RESUMO
Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical "interferon signature genes" that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry.
Assuntos
Redes Reguladoras de Genes , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Conjuntos de Dados como Assunto , Humanos , Janus Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Interferon alfa e beta/metabolismoRESUMO
Small-molecule inhibitors of the Janus kinase family (JAKis) are clinically efficacious in multiple autoimmune diseases, albeit with increased risk of certain infections. Their precise mechanism of action is unclear, with JAKs being signaling hubs for several cytokines. We assessed the in vivo impact of pan- and isoform-specific JAKi in mice by immunologic and genomic profiling. Effects were broad across the immunogenomic network, with overlap between inhibitors. Natural killer (NK) cell and macrophage homeostasis were most immediately perturbed, with network-level analysis revealing a rewiring of coregulated modules of NK cell transcripts. The repression of IFN signature genes after repeated JAKi treatment continued even after drug clearance, with persistent changes in chromatin accessibility and phospho-STAT responsiveness to IFN. Thus, clinical use and future development of JAKi might need to balance effects on immunological networks, rather than expect that JAKis affect a particular cytokine response and be cued to long-lasting epigenomic modifications rather than by short-term pharmacokinetics.
Assuntos
Citocinas/metabolismo , Inibidores de Janus Quinases/farmacologia , Janus Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Citocinas/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/imunologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Fenômenos Imunogenéticos/genética , Janus Quinases/genética , Janus Quinases/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologiaRESUMO
Mycobacterium tuberculosis (MTB) is one of the most successful pathogens in human history and remains a global health challenge. MTB has evolved a plethora of strategies to evade the immune response sufficiently to survive within the macrophage in a bacterial-immunological equilibrium, yet causes sufficient immunopathology to facilitate its transmission. This review highlights MTB as the driver of disease pathogenesis and presents evidence of the mechanisms by which MTB manipulates the protective immune response into a pathological productive infection.
Assuntos
Evasão da Resposta Imune , Mycobacterium tuberculosis/imunologia , Tuberculoma/imunologia , Animais , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Tuberculoma/microbiologiaRESUMO
Atorvastatin is used to control cholesterol and lipid levels in hyperlipidaemic and hypercholesterolaemic patients. Myopathy and hepatotoxicity, however, have been reported as side effects in a small percentage of statin users. This study aimed to investigate the cytotoxicity and the effect of atorvastatin on microRNA expression in HepG2 cells. The methylthiazol tetrazolium assay was used to assess hepatocyte viability and at 20 µM atorvastatin (24 h) treatment were 82 ± 1.5% viable (P = 0.0002). Levels of intracellular ATP in cells treated with 20 µM atorvastatin were reduced by 1.25-fold, P = 0.002. Cytotoxicity, measured by the release of intracellular lactate dehydrogenase, was increased from 0.95 ± 0.29 units in control cells to 1.12 ± 0.02 units (P = 0.002) in atorvastatin treated cells. A panel of 84-miRNA species was used to evaluate the effect of atorvastatin on miRNA expression. MiR-124a was significantly up-regulated by atorvastatin (12.94-fold). A significant decrease in GAMT expression (3.54-fold) was observed in atorvastatin treated cells following quantitative PCR analysis. In addition, western blotting data showed GAMT protein levels were significantly lower than the controls (3.02-fold) and analysis of creatine levels in treated cells showed a significant decrease in the atorvastatin treated culture supernatant compared to control culture supernatant (32.33 ± 3.51 µM/l vs. 59.67 ± 1.52µM/l, P = 0.0056). This is the first study to show that atorvastatin up-regulates miR-124a levels and consequently modulates GAMT expression in hepatocytes.
Assuntos
Anticolesterolemiantes/farmacologia , Atorvastatina/farmacologia , Guanidinoacetato N-Metiltransferase/genética , Guanidinoacetato N-Metiltransferase/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas , Trifosfato de Adenosina/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Creatinina/metabolismo , Células Hep G2 , Humanos , Regulação para CimaRESUMO
Mice that lack the genes for IL-27, or the IL-27 receptor, and infected with Toxoplasma gondii develop T cell-mediated pathology. Here, studies were performed to determine the impact of endogenous IL-27 on the immune response to T. gondii in wild-type (WT) mice. Analysis of infected mice revealed the early production of IL-27p28 by a subset of Ly6Chi, inflammatory monocytes, and sustained IL-27p28 production at sites of acute and chronic infection. Administration of anti-IL-27p28 prior to infection resulted in an early (day 5) increase in levels of macrophage and granulocyte activation, as well as enhanced effector T cell responses, as measured by both cellularity, cytokine production, and transcriptional profiling. This enhanced acute response led to immune pathology, while blockade during the chronic phase of infection resulted in enhanced T cell responses but no systemic pathology. In the absence of IL-27, the enhanced monocyte responses observed at day 10 were a secondary consequence of activated CD4+ T cells. Thus, in WT mice, IL-27 has distinct suppressive effects that impact innate and adaptive immunity during different phases of this infection. IMPORTANCE: The molecule IL-27 is critical in limiting the immune response to the parasite Toxoplasma gondii. In the absence of IL-27, a lethal, overactive immune response develops during infection. However, when exactly in the course of infection this molecule is needed was unclear. By selectively inhibiting IL-27 during this parasitic infection, we discovered that IL-27 was only needed during, but not prior to, infection. Additionally, IL-27 is only needed in the active areas in which the parasite is replicating. Finally, our work found that a previously unstudied cell type, monocytes, was regulated by IL-27, which contributes further to our understanding of the regulatory networks established by this molecule.
Assuntos
Interleucina-27 , Toxoplasma , Toxoplasmose , Animais , Camundongos , Interleucina-27/metabolismo , Camundongos Endogâmicos C57BL , Monócitos , Linfócitos T , Toxoplasmose/parasitologiaRESUMO
Inhibitory receptors (IR) are a diverse group of cell surface molecules that modulate T cell activation, but there are gaps in our knowledge of the cell-extrinsic factors that regulate their expression. The present study found that in vivo overexpression of IL-27 in mice led to increased T cell expression of PD-L1, LAG-3, TIGIT, and TIM-3. In vitro, TCR stimulation alone promoted expression of multiple IRs, whereas IL-27 alone induced expression of PD-L1. However, the combination of intermediate TCR stimulation and IL-27 resulted in synergistic induction of LAG-3, CTLA-4, and TIGIT. In vivo, infection with Toxoplasma gondii resulted in parasite-specific effector T cells that expressed high levels of IR, and at local sites of infection where IL-27 production was highest, IL-27 was required for maximal effector cell expression of PD-L1, LAG-3, CTLA-4, and TIGIT. Together, these results affirm the critical role of TCR signals in the induction of IR expression but find that during infection, IL-27 promotes T cell expression of IR.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptores Coestimuladores e Inibidores de Linfócitos T/metabolismo , Interleucinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/parasitologia , Antígeno CTLA-4/metabolismo , Receptores Coestimuladores e Inibidores de Linfócitos T/genética , Feminino , Interleucinas/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/genética , Receptores Imunológicos/metabolismo , Baço/patologia , Toxoplasma , Toxoplasmose/imunologia , Transcriptoma , TransfecçãoRESUMO
BACKGROUND: Sirtuin 1 (SIRT1), a class III histone deacetylase, has been identified as a candidate molecule affecting the epigenetic mechanisms of cardiovascular disease (CVD). Previous studies have shown that some SIRT1 single-nucleotide polymorphisms (SNPs) are associated with body mass index, diabetes, blood pressure, cholesterol metabolism and coronary artery calcification. We investigated two A>G SIRT1 SNPs, rs1467568 and rs7895833, in young South African (SA) Indians with coronary artery disease (CAD) and compared them to Indian and black controls. METHODS: For rs1467568, a total of 287 subjects were recruited into this study (104 CAD patients, 99 age-, gender- and race-matched controls, and 84 age- and gender-matched black controls). For rs7895833, a total of 281 subjects were recruited into this study (100 CAD patients, 99 age-, gender- and race-matched controls, and 82 age- and gender-matched black controls). All patients were male, of Indian ethnicity, stable CAD confirmed on angiography, mean age 37.5 years; range 24-45. All subjects were genotyped using TaqMan SNP genotyping assays. RESULTS: The variant allele for both SNPs was found at a higher frequency in the total Indian group compared to the total black population (rs1467568: 41 vs 18.5%, respectively, p < 0.0001, OR = 3.190, 95% CI: 2.058-40943; and rs7895833: 41 vs 22%, respectively, p < 0.0001, OR = 2.466, 95% CI: 1.620- 3.755). Indian controls presented with a higher frequency for both SNPs compared to black controls (rs1467568: 40 vs 18.5%, respectively, p < 0.0001, OR = 2.996, 95% CI: 1.850- 4.853; and rs7895833: 41 vs 22%, respectively, p < 0.0001, OR = 2.513, 95% CI: 1.578-4.004). No difference was seen in the distribution of both SNPs between CAD patients and either control group. We did not observe any association between the SNPs and clinical parameters in CAD patients and controls. CONCLUSION: Both SNP variant alleles occurred more frequently in SA Indians than in SA blacks. A larger study group and further analysis is required to assess whether these SIRT1 SNPs may serve as risk factors that contribute to Indians developing early-onset CAD.
Assuntos
Doença da Artéria Coronariana/genética , Polimorfismo de Nucleotídeo Único , Sirtuína 1/genética , Adulto , Idade de Início , População Negra/genética , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/etnologia , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Índia/etnologia , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fenótipo , Fatores de Risco , África do Sul/epidemiologia , Adulto JovemRESUMO
BACKGROUND AND AIM: Tumor protein p53 (p53), classically referred to as a tumor suppressor gene, is involved in cell cycle regulation and may be related to atherosclerosis by affecting smooth muscle cell proliferation, a feature of atherogenesis. A polymorphism at codon 72 (rs1042522) results in functional variability and hence plays a role in the pathophysiology of coronary artery disease (CAD). This polymorphism has been well established for its role in cancer and has only recently been investigated in CAD. Limited data is available on South Africans (SA) of Indian ancestry. We examined associations of this polymorphism and clinical markers in a cohort of young SA Indian CAD patients. METHODS: A total of 284 subjects were recruited into this study which included 100 CAD patients (diagnosed on angiography, mean age 37.5, range 24-45years), 100 age- and sex-matched Indian controls and 84 age- and sex-matched Black controls. Polymorphic variants were assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Data for clinical markers were obtained from pathology reports. RESULTS: Genotype distribution differed significantly between CAD patients and Indian controls (Pro/Pro, Pro/Arg, Arg/Arg: 24%, 48%, 28% vs. 30%, 61%, 9% respectively, p=0.0025). There was a significant genotype distribution between Indian and Black controls (Pro/Pro, Pro/Arg, Arg/Arg: 30%, 61%, 9% vs. 45.2% 40.5%, 14.3% respectively, p=0.0212). A significantly higher frequency of the p53 Arg72 allele was found in CAD patients compared to controls (52% vs. 39.5% respectively, p=0.0159). The variant allele was slightly higher in Indian controls (39.5%) compared to Black controls (34.5%), but this did not reach statistical significance (p=0.3324). The levels of total cholesterol, LDL, HDL, triglycerides, fasting glucose, fasting insulin and %HbA1c were not significantly influenced by the p53 genotypic variants. CONCLUSION: Although the p53 codon 72 SNP is not associated with clinical markers of disease in CAD, the higher frequency of the variant allele in SA Indians may be a contributing factor for this population having an increased risk of developing premature CAD.
Assuntos
Doença da Artéria Coronariana/genética , Polimorfismo de Nucleotídeo Único , Proteína Supressora de Tumor p53/genética , Adulto , Substituição de Aminoácidos , Arginina/genética , Povo Asiático , População Negra , Estudos de Casos e Controles , Doença da Artéria Coronariana/etnologia , Humanos , África do SulRESUMO
T-helper (Th) 17 cells are a pro-inflammatory subset of CD4(+) effector T-cells critical in mucosal immunity. Imbalances in Th17 cell proportion have been implicated in the pathogenesis of several diseases; however, this has not been adequately explored in tuberculosis (TB) and human immunodeficiency virus (HIV) co-infection. Since Th17 cells are predominantly mucosally associated, we assessed Th17 proportion and associated microenvironment in pleural effusions from patients co-infected with TB/HIV. Our results show that TB(+)HIV(+) pleurisy results in significantly reduced frequency of CD4(+)IL-17(+)RORC(+)STAT3(+) Th17 cells compared to TB(-)HIV(-)ex vivo (p = 0.0054) and was confirmed in conditioned media studies in vitro (p = 0.0001). This was not associated with alterations in Th17 polarising cytokines IL-6, IL-21 and IL-23 or changes in Th17 signature cytokines IL-17A and F. However, the mRNA expression of Th17 signalling molecules, IL-6 (p = 0.0022), IL-6R (p = 0.0247), IL-1ß (p = 0.0022) and signal transducer and activator (STAT) 3 (p = 0.0022) were significantly upregulated. Notably, TB(+)HIV(+) pleural fluid contained significantly higher concentrations of IL-1ß (p = 0.0008), IL-22 (p = 0.0115), IL-31 (p = 0.0210), TNF-α (p = 0.0251) and IFN-γ (p = 0.0026) than TB(-)HIV(-) pleural fluid ex vivo. Taken together, this suggests a reduced portion of Th17 lymphocytes in TB/HIV pleurisy is independent of locally mediated cytokine polarisation.
Assuntos
Microambiente Celular , Coinfecção , Citocinas/imunologia , Infecções por HIV/imunologia , Células Th17/imunologia , Tuberculose Pleural/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Idoso , Contagem de Linfócito CD4 , Estudos de Casos e Controles , Células Cultivadas , Citocinas/genética , Feminino , Regulação da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Derrame Pleural/imunologia , Derrame Pleural/microbiologia , Derrame Pleural/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Th17/microbiologia , Células Th17/virologia , Tuberculose Pleural/genética , Tuberculose Pleural/microbiologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/microbiologia , Adulto JovemRESUMO
BACKGROUND: Hyperglycemia exacerbates the production of mitochondrial reactive oxygen species and this contributes to a variety of pathological conditions. Sirtuin 3 (SIRT3) has been shown to play a role in decreasing oxidative stress and improving disease outcomes by regulating antioxidant defense. Our understanding of molecular events during oxidative stress under chronic hyperglycemia in the liver is limited. We postulated that SIRT3 may play a role in antioxidant defense under hyperglycemic conditions in HepG2 cells. METHODS: Cell viability was determined in HepG2 and human embryonic kidney (HEK) 293 cells cultured in the presence of 5.5 mM glucose (control), 19.9 mM mannitol (osmotic control), 10 mM glucose and 30 mM glucose (hyperglycemic), and nicotinamide (NAM) (10 mM) at 24-hr and 72-hr time points. SIRT3, peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and cyclic adenosoine monophosphate (cAMP) response element binding protein (pCREB) protein expression were measured via western blotting. Mitochondrial antioxidant enzymes glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), and uncoupling protein 2 (UCP2) and mitochondrial DNA (mtDNA) repair enzyme 8-oxoguanine glycosylase (OGG1) were evaluated via quantitative PCR (qPCR). RESULTS: Increased cell viability and protein expression of SIRT3, pCREB, and PGC-1α were observed under hyperglycemic conditions at 24 hr. These were further elevated at the 72-hr time point. We also observed higher fold changes of SIRT3, GPx1, SOD2, UCP2, and OGG1 in the treated groups. Treatment with NAM decreased protein and gene expression of SIRT3, pCREB, PGC-1α, GPx1, SOD2, UCP2, and OGG1 at both time points in the hyperglycemic groups. CONCLUSIONS: Our data may allude to the relationship that has been established between SIRT3 and PGC-1α with regard to increased antioxidant defense during oxidative stress. This suggests that SIRT3 may play a role in increasing antioxidant defense and conferring resistance to oxidative stress-induced damage under hyperglycemic conditions in the human hepatoma cell line.
Assuntos
Antioxidantes/metabolismo , Hepatócitos/enzimologia , Hiperglicemia/enzimologia , Estresse Oxidativo , Sirtuína 3/metabolismo , Sobrevivência Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Hiperglicemia/genética , Hiperglicemia/patologia , Mitocôndrias Hepáticas/enzimologia , Niacinamida/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , RNA Mensageiro/metabolismo , Sirtuína 3/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium sp., a common contaminant of maize. FB1 inhibits sphingolipid biosynthesis, alters sphingosine/sphinganine ratios and modifies cell survival and cell death processes at varying propensities at both species- and tissue-specific level. We investigated the effect of FB1 on the apoptotic pathway in human hepatoma (HepG2) cells. We measured: (i) the level of cell proliferation and cell death mechanism of HepG2 cells (MTT assay, annexin V and propidium iodide staining, JC-1 assay, γH2AX and cleaved PARP and Hoechst staining); (ii) initiator and executioner caspase activity (luminometric enzyme activity assays); (iii) regulation of mRNA expression of pro- and anti- apoptotic molecules using an apoptosis array (qPCR) and (iv) levels of significantly altered apoptosis-related proteins (Western blotting) following a 24 h incubation. FB1 caused a dose-dependent decrease in cell viability with an inhibitory concentration for 50% of cell growth at 200 µM. FACS data showed FB1 induced a 2.5-fold increase in annexin V staining, however, caspase activity and mitochondrial depolarization was not significantly influenced. Cleaved PARP and γH2AX were significantly lower in treated cells with minimal DNA condensation and fragmentation observed with the Hoechst stain. BIRC-8/ILP-2 was most significantly up-regulated (8-fold; apoptosis array). ILP2 protein levels were elevated (2.3-fold) with a corresponding decrease in Smac/DIABLO protein levels (1.7-fold). Further analysis showed a dose-dependent increase in BIRC-8/ILP-2 mRNA and protein expression in HepG2 cells. We conclude that FB1 modulates apoptosis in a complex dose-dependent regulation of pro- and anti-apoptotic molecules.
