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Alterations in the gut-microbiome-brain axis are increasingly being recognized to be involved in Alzheimer's disease (AD) pathogenesis. However, the functional consequences of enteric dysbiosis linking gut microbiota and brain pathology in AD progression remain largely undetermined. The present work investigated the causal role of age-associated temporal decline in butyrate-producing bacteria and butyrate in the etiopathogenesis of AD. Longitudinal metagenomics, neuropathological, and memory analyses were performed in the 3×Tg-AD mouse model. Metataxonomic analyses showed a significant temporal decline in the alpha diversity marked by a decrease in butyrate-producing bacterial communities and a concurrent reduction in cecal butyrate production. Inferred metagenomics analysis identified the bacterial acetyl-CoA pathway as the main butyrate synthesis pathway impacted. Concomitantly, there was an age-associated decline in the transcriptionally permissive acetylation of histone 3 at lysines 9 and 14 (H3K9/K14-Ac) in hippocampal neurons. Importantly, these microbiome-gut-brain changes preceded AD-related neuropathology, including oxidative stress, tau hyperphosphorylation, memory deficits, and neuromuscular dysfunction, which manifest by 17-18 months. Initiation of oral administration of tributyrin, a butyrate prodrug, at 6 months of age mitigated the age-related decline in butyrate-producing bacteria, protected the H3K9/K14-Ac status, and attenuated the development of neuropathological and cognitive changes associated with AD pathogenesis. These data causally implicate age-associated decline in butyrate-producing bacteria as a key pathogenic feature of the microbiome-gut-brain axis affecting the onset and progression of AD. Importantly, the regulation of butyrate-producing bacteria and consequent butyrate synthesis could be a significant therapeutic strategy in the prevention and treatment of AD.
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Enfermedad de Alzheimer , Bacterias , Butiratos , Modelos Animales de Enfermedad , Disbiosis , Microbioma Gastrointestinal , Trastornos de la Memoria , Animales , Butiratos/metabolismo , Ratones , Enfermedad de Alzheimer/microbiología , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Trastornos de la Memoria/microbiología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Bacterias/clasificación , Bacterias/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Disbiosis/microbiología , Hipocampo/metabolismo , Hipocampo/patología , Ratones Transgénicos , Masculino , Progresión de la Enfermedad , Eje Cerebro-Intestino/fisiología , Encéfalo/metabolismo , Encéfalo/patologíaRESUMEN
BACKGROUND: Both alcohol consumption and HIV infection are associated with worse brain, cognitive, and clinical outcomes in older adults. However, the extent to which brain and cognitive dysfunction is reversible with reduction or cessation of drinking is unknown. OBJECTIVE: The 30-Day Challenge study was designed to determine whether reduction or cessation of drinking would be associated with improvements in cognition, reduction of systemic and brain inflammation, and improvement in HIV-related outcomes in adults with heavy drinking. METHODS: The study design was a mechanistic experimental trial, in which all participants received an alcohol reduction intervention followed by repeated assessments of behavioral and clinical outcomes. Persons were eligible if they were 45 years of age or older, had weekly alcohol consumption of 21 or more drinks (men) or 14 or more drinks (women), and were not at high risk of alcohol withdrawal. After a baseline assessment, participants received an intervention consisting of contingency management (money for nondrinking days) for at least 30 days followed by a brief motivational interview. After this, participants could either resume drinking or not. Study questionnaires, neurocognitive assessments, neuroimaging, and blood, urine, and stool samples were collected at baseline, 30 days, 90 days, and 1 year after enrollment. RESULTS: We enrolled 57 persons with heavy drinking who initiated the contingency management protocol (mean age 56 years, SD 4.6 years; 63%, n=36 male, 77%, n=44 Black, and 58%, n=33 people with HIV) of whom 50 completed 30-day follow-up and 43 the 90-day follow-up. The planned study procedures were interrupted and modified due to the COVID-19 pandemic of 2020-2021. CONCLUSIONS: This was the first study seeking to assess changes in brain (neuroimaging) and cognition after alcohol intervention in nontreatment-seeking people with HIV together with people without HIV as controls. Study design strengths, limitations, and lessons for future study design considerations are discussed. Planned analyses are in progress, after which deidentified study data will be available for sharing. TRIAL REGISTRATION: ClinicalTrials.gov NCT03353701; https://clinicaltrials.gov/study/NCT03353701. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/53684.
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PURPOSE: Chemokine-driven leukocyte infiltration and sustained inflammation contribute to alcohol-associated liver disease (ALD). Elevated hepatic CCL2 expression, seen in ALD, is associated with disease severity. However, mechanisms of CCL2 regulation are not completely elucidated. Post-translational modifications (PTMs) of proteins, particularly acetylation, modulate gene expression. This study examined the acetylation changes of promoter-associated histone-H3 and key transcription factor-NFκB in regulating hepatic CCL2 expression and subsequent inflammation and injury. Further, the effect of therapeutic modulation of the acetylation state by tributyrin (TB), a butyrate prodrug, was assessed. METHODS: Hepatic CCL2 expression was assessed in mice fed control (PF) or an ethanol-containing Lieber-DeCarli (5% v/v, EF) diet for 7 weeks with or without oral administration of tributyrin (TB, 2 g/kg, 5 days/week). A chromatin immunoprecipitation (ChIP) assay evaluated promoter-associated modifications. Nuclear association between SIRT1, p300, and NFκB-p65 and acetylation changes of p65 were determined using immunoprecipitation and Western blot analyses. A Student's t-test and one-way ANOVA determined the significance. RESULTS: Ethanol significantly increased promoter-associated histone-H3-lysine-9 acetylation (H3K9Ac), reflecting a transcriptionally permissive state with a resultant increase in hepatic CCL2 mRNA and protein expression. Moreover, increased lysine-310-acetylation of nuclear RelA/p65 decreased its association with SIRT1, a class III HDAC, but concomitantly increased with p300, a histone acetyltransferase. This further led to enhanced recruitment of NF-κB/p65 and RNA polymerase-II to the CCL2 promoter. Oral TB administration prevented ethanol-associated acetylation changes, thus downregulating CCL2 expression, hepatic neutrophil infiltration, and inflammation/ injury. CONCLUSION: The modulation of a protein acetylation state via ethanol or TB mechanistically regulates hepatic CCL2 upregulation in ALD.
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Hepatitis , Histonas , Ratones , Animales , Histonas/metabolismo , FN-kappa B/metabolismo , Etanol , Lisina/metabolismo , Sirtuina 1/metabolismo , Acetilación , Procesamiento Proteico-Postraduccional , InflamaciónRESUMEN
BACKGROUND: Osteopontin (OPN) is a proinflammatory cytokine that has been recently implicated in neuroinflammation and neurodegeneration. We hypothesized that an increase in plasma osteopontin is a deleterious neuroinflammatory marker in people with dementia and cerebral small vessel disease (CSVD). METHODS: A pilