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1.
Gene ; 741: 144539, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32160960

RESUMO

microRNAs (miRNAs) are involved in the physiological and pathophysiological processes of diabetes and its microvascular and macrovascular complications. Hence, the aim of the study was to investigate whether miR-499-3p played an important role in diabetic retinopathy. Diabetic retinopathy was developed in rats by intraperitoneal injection of streptozocin (STZ), followed by collection of retinal tissues and preparation of retinal cells. Immunohistochemical staining was used to detect expression of interferon alpha 2 (IFNA2). RT-qPCR was used to determine the expression of miR-499-3p. Bioinformatics website and dual luciferase reporter gene assay were used to validate the targeting relationship between miR-499-3p and IFNA2. Gain- and loss-of-function assays were performed to explore the functional roles of aberrantly expressed miR-499-3p and IFNA2 in retinal cell proliferation by MTT, and apoptosis by flow cytometry. In retinal tissues and cells of diabetic rats, IFNA2 expression was reduced, and miR-499-3p expression increased to activate the toll-like receptor 4 (TLR4) signaling pathway. IFNA2 was a target gene of miR-499-3p and negatively regulated by miR-499-3p. Further, downregulated miR-499-3p promoted retinal cell proliferation while suppressing apoptosis to alleviate diabetic retinopathy. All in all, miR-499-3p promoted retinopathy by enhancing activation of the TLR4 signaling pathway, which provides a new therapeutic target for diabetic retinopathy.


Assuntos
Retinopatia Diabética/genética , Interferon-alfa/genética , MicroRNAs/genética , Receptor 4 Toll-Like/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Ratos , Retina/metabolismo , Retina/patologia , Transdução de Sinais/genética
2.
Pol J Microbiol ; 68(4): 439-447, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880888

RESUMO

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes - Typhimurium, Enteritidis, and Choleraesuis - in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy - significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes ­ Typhimurium, Enteritidis, and Choleraesuis ­ in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy ­ significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.


Assuntos
Aneurisma Aórtico/microbiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Aneurisma Aórtico/genética , Aneurisma Aórtico/imunologia , Linhagem Celular , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Antígeno Ki-1/genética , Antígeno Ki-1/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , Plasmídeos/genética , Plasmídeos/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Sorogrupo , Virulência
3.
J Biotechnol ; 303: 46-52, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31336133

RESUMO

Different strategies have been developed and successfully applied to biotherapeutics in order to improve their in vivo efficacy. The genetic fusion to natural or synthetic glycosylated peptides constitutes a promising strategy since it conserves the protein sequence and results in the improvement of the pharmacokinetic properties. The ANITVNITV peptide described by Perlmann and coworkers presents 9 amino acids and 2 potential N-glycosylation sites. Its fusion to FSH resulted in the increase of the molecular mass and negative charge of the protein. Consequently, the pharmacokinetics was considerably improved. The aim of the present study was to compare the influence of ANITVNITV peptide fusion on the physicochemical, biological and pharmacokinetic properties of native hIFN-α2b (IFNwt), which contains a single O-glycosylation site, and a hyperglycosylated variant (IFN4N), that bears, in addition, 4 N-linked glycans. The resulting molecules, IFNwtNter and IFN4NNter, evidenced a higher molecular mass and negative charge compared to IFNwt and IFN4N, respectively. Therefore, the pharmacokinetic properties of the new molecules were significantly improved. The molecules obtained by the synthetic peptide fusion strategy evidenced a decrease in their in vitro antiviral specific biological activities (SBA). However, in vitro antiproliferative SBA was differentially modified for IFNwtNter and IFN4NNter in comparison with the parental molecules. For IFNwtNter, a reduction in the antiproliferative SBA was also observed. Remarkably, the addition of the ANITVNITV peptide to the N-terminus of IFN4N had a positive impact on its growth-inhibitory activity. This feature together with its improved pharmacokinetics encourages the development of IFN4NNter as an IFN-α based biobetter.


Assuntos
Interferon-alfa/genética , Interferon-alfa/farmacocinética , Peptídeos/química , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetulus , Cães , Variação Genética , Glicosilação , Humanos , Interferon-alfa/química , Células Madin Darby de Rim Canino , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética
4.
EBioMedicine ; 45: 328-340, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31300344

RESUMO

BACKGROUND: TLR9 agonists are being developed as immunotherapy against malignancies and infections. TLR9 is primarily expressed in B cells and plasmacytoid dendritic cells (pDCs). TLR9 signalling may be critically important for B cell activity in lymph nodes but little is known about the in vivo impact of TLR9 agonism on human lymph node B cells. As a pre-defined sub-study within our clinical trial investigating TLR9 agonist MGN1703 (lefitolimod) treatment in the context of developing HIV cure strategies (NCT02443935), we assessed TLR9 agonist-mediated effects in lymph nodes. METHODS: Participants received MGN1703 for 24 weeks concurrent with antiretroviral therapy. Seven participants completed the sub-study including lymph node resection at baseline and after 24 weeks of treatment. A variety of tissue-based immunologic and virologic parameters were assessed. FINDINGS: MGN1703 dosing increased B cell differentiation; activated pDCs, NK cells, and T cells; and induced a robust interferon response in lymph nodes. Expression of Activation-Induced cytidine Deaminase, an essential regulator of B cell diversification and somatic hypermutation, was highly elevated. During MGN1703 treatment IgG production increased and antibody glycosylation patterns were changed. INTERPRETATION: Our data present novel evidence that the TLR9 agonist MGN1703 modulates human lymph node B cells in vivo. These findings warrant further considerations in the development of TLR9 agonists as immunotherapy against cancers and infectious diseases. FUND: This work was supported by Aarhus University Research Foundation, the Danish Council for Independent Research and the NovoNordisk Foundation. Mologen AG provided study drug free of charge.


Assuntos
Diferenciação Celular/efeitos dos fármacos , DNA/administração & dosagem , Infecções por HIV/tratamento farmacológico , Receptor Toll-Like 9/genética , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Interferon-alfa/genética , Linfonodos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Receptor Toll-Like 9/agonistas
5.
Arh Hig Rada Toksikol ; 70(2): 104-108, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31246567

RESUMO

Individuals chronically exposed to low-level ionising radiation (IR) run the risk of harmful and long-term adverse health effects, including gene mutations and cancer development. The search for reliable biomarkers of IR exposure in human population is still of great interest, as they may have a great implementation potential for the surveillance of occupationally exposed individuals. In this context, and considering previous literature, this study aimed to identify mutations in the human interferon alpha-2b (hIFNα-2b) as a potential biomarker of occupational chronic low-dose IR exposure linking low-IR exposure to the effects on haematopoiesis and reduced immunity. The analysis was performed in the genomic DNA of 51 uranium miners and 38 controls from Kazakhstan, and in 21 medical radiology workers and 21 controls from Italy. hIFNα-2b gene mutations were analysed with the real-time polymerase chain reaction (PCR) or Sanger sequencing. However, none of the investigated workers had the hIFNα-2b mutation. This finding highlights the need for further research to identify biomarkers for early detection of health effects associated with chronic low-dose IR exposure.


Assuntos
Minas de Carvão , Biomarcadores Ambientais/genética , Interferon-alfa/genética , Interferon-alfa/efeitos da radiação , Mutação/efeitos da radiação , Doenças Profissionais/genética , Exposição à Radiação/efeitos adversos , Radiação Ionizante , Adulto , Humanos , Itália , Cazaquistão , Masculino , Pessoa de Meia-Idade , Exposição Ocupacional
6.
Biomed Res Int ; 2019: 8084109, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179333

RESUMO

Shenqi Fuzheng Injection (SFI) is a traditional Chinese medicine injection with anticancer properties and is mainly composed of ginseng and astragalus. Its efficacy has been confirmed in clinical trials, but the mechanism remains unclear. We investigated the effect of SFI on vascular endothelial growth factor (VEGF) gene expression in hepatocellular carcinoma (HCC) cells and identified its possible mechanism of synergistic effects when combined with the chemotherapeutic drug interferon (IFN-) α. An MTT assay was used to measure the inhibition effects of low-dose IFN-α (6000 IU) with or without SFI (0.5 g/L) on the HCC cell line MHCC97. VEGF-silenced MHCC97L-mir200 cell lines were prepared using lentiviral vectors and evaluated by real-time PCR to determine the inhibition effect. We examined MHCC97L-mir200 and MHCC97L cells by MTT assay, using IFN-α alone or in combination with SFI. The inhibition ratio of IFN-α (6000 IU) was -29.5%, while that for IFN-α (6000 IU) + SFI (0.5 g/L) was 17.0%, which was significantly higher than that for the IFN-α group (P < 0.01). The VEGF gene was silenced successfully in MHCC97-L cells. After interference of VEGF, the inhibition by SFI and IFN-α in MHCC97L-mir200 did not differ from that in MHCC97-L cells (P > 0.05). SFI can reduce the expression of VEGF in HCC, which can increase the efficacy of IFN-α, providing a theoretical basis for clinical application.


Assuntos
Carcinoma Hepatocelular/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Interferon-alfa/biossíntese , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Humanos , Interferon-alfa/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/genética
7.
Bull Exp Biol Med ; 167(1): 111-115, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31177454

RESUMO

Rat glioma cell line C6 expressing human poliovirus receptor (PVR) and susceptible to polioviruses (C6-PVR-BFP) was used to produce a clone with knockout of IFNα/ß (Ifnar1) receptor subunit 1 gene (Ifnar1). The sensitivity of C6-PVR-BFP cells to the vaccine strain of poliovirus type 3 (PV3) depended on the signaling pathways of the cell response to type 1 IFN. Using the model of subcutaneous tumor xenografts, we demonstrated oncolytic activity of PV3 against C6-PVR-BFP cells that depended on the expression of PVR and increased considerably upon disturbances in IFN response pathways.


Assuntos
Glioma/terapia , Glioma/virologia , Terapia Viral Oncolítica/métodos , Poliovirus/fisiologia , Animais , Linhagem Celular Tumoral , Glioma/metabolismo , Interferon-alfa/genética , Interferon beta/genética , Camundongos , Vírus Oncolíticos/fisiologia , Ratos , Ratos Mutantes , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo
8.
Vet Immunol Immunopathol ; 212: 15-22, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31213247

RESUMO

Marek's disease virus (MDV), an α-herpesvirus targeting avian species, causes fatal Marek's disease (MD) in chickens. The host interferon (IFN) responses play a key role in resisting viral infection. However, host IFN responses following MDV infection in the chicken central immune organs (thymus and bursa of Fabricius), which contain numerous MDV target cells, is poorly understood. In this study, we performed animal experiments in specific pathogen-free chickens infected with two virulent MDV strains (BS/15 and Md5) or without infection as negative controls. Specifically, the type I IFN (IFN-α and IFN-ß) transcriptional and proteomic expression levels at 7, 10, 14, 17, and 21 days post infection (dpi) were detected and analyzed. Our results indicated that the mRNA and protein expression levels of IFN-α and IFN-ß in the thymus and bursa of Fabricius were mainly downregulated in cytolytic infection (such as 10 dpi) and reactivation (such as 17 dpi) stages, but not the latent (such as 14 dpi) stage of MDV infection, which was determined by comprehensively analyzing the MDV viral load and immune organ damage caused by MDV infection. These data suggest that MDV could inhibit the expression of host type I IFNs, which may be involved in the MDV-induced host immunosuppression and contribute to the immune escape of MDV from host immunity. Furthermore, we found that the downregulated expression of the host type I IFNs induced by BS/15 and Md5 infection was significantly different, which we speculated may be related to the diverse virulence and pathogenicity of MDV strains. In conclusion, our study demonstrated that MDV mostly inhibited the expression of type I IFNs in infected hosts, which may be associated to its pathogenesis.


Assuntos
Interferon Tipo I/imunologia , Doença de Marek/imunologia , Doenças das Aves Domésticas/imunologia , Animais , Bolsa de Fabricius/imunologia , Galinhas , Expressão Gênica , Herpesvirus Galináceo 2 , Interferon Tipo I/genética , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Doenças das Aves Domésticas/virologia , Proteômica , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Timo/imunologia , Carga Viral , Virulência
9.
Vet Microbiol ; 234: 77-82, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213275

RESUMO

Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, prokaryotic expression recombination chicken interferon-α (rchIFN-α) was used as vaccine adjuvant.In this study chIFN-α was used as adjuvant in inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single AI H9N2 vaccine. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were challenged with a high dose of LPAI H9N2 virus. Combined administration rchIFN-α showed markedly enhanced protection compared to single administration of the vaccine, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in challenged chickens. Our results indicate the value of combined administration of rchIFN-α to generate an effective immunization strategy in chickens against LPAI H9N2.


Assuntos
Imunogenicidade da Vacina , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Interferon-alfa/genética , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais/sangue , Galinhas , Imunidade Celular , Imunidade Humoral , Vírus da Influenza A Subtipo H9N2/imunologia , Vacinas contra Influenza/genética , Influenza Aviária/imunologia , Interferon-alfa/imunologia , Organismos Livres de Patógenos Específicos , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Eliminação de Partículas Virais
10.
Acta Virol ; 63(2): 235-239, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230454

RESUMO

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are among the most dangerous pathogens globally. Infection with HCV has been reported in a high percentage of HIV patients. Viruses are obligate intracellular pathogens and their survival is associated with their capability to subvert antiviral defenses of cells and to improve cellular processes required for their replication. The aim of this study was to compare the expression rate of the key gene for autophagy process, Beclin-1, as a cellular response to viral infections, and its effect on IFN-α expression in both HCV and HCV/HIV patient groups. In this study, a total number of 40 samples of peripheral blood mononuclear cells (PBMCs) including 20 HCV and 20 HCV/HIV patients before treatment were evaluated. The HCV viral load in both groups was evaluated by semi quantitative real-time PCR. The level of Beclin-1 and IFN-α gene expression was examined in all samples by semi quantitative real-time PCR assay. The median viral load was 8.3×105 copies/ml in HCV group and 2.1×106 copies/ml in HCV/HIV patients. While the expression level of Beclin-1 gene in HCV group was significantly higher, the level of IFN-α expression was lower compared to the HCV/HIV group (P  Keywords: autophagy; Beclin gene; HCV; HIV; IFN gene.


Assuntos
Antivirais , Autofagia , Infecções por HIV , Hepacivirus , Hepatite C , Interferon-alfa , Proteína Beclina-1/genética , Infecções por HIV/complicações , Hepacivirus/fisiologia , Hepatite C/complicações , Hepatite C/virologia , Humanos , Interferon-alfa/genética , Leucócitos Mononucleares , Carga Viral
11.
J Sci Food Agric ; 99(13): 6076-6083, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31233219

RESUMO

BACKGROUND: This study was conducted to evaluate the health benefits to weaning pigs, raised under low sanitary conditions, of dietary supplementation with a multi-strain yeast fraction product (Cyberlindnera jadinii and Saccharomyces cerevisiae). In total, 160 weaning pigs (7.21 ± 1.05 kg) were randomly allotted to two dietary treatments in a 6-week feeding trial. The dietary treatments included a corn-soybean meal-based basal diet (CON) and CON + 2 g kg-1 multi-strain yeast fraction product (MsYF) during weeks 1-2 and 0.4 g kg-1 MsYF during weeks 3-6. RESULTS: The MsYF supplementation increased (P < 0.05) body weight (BW) at day 42 and average daily gain (ADG) during days 1-14 and days 1-42 (P < 0.05) compared to CON. The total tract digestibility of dry matter (DM), fecal Lactobacillus counts, and serum immunoglobulin G (IgG) concentration at day 42 were higher (P < 0.05) in pigs fed a MsYF supplemented diet. The concentration of serum haptoglobin in pigs receiving a MsYF-supplemented diet was higher (P < 0.05) at days 7, 14, and 42 than those receiving CON. The mRNA expression for INF-γ and TNF-α genes were lower (P < 0.05) at days 14 and 7 respectively and the expression of IL-6 and TLR-2 genes was lower (P < 0.01) at days 7 and 14 in pigs fed an MsFY supplemented diet than those fed CON. CONCLUSION: Supplementation with a multi-strain yeast fraction product had a positive effect on ADG during the early post-weaning period and led to better health in weaning pigs. © 2019 Society of Chemical Industry.