Assuntos
Apoptose/efeitos dos fármacos , Fumonisinas/toxicidade , Proteínas Inibidoras de Apoptose/metabolismo , Fígado/efeitos dos fármacos , Micotoxinas/toxicidade , Proteínas Reguladoras de Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Relação Dose-Resposta a Droga , Células Hep G2 , Histonas/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Fígado/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
Methylenetetrahydrofolate reductase (MTHFR) reduces 5',10'-methylenetetrahydrofolate to 5'-methyltetrahydrofolate, and is involved in remethylation of homocysteine to methionine, two important reactions involved in folate metabolism and methylation pathways. The common MTHFR C677T single nucleotide polymorphism (SNP) (rs1801133) has been associated with raised levels of homocysteine, a well known risk factor for coronary artery disease (CAD). CAD is a major cause of mortality worldwide. The age of onset of this chronic disorder is on the decline, particularly in the Indian population. Indians in South Africa (SA) have a higher prevalence of premature CAD compared to Black South Africans. The MTHFR C677T SNP has not been investigated in the SA Indian population. The present study therefore investigated the MTHFR C677T SNP in young SA Indian males with CAD compared to young Indian and Black male controls. A total of 290 subjects were recruited into this study which included 106 CAD patients (diagnosed on angiography, mean age 37.5, range 24-45 years), 100 Indian male controls (mean age 37.5, range 28-45 years), and 84 Black male controls (mean age 36.4, range 25-45). Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) was used to genotype CAD patients and healthy controls. Data for clinical markers were obtained from pathology reports. There was a significant association between the 677 MTHFR variant (T) allele and CAD patients compared to the healthy Indian controls (p=0.0353, OR=2.105 95% CI 1.077-4.114). Indian controls presented with a higher frequency of the variant allele compared to Black controls (7% vs. 2% respectively, p=0.0515 OR=3.086 95% CI 0.9958-9.564). The MTHFR C677T SNP did not influence levels of total cholesterol, LDL, HDL, triglycerides, fasting glucose, fasting insulin, HbA1c or hsCRP. The higher frequency of the MTHFR 677 variant allele in South African Indians may be a contributing factor to the higher risk profile for the development of premature CAD in Indians.
Assuntos
Doença da Artéria Coronariana/genética , Predisposição Genética para Doença/genética , Indígenas Sul-Americanos/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo de Nucleotídeo Único , Adulto , População Negra/genética , Doença da Artéria Coronariana/etnologia , Frequência do Gene , Predisposição Genética para Doença/etnologia , Genótipo , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Fatores de Risco , África do Sul , Adulto JovemRESUMO
Fumonisin B1 (FB1), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in cancer promoting activity in animals and humans. FB1 disrupts DNA methylation and chromatin modifications in human hepatoma (HepG2) cells. We investigated the effect of FB1 on enzymes, DNA methyltransferases and demethylases, involved in chromatin maintenance and gross changes in structural integrity of DNA in HepG2 cells. We measured: (i) the expression of 84 key genes encoding enzymes known to modify genomic DNA and histones (superarray and qPCR); (ii) protein expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and the major demethylase (MBD2) (western blotting); (iii) degree of DNA methylation by FACS using anti-5-MeCyt and (iv) DNA migration (single cell gel electrophoresis). FB1 significantly decreased the methyltransferase activities of DNMT1, DNMT3A and DNMT3B, and significantly up regulated the demethylases (MBD2 expression and activity, and KDM5B and KDM5C expression). FACS data showed FB1 significantly increased DNA hypomethylation and resulted in gross changes in structural DNA as evidenced by the Comet assay. We conclude that FB1 induces global DNA hypomethylation and histone demethylation that causes chromatin instability and may lead to liver tumourigenesis.
