RESUMO
BACKGROUND AND OBJECTIVES: Available information about infection after spine instrumentation (IASI) and its management are scarce. We aimed to analyse DAIR (debridement, antibiotics and implant retention) prognosis and evaluate effectiveness of short antibiotic courses on early forms. METHODS: Multicentre retrospective study of patients with IASI managed surgically (January 2010-December 2016). Risk factors for failure were analysed by multivariate Cox regression and differences between short and long antibiotic treatment were evaluated with a propensity score-matched analysis. RESULTS: Of the 411 IASI cases, 300 (73%) presented in the first month after surgery, 48 in the second month, 22 in the third and 41 thereafter. Infections within the first 2 months (early cases) occurred mainly to older patients, with local inflammatory signs and predominance of Enterobacteriaceae, unlike those in the later periods. When managed with DAIR, prognosis of early cases was better than later ones (failure rate 10.4% versus 26.1%, respectively; P = 0.02). Risk factors for DAIR failure in early cases were female sex, Charlson Score, large fusions (>6 levels) and polymicrobial infections (adjusted HRs of 2.4, 1.3, 2.6 and 2.26, respectively). Propensity score matching proved shorter courses of antibiotics (4-6 weeks) as effective as longer courses (failure rates 11.4% and 10.5%, respectively; P = 0.870). CONCLUSIONS: IASIs within the first 2 months could be managed effectively with DAIR and shorter antibiotic courses. Clinicians should be cautious when faced with patients with comorbidities, large fusions and/or polymicrobial infections.
Assuntos
Infecções Relacionadas à Prótese , Antibacterianos/uso terapêutico , Desbridamento , Feminino , Humanos , Infecções Relacionadas à Prótese/tratamento farmacológico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Collateral growth in patients with coronary artery disease (CAD) is highly heterogeneous. Although multiple factors are thought to play a role in collateral development, the contribution of genetic factors to coronary collateral circulation (CCC) is largely unknown. The goal of this study was to assess whether functional single nucleotide polymorphisms (SNPs) in genes involved in vascular growth are associated with CCC. METHODS: 677 consecutive CAD patients were enrolled in the study and their CCC was assessed by the Rentrop method. 22 SNPs corresponding to 10 genes involved in postischemic neovascularization were genotyped and multivariate logistic regression models were adjusted using clinically relevant variables to estimate odds ratios and used to examine associations of allelic variants, genotypes and haplotypes with CCC. RESULTS: Statistical analysis showed that the HIF1A rs11549465 and rs2057482; VEGFA rs2010963, rs1570360, rs699947, rs3025039 and rs833061; KDR rs1870377, rs2305948 and rs2071559; CCL2 rs1024611, rs1024610, rs2857657 and rs2857654; NOS3 rs1799983; ICAM1 rs5498 and rs3093030; TGFB1 rs1800469; CD53 rs6679497; POSTN rs3829365 and rs1028728; and LGALS2 rs7291467 polymorphisms, as well as their haplotype combinations, were not associated with CCC (p < 0.05). CONCLUSIONS: We could not validate in our cohort the association of the NOS3 rs1799983, HIF1A rs11549465, VEGFA rs2010963 and rs699947, and LGALS2 rs7291467 variants with CCC reported by other authors. A validated SNP-based genome-wide association study is required to identify polymorphisms influencing CCC.
Assuntos
Circulação Colateral , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/fisiopatologia , Circulação Coronária , Neovascularização Fisiológica , Polimorfismo de Nucleotídeo Único , Idoso , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: Hypoxia is required for the development of the cardiovascular system. Tissue adaptation to low oxygen is mediated through hypoxia-inducible factor 1. Hypoxia-driven gradients of vascular endothelial growth factor within the heart drive vessel tip sprouting and the angiogenic phase of vasculogenesis. We hypothesized that functional variants of the HIF1A C85T single nucleotide polymorphism (SNP) are associated with the number of coronary artery branches in humans. METHODS: Coronary artery branching in 88 individuals was assessed by dynamic counting of the arterial branches detected in coronary angiograms. Values were classified on the basis of the branches emerging from the right and left coronary arteries. HIF1A C85T genotypes were determined using TaqMan-based assays. A generalized linear model was used to measure the effect of each SNP on the response variables. RESULTS: The presence of the T allele in the HIF1A C85T SNP was associated with few branches of the coronary arteries: 81.03 ± 1.79 for individuals with the CC genotype versus 74.09 ± 2.48 for T-carrying ones (p = 0.042). CONCLUSIONS: The functionality of HIF1A may influence the degree of branching of the human coronary tree. We propose that the HIF1A C85T SNP is a genetic marker that determines interindividual differences in the human coronary artery pattern.
