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1.
Nat Commun ; 12(1): 4988, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404781

RESUMO

Glycans are fundamental cellular building blocks, involved in many organismal functions. Advances in glycomics are elucidating the essential roles of glycans. Still, it remains challenging to properly analyze large glycomics datasets, since the abundance of each glycan is dependent on many other glycans that share many intermediate biosynthetic steps. Furthermore, the overlap of measured glycans can be low across samples. We address these challenges with GlyCompare, a glycomic data analysis approach that accounts for shared biosynthetic steps for all measured glycans to correct for sparsity and non-independence in glycomics, which enables direct comparison of different glycoprofiles and increases statistical power. Using GlyCompare, we study diverse N-glycan profiles from glycoengineered erythropoietin. We obtain biologically meaningful clustering of mutant cell glycoprofiles and identify knockout-specific effects of fucosyltransferase mutants on tetra-antennary structures. We further analyze human milk oligosaccharide profiles and find mother's fucosyltransferase-dependent secretor-status indirectly impact the sialylation. Finally, we apply our method on mucin-type O-glycans, gangliosides, and site-specific compositional glycosylation data to reveal tissues and disease-specific glycan presentations. Our substructure-oriented approach will enable researchers to take full advantage of the growing power and size of glycomics data.


Assuntos
Vias Biossintéticas , Glicômica , Polissacarídeos/biossíntese , Transporte Biológico , Vias Biossintéticas/genética , Análise por Conglomerados , Análise de Dados , Eritropoetina/metabolismo , Fucosiltransferases/genética , Gangliosídeos , Técnicas de Inativação de Genes , Glicosilação , Humanos , Mucinas
2.
FASEB J ; 35(9): e21788, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34425031

RESUMO

Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n = 3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.


Assuntos
Técnicas de Cultura de Células , Modelos Animais de Doenças , Retardo do Crescimento Fetal/metabolismo , Feto/metabolismo , Hipóxia/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Animais , Eritropoetina/metabolismo , Peso Fetal , Feto/química , Células Hep G2 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Microscopia de Fluorescência , Tamanho do Órgão , Papio , Fosforilação , Proteína Quinase C-alfa/metabolismo , Receptores da Eritropoetina/metabolismo , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Chem Biol Interact ; 347: 109617, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34391751

RESUMO

PURPOSE: This study was designed to investigate the mechanism of Dapagliflozin (Dapa) cardioprotection against diabetic cardiomyopathy (DCM). Structural and functional changes in the heart as well as decrease of erythropoietin (EPO) levels were reported in DCM. EPO simultaneously activates three pathways: the Janus-activated kinase-signal transducer and activator of transcription (JAK2/STAT5), phosphatidylinositol-3-kinase-Akt (PI3K/Akt), and extracellular signal-related kinase (ERK/MAPK) cascades, that result in proliferation and differentiation of cardiac cells. METHODS AND RESULTS: DCM was induced by a high fat diet for 10 weeks followed by administration of streptozotocin. After confirmation of diabetes, rats were divided randomly to 5 groups: Group 1; normal control group, Group 2; untreated diabetic group and Groups (3-5); diabetic groups received Dapa daily (0.75 mg, 1.5 or 3 mg/Kg, p.o) respectively for a month. At the end of the experiment, full anaesthesia was induced in all rats using ether inhalation and ECG was recorded. Blood samples were collected then rats were sacrificed and their heart were dissected out and processed for biochemical and histopathological studies. Untreated diabetic rats showed abnormal ECG pattern, elevation of serum cardiac enzymes, decrease EPO levels, downregulation of P-Akt, P-JAK2 and pMAPK pathways, abnormal histological structure of the heart and increase immunostaining intensity of P53 and TNF α in the cardiomyocytes. Dapa in a dose dependent manner attenuated the alterations in the previously mentioned parameters. CONCLUSION: The cardioprotective effect of Dapa could be mediated by increasing EPO levels and activation of P-Akt, P-JAK2 and pMAPK signalling cascades which in turn decrease apoptosis.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Cardiotônicos/uso terapêutico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/tratamento farmacológico , Glucosídeos/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Eletrocardiografia/efeitos dos fármacos , Eritropoetina/sangue , Eritropoetina/metabolismo , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Wistar , Estreptozocina
4.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281163