Assuntos
Carcinógenos Ambientais/toxicidade , Metilação de DNA/efeitos dos fármacos , Fumonisinas/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Western Blotting , Cromatina/efeitos dos fármacos , Ensaio Cometa , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Células Hep G2 , Histona Desmetilases/metabolismo , Humanos , Regulação para Cima/efeitos dos fármacos , DNA Metiltransferase 3BRESUMO
Fumonisin B1 (FB1), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in hepatocellular carcinoma promoting activity in humans and animals. MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression via translational repression. Human cytochrome P450 (CYP1B1) is highly expressed in oestrogen target tissues and catalyzes the metabolic activation of many procarcinogens. The aim of our study was to investigate the effect of FB1 on miR-27b suppression and its effect on CYP1B1 modulation in a human hepatoma cell line (HepG2). MiR27b and CYP1B1 expressions were evaluated in HepG2 cells by quantitative PCR. In order to directly assess the effect of miR-27b on CYP1B1 mRNA levels, cells were transfected with the mimic to miR-27b. CYP1B1 protein expression was measured using Western blot. FB1 significantly down-regulated (11-fold) expression of miR-27b in HepG2 cells; whilst CYP1B1 mRNA and protein expression was significantly upregulated by 1.8-fold and 2.6-fold, respectively. CYP1B1 is post-transcriptionally regulated by miR-27b after HepG2 exposure to FB1. FB1-induced modulation of miR-27b in hepatic cells may be an additional mode of hepatic neoplastic transformation.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Carcinógenos Ambientais/toxicidade , Indução Enzimática/efeitos dos fármacos , Fumonisinas/toxicidade , Hepatócitos/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Análise por Conglomerados , Citocromo P-450 CYP1B1 , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , Mimetismo Molecular , Proteínas de Neoplasias/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Modulation of nuclear factor KappaB (NF-κB) activation may play a role in regulating inflammatory conditions associated with coronary artery disease (CAD). MicroRNA-146a (miR-146a) primarily targets interleukin-1 receptor-associated kinase 1 (IRAK-1) and tumour necrosis factor receptor associated factor 6 (TRAF-6), which results in inhibition of NF-κB via the TLR pathway. This study investigated the influence of the miR-146a GC rs2910164 on miR-146a expression in young South African Indians with CAD. CAD patients and controls were genotyped by PCR-RFLP and miRNA-146a levels were measured by qPCR. IRAK-1, TRAF-6 and NF-κB expression was determined by Western blot. No differences in genotypic frequency was found (GG: 45 vs. 47%, GC: 46 vs. 41%, CC: 9 vs. 12%) in controls and patients respectively (odds ratio = 1.025; 95% confidence interval 0.6782-1.550; p = 0.9164). Significantly higher levels of miR-146a was associated with CAD patients with the CC genotype (6.25-fold increase relative to controls and patients with the wildtype variant, p < 0.0001). Significantly lower levels of IRAK-1 (0.38 ± 0.02; p = 0.0072) and TRAF-6 (0.44 ± 0.02; p = 0.0146) was found in CAD patients with the CC genotype. The lowest levels of NF-κB and C-reactive protein were found in patients with the homozygous C allele compared to the heterozygous GC and wildtype variants. We propose a role for miR-146a in TLR signalling through a negative feedback mechanism involving the attenuation of NF-κB by down-regulation of IRAK-1 and TRAF-6. Our observations implicate miR-146a as a target for lowering inflammation in CAD patients.
Assuntos
Doença da Artéria Coronariana/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , MicroRNAs/metabolismo , Polimorfismo Genético , Fator 6 Associado a Receptor de TNF/metabolismo , Adulto , Alelos , Proteína C-Reativa/análise , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Regulação para Baixo , Genótipo , Homozigoto , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Masculino , MicroRNAs/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Razão de Chances , RNA Mensageiro/metabolismo , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
BACKGROUND: Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Promoter polymorphisms of these genes have been linked to chronic diseases including heart disease and type II diabetes mellitus in several populations. This is the first investigation of the UCP2 -866G/A rs659366 and UCP3 -55C/T rs1800849 polymorphisms in young South African (SA) Indians with coronary artery disease (CAD). METHODS: A total of 300 subjects were recruited into this study of which 100 were SA Indian males with CAD, 100 age- (range 24-45 years), gender- and race-matched controls and 100 age-matched black SA males. The frequency of the UCP2 -866G/A and UPC3 -55C/T genotypes was assessed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). RESULTS: The heterozygous UCP2 -866G/A and homozygous UCP3 -55C/C genotypes occurred at highest frequency in CAD patients (60% and 64%, respectively) compared to SA Indian controls (52% and 63%) and SA Black controls (50% and 58%). The UCP2 -886G/A (OR=1.110; 95% CI=0.7438-1.655; p=0.6835) and UCP3 -55C/T (OR=0.788; 95% CI=0.482-1.289; p=0.382) polymorphisms did not influence the risk of CAD. The rare homozygous UCP3 -55T/T genotype was associated with highest fasting glucose (11.87 ± 3.7 mmol/L vs. C/C:6.11 ± 0.27 mmol/L and C/T:6.48 ± 0.57 mmol/L, p=0.0025), HbA1c (10.05 ± 2.57% vs. C/C:6.44 ± 0.21% and C/T:6.76 ± 0.35%, p=0.0006) and triglycerides (6.47 ± 1.7 mmol/L vs. C/C:2.33 ± 0.17 mmol/L and C/T:2.06 ± 0.25 mmol/L, p<0.0001) in CAD patients. CONCLUSION: The frequency of the UCP2 -866G/A and UCP3 -55C/T polymorphisms was similar in our SA Indian and SA Black groups. The presence of the UCP2 -866G/A and UCP3 -55C/T polymorphisms does not influence the risk of CAD in young South African Indian CAD patients.
Assuntos
Doença da Artéria Coronariana/genética , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Adulto , População Negra/genética , Glicemia/análise , Estudos de Casos e Controles , Doença da Artéria Coronariana/etnologia , Frequência do Gene , Estudos de Associação Genética , Genótipo , Hemoglobinas Glicadas/análise , Humanos , Índia/etnologia , Masculino , Pessoa de Meia-Idade , Razão de Chances , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de Risco , África do Sul/epidemiologia , Triglicerídeos/análise , Proteína Desacopladora 2 , Proteína Desacopladora 3 , Adulto JovemRESUMO
BACKGROUND: Interleukin-6 (IL-6) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases such as coronary artery disease (CAD). The -174 IL-6 G/C promoter polymorphism influences mRNA levels and protein expression and is implicated in CAD. The Indian population in South Africa, unlike the black community, has a high prevalence of premature CAD. This polymorphism has not been fully explored in this population. The present study assessed the -174 IL-6 G/C polymorphism in young Indian patients with angiographically documented CAD and compared them with age- and gender-matched Indian and black control subjects. METHODS: Polymorphic variants were assessed by polymerase chain reaction-restriction fragment length polymorphism, and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The -174 IL-6 C allele was found with a higher frequency (23%) in the total Indian group compared to 2% in the black participants [P<0.0001, odds ratio (OR)=0.05, 95% confidence interval (CI) 0.018-0.14). The difference in frequency was more pronounced when Indian controls were compared to black controls (29% vs. 2%, respectively) (P<0.0001, OR=0.05, 95% CI 0.02-0.17). A significant association between the -174 IL-6 G allele and CAD was found in Indian patients compared to Indian controls (84% in cases vs. 71% in Indian controls; P=0.043, OR=0.47 95% CI 0.23-0.95). Levels of IL-6 in circulation were higher in black controls (6.62±0.63 pg/mL) compared to Indian controls (2.51±0.57 pg/mL) and CAD patients (1.46±0.36 pg/mL) (P<0.0001). Levels of IL-6 were higher in all groups with homozygous -174 IL-6 C alleles, but only significant in the healthy Indian control group (GG 3.73±0.94 pg/mL vs. GC/CC 0.89±0.5 pg/mL, P=0.0001). CONCLUSION: The presence of the IL-6 -174 G allele influences levels of IL-6 and increases the risk of CAD in South African Indians.