Assuntos
Vasos Coronários/anatomia & histologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Angiografia Coronária , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/genética , Adulto JovemRESUMO
Fructose 2,6-bisphosphate (Fru-2,6-P2) is an important metabolite that controls glycolytic and gluconeogenic pathways in several cell types. Its synthesis and degradation are catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2). Four genes, designated Pfkfb1-4, codify the different PFK-2 isozymes. The Pfkfb3 gene product, ubiquitous PFK-2 (uPFK-2), has the highest kinase/bisphosphatase activity ratio and is associated with proliferation and tumor metabolism. A transgenic mouse model that overexpresses uPFK-2 under the control of the phosphoenolpyruvate carboxykinase promoter was designed to promote sustained and elevated Fru-2,6-P2 levels in the liver. Our results demonstrate that in diet-induced obesity, high Fru-2,6-P2 levels in transgenic livers caused changes in hepatic gene expression profiles for key gluconeogenic and lipogenic enzymes, as well as an accumulation of lipids in periportal cells, and weight gain.
Assuntos
Fígado/fisiologia , Fosfofrutoquinase-2/metabolismo , Aumento de Peso/fisiologia , Animais , Camundongos , Camundongos Transgênicos , Fosfofrutoquinase-2/genética , Regulação para CimaRESUMO
The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P(2)), a signalling molecule that controls the balance between glycolysis and gluconeogenesis in several cell types. Four genes, designated Pfkfb1-4, code several PFK-2 isozymes that differ in their kinetic properties, molecular masses, and regulation by protein kinases. In rat tissues, Pfkfb3 gene accounts for eight splice variants and two of them, ubiquitous and inducible PFK-2 isozymes, have been extensively studied and related to cell proliferation and tumour metabolism. Here, we characterize a new kidney- and liver-specific Pfkfb3 isozyme, a product of the RB2K3 splice variant, and demonstrate that its expression, in primary cultured hepatocytes, depends on hepatic cell proliferation and dedifferentiation. In parallel, our results provide further evidence that ubiquitous PFK-2 is a crucial isozyme in supporting growing and proliferant cell metabolism.
Assuntos
Hepatócitos/citologia , Hepatócitos/enzimologia , Rim/citologia , Rim/enzimologia , Fosfofrutoquinase-2/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Isoenzimas/metabolismo , Masculino , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
To investigate the potential of antibody derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed a small immune protein (SIP) in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The epsilonSIP molecule consisted of a single-chain antibody (scFv) specific for the porcine coronavirus transmissible gastroenteritis virus (TGEV) linked to the epsilon-CH4 domain from human immunoglobulin E (IgE). In some constructs, the sequence encoding the epsilonSIP molecule was flanked by the leader peptide from the original murine antibody at its N-terminus and an endoplasmic reticulum retention signal (HDEL) at its C-terminus to allow the expressed protein to be directed to, and retained within, the endoplasmic reticulum. Western blot analysis of samples from Nicotiana clevelandii or cowpea tissue infected with constructs revealed the presence of SIP molecules which retained their ability to dimerize. The analysis of crude plant extracts revealed that the plant-expressed epsilonSIP molecules could bind to and neutralize TGEV in tissue culture, the levels of binding and neutralization reflecting the level of expression. Oral administration of crude extracts from SIP-expressing plant tissue to 2-day-old piglets demonstrated that the extracts which showed the highest levels of in vitro neutralization could also provide in vivo protection against challenge with TGEV.
Assuntos
Anticorpos Antivirais/imunologia , Gastroenterite Suína Transmissível/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Vacinas Virais/uso terapêutico , Administração Oral , Animais , Anticorpos Antivirais/administração & dosagem , Gastroenterite Suína Transmissível/mortalidade , Vetores Genéticos , Humanos , Imunização Passiva/métodos , Imunoglobulina E/imunologia , Testes de Neutralização , Extratos Vegetais/imunologia , Extratos Vegetais/uso terapêutico , Folhas de Planta/imunologia , Recombinação Genética , Suínos , Vírus da Gastroenterite Transmissível/genética , Vacinas Sintéticas/uso terapêuticoRESUMO
A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.
Assuntos
Síndrome de Angelman/patologia , Desenvolvimento Embrionário/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Cerebelo/patologia , Cerebelo/ultraestrutura , Feminino , Fibroblastos , Fatores de Troca do Nucleotídeo Guanina/genética , Heterozigoto , Homozigoto , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Gravidez , Células de Purkinje/patologia , Células de Purkinje/ultraestrutura , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The results presented demonstrate the expression of pfkfb4 gene in adult testis and in a mouse spermatogonia germ cell line (GC-1spg). The genomic organization of the human pfkfb4 gene shows the existence of 14 exons and 13 introns, spanning 45 kb. A detailed analysis of the 5'-flanking region by transient transfection assays with different 5'-deletion promoter constructs in GC-1spg and mouse sertoli cells (TM-4), allows us to define the minimal promoter unit, containing several GC-rich and ETF sequences along the first -141 nucleotides involved in basal expression. This gene is activated by serum and chemical hypoxia (CoCl(2) treatment) whereas beta-estradiol decreases its expression.
Assuntos
Região 5'-Flanqueadora/genética , Fosfofrutoquinase-2/genética , Regiões Promotoras Genéticas/genética , Espermatogônias/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Éxons/genética , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Células Germinativas/metabolismo , Humanos , Íntrons/genética , Luciferases/genética , Masculino , Camundongos , Dados de Sequência Molecular , Fosfofrutoquinase-2/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Deleção de Sequência/genética , Espermatogônias/citologia , Testículo/química , Testículo/metabolismo , Distribuição TecidualRESUMO
6-Phosphofructo-2-kinase catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase. These properties confer to this bifunctional enzyme a key role in the control of glycolysis and gluconeogenesis. Several mammalian isozymes generated by alternative splicing from four genes, designated pfkfb1-4, have been identified. The results presented in this study demonstrate the expression of the pfkfb3 gene in C2C12 cells and its downregulation during myogenic cell differentiation. We also show that the decrease of ubiquitous 6-phosphofructo-2-kinase isozyme levels, product of pfkfb3 gene, is due to its enhanced degradation through the ubiquitin-proteasome proteolytic pathway.
Assuntos
Diferenciação Celular/fisiologia , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Mioblastos/metabolismo , Fosfofrutoquinase-2/metabolismo , Ubiquitina/metabolismo , Animais , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leupeptinas/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Mioblastos/citologia , Fosfofrutoquinase-2/genética , Complexo de Endopeptidases do Proteassoma , Proteínas/genética , Proteínas/metabolismoRESUMO
INTRODUCTION AND OBJECTIVES: Urokinase-type plasminogen activator, which is encoded by the PLAU gene, plays a prominent role during collateral arterial growth. We investigated whether the PLAU P141L (C > T) polymorphism, which causes a mutation in the kringle domain of the protein, is associated with coronary collateral circulation in a cohort of 676 patients with coronary artery disease. METHODS: The polymorphism was genotyped in blood samples using a TaqMan-based genotyping assay, and collateral circulation was assessed by the Rentrop method. Multivariate logistic regression models adjusted by clinically relevant variables to estimate odds ratios were used to examine associations of PLAU P141L allelic variants and genotypes with collateral circulation. RESULTS: Patients with poor collateral circulation (Rentrop 0-1; n = 547) showed a higher frequency of the TT genotype than those with good collateral circulation (Rentrop 2-3; n = 129; P = .020). The T allele variant was also more common in patients with poor collateral circulation (P = .006). The odds ratio of having poorly developed collaterals in patients bearing the T allele (adjusted for clinically relevant variables) was statistically significant under the dominant model (odds ratio = 1.83 [95% confidence interval, 1.16-2.90]; P = .010) and the additive model (odds ratio = 1.73 [95% confidence interval, 1.14-2.62]; P = .009). CONCLUSIONS: An association was found between coronary collateral circulation and the PLAU P141L polymorphism. Patients with the 141L variant are at greater risk of developing poor coronary collateral circulation.
Assuntos
Circulação Colateral/genética , Doença das Coronárias/genética , Polimorfismo de Nucleotídeo Único/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Idoso , Circulação Colateral/efeitos da radiação , Angiografia Coronária , Feminino , Estudos de Associação Genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
Cattle and sheep that had received a primary course of vaccination with an inactivated bluetongue virus serotype 8 (BTV-8) vaccine were booster vaccinated 6 or 12 months later with the homologous vaccine or an alternative inactivated BTV-8 vaccine and neutralising antibody responses were determined. Antibody titres to the alternative vaccine were significantly higher than to the homologous vaccine (P=0.013) in cattle. There was no significant difference between the antibody responses to alternative and homologous vaccines in sheep. These data indicate that cattle and sheep primed with one inactivated BTV-8 vaccine may be effectively boosted with an alternative commercial inactivated BTV-8 vaccine.
Assuntos
Anticorpos Neutralizantes/sangue , Vírus Bluetongue/imunologia , Bluetongue/prevenção & controle , Imunização Secundária/veterinária , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Bluetongue/imunologia , Bovinos , Feminino , Masculino , Ovinos , Vacinas de Produtos InativadosRESUMO
Due to the vast economic consequences of classical swine fever (CSF) outbreaks, emergency vaccination plans are under discussion in European Union Member States. However, animals vaccinated with the conventional C-strain vaccine are subject to trade restrictions. To ease these restrictions, potent marker vaccines are required. One promising candidate is the chimeric pestivirus CP7_E2alf. For emergency vaccination in a CSF outbreak scenario, early onset of immunity is required. Here, the studies performed with a CP7_E2alf virus stock produced under good manufacturing conditions (GMP) are reported. In challenge experiments, CP7_E2alf induced full clinical protection 1 week after intramuscular vaccination, and 2 weeks after oral immunization. Furthermore, even after application of diluted vaccine preparations complete protection could be achieved if challenge infection was carried out 4 weeks after vaccination. In conclusion, GMP-produced CP7_E2alf proved to be a suitable marker vaccine candidate - also for emergency vaccination - both after intramuscular and oral application.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Vacinas Virais/imunologia , Administração Oral , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Peste Suína Clássica/imunologia , Vírus da Febre Suína Clássica/genética , Injeções Intramusculares , Mutação , RNA Viral/genética , Análise de Sequência de RNA , SuínosRESUMO
The ubiquitous isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (uPFK-2), a product of the Pfkfb3 gene, plays a crucial role in the control of glycolytic flux. In this study, we demonstrate that Pfkfb3 gene expression is increased in streptozotocin-induced diabetic mouse liver. The Pfkfb3/-3566 promoter construct linked to the luciferase reporter gene was delivered to the liver via hydrodynamic gene transfer. This promoter was upregulated in streptozotocin-induced diabetic mouse liver compared with transfected healthy cohorts. In addition, increases were observed in Pfkfb3 mRNA and uPFK-2 protein levels, and intrahepatic fructose-2,6-bisphosphate concentration. During streptozotocin-induced diabetes, phosphorylation of both p38 mitogen-activated protein kinase and Akt was detected, together with the overexpression of the proliferative markers cyclin D and E2F. These findings indicate that uPFK-2 induction is coupled to enhanced hepatocyte proliferation in streptozotocin-induced diabetic mouse liver. Expression decreased when hepatocytes were treated with either rapamycin or LY 294002. This shows that uPFK-2 regulation is phosphoinositide 3-kinase-Akt-mammalian target of rapamycin dependent. These results indicate that fructose-2,6-bisphosphate is essential to the maintenance of the glycolytic flux necessary for providing energy and biosynthetic precursors to dividing cells.
Assuntos
Proliferação de Células , Fígado/enzimologia , Fosfofrutoquinase-2/genética , Transdução de Sinais , Transcrição Gênica , Animais , Diabetes Mellitus Experimental , Frutosedifosfatos/análise , Glicólise , Camundongos , RNA Mensageiro/análise , Regulação para CimaRESUMO
Introducción y objetivos El gen PLAU, que codifica para el activador del plasminógeno tipo urocinasa, desempeña un papel destacado en el crecimiento colateral. Se ha investigado si el polimorfismo PLAUP141L ( C > T), que causa una mutación en el dominio kringle de la proteína, se asocia con la circulación colateral coronaria en una cohorte de 676 pacientes con enfermedad arterial coronaria. Métodos Se genotipificó el polimorfismo de muestras de sangre mediante prueba basada en TaqMan, y la circulación colateral se evaluó por el método Rentrop. Las asociaciones de las variantes alélicas y los genotipos con la circulación colateral se examinaron mediante modelos de regresión logística multivariable ajustados por las variables clínicamente relevantes. Resultados Los pacientes con circulación colateral deficiente (Rentrop 0-1; n = 547) presentaron mayor frecuencia del genotipo TT que aquellos con buena circulación colateral (Rentrop 2-3; n = 129; p = 0,020). Por otra parte, el alelo T fue más frecuente en los paciente con circulación deficiente (p = 0,006). La odds ratio de los portadores del alelo T de presentar una circulación colateral deficiente (ajustada por variables clínicamente relevantes) fue estadísticamente significativa en el modelo dominante (odds ratio = 1,83 [intervalo de confianza del 95%, 1,16-2,90]; p = 0,010) o el aditivo (odds ratio = 1,73 [intervalo de confianza del 95%, 1,14-2,62]; p = 0,009). Conclusiones Se demuestra una asociación entre la circulación colateral coronaria y el polimorfismo PLAUP141L. Los pacientes con la variante 141L tienen mayor riesgo de tener una circulación colateral deficiente (AU)
Introduction and objectives Urokinase-type plasminogen activator, which is encoded by the PLAU gene, plays a prominent role during collateral arterial growth. We investigated whether the PLAU P141L (C > T) polymorphism, which causes a mutation in the kringle domain of the protein, is associated with coronary collateral circulation in a cohort of 676 patients with coronary artery disease. Methods The polymorphism was genotyped in blood samples using a TaqMan-based genotyping assay, and collateral circulation was assessed by the Rentrop method. Multivariate logistic regression models adjusted by clinically relevant variables to estimate odds ratios were used to examine associations of PLAU P141L allelic variants and genotypes with collateral circulation. Results Patients with poor collateral circulation (Rentrop 0-1; n = 547) showed a higher frequency of the TT genotype than those with good collateral circulation (Rentrop 2-3; n = 129; P = .020). The T allele variant was also more common in patients with poor collateral circulation (P = .006). The odds ratio of having poorly developed collaterals in patients bearing the T allele (adjusted for clinically relevant variables) was statistically significant(..) (AU)
Assuntos
Humanos , Doença da Artéria Coronariana/genética , Polimorfismo de Nucleotídeo Único/genética , Ativador de Plasminogênio Tipo Uroquinase/análise , Circulação Colateral/fisiologia , Estudos de Associação Genética , GenótipoRESUMO
To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full-length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The alphaSIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the alpha-CH3 domain from human IgA. To express the full-length IgA, the individual light and heavy chains from the TGEV-specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co-infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant-expressed alphaSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody-expressing plant tissue to 2-day-old piglets showed that both the alphaSIP and full-length IgA molecules can provide in vivo protection against TGEV.
Assuntos
Anticorpos/imunologia , Comovirus/genética , Coronavirus/imunologia , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Proteínas de Plantas/imunologia , Potexvirus/genética , Animais , Anticorpos/genética , Anticorpos/metabolismo , Vetores Genéticos/genética , Imunoglobulina A/genética , Região Variável de Imunoglobulina/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Suínos , Transfecção/métodosRESUMO
The up-regulation of glycolysis to enhance the production of energy under reduced pO(2) is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1-4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5'-flanking region. Extensive analysis of the 5' pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1alpha null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.
Assuntos
Hipóxia Celular/fisiologia , Regiões Promotoras Genéticas , Proteínas/genética , Proteínas/fisiologia , Fatores de Transcrição/química , Animais , Sítios de Ligação , Biotinilação , Western Blotting , Linhagem Celular , Proliferação de Células , Cobalto/química , Desferroxamina/química , Elementos Facilitadores Genéticos , Frutosedifosfatos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Genes Reporter , Glicina/química , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Quelantes de Ferro/farmacologia , Luciferases/metabolismo , Camundongos , Modelos Genéticos , Oligonucleotídeos/química , Oxigênio/metabolismo , Fosfofrutoquinase-2 , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Interferência de RNA , Receptores de Estrogênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Software , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Regulação para CimaRESUMO
In vitro cytokine profiles of peripheral blood mononuclear cells (PBMC) from pigs with postweaning multisystemic wasting syndrome (PMWS) and healthy pigs were determined in response to recall viral antigens (porcine circovirus type 2; PCV2), mitogens (phytohaemagglutinin) or superantigens (staphylococcal enterotoxin B). PBMC from PMWS-affected pigs, in contrast to those from healthy pigs, responded to recall PCV2 antigen by releasing IL-10 and IFN-gamma, but they were less able or even unable to produce IL-4, IL-2 or IFN-gamma upon challenge with mitogen or superantigen. Moreover, only PCV2 had the ability to downregulate or suppress the release of IL-4 and IL-2 from PBMC from both healthy and diseased animals, and to stimulate the production of pro-inflammatory cytokines (IL-1beta, IL-8). In conclusion, the immune system cells of PMWS pigs have a diminished ability to perform their immunological functions upon viral or immunostimulatory molecules. In addition, PCV2 can alter the functionality of PBMC in both healthy and PMWS pigs.
Assuntos
Antígenos Virais/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Citocinas/biossíntese , Leucócitos Mononucleares/imunologia , Doenças dos Suínos/imunologia , Síndrome de Emaciação/veterinária , Animais , Infecções por Circoviridae/imunologia , Citocinas/análise , Enterotoxinas/imunologia , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Fito-Hemaglutininas/imunologia , Superantígenos/imunologia , Suínos , Doenças dos Suínos/sangue , Síndrome de Emaciação/imunologiaRESUMO
Porcine circovirus type 2 (PCV2) is considered as the causative agent of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs, where the virus is ubiquitous as evidenced by serological surveys. We present the results of the first nationwide sero-survey on the presence of PCV2 antibodies in European wild boars, and report the first PMWS case in a wild boar from Spain. Sera from 656 hunter harvested wild boars from 45 different geographical sites and 22 additional imported animals were analysed by means of an immunoperoxidase monolayer assay (IPMA). We also examined the tissues from 55 healthy and one diseased wild boars for the presence of PCV2 nucleic acid and PMWS lesions by in situ hybridisation and histopathology, respectively. Additionally, abundance estimates of wild boars and field interviews were carried out on 30 sampling sites. The prevalence of medium to high PCV2 serological titres among the examined wild boars was 47.89 +/- 1.9%. Seropositive wild boars appeared in all but one of the geographical regions analysed. Seroprevalence and titre of PCV2 antibodies were closely related to the management of the wild boar populations. Wild boars from intensively managed, farm-like populations had higher prevalence than wild boars living in more natural situations. The effect of wild boar abundance and management on PCV2 antibody prevalence was further evidenced by the high correlation existing between the relative abundance estimates of animals and the percentage of wild boars with medium to high levels of PCV2 antibodies. PCV2 nucleic acid was detected in the tissues of three wild boars. One of these was diagnosed as PMWS. The results, in addition to information on piglet mortalities, suggest a potential role of PMWS in piglet mortality in intensively managed wild boar populations.
Assuntos
Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Animais , Animais Selvagens , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Feminino , Masculino , Prevalência , Espanha/epidemiologia , SuínosRESUMO
The objective of this study was to evaluate the effect of a commercial vaccine adjuvant on the clinical and pathological outcome of PCV2 experimentally infected 8 to 9-week-old conventional pigs. Forty-four pigs were divided into four groups: non-infected control pigs, pigs that received a vaccine adjuvant, pigs inoculated with PCV2, and pigs inoculated with PCV2 together with the vaccine adjuvant. Infection was monitored until 69 days post-inoculation (PI). Some PCV2 inoculated pigs had hyperthermia, but no other clinical signs were recorded. No characteristic PMWS gross or microscopic lesions were observed in any of the pigs. PCV2 DNA was detected in lymphoid tissues by in situ hybridisation in 6 PCV2 inoculated pigs on day 69 PI. All PCV2 inoculated pigs seroconverted between days 21 and 49 PI, shortly after viremia detection. Moreover, viremia was detected between days 7 and 69 PI using PCR. A peak of the virus load was detected by real-time quantitative PCR between days 14 and 21 PI. There were no significant differences in the proportion of PCV2 positive serum and in the viral load between PCV2 and PCV2 + adjuvant inoculated pigs. Although PMWS was not reproduced in neither PCV2 nor PCV2 + adjuvant inoculated pigs, viremia detection and seroconversion indicated that all PCV2 inoculated pigs developed a chronic long-term asymptomatic infection. An increase of PCV2 replication was not observed in pigs inoculated with the adjuvant. These results indicate that the principle of immunostimulation may not be applicable under the experimental conditions used, suggesting that not all adjuvants used in commercial vaccines are capable of triggering mechanisms for PMWS development.