RESUMO

Erythropoietin (EPO) acts on multiple tissues through its receptor EPOR, a member of a cytokine class I receptor superfamily with pleiotropic effects. The interaction of EPO and EPOR triggers the activation of several signaling pathways that induce erythropoiesis, including JAK2/STAT5, PI3K/AKT, and MAPK. The canonical EPOR/JAK2/STAT5 pathway is a known regulator of differentiation, proliferation, and cell survival of erythroid progenitors. In addition, its role in the protection of other cells, including cancer cells, is under intense investigation. The involvement of EPOR/JAK2/STAT5 in other processes such as mRNA splicing, cytoskeleton reorganization, and cell metabolism has been recently described. The transcriptomics, proteomics, and epigenetic studies reviewed in this article provide a detailed understanding of EPO signalization. Advances in this area of research may be useful for improving the efficacy of EPO therapy in hematologic disorders, as well as in cancer treatment.


Assuntos
Eritropoetina/metabolismo , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT5/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Epigenômica/métodos , Eritropoese/efeitos dos fármacos , Eritropoetina/fisiologia , Humanos , Janus Quinase 2/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores da Eritropoetina/metabolismo , Receptores da Eritropoetina/fisiologia , Fator de Transcrição STAT5/genética , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Transcriptoma/genética
5.
Nature ; 596(7871): 291-295, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321659

RESUMO

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Edição de Genes/métodos , Terapia Genética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Eritropoetina/biossíntese , Eritropoetina/genética , Eritropoetina/metabolismo , Éxons/genética , Feminino , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular Espinal/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Progranulinas/biossíntese , Progranulinas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
6.
PLoS One ; 16(6): e0253458, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34185793

RESUMO

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , Dopa Descarboxilase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Enzima de Conversão de Angiotensina 2/genética , Área Sob a Curva , Descarboxilases de Aminoácido-L-Aromático , COVID-19/virologia , Dopa Descarboxilase/genética , Regulação para Baixo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Curva ROC , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Regulação para Cima , Carga Viral
7.
Exp Hematol ; 99: 12-20.e3, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34077792

RESUMO

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) is a glycoprotein cytokine that plays a major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration of Epo in bone marrow is hypothesized to be suboptimal, and the survival of erythroid cells has been suggested to depend on Epo sensitivity. However, the key factors that control Epo sensitivity remain unknown. Two types of transferrin receptors (TfRs), TfR1 and TfR2, are known to play a role in iron uptake in erythroid cells. Here, we hypothesized that TfRs may additionally modulate Epo sensitivity during erythropoiesis by modulating Epo receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) erythroid cell and human erythroid progenitor cell models, we report that iron-loaded transferrin, that is, holo-transferrin (holo-Tf), synergizes with suboptimal Epo levels to improve erythroid cell survival, proliferation, and differentiation. This is accomplished via the major signaling pathways of erythropoiesis, which include signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, we found that this cooperation is improved by, but does not require, the internalization of TfR1. Interestingly, we observed that loss of TfR2 stabilizes EpoR levels and abolishes the beneficial effects of holo-Tf. Overall, these data reveal novel signaling properties of TfRs, which involve the regulation of erythropoiesis through EpoR signaling.


Assuntos
Antígenos CD/metabolismo , Proliferação de Células/efeitos dos fármacos , Eritroblastos/metabolismo , Eritropoetina/farmacologia , Ferro/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores da Transferrina/metabolismo , Transferrina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eritropoetina/metabolismo , Humanos , Ferro/metabolismo , Transferrina/metabolismo
8.
Biochem Biophys Res Commun ; 557: 143-150, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33865222

RESUMO

Hypoxia-inducible factor 2 (HIF-2), is essential for cellular response to hypoxia and holds an important role in erythropoiesis, angiogenesis, tissue invasion and metastasis, thus, constituting an important therapeutic target. Maximal HIF-2 transcriptional activation requires HIF-2α phosphorylation by ERK1/2 that impairs its CRM1-mediated nuclear export. Herein, we reveal a novel interaction of HIF-2α with Reptin52, a multifunctional protein involved in cellular functions orchestrated both in the nucleus and the cytoplasm. HIF-2α and Reptin52 interact both in nuclear and cytoplasmic fractions, however, ERK1/2 pathway inactivation seems to favour their association in the cytoplasm. Notably, we demonstrate that Reptin52 reduces HIF-2 transcriptional activity, which results in decreased EPO secretion under hypoxia, by impairing HIF-2α stability via a non-canonical PHD-VHL-proteasome independent mechanism. This interaction represents a novel HIF-2 fine tuning mechanism that allows for distinct HIF1/2 isoforms regulation.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Eritropoetina/metabolismo , Regulação da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/genética , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatografia Líquida , Citoplasma/genética , Citoplasma/metabolismo , DNA Helicases/genética , Eritropoese/genética , Eritropoetina/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em Tandem
9.
J Immunol ; 206(8): 1752-1764, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33811103

RESUMO

Macrophages play an important role in the pathogenesis of systemic lupus erythematosus-associated diffuse alveolar hemorrhage (DAH). The immunomodulation of macrophage responses might be a potential approach for the prevention and treatment of DAH. Erythropoietin (EPO) could regulate macrophage bioactivities by binding to the EPO receptor expressing on macrophages. This study assessed the effects of EPO on DAH protection using an immune-mediated DAH murine model with macrophages as the major contributor. A DAH murine model was established in female C57BL/6 mice by an i.p. injection of pristane. We found that EPO administration alleviates DAH by reducing pulmonary macrophages recruitment and promoting phenotype switch toward M2 macrophages in vivo. EPO drove macrophages to the anti-inflammatory phenotype in the primary murine bone marrow-derived macrophages and macrophages cell line RAW 264.7 with LPS, IFN-γ, and IL-4 in vitro. Moreover, EPO treatment increases the expression of EPOR and decreases the expression of miR-494-3p, resulting in increased phosphorylation of JAK2 and STAT3. In conclusion, EPO can be a potential therapeutic agent in DAH by reducing cell apoptosis and regulating macrophage polarization through the EPOR/JAK2/STAT3 axis. Further studies are also needed to validate the direct target of miR-494-3p in regulating JAK2/STAT3 signaling transduction.


Assuntos
Eritropoetina/metabolismo , Hemorragia/imunologia , Pneumopatias/imunologia , Macrófagos Alveolares/imunologia , Alvéolos Pulmonares/patologia , Animais , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Humanos , Janus Quinase 2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Receptores da Eritropoetina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Terpenos
10.
Biomolecules ; 11(4)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920974

RESUMO

We systematically reviewed published translational research on gene-based therapy for retinal ganglion cell (RGC) neuroprotection. A search was conducted on Entrez PubMed on 23 December 2020 using the keywords "gene therapy", "retinal ganglion cell" and "neuroprotection". The initial search yielded 82 relevant articles. After restricting publications to those with full text available and in the English language, and then curating for only original articles on gene-based therapy, the final yield was 18 relevant articles. From the 18 papers, 17 of the papers utilized an adeno-associated viral (AAV) vector for gene therapy encoding specific genes of interest. Specifically, six of the studies utilized an AAV vector encoding brain-derived neurotrophic factor (BDNF), two of the studies utilized an AAV vector encoding erythropoietin (EPO), the remaining 10 papers utilized AAV vectors encoding different genes and one microRNA study. Although the literature shows promising results in both in vivo and in vitro models, there is still a significant way to go before gene-based therapy for RGC neuroprotection can proceed to clinical trials. Namely, the models of injury in many of the studies were more acute in nature, unlike the more progressive and neurodegenerative pathophysiology of diseases, such as glaucoma. The regulation of gene expression is also highly unexplored despite the use of AAV vectors in the majority of the studies reviewed. It is also expected that with the successful launch of messenger ribonucleic acid (mRNA)-based vaccinations in 2020, we will see a shift towards this technology for gene-based therapy in glaucoma neuroprotection.