Assuntos
Doença da Artéria Coronariana/etnologia , Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Interleucina-6/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idade de Início , População Negra/etnologia , População Negra/genética , Estudos de Casos e Controles , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição/fisiologia , Prevalência , Regiões Promotoras Genéticas/genética , Fatores de Risco , África do Sul/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in coronary artery disease (CAD). OBJECTIVES: We aimed to assess 2 common polymorphic variant isoforms in GSTM1 and GSTP1 of GST in young CAD patients. METHODS: All patients (N=102) were South Africans of Indian ancestry, a population associated with high CAD risk. A corresponding age-, sex- and race-matched control group (N=100) was also recruited. Frequency of the GSTM1 +/0 (v. +/0 and 0/0) and GSTP1 A105/G105 (v. wild-type A105/A105) genotypes was assessed by differential polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP), respectively. RESULTS: The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% v. 18% and 65% v. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (OR=2.593; 95% CI 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 (odds ratio (OR)=0.6011; 95% confidence interval (CI) 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 RR=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221). CONCLUSION: Our findings support the association of genotypes GSTM1 0/0 and GSTP1 A105/A105 and smoking with CAD.
Assuntos
Povo Asiático/genética , Doença da Artéria Coronariana/genética , Glutationa Transferase/genética , Fumar/genética , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Índia/etnologia , Masculino , Polimorfismo Genético , Fumar/efeitos adversos , África do Sul/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease, which causes synovial damage. Persistence of lymphocyte infiltrates in the rheumatoid synovium has been attributed to abnormal apoptosis. While not comprehensively investigated, perturbations in peripheral blood lymphocyte (PBL) apoptosis may also be involved in perpetuation of autoimmune processes in RA. METHODS: We investigated total, CD4+ and CD19+ PBL apoptosis in our study cohort by monitoring the translocation of phosphatidylserine using the Annexin-V assay. To examine the role of death receptor mediated apoptosis as well as activation-induced-cell-death (AICD), PBLs were labeled with CD95/Fas and CD69 markers and enumerated by flow cytometry. Proteolytic activity of initiator and executioner caspases was determined by luminometry. DNA fragmentation assays were used to examine whether apoptotic signals were transduced to the nucleus. Quantitative PCR arrays were used to investigate apoptotic pathways associated with RA-PBLs. Since heat-shock-protein-70 (HSP70) is an inducible protein which modulates apoptotic signals, we determined HSP70 levels by intra-cellular flow cytometry and western blots. RESULTS: The RA-PBLs showed signs of elevated apoptosis whilst in circulation. These include increases in the loss of plasma membrane asymmetry, indicated by increased externalization of phosphatidylserine (especially in B-lymphocytes). RA-PBLs showed a bias to CD95/Fas mediated apoptotic pathways, but low levels of the CD69 marker suggested that this was not associated with immune activation. Although downstream markers of apoptosis such as caspase-3/7 activity, were increased, no DNA fragmentation was observed in RA-PBLs. Interestingly, elevated levels of apoptosis did not correlate with absolute lymphocyte counts in RA patients. Levels of HSP70 were highly elevated in RA-PBLs compared to controls. CONCLUSION: The results suggest that while apoptosis may be initiated in RA-PBLs, they may lack commitment to fully executing the apoptotic program. This may be related to inhibition on apoptotic transduction by HSP70. This study provides evidence that abnormalities in RA-PBLs apoptosis may occur whilst still in circulation and may contribute to pathogenesis of the disease.
RESUMO
The p53 tumor-suppressor protein plays an integral role in apoptosis. Perturbations in peripheral lymphocyte (PL) apoptosis may be associated with rheumatoid arthritis (RA). Polymorphisms at codon 72 of p53 (arginine (Arg72) to proline transition) confers differences in mitochondrial translocation and apoptosis inducing capabilities of p53 in vitro. We examined associations of this polymorphism with PL apoptosis, mitochondrial depolarization, and clinical markers of disease activity in a cohort of black South African RA patients. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. PL apoptosis was measured using the annexin-V assay and mitochondrial membrane potential with the JC-1 assay. Clinical and laboratory parameters were recorded for all patients. Statistical differences in these parameters were investigated according to genotype. Genotype distribution did not differ significantly between RA patients and controls (Arg/Arg, Arg/Pro, Pro/Pro: 12%, 46%, and 42% versus 3%, 34%, and 63%), despite significantly higher frequency of the Arg72 allele in patients (p = 0.0406). There was no significant difference in PL apoptosis and mitochondrial depolarization based on p53 codon 72 genotype. In addition, clinical markers of disease activity were not significantly different between genotypes. We conclude that p53 codon 72 genotype does not influence PL apoptosis or mitochondrial depolarization and is not associated with clinical markers of disease in RA.