Assuntos
Saccharomyces cerevisiae/química , Suínos/crescimento & desenvolvimento , Leveduras/química , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Suplementos Nutricionais/análise , Fezes/microbiologia , Microbioma Gastrointestinal , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Higiene , Interferon-alfa/genética , Interferon-alfa/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Suínos/genética , Suínos/imunologia , Suínos/microbiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Desmame
12.
Prep Biochem Biotechnol ; 49(8): 735-743, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31135267

RESUMO

Type I interferons (IFNs) are homologous cytokines that bind to a cell surface receptor and establish signaling pathways that motivate immune responses. The purpose of the current study is to assess the activity of a novel-engineered IFN-α2b. The crystallographic structure of IFN-α2b and its receptors was acquired from Protein Data Bank. Various amino acid substitutions were designed based on structural properties and other biological characteristics of residues to find the most effective amino acid on IFN affinity to advanced activities. The IFN-α2b mutants and receptors have been modeled and the interactions between two proteins have been studied as in silico by protein-protein docking for both mutants and native forms. The proper nucleic acid sequence IFN-α2 (T79Q) has been prepared based on the selected mutant. The modified IFN gene was cloned in pcDNA 3.1(-) and introduced to Chinese Hamster Ovary (CHO) cell line. Antiviral and antiproliferative assays of native and IFN-α2 (T79Q) proteins were performed in vitro. The results showed two-fold increasing in IFN-α2 (T79Q) activity (antiviral and antiproliferative activity) in comparison to native IFN-α2b. This engineered IFN-α2b may have significant novel therapeutic applications and in silico studies can be an influential method for practical research function and structure of these molecules.


Assuntos
Substituição de Aminoácidos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Engenharia de Proteínas , Receptores de Interferon/metabolismo , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Células HeLa , Humanos , Interferon-alfa/química , Interferon-alfa/farmacologia , Simulação de Acoplamento Molecular , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
13.
Immunol Invest ; 48(8): 781-793, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31062637

RESUMO

Dendritic cells (DCs) play a major role in regulating immune responses, but the aberrant phenotype and function of defective DCs in adult acute lymphoblastic leukemia (ALL) remain unclear. Here, B lineage ALL (B-ALL) patients were divided into groups according to different standards. By course of disease: newly diagnosed (ND), complete remission (CR), consolidation (CONS). By stratification: high risk (HR), standard risk (SR). By minimal residual disease (MRD): MRD positive(MRD+), MRD negative (MRD-). The proportion of plasmacytoid DC(pDC) and myeloid DC(mDC) were compared within these standards. The costimulatory molecule levels of pDC, mDC in ND and CR were measured and the function of peripheral blood monocyte-derived DC(MoDC)s were examined. We found proportions of pDC and mDC in ND were both lower compared to control group and gradually increased after CR. In HR and MRD+, the proportions were also lower compared to SR and MRD- at CR stage, respectively; but there were no difference between these comparisons when newly diagnosed. In ND, both CD80, CD86 levels in pDC, mDC were higher while the levels in activated MoDCs were lower when compared to control and CR group, respectively. The dextran uptake of MoDCs, T cell proliferation promoting ability, IL-12, BAFF, INF-α levels in supernatant and their mRNA relative expression in activated MoDCs in ND were also lower than those in control and CR group. So, DCs in B-ALL display suppressed status in phenotype and function,which would be gradually restored after effective chemotherapy. pDC and mDC could respond to patient condition, DCs proportion may be useful for monitoring disease progression.


Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Monócitos/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Adulto , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Fator Ativador de Células B/metabolismo , Proliferação de Células/genética , Células Cultivadas , Células Dendríticas/metabolismo , Feminino , Expressão Gênica/imunologia , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Neoplasia Residual/genética , Neoplasia Residual/imunologia , Neoplasia Residual/metabolismo , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Indução de Remissão
14.
Mol Med Rep ; 20(1): 225-235, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115544

RESUMO

Psoriasis is an immune­mediated cutaneous disorder with a high incidence and prevalence. Patients with psoriasis may experience irritation, pain and psychological problems. The cause and underlying molecular etiology of psoriasis remains unknown. In an attempt to achieve a more comprehensive understanding of the molecular pathogenesis of psoriasis, the gene expression profiles of 175 pairs of lesional and corresponding non­lesional skin samples were downloaded from 5 data sets in the Gene Expression Omnibus (GEO) database. Integrated differentially expressed genes (DEGs) were obtained with the use of R software. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed using the DAVID online analysis tool. The protein­protein interaction (PPI) network was constructed on the STRING platform and hub genes were calculated with the use of Cytoscape software. Finally, GEO2R was used to determine the expression of the hub genes in scalp psoriasis. A total of 373 genes from the 5 data sets were identified as DEGs, including 277 upregulated and 96 downregulated genes. GO analysis revealed that immune responses and epidermal differentiation/development were the most enriched terms in biological processes, extracellular space/matrix was the most enriched term in cellular components, and endopeptidase inhibitor activity was the most enriched term in molecular functions. In the KEGG pathway enrichment, DEGs were mainly enriched in the metabolic and viral infection­associated pathways. A total of 17 hub genes were calculated, including CSK2, CDC45, MCM10, SPC25, NDC80, NUF2, AURKA, CENPE, RRM2, DLGP5, HMMR, TTK, IFIT1, RSAD2, IFI6, IFI27 and ISG20, among which interferon­α­inducible genes were revealed to display a similar expression pattern as that obtained in scalp psoriasis. This comprehensive bioinformatic re­analysis of GEO data provides new insights on the molecular pathogenesis of psoriasis and the identification of potential therapeutic targets for the treatment of psoriasis.


Assuntos
Biologia Computacional , Interferon-alfa/genética , Psoríase/genética , Regulação da Expressão Gênica/genética , Ontologia Genética , Redes Reguladoras de Genes/genética , Humanos , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , Psoríase/metabolismo , Psoríase/patologia , Couro Cabeludo/metabolismo , Couro Cabeludo/patologia , Software
15.
Vet Immunol Immunopathol ; 210: 55-59, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30947980

RESUMO

Virulent strains of Escherichia coli (Avian Pathogenic E. Coli: APEC) can cause initial infection of the respiratory tract in chickens potentially leading to systemic infection called colibacillosis, which remains a major cause of economic losses in the poultry industry. The role of epithelial lung cells as first targets of APEC and in initiating the innate immune response is unclear and was investigated in this study. APEC was able to adhere and subsequently invade cells from the chicken lung epithelial CLEC213 cell line exhibiting pneumocyte type II-like characteristics. Invasion was confirmed using confocal microscopy after infection with GFP-labelled APEC. Moreover, the infection resulted in a significant increase in IL-8 gene expression, a chemo-attractant of macrophages and heterophils. Gene expression of interferon α and ß were not significantly upregulated and chicken Surfactant Protein A, also did not show a significant upregulation on either gene or protein level. The immune response of CLEC213 cells towards APEC was shown to be similar to stimulation with E. coli LPS. These results establish CLEC213 cells as a novel model system for studying bacterial infection of the lung epithelium and show that these cells may play a role in the initial innate response towards bacterial pathogens.


Assuntos
Células Epiteliais Alveolares/microbiologia , Escherichia coli/patogenicidade , Pulmão/citologia , Células Epiteliais Alveolares/imunologia , Animais , Doenças das Aves/microbiologia , Linhagem Celular , Galinhas , Escherichia coli/imunologia , Expressão Gênica , Interferon-alfa/genética , Interferon beta/genética , Interleucina-8/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Regulação para Cima , Virulência
16.
J Immunol ; 202(9): 2529-2534, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30936294

RESUMO

Systemic lupus erythematosus severity correlates with elevated serum levels of type I IFNs, cytokines produced in large quantities by plasmacytoid dendritic cells (pDC) in response to engagement of TLR7 and TLR9 with endocytosed nucleic acids. B cell adaptor for PI3K (BCAP) promoted many aspects of TLR7-driven lupus-like disease, including Isg15 and Ifit1 expression in blood and an immature pDC phenotype associated with higher IFN production. BCAP-/- mice produced significantly less serum IFN-α than wild-type mice after injection of TLR9 agonist, and BCAP promoted TLR7 and TLR9-induced IFN-α production specifically in pDC. TLR-induced IFN-α production in pDC requires DOCK2-mediated activation of Rac1 leading to activation of IKKα, a mechanism we show was dependent on BCAP. BCAP-/- pDC had decreased actin polymerization and Rac1 activation and reduced IKKα phosphorylation upon TLR9 stimulation. We show a novel role for BCAP in promoting TLR-induced IFN-α production in pDC and in systemic lupus erythematosus pathogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Células Dendríticas/imunologia , Interferon-alfa/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/imunologia , Plasmócitos/imunologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/patologia , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/imunologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Interferon-alfa/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Plasmócitos/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética , Ubiquitinas/genética , Ubiquitinas/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia
17.
J Cutan Med Surg ; 23(4): 370-379, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31010295

RESUMO

BACKGROUND: 5-aminolevulinic acid photodynamic therapy (PDT) for genital warts is effective, safe, and can prevent recurrence. It is believed that PDT can induce immune responses, but the mechanism is not completely understood. OBJECTIVES: The objectives of this article are to confirm the effect of PDT for genital warts on local immunity and to investigate the recruitment and significance of immune cells in tissues. METHODS: Local immune changes in T lymphocytes (CD3+, CD4+, CD8+), plasmacytoid dendritic cells (pDCs) (CD123+), and myeloid dendritic cells (CD1a+) after PDT in patients were evaluated by immunohistochemistry staining. Changes in mRNA levels of IFN-γ, IFN-α, IFN-ß, interferon-stimulated gene 15 kDa (ISG-15), Mx2, Toll-like receptor 9 (TLR9), and interferon regulatory factor 7 (IRF7) were analyzed by real-time quantitative polymerase chain reaction. RESULTS: At 4 hours after PDT, CD4+ increased, accompanied by increased levels of mRNA expression of IFN-γ, but CD4+ and mRNA expression levels of IFN-γ were decreased at 24 hours after PDT. CD123+ pDCs showed an increasing trend. CD1a+ LCs in the epidermis gradually decreased, and DCs in the epidermis gradually increased. CD3+ infiltrated and migrated to the superficial dermis, but CD8+ did not change significantly after PDT. The mRNA expression levels of IFN-α, IFN-ß, ISG-15, Mx2, TLR9, and IRF7 showed an increasing trend after PDT. As compared with the patients without significantly increased IFN-α and IFN-ß after PDT sessions, patients with significant increases needed fewer sessions of PDT for remission. CONCLUSIONS: PDT for genital warts can activate T lymphocyte-mediated, DC-related, and pDC-related immunity. The clinical efficacy of PDT for genital warts may be related to the increased levels of IFN-α and IFN-ß after treatment.


Assuntos
Ácido Aminolevulínico/farmacologia , Condiloma Acuminado/tratamento farmacológico , Epiderme/imunologia , Células de Langerhans/imunologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Adulto , Ácido Aminolevulínico/uso terapêutico , Antígenos CD1/metabolismo , Complexo CD3/metabolismo , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Citocinas/genética , Epiderme/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Fator Regulador 7 de Interferon/genética , Interferon-alfa/genética , Interferon beta/genética , Interferon gama/genética , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus/genética , Fármacos Fotossensibilizantes/uso terapêutico , RNA Mensageiro/metabolismo , Receptor Toll-Like 9/genética , Ubiquitinas/genética , Adulto Jovem
18.
Front Biosci (Landmark Ed) ; 24: 790-797, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844713

RESUMO

We previously reported a natural antisense (AS) RNA as an important modulator of human interferon-Alpha1 (IFNA1) mRNA levels. Here, we identified the guinea pig (Cavia porcellus) IFNA1 gene to enable a proof-of-concept experiment to be performed to confirm that the AS-mRNA regulatory axis exerts in vivo control over innate immunity. We selected a guinea pig model system for influenza virus infection because guinea pigs encode a functional Mx1 gene, an important anti-viral effector in the type I interferon pathway. We identified 15 guinea pig IFNA1 gene candidates upon bioinformatic analysis and selected the three candidates with the highest sequence homology to Homo sapiens, Mus musculus and Marmota himalayana IFNA1. The anti-viral activity of guinea pig IFN-Alpha1 protein against influenza virus A/Puerto Rico/8/34- or endomyocarditis virus-infection was then determined for the three gene candidates. We identified cpIFNA1 as the candidate with the highest sequence homologies and best anti-viral effects. cpIFNA1 will enable us to perform a proof-of-concept experiment to verify that IFN-Alpha1 AS increases cpIFNA1 mRNA levels, resulting in inhibition of influenza virus proliferation in vivo.


Assuntos
Vacinas contra Influenza , Interferon-alfa/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/terapia , RNA Antissenso/genética , Animais , Linhagem Celular , Biologia Computacional , Cães , Cobaias , Humanos , Imunidade Inata , Influenza Humana/imunologia , Influenza Humana/terapia , Interferon-alfa/metabolismo , Células Madin Darby de Rim Canino , Marmota , Camundongos , Oligonucleotídeos Antissenso/genética , Orthomyxoviridae , Plasmídeos/metabolismo
19.
Front Biosci (Landmark Ed) ; 24: 798-818, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844714

RESUMO

We reported a natural antisense (AS) long non-coding RNA as an important modulator of interferon-Alpha1 (IFNA1) mRNA levels. We showed that IFN-Alpha1 AS promotes IFNA1 mRNA stability by transient duplex formation and inhibition of miR-1270-induced mRNA decay. Here, we performed a proof-of-concept experiment to verify that the AS-mRNA regulatory axis exerts in vivo control of innate immunity. We established a model system for influenza virus infection using guinea pig, which encodes a functional MX1 gene for the type I IFN pathway. This system allowed us to investigate the effects of antisense oligoribonucleotides representing functional domains of guinea pig IFN-Alpha1 AS on gpIFNA1 mRNA levels and, consequently, on viral proliferation in the respiratory tract of influenza virus-infected animals. We demonstrated that pulmonary-administered asORNs inhibited the proliferation of the virus in the animals by modulating IFNA1 mRNA levels. These results indicate that, in light of the proposed actions, asORNs may modulate the level of IFNA1 mRNA in vivo, indicating that IFN-Alpha1 AS plays a pivotal role in determining the outcome of type I IFN responses.


Assuntos
Interferon-alfa/genética , Infecções por Orthomyxoviridae/imunologia , RNA Antissenso/genética , Animais , Células Cultivadas , Cães , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Cobaias , Imunidade Inata , Vírus da Influenza A/fisiologia , Interferon-alfa/metabolismo , Cinética , Células Madin Darby de Rim Canino , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição
20.
Prep Biochem Biotechnol ; 49(2): 192-201, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30734625

RESUMO

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.


Assuntos
Antivirais/farmacologia , Proteínas Aviárias/farmacologia , Galinhas/genética , Vírus da Influenza A Subtipo H9N2/efeitos dos fármacos , Influenza Aviária/tratamento farmacológico , Interferon-alfa/farmacologia , Animais , Antivirais/química , Antivirais/isolamento & purificação , Antivirais/metabolismo , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/isolamento & purificação , Escherichia coli/genética , Interferon-alfa/química , Interferon-alfa/genética , Interferon-alfa/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Tiorredoxinas/farmacologia
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