, were assayed 6 h and 24 h after poly(I:C) injection using multiplexed bead-based immunoassay (Milliplex Map) and processed in a Luminex 100 IS instrument. RESULTS: Maternal exposure to poly(I:C) at gestational day 16 induced a significant increase in cytokines interleukin (IL)-1ß, IL-7 and IL-13; chemokines monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, interferon gamma-induced protein (IP)-10 and monokine induced by IFN-gamma (MIG); and in the colony stimulating factor vascular endothelial growth factor (VEGF) in the fetal brain. IL-1ß showed the highest concentration levels in fetal brains and was the only cytokine significantly up-regulated 24 h after maternal poly(I:C) injection, suggesting that IL-1ß may have a deleterious impact on central nervous system development. In contrast, poly(I:C) treatment of postnatal day 4 pups induced a pronounced rise in chemokines and colony stimulating factors in their brains instead of the pro-inflammatory cytokine IL-1ß. CONCLUSIONS: This study identified a significant increase in the concentration levels of the cytokines IL-1ß and IL-13, the chemokine MCP-1 and the colony stimulating factor VEGF in the developing central nervous system during activation of an innate immune response, suggesting that these factors are mediators of the noxious effects of maternal immune activation on central nervous system development, with potential long-lasting effects on animal behavior.


Assuntos
Encéfalo/imunologia , Quimiocina CCL2/biossíntese , Interleucina-13/biossíntese , Interleucina-1beta/biossíntese , Polinucleotídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Animais Recém-Nascidos , Encéfalo/embriologia , Encéfalo/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/fisiologia , Fatores Estimuladores de Colônias/biossíntese , Fatores Estimuladores de Colônias/genética , Fatores Estimuladores de Colônias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Imunidade Inata/genética , Interleucina-13/genética , Interleucina-13/fisiologia , Interleucina-1beta/genética , Interleucina-1beta/fisiologia , Interleucina-7/biossíntese , Interleucina-7/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/imunologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/fisiologia
17.
J Bone Miner Metab ; 30(4): 408-18, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22105655

RESUMO

To better define the biologic function of membrane-bound CSF1 (mCSF1) in vivo, we have generated mCSF1 knockout (k/o) mice. Spinal bone density (BMD) was 15.9% higher in k/o mice compared to wild-type (wt) controls (P < 0.01) and total BMD was increased by 6.8% (P < 0.05). A higher mean femur BMD was also observed but did not reach statistical significance (6.9% P = NS). The osteoclastogenic potential of bone marrow isolated from mCSF1 k/o mice was reduced compared to wt marrow. There were no defects in osteoblast number or function suggesting that the basis for the high bone mass phenotype was reduced resorption. In addition to a skeletal phenotype, k/o mice had significantly elevated serum triglyceride levels (123 ± 7 vs. 88 ± 3.2 mg/dl; k/o vs. wt, P < 0.001), while serum cholesterol levels were similar (122 ± 6 vs. 116 ± 6 mg/dl; k/o vs. wt, P = NS). One month after surgery, 5-month-old k/o and wt female mice experienced the same degree of bone loss following ovariectomy (OVX). OVX induced a significant fourfold increase in the expression of the soluble CSF1 isoform (sCSF1) in the bones of wt mice while expression of mCSF1 was unchanged. These findings indicate that mCSF1 is essential for normal bone remodeling since, in its absence, BMD is increased. Membrane-bound CSF1 does not appear to be required for estrogen-deficiency bone loss while in contrast; our data suggest that sCSF1 could play a key role in this pathologic process. The reasons why mCSF1 k/o mice have hypertriglyceridemia are currently under study.