Assuntos
Terapia Genética/métodos , Glaucoma/terapia , Células Ganglionares da Retina/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dependovirus/genética , Eritropoetina/genética , Eritropoetina/metabolismo , Humanos , Células Ganglionares da Retina/fisiologia
11.
Front Immunol ; 12: 658715, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33927725

RESUMO

Endogenous mechanisms underlying bacterial infection resolution are essential for the development of novel therapies for the treatment of inflammation caused by infection without unwanted side effects. Herein, we found that erythropoietin (EPO) promoted the resolution and enhanced antibiotic actions in Escherichia coli (E. coli)- and Staphylococcus aureus (S. aureus)-initiated infections. Levels of peritoneal EPO and macrophage erythropoietin receptor (EPOR) were elevated in self-limited E. coli-initiated peritonitis. Myeloid-specific EPOR-deficient mice exhibited an impaired inflammatory resolution and exogenous EPO enhanced this resolution in self-limited infections. Mechanistically, EPO increased macrophage clearance of bacteria via peroxisome proliferator-activated receptor γ (PPARγ)-induced CD36. Moreover, EPO ameliorated inflammation and increased the actions of ciprofloxacin and vancomycin in resolution-delayed E. coli- and S. aureus-initiated infections. Collectively, macrophage EPO signaling is temporally induced during infections. EPO is anti-phlogistic, increases engulfment, promotes infection resolution, and lowers antibiotic requirements.


Assuntos
Antibacterianos/farmacologia , Eritropoetina/metabolismo , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli/efeitos dos fármacos , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Farmacorresistência Bacteriana , Infecções por Escherichia coli/tratamento farmacológico , Interações Hospedeiro-Patógeno , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , PPAR gama/metabolismo , Peritonite/tratamento farmacológico , Peritonite/metabolismo , Peritonite/microbiologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Transdução de Sinais , Infecções Estafilocócicas/tratamento farmacológico
12.
Crit Care Med ; 49(4): e394-e403, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33566466

RESUMO

OBJECTIVES: Mannitol and hypertonic saline are used to treat raised intracerebral pressure in patients with traumatic brain injury, but their possible effects on kidney function and mortality are unknown. DESIGN: A post hoc analysis of the erythropoietin trial in traumatic brain injury (ClinicalTrials.gov NCT00987454) including daily data on mannitol and hypertonic saline use. SETTING: Twenty-nine university-affiliated teaching hospitals in seven countries. PATIENTS: A total of 568 patients treated in the ICU for 48 hours without acute kidney injury of whom 43 (7%) received mannitol and 170 (29%) hypertonic saline. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We categorized acute kidney injury stage according to the Kidney Disease Improving Global Outcome classification and defined acute kidney injury as any Kidney Disease Improving Global Outcome stage-based changes from the admission creatinine. We tested associations between early (first 2 d) mannitol and hypertonic saline and time to acute kidney injury up to ICU discharge and death up to 180 days with Cox regression analysis. Subsequently, acute kidney injury developed more often in patients receiving mannitol (35% vs 10%; p < 0.001) and hypertonic saline (23% vs 10%; p < 0.001). On competing risk analysis including factors associated with acute kidney injury, mannitol (hazard ratio, 2.3; 95% CI, 1.2-4.3; p = 0.01), but not hypertonic saline (hazard ratio, 1.6; 95% CI, 0.9-2.8; p = 0.08), was independently associated with time to acute kidney injury. In a Cox model for predicting time to death, both the use of mannitol (hazard ratio, 2.1; 95% CI, 1.1-4.1; p = 0.03) and hypertonic saline (hazard ratio, 1.8; 95% CI, 1.02-3.2; p = 0.04) were associated with time to death. CONCLUSIONS: In this post hoc analysis of a randomized controlled trial, the early use of mannitol, but not hypertonic saline, was independently associated with an increase in acute kidney injury. Our findings suggest the need to further evaluate the use and choice of osmotherapy in traumatic brain injury.


Assuntos
Injúria Renal Aguda/metabolismo , Lesões Encefálicas Traumáticas/terapia , Diuréticos Osmóticos/uso terapêutico , Eritropoetina/metabolismo , Manitol/uso terapêutico , Solução Salina Hipertônica/uso terapêutico , Injúria Renal Aguda/etiologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Diuréticos Osmóticos/efeitos adversos , Feminino , Hidratação/métodos , Humanos , Pressão Intracraniana/efeitos dos fármacos , Masculino , Manitol/efeitos adversos , Resultado do Tratamento
13.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33566111

RESUMO

In the embryo, the first hematopoietic cells derive from the yolk sac and are thought to be rapidly replaced by the progeny of hematopoietic stem cells. We used three lineage-tracing mouse models to show that, contrary to what was previously assumed, hematopoietic stem cells do not contribute significantly to erythrocyte production up until birth. Lineage tracing of yolk sac erythromyeloid progenitors, which generate tissue resident macrophages, identified highly proliferative erythroid progenitors that rapidly differentiate after intra-embryonic injection, persisting as the major contributors to the embryonic erythroid compartment. We show that erythrocyte progenitors of yolk sac origin require 10-fold lower concentrations of erythropoietin than their hematopoietic stem cell-derived counterparts for efficient erythrocyte production. We propose that, in a low erythropoietin environment in the fetal liver, yolk sac-derived erythrocyte progenitors efficiently outcompete hematopoietic stem cell progeny, which fails to generate megakaryocyte and erythrocyte progenitors.


Assuntos
Desenvolvimento Embrionário/genética , Eritrócitos/metabolismo , Eritropoese , Células Progenitoras de Megacariócitos/metabolismo , Saco Vitelino/fisiologia , Animais , Linhagem da Célula/genética , Eritropoetina/metabolismo , Feminino , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteínas Proto-Oncogênicas c-myb/deficiência , Proteínas Proto-Oncogênicas c-myb/genética
14.
Sci Rep ; 11(1): 1491, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452310

RESUMO

Recombinant human erythropoietin (rHuEPO) is a biopharmaceutical drug given to patients who have a low hemoglobin related to chronic kidney disease, cancer or anemia. However, some patients repeatedly receiving rHuEPO develop anti-rHuEPO neutralizing antibodies leading to the development of pure red cell aplasia (PRCA). The immunogenic antibody response activated by rHuEPO is believed to be triggered by T-cells recognizing EPO epitopes bound to MHC molecules displayed on the cell surface of APCs. Previous studies have reported an association between the development of anti-rHuEpo-associated PRCA and the HLA-DRB1*09 gene, which is reported to be entrenched in the Thai population. In this study, we used computational design to screen for immunogenic hotspots recognized by HLA-DRB1*09, and predicted seventeen mutants having anywhere between one through four mutations that reduce affinity for the allele, without disrupting the structural integrity and bioactivity. Five out of seventeen mutants were less immunogenic in vitro while retaining similar or slightly reduced bioactivity than rHuEPO. These engineered proteins could be the potential candidates to treat patients who are rHuEpo-dependent and express the HLA-DRB1*09 allele.


Assuntos
Eritropoetina/imunologia , Eritropoetina/metabolismo , Alelos , Anemia/tratamento farmacológico , Formação de Anticorpos/genética , Técnicas de Cultura de Células , Linhagem Celular , Eritropoetina/genética , Humanos , Complexo Principal de Histocompatibilidade/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Aplasia Pura de Série Vermelha/tratamento farmacológico , Aplasia Pura de Série Vermelha/imunologia , Aplasia Pura de Série Vermelha/fisiopatologia , Diálise Renal
15.
Blood Cells Mol Dis ; 88: 102536, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33450539

RESUMO

In a two-part process, we assessed elements of the principal hormonal pathway regulating iron homeostasis in human neonates. Part 1: Quantifying erythropoietin (Epo), erythroferrone (ERFE), hepcidin, and relevant serum and erythrocytic iron-related metrics in umbilical cord blood from term (n = 13) and preterm (n = 10) neonates, and from neonates born to mothers with diabetes and obesity (n = 13); Part 2: Quantifying serum Epo, ERFE, and hepcidin before and following darbepoetin administration. Part 1: We measured Epo, ERFE and hepcidin in all cord blood samples. Epo and ERFE levels did not differ between the three groups. Preterm neonates had the lowest hepcidin levels, while neonates born to diabetic women with a very high BMI had the lowest ferritin and RET-He levels. Part 2: Following darbepoetin dosing, ERFE levels generally increased (p < 0.05) and hepcidin levels generally fell (p < 0.05). Our observations suggest that the Epo/ERFE/hepcidin axis is intact in the newborn period.


