RESUMO
RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.
Assuntos
Quitridiomicetos , Técnicas de Silenciamento de Genes , Glutamato-Cisteína Ligase , Interferência de RNA , RNA Mensageiro , RNA Interferente Pequeno , RNA Interferente Pequeno/genética , RNA Mensageiro/genética , Animais , Quitridiomicetos/genética , Quitridiomicetos/patogenicidade , Glutamato-Cisteína Ligase/genética , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Anfíbios/microbiologia , Glutationa/metabolismoRESUMO
The aim of this study is to determine the acute effects of resistance and plyometric training on sprint and change of direction (COD) performance in healthy adults and adolescents. A systematic literature search was conducted via Medline, Cinahl, Scopus and SportDiscus databases for studies that investigated: 1) healthy male, female adults, or adolescents; and 2) measured sprint or change of direction performance following resistance and plyometric exercises. Studies were excluded if: 1) resistance or plyometric exercises was not used to induce muscle damage; 2) conducted in animals, infants, elderly; 3) sprint performance and/or agility performance was not measured 24 h post muscle damaging protocol. Study appraisal was completed using the Kmet Quality Scoring for Quantitative Study tool. Forest plots were generated to quantitatively analyse data and report study statistics for statistical significance and heterogeneity. The included studies (n = 20) revealed sprint and COD performance was significantly impaired up to 72 hr following resistance and plyometric exercises; both protocols significantly increased creatine kinase (CK), delayed-onset muscle soreness (DOMS) and decreased countermovement jump (CMJ) up to 72 hr. The systematic review of 20 studies indicated that resistance and plyometric training significantly impaired sprint and COD performance up to 72 hours post-exercise. Both training protocols elevated exercise-induced muscle damage (EIMD) markers (CK, DOMS) and decreased CMJ performance within the same timeframe.
RESUMO
Tuberculosis (TB) claims 1.5 million lives per year. This situation is largely due to the low efficacy of the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG) against pulmonary TB. The metabolic disease type 2 diabetes (T2D) is a risk factor for TB and the mechanisms underlying increased TB susceptibility in T2D are not well understood. Furthermore, it is unknown if new TB vaccines will provide protection in the context of T2D. Here we used a diet-induced murine model of T2D to investigate the underlying mechanisms of TB/T2D comorbidity and to evaluate the protective capacity of two experimental TB vaccines in comparison to conventional BCG. Our data reveal a distinct immune dysfunction that is associated with diminished recognition of mycobacterial antigens in T2D. More importantly, we provide compelling evidence that mucosal delivery of recombinant BCG strains expressing the Mycobacterium tuberculosis (Mtb) ESX-1 secretion system (BCG::RD1 and BCG::RD1 ESAT-6 ∆92-95) are safe and confer superior immunity against aerosol Mtb infection in the context of T2D. Our findings suggest that the remarkable anti-TB immunity by these recombinant BCG strains is achieved via augmenting the numbers and functional capacity of antigen presenting cells in the lungs of diabetic mice.
Assuntos
Antígenos de Bactérias/farmacologia , Proteínas de Bactérias/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Vacina BCG , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Diabetes Mellitus Experimental , Modelos Animais de Doenças , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Vacinas contra a Tuberculose/imunologia , VacinaçãoRESUMO
Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic platforms, including molecular assays with quantitative PCR (qPCR) and RNA-sequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings.
Assuntos
RNA , Succinato Desidrogenase , Biomarcadores , Coleta de Amostras Sanguíneas/métodos , Perfilação da Expressão Gênica/métodos , RNA/genética , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , Succinato Desidrogenase/genética , Proteína de Ligação a TATA-Box/genética , TemperaturaRESUMO
Comorbid type 2 diabetes poses a great challenge to the global control of tuberculosis. Here, we assessed the efficacy of metformin (MET), an antidiabetic drug, in mice infected with a very low dose of Mycobacterium tuberculosis In contrast to diabetic mice, infected nondiabetic mice that received the same therapeutic concentration of MET presented with significantly higher disease burden. This warrants further studies to investigate the disparate efficacy of MET against tuberculosis in diabetic and nondiabetic individuals.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Metformina , Mycobacterium tuberculosis , Tuberculose , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Tuberculose/tratamento farmacológicoRESUMO
Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.
Assuntos
Doenças Autoimunes/etiologia , Interferon gama/metabolismo , Interleucina-17/metabolismo , Cardiopatia Reumática/etiologia , Infecções Estreptocócicas/patologia , Streptococcus/imunologia , Animais , Antígenos de Bactérias/imunologia , Doenças Autoimunes/microbiologia , Doenças Autoimunes/fisiopatologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Modelos Animais de Doenças , Feminino , Doenças das Valvas Cardíacas/etiologia , Doenças das Valvas Cardíacas/microbiologia , Doenças das Valvas Cardíacas/fisiopatologia , Miocardite/etiologia , Miocardite/microbiologia , Miocardite/fisiopatologia , Ratos Endogâmicos Lew , Cardiopatia Reumática/microbiologia , Cardiopatia Reumática/fisiopatologia , Streptococcus/patogenicidadeRESUMO
OBJECTIVE: Sclerostin (SOST) has been identified as an important regulator of bone formation; however, it has not been previously implicated in arterial disease. The aim of this study was to assess the role of SOST in aortic aneurysm (AA) and atherosclerosis using human samples, a mouse model, and in vitro investigations. APPROACH AND RESULTS: SOST protein was downregulated in human and mouse AA samples compared with controls. Transgenic introduction of human SOST in apolipoprotein E-deficient (ApoE-/-) mice (SOSTTg .ApoE-/-) and administration of recombinant mouse Sost inhibited angiotensin II-induced AA and atherosclerosis. Serum concentrations of several proinflammatory cytokines were significantly reduced in SOSTTg .ApoE-/- mice. Compared with controls, the aortas of mice receiving recombinant mouse Sost and SOSTTg .ApoE-/- mice showed reduced matrix degradation, reduced elastin breaks, and preserved collagen. Decreased inflammatory cell infiltration and a reduction in the expression of wingless-type mouse mammary virus integration site/ß-catenin responsive genes, including matrix metalloproteinase-9, osteoprotegerin, and osteopontin, were observed in the aortas of SOSTTg .ApoE-/- mice. SOST expression was downregulated and the wingless-type mouse mammary virus integration site/ß-catenin pathway was activated in human AA samples. The cytosine-phosphate-guanine islands in the SOST gene promoter showed significantly higher methylation in human AA samples compared with controls. Incubation of vascular smooth muscle cells with the demethylating agent 5-azacytidine resulted in upregulation of SOST, suggesting that SOST is epigenetically regulated. CONCLUSIONS: This study identifies that SOST is expressed in the aorta and downregulated in human AA possibly because of epigenetic silencing. Upregulating SOST inhibits AA and atherosclerosis development, with potential important implications for treating these vascular diseases.
Assuntos
Angiotensina II , Aneurisma Aórtico/prevenção & controle , Aterosclerose/prevenção & controle , Proteínas Morfogenéticas Ósseas/metabolismo , Glicoproteínas/administração & dosagem , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Idoso de 80 Anos ou mais , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aneurisma Aórtico/induzido quimicamente , Aneurisma Aórtico/genética , Aneurisma Aórtico/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Células Cultivadas , Citocinas/metabolismo , Epigênese Genética/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Marcadores Genéticos/genética , Predisposição Genética para Doença , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fenótipo , Remodelação Vascular/efeitos dos fármacosRESUMO
Neurologic melioidosis is a serious, potentially fatal form of Burkholderia pseudomallei infection. Recently, we reported that a subset of clinical isolates of B. pseudomallei from Australia have heightened virulence and potential for dissemination to the central nervous system. In this study, we demonstrate that this subset has a B. mallei-like sequence variation of the actin-based motility gene, bimA. Compared with B. pseudomallei isolates having typical bimA alleles, isolates that contain the B. mallei-like variation demonstrate increased persistence in phagocytic cells and increased virulence with rapid systemic dissemination and replication within multiple tissues, including the brain and spinal cord, in an experimental model. These findings highlight the implications of bimA variation on disease progression of B. pseudomallei infection and have considerable clinical and public health implications with respect to the degree of neurotropic threat posed to human health.