Assuntos
Osso e Ossos/metabolismo , Fatores Estimuladores de Colônias/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Animais , Densidade Óssea , Osso e Ossos/patologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Fatores Estimuladores de Colônias/química , Fatores Estimuladores de Colônias/genética , Feminino , Humanos , Hipertrigliceridemia/etiologia , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/sangue , Osteoporose/metabolismo , Osteoporose/patologia , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Caracteres Sexuais , Solubilidade , Regulação para Cima
18.
Scand J Immunol ; 73(4): 338-43, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21231951

RESUMO

The nuclear factor NF-{kappa}B (NFκB) is involved in the regulation of innate immunity and in particular, inflammatory genes. It is associated with the pathogenesis of many chronic diseases such as coronary heart disease (CHD). It is believed that individual susceptibility to CHD might be affected by differences in gene transcription and therefore gene expression in circulating cell populations such as leucocytes is of interest. The aim of this study was to investigate whether the total white blood cell population (leucocytes) could be used to study the effect of lipopolysaccharide (LPS) treatment on the expression of genes of the NFκB pathway. Gene expression of the NFκB pathway was examined in total leucocyte, monocyte and neutrophil populations. The majority of the 84 genes examined were up-regulated after treatment with LPS for 12 h in all cell populations examined. The total leucocyte population behaved in a similar manner to both neutrophils and monocytic cells, indicating that it alone could be used in studies, therefore avoiding cell separation, which is time-consuming and can result in cell activation. Furthermore, in clinical studies, it enables a larger number of patient samples to be studied simultaneously, while also reducing the amount of blood required from each. This will provide enough starting material for use with molecular techniques, such as chromatin immunoprecipitation (ChIP) and ChIP-sequencing, and allow large-scale gene expression studies of the NFκB pathway in patients with chronic and acute inflammation with established CHD.


Assuntos
Leucócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Adulto , Quimiocina CCL2/genética , Fatores Estimuladores de Colônias/genética , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Receptor beta de Linfotoxina/genética , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores Toll-Like/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genética , Regulação para Cima/imunologia
19.
Nat Med ; 16(5): 571-9, 1p following 579, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20436485

RESUMO

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell-derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.


Assuntos
Fatores Estimuladores de Colônias/genética , Linfoma/genética , Fator Estimulador de Colônias de Macrófagos/genética , Proto-Oncogenes/genética , Sequências Repetidas Terminais , Expressão Gênica , Doença de Hodgkin/genética , Humanos , Linfoma de Células B/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proto-Oncogene Mas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
20.
Nat Med ; 16(5): 580-5, 1p following 585, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20418886

RESUMO

Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. Cancer stem cell eradication is thought to be crucial for successful anticancer therapy. Using an acute myeloid leukemia (AML) model induced by the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. The MOZ fusion proteins MOZ-TIF2 and MOZ-CBP interacted with the transcription factor PU.1 to stimulate the expression of macrophage colony-stimulating factor receptor (CSF1R, also known as M-CSFR, c-FMS or CD115). Studies using PU.1-deficient mice showed that PU.1 is essential for the ability of MOZ-TIF2 to establish and maintain AML stem cells. Cells expressing high amounts of CSF1R (CSF1R(high) cells), but not those expressing low amounts of CSF1R (CSF1R(low) cells), showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the CSF1R promoter, we cured AML by ablation of CSF1R(high) cells. Moreover, induction of AML was suppressed in CSF1R-deficient mice and CSF1R inhibitors slowed the progression of MOZ-TIF2-induced leukemia. Thus, in this subtype of AML, leukemia stem cells are contained within the CSF1R(high) cell population, and we suggest that targeting of PU.1-mediated upregulation of CSF1R expression might be a useful therapeutic approach.


Assuntos
Leucemia Mieloide Aguda/genética , Coativador 2 de Receptor Nuclear/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores de Fator Estimulador de Colônias/genética , Transativadores/genética , Regulação para Cima , Animais , Diferenciação Celular/genética , Fatores Estimuladores de Colônias/genética , Genes , Humanos , Leucemia/genética , Fator Estimulador de Colônias de Macrófagos/genética , Camundongos , Camundongos Transgênicos , Coativador 2 de Receptor Nuclear/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Proteínas Recombinantes de Fusão/metabolismo , Dedos de Zinco/genética
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