Assuntos
Eritropoetina/sangue , Hepcidinas/sangue , Hormônios Peptídicos/sangue , Transdução de Sinais , Eritropoetina/metabolismo , Feminino , Sangue Fetal/metabolismo , Hepcidinas/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Obesidade/sangue , Obesidade/metabolismo , Hormônios Peptídicos/metabolismo , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/metabolismo , Gravidez em Diabéticas/sangue , Gravidez em Diabéticas/metabolismo , Nascimento Prematuro/sangue , Nascimento Prematuro/metabolismo
16.
J Cell Physiol ; 236(6): 4515-4527, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33442879

RESUMO

Ischemic preconditioned (IP) neurons protect astrocytes against ischemia/reperfusion (I/R)-induced injury by inhibiting oxidative stress. However, the relevant mechanisms are unknown. Based on the role of nuclear factor-κB (NF-κB) in cell survival and adaption to oxidative stress, we hypothesized that NF-κB might be associated with astroprotection induced by IP neurons via upregulation of antioxidant enzymes. Here, we investigated the effects of IP neurons on NF-κB activation, cell viability, reactive oxygen species (ROS), expression of antioxidant enzymes, erythropoietin (EPO), and tumor necrosis factor α (TNF-α), in the presence or absence of BAY11-7082 (an NF-κB inhibitor), anti-EPO, and anti-TNF-α antibodies, in astrocytes treated with or without I/R. We found that IP neurons could keep NF-κB activation at a relatively higher but beneficial level, and in turn, upregulated the activity of antioxidant enzymes and hence enhanced cell viability and reduced ROS in I/R treated astrocytes. The results collectively indicated that IP neurons are able to significantly inhibit the I/R-induced NF-κB overactivation, probably via EPO and TNF-α, being essential for IP neuron-induced astroprotection under the conditions of I/R. We concluded that NF-κB-mediated antioxidative stress is one of the mechanisms by which IP neurons protect astrocytes against I/R injury.


Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , NF-kappa B/metabolismo , Neurônios/metabolismo , Comunicação Parácrina , Traumatismo por Reperfusão/prevenção & controle , Animais , Antioxidantes/metabolismo , Astrócitos/enzimologia , Astrócitos/patologia , Hipóxia Celular , Células Cultivadas , Córtex Cerebral/patologia , Meios de Cultivo Condicionados/metabolismo , Eritropoetina/metabolismo , Glucose/deficiência , Neurônios/patologia , Estresse Oxidativo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
17.
Life Sci ; 269: 119075, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33465391

RESUMO

BACKGROUND: Erythropoietin (EPO) is an essential growth factor for erythroid cells and is mainly secreted from the kidneys and subsidiarily from the livers of adult mammals in an anemia/hypoxia-inducible manner. AIM AND METHOD: To elucidate the regulatory mechanisms of stress-inducible and cell type-specific Epo gene transcription, the rate-limiting step of EPO production, we investigated the sufficiency of a 180-kb genomic fragment flanking the mouse Epo gene locus for recapitulating endogenous Epo gene function by a transgene complementation strategy. KEY FINDINGS: While Epo gene-deficient mice exhibited lethal anemia in utero with defects in erythroblast proliferation and maturation, Epo-knockout mice integrated with the 180-kb Epo transgene showed normal erythropoiesis throughout life. In the transgene-rescued mice, liver-specific deletion of the transgene by the Cre-loxP recombination system caused neonatal anemia with erythropoietic defects in the liver but not in the spleen, indicating the essential function of hepatic EPO on normal erythropoiesis in the liver, which is the major erythropoietic site in late embryonic and neonatal stages. SIGNIFICANCE: These results demonstrate that the 180 kb Epo gene flanking region contains the fully functional Epo gene unit and that EPO from the liver dominantly stimulates hepatic erythropoiesis but contributes less to erythropoiesis in other organs.


Assuntos
Anemia/prevenção & controle , Eritropoese , Eritropoetina/metabolismo , Fígado/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transgenes/fisiologia , Anemia/genética , Anemia/patologia , Animais , Eritropoetina/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos
18.
Biotechnol J ; 16(5): e2000455, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33471394

RESUMO

Neurological disorders affect millions of people causing behavior-cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.


Assuntos
Eritropoetina , Animais , Cricetinae , Eritropoetina/metabolismo , Glicosilação , Hematopoese , Humanos , Plasticidade Neuronal , Polissacarídeos
19.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467674

RESUMO

Erythropoiesis regulation is essential in normal physiology and pathology, particularly in myelodysplastic syndromes (MDS) and ß-thalassemia. Several signaling transduction processes, including those regulated by inositides, are implicated in erythropoiesis, and the latest MDS or ß-thalassemia preclinical and clinical studies are now based on their regulation. Among others, the main pathways involved are those regulated by transforming growth factor (TGF)-ß, which negatively regulates erythrocyte differentiation and maturation, and erythropoietin (EPO), which acts on the early-stage erythropoiesis. Also small mother against decapentaplegic (SMAD) signaling molecules play a role in pathology, and activin receptor ligand traps are being investigated for future clinical applications. Even inositide-dependent signaling, which is important in the regulation of cell proliferation and differentiation, is specifically associated with erythropoiesis, with phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K) as key players that are becoming increasingly important as new promising therapeutic targets. Additionally, Roxadustat, a new erythropoiesis stimulating agent targeting hypoxia inducible factor (HIF), is under clinical development. Here, we review the role and function of the above-mentioned signaling pathways, and we describe the state of the art and new perspectives of erythropoiesis regulation in MDS and ß-thalassemia.


Assuntos
Eritropoese , Síndromes Mielodisplásicas/metabolismo , Transdução de Sinais , Talassemia beta/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Ensaios Clínicos como Assunto , Eritropoetina/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Hematínicos/uso terapêutico , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Isoquinolinas/farmacologia , Ligantes , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fosfolipases Tipo C/metabolismo
20.
Muscle Nerve ; 63(2): 268-272, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33205838

RESUMO

BACKGROUND: Erythropoietin (EPO) promotes myelination and functional recovery in rodent peripheral nerve injury (PNI). While EPO receptors (EpoR) are present in Schwann cells, the role of EpoR in PNI recovery is unknown because of the lack of EpoR antagonists or Schwann cell-specific EpoR knockout animals. METHODS: Using the Cre-loxP system, we developed a myelin protein zero (Mpz) promoter-driven knockout mouse model of Schwann cell EpoR (MpzCre-EpoRflox/flox , Mpz-EpoR-KO). Mpz-EpoR-KO and control mice were assigned to sciatic nerve crush injury followed by EPO treatment. RESULTS: EPO treatment significantly accelerated functional recovery in control mice in contrast to significantly reduced functional recovery in Mpz-EpoR-KO mice. Significant muscle atrophy was found in the injured hindlimb of EPO-treated Mpz-EpoR-KO mice but not in EPO-treated control mice. CONCLUSIONS: These preliminary findings provide direct evidence for an obligatory role of Schwann-cell specific EpoR for EPO-induced functional recovery and muscle atrophy following PNI.


Assuntos
Eritropoetina/metabolismo , Atrofia Muscular/genética , Traumatismos dos Nervos Periféricos/genética , Receptores da Eritropoetina/genética , Recuperação de Função Fisiológica/genética , Células de Schwann/metabolismo , Nervo Isquiático/lesões , Animais , Lesões por Esmagamento/complicações , Lesões por Esmagamento/genética , Lesões por Esmagamento/metabolismo , Camundongos , Camundongos Knockout , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/metabolismo , Receptores da Eritropoetina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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