Assuntos
Burkholderia pseudomallei/genética , Infecções Bacterianas do Sistema Nervoso Central/microbiologia , Variação Genética , Melioidose/microbiologia , Proteínas dos Microfilamentos/genética , Animais , Austrália , Burkholderia mallei/genética , Burkholderia pseudomallei/isolamento & purificação , Infecções Bacterianas do Sistema Nervoso Central/mortalidade , Infecções Bacterianas do Sistema Nervoso Central/patologia , Doenças Transmissíveis Emergentes/microbiologia , Doenças Transmissíveis Emergentes/mortalidade , Doenças Transmissíveis Emergentes/patologia , Modelos Animais de Doenças , Progressão da Doença , Mormo/microbiologia , Humanos , Melioidose/mortalidade , Melioidose/patologia , Camundongos , Mucosa Nasal/microbiologia , Fagócitos/imunologia , Fagócitos/microbiologia , Virulência/genéticaRESUMO
OBJECTIVE: Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Activity of the local kallikrein-kinin system may be important in cardiovascular disease. The effect of kinin B2 receptor (B2R) agonist and antagonist peptides on experimental AAA was investigated. APPROACH AND RESULTS: AAA was induced in apolipoprotein E-deficient mice via infusion of angiotensin II (1.0 µg/kg per minute SC). B2R agonists or antagonists were given via injection (2 mg/kg IP) every other day. The B2R agonist (B9772) promoted aortic rupture in response to angiotensin II associated with an increase in neutrophil infiltration of the aorta in comparison to controls. Mice receiving a B2R/kinin B1 receptor antagonist (B9430) were relatively protected from aortic rupture. Neutrophil depletion abrogated the ability of the B2R agonist to promote aortic rupture. Progression of angiotensin II-induced aortic dilatation was inhibited in mice receiving a B2R antagonist (B9330). Secretion of metalloproteinase-2 and -9, osteoprotegerin, and osteopontin by human AAA explant was reduced in the presence of the B2R antagonist (B9330). B2R agonist and antagonist peptides enhanced and inhibited, respectively, angiotensin II-induced neutrophil activation and aortic smooth muscle cell inflammatory phenotype. The B2R antagonist (B9330; 5 µg) delivered directly to the aortic wall 1 week post-AAA induction with calcium phosphate in a rat model reduced aneurysm growth associated with downregulation of aortic metalloproteinase-9. CONCLUSIONS: B2R signaling promotes aortic rupture within a mouse model associated with the ability to stimulate inflammatory phenotypes of neutrophils and vascular smooth muscle cells. B2R antagonism could be a potential therapy for AAA.
Assuntos
Angiotensina II , Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Ruptura Aórtica/metabolismo , Apolipoproteínas E/deficiência , Receptor B2 da Bradicinina/metabolismo , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/prevenção & controle , Ruptura Aórtica/genética , Ruptura Aórtica/patologia , Ruptura Aórtica/prevenção & controle , Apolipoproteínas E/genética , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Fosfatos de Cálcio , Dilatação Patológica , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Knockout , Ativação de Neutrófilo/efeitos dos fármacos , Osteopontina/metabolismo , Osteoprotegerina/metabolismo , Fenótipo , Ratos Sprague-Dawley , Receptor B2 da Bradicinina/agonistas , Transdução de Sinais , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Diabetes has been recognized as an important risk factor for a variety of intracellular bacterial infections, but research into the dysregulated immune mechanisms contributing to the impaired host-pathogen interactions is in its infancy. Diabetes is characterized by a chronic state of low-grade inflammation due to activation of pro-inflammatory mediators and increased formation of advanced glycation end products. Increased oxidative stress also exacerbates the chronic inflammatory processes observed in diabetes. The reduced phagocytic and antibacterial activity of neutrophils and macrophages provides an intracellular niche for the pathogen to replicate. Phagocytic and antibacterial dysfunction may be mediated directly through altered glucose metabolism and oxidative stress. Furthermore, impaired activation of natural killer cells contributes to decreased levels of interferon-γ, required for promoting macrophage antibacterial mechanisms. Together with impaired dendritic cell function, this impedes timely activation of adaptive immune responses. Increased intracellular oxidation of antigen-presenting cells in individuals with diabetes alters the cytokine profile generated and the subsequent balance of T-cell immunity. The establishment of acute intracellular bacterial infections in the diabetic host is associated with impaired T-cell-mediated immune responses. Concomitant to the greater intracellular bacterial burden and potential cumulative effect of chronic inflammatory processes, late hyper-inflammatory cytokine responses are often observed in individuals with diabetes, contributing to systemic pathology. The convergence of intracellular bacterial infections and diabetes poses new challenges for immunologists, providing the impetus for multidisciplinary research.
Assuntos
Infecções Bacterianas/epidemiologia , Infecções Bacterianas/imunologia , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/imunologia , Imunidade Ativa/imunologia , Estresse Oxidativo/imunologia , Células Dendríticas/imunologia , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Inflamação/imunologia , Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Fatores de Risco , Linfócitos T/imunologiaRESUMO
The death rate for neurologic melioidosis is high. Whether certain Burkholderia pseudomallei strains are more likely than other strains to cause central nervous system infection and whether route of infection influences the neurotropic threat remain unclear. Therefore, we compared the virulence and dissemination of Australian clinical isolates collected during October 1989-October 2012 from patients with neurologic and nonneurologic melioidosis after intranasal and subcutaneous infection of mice in an experimental model. We did not observe neurotropism as a unique characteristic of isolates from patients with neurologic melioidosis. Rather, a distinct subset of B. pseudomallei strains appear to have heightened pathogenic potential for rapid dissemination to multiple tissues, including the central nervous system, irrespective of the infection route. This finding has valuable public health ramifications for initiating appropriate and timely therapy after exposure to systemically invasive B. pseudomallei strains. Increasing understanding of B. pseudomallei pathology and its influencing factors will further reduce illness and death from this disease.
Assuntos
Burkholderia pseudomallei/patogenicidade , Infecções Bacterianas do Sistema Nervoso Central/microbiologia , Melioidose/microbiologia , Adolescente , Adulto , Idoso , Animais , Carga Bacteriana , Sistema Nervoso Central/microbiologia , Sistema Nervoso Central/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Virulência , Adulto JovemAssuntos
Miocardite , Cardiopatia Reumática , Animais , Interferon gama , Interleucina-17 , Ratos , Ratos Endogâmicos Lew , StreptococcusRESUMO
Burkholderia pseudomallei, the etiological agent for melioidosis, is an important cause of community-acquired sepsis in northern Australia and northeast Thailand. Due to the rapid dissemination of disease in acute melioidosis, we hypothesized that dendritic cells (DC) could act as a vehicle for dissemination of B. pseudomallei. Therefore, this study investigated the effect of B. pseudomallei infection on DC migration capacity and whether migration of DC enabled transportation of B. pseudomallei from the site of infection. B. pseudomallei stimulated significantly increased migration of bone marrow-derived DC (BMDC), both in vitro and in vivo, compared to uninfected BMDC. Furthermore, migration of BMDC enabled significantly increased in vitro trafficking of B. pseudomallei and in vivo dissemination of B. pseudomallei to secondary lymphoid organs and lungs of C57BL/6 mice. DC within the footpad infection site of C57BL/6 mice also internalized B. pseudomallei and facilitated dissemination. Although DC have previously been shown to kill intracellular B. pseudomallei in vitro, the findings of this study demonstrate that B. pseudomallei-infected DC facilitate the systemic spread of this pathogen.
Assuntos
Burkholderia pseudomallei/imunologia , Burkholderia pseudomallei/fisiologia , Movimento Celular , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Melioidose/imunologia , Melioidose/microbiologia , Animais , Modelos Animais de Doenças , Endocitose , Pulmão/microbiologia , Tecido Linfoide/microbiologia , Melioidose/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior.
Assuntos
Diagnóstico por Imagem , Sistema Imunitário/patologia , Linfonodos/imunologia , Linfonodos/patologia , Doenças Linfáticas/patologia , Animais , Apresentação de Antígeno/imunologia , Citometria de Fluxo , Imunofluorescência , Proteínas de Fluorescência Verde/metabolismo , Estudos Longitudinais , Doenças Linfáticas/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fótons , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologiaRESUMO
AAA (abdominal aortic aneurysm) is an important cause of sudden death in older adults, but there is no current effective drug therapy for this disease. The UCNs (urocortins1-3) and their receptors: CRFR (corticotrophin-releasing factor receptor)-1 and -2 have been implicated in various CVDs (cardiovascular diseases). We assessed the relative expression of UCN1-3 in AAA by qRT-PCR (quantitative reverse transcription-PCR) and ELISA, and examined in vitro how UCN2 affects human aortic VSMC (vascular smooth muscle cell) Akt phosphorylation, pro-inflammatory cytokine IL (interleukin)-6 secretion, proliferation, cell cycle and apoptosis. UCN2 and CRFR2 expression were significantly up-regulated in biopsies from the AAA body. AAA body biopsies released high amounts of UCN2 in vitro. Median plasma UCN2 concentrations were 2.20 ng/ml (interquartile range 1.14-4.55 ng/ml, n=67) in AAA patients and 1.11 ng/ml (interquartile range 0.76-2.55 ng/ml, n=67) in patients with non-aneurysmal PAD (peripheral artery disease) (P=0.001). Patients with UCN2 in the highest quartile had a 4.12-fold (95% confidence interval, 1.37-12.40) greater prevalence of AAA independent of other risk factors, P=0.012. In vitro, UCN2 significantly inhibited VSMC Akt phosphorylation and proliferation in a dose-dependent manner. UCN2 induced VSMC G1 cell-cycle arrest and increased IL-6 secretion over 24 h. The CRFR2 antagonist astressin-2B significantly abrogated the effects of UCN2 on VSMCs. In conclusion, UCN2 is significantly associated with AAA and inhibits VSMC proliferation by inducing a G1 cell cycle arrest suggesting a plausible regulatory role in AAA pathogenesis.
Assuntos
Aneurisma da Aorta Abdominal/fisiopatologia , Hormônio Liberador da Corticotropina/fisiologia , Músculo Liso Vascular/patologia , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Urocortinas/fisiologia , Proliferação de Células , Células Cultivadas , Hormônio Liberador da Corticotropina/sangue , Humanos , Interleucina-8/metabolismo , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Urocortinas/sangueRESUMO
OBJECTIVE: We aimed to determine the effect of mechanistic target of rapamycin inhibitor everolimus on abdominal aortic aneurysm within the angiotensin II (A2)-infused apolipoprotein E-deficient mouse model. APPROACH AND RESULTS: Abdominal aortic aneurysm was induced via subcutaneous infusion of A2. Flow cytometry demonstrated increased circulating and aortic C-C chemokine receptor 2 (CCR2) monocytes during A2 infusion. The number of CCR2 monocytes present within the aorta was positively correlated with suprarenal aortic diameter. Simultaneous infusion of everolimus via a second subcutaneous osmotic micropump inhibited A2-induced aortic dilatation. Using flow cytometry and Western blot analysis, decreased aortic dilatation was associated with reduced development of CCR2 bone marrow monocytes, fewer numbers of circulating CCR2 monocytes, and lower aortic CCR2 concentration. In vitro, everolimus inhibited A2-stimulated production of interferon (IFN)-γ and IFNγ-induced CCR2 expression in apolipoprotein E-deficient mouse bone marrow monocytes. Further, everolimus diminished IFNγ/lipopolysaccharide-stimulated M1 polarization in apolipoprotein E-deficient mouse bone marrow monocyte-differentiated macrophages. CONCLUSIONS: Systemic administration of everolimus limits aortic aneurysm in the A2-infused apolipoprotein E-deficient mouse model via suppressed development of bone marrow CCR2 monocytes and reduced egress of these cells into the circulation.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Apolipoproteínas E/deficiência , Monócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores CCR2/metabolismo , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Angiotensina II , Animais , Aorta Abdominal/enzimologia , Aorta Abdominal/imunologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/patologia , Apolipoproteínas E/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Everolimo , Citometria de Fluxo , Bombas de Infusão Implantáveis , Infusões Subcutâneas , Interferon gama/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/enzimologia , Monócitos/imunologia , Monócitos/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Batrachochytrium dendrobatidis (Bd) is a lethal amphibian pathogen, partly due to its ability to evade the immune system of susceptible frog species. In many pathogenic fungi, the antioxidant glutathione is a virulence factor that helps neutralise oxidative stressors generated from host immune cells, as well as other environmental stressors such as heavy metals. The role of glutathione in stress tolerance in Bd has not been investigated. Here, we examine the changes in the glutathione pool after stress exposure and quantify the effect of glutathione depletion on cell growth and stress tolerance. Depletion of glutathione repressed growth and release of zoospores, suggesting that glutathione is essential for life cycle completion in Bd. Supplementation with <2 mM exogenous glutathione accelerated zoospore development, but concentrations >2 mM were strongly inhibitory to Bd cells. While hydrogen peroxide exposure lowered the total cellular glutathione levels by 42 %, glutathione depletion did not increase the sensitivity to hydrogen peroxide. Exposure to cadmium increased total cellular glutathione levels by 93 %. Glutathione-depleted cells were more sensitive to cadmium, and this effect was attenuated by glutathione supplementation, suggesting that glutathione plays an important role in cadmium tolerance. The effects of heat and salt were exacerbated by the addition of exogenous glutathione. The impact of glutathione levels on Bd stress sensitivity may help explain differences in host susceptibility to chytridiomycosis and may provide opportunities for synergistic therapeutics.
Assuntos
Batrachochytrium , Cádmio , Glutationa , Peróxido de Hidrogênio , Glutationa/metabolismo , Cádmio/toxicidade , Animais , Batrachochytrium/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Micoses/microbiologia , Micoses/veterinária , Micoses/metabolismo , Anfíbios/microbiologiaRESUMO
There are currently no acceptable treatments to limit progression of abdominal aortic aneurysm (AAA). Increased serum concentrations of high-density lipoprotein (HDL) are associated with reduced risk of developing an AAA. The present study aimed to assess the effects of fenofibrate on aortic dilatation in a mouse model of AAA. Male low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice were maintained on a high-fat diet for 3 weeks followed by 6 weeks of oral administration of vehicle or fenofibrate. From 14 to 18 weeks of age, all mice were infused with angiotensin II (AngII). At 18 weeks of age, blood and aortas were collected for assessment of serum lipoproteins, aortic pathology, aortic Akt1 and endothelial nitric oxide synthase (eNOS) activities, immune cell infiltration, eNOS and inducible NOS (iNOS) expression, sphingosine 1 phosphate (S1P) receptor status, and apoptosis. Mice receiving fenofibrate had reduced suprarenal aortic diameter, reduced aortic arch Sudan IV staining, higher serum HDL levels, increased serum S1P concentrations, and increased aortic Akt1 and eNOS activities compared with control mice. Macrophages, T lymphocytes, and apoptotic cells were less evident and eNOS, iNOS, and S1P receptors 1 and 3 were up-regulated in aortas from mice receiving fenofibrate. The present findings suggest that fenofibrate antagonizes AngII-induced AAA and atherosclerosis by up-regulating serum HDL and S1P levels, with associated activation of NO-producing enzymes and reduction of aortic inflammation.
Assuntos
Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/patologia , Progressão da Doença , Fenofibrato/uso terapêutico , Lipoproteínas HDL/sangue , Lisofosfolipídeos/sangue , Esfingosina/análogos & derivados , Angiotensina II , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aorta/patologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Aneurisma da Aorta Abdominal/complicações , Aneurisma da Aorta Abdominal/tratamento farmacológico , Apoptose/efeitos dos fármacos , Compostos Azo/metabolismo , Dilatação Patológica/sangue , Dilatação Patológica/complicações , Dilatação Patológica/tratamento farmacológico , Dilatação Patológica/patologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Fenofibrato/farmacologia , Inflamação/sangue , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/patologia , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/sangue , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacosRESUMO
Reference genes are frequently used for the normalization of quantitative reverse transcriptase PCR (qRTPCR) data in gene expression studies. Staphylococcus aureus is one of the most common causes of biofilm-related infections. Savirin and ticagrelor show in vitro as well as in vivo antibiofilm activity against S. aureus. The main aim of this study was to identify the most stably expressed reference genes to study the effect of these molecules on genes in a strong biofilm producing S. aureus isolate isolated from biofilm-related infection. Quantitative real-time PCR was performed by using relative quantification method. Four different algorithms, delta Ct, normfinder, bestkeeper, and genorm, followed by a comprehensive analysis was used to identify the most stable reference genes from a list of sixteen different candidate reference genes. All four algorithms reported different results, with some comparable findings among some methods. In the comprehensive analysis of the results of all the algorithms used, the most stable reference genes found were spa, rpoD, and pyk for savirin treatment experiment and gapdH, gyrA, and gmk for ticagrelor treatment experiment. The optimal number of reference genes required was two for both the experimental conditions. Despite having some drawbacks, each algorithm can reliably determine an appropriate reference gene independently. However, based on consensus ranking and the required optimal number of reference genes reported, spa and rpoD were the most appropriate reference genes for savirin treatment experiment, and gapdH and gyrA were most appropriate for ticagrelor treatment experiment. This study provides baseline data on reference genes to study the effect of savirin or ticagrelor treatment on the expression of potential reference genes in S. aureus. We recommend prior re-validation of reference genes on a case-by-case basis before they can be used.
RESUMO
Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Global research efforts to improve TB control are hindered by insufficient understanding of the role that antibodies play in protective immunity and pathogenesis. This impacts knowledge of rational and optimal vaccine design, appropriate diagnostic biomarkers, and development of therapeutics. Traditional approaches for the prevention and diagnosis of TB may be less efficacious in high prevalence, remote, and resource-poor settings. An improved understanding of the immune response to the causative agent of TB, Mycobacterium tuberculosis (Mtb), will be crucial for developing better vaccines, therapeutics, and diagnostics. While memory CD4+ T cells and cells and cytokine interferon gamma (IFN-g) have been the main identified correlates of protection in TB, mounting evidence suggests that other types of immunity may also have important roles. TB serology has identified antibodies and functional characteristics that may help diagnose Mtb infection and distinguish between different TB disease states. To date, no serological tests meet the World Health Organization (WHO) requirements for TB diagnosis, but multiplex assays show promise for improving the sensitivity and specificity of TB serodiagnosis. Monoclonal antibody (mAb) therapies and serum passive infusion studies in murine models of TB have also demonstrated some protective outcomes. However, animal models that better reflect the human immune response to Mtb are necessary to fully assess the clinical utility of antibody-based TB prophylactics and therapeutics. Candidate TB vaccines are not designed to elicit an Mtb-specific antibody response, but evidence suggests BCG and novel TB vaccines may induce protective Mtb antibodies. The potential of the humoral immune response in TB monitoring and control is being investigated and these studies provide important insight into the functional role of antibody-mediated immunity against TB. In this review, we describe the current state of development of antibody-based clinical tools for TB, with a focus on diagnostic, therapeutic, and vaccine-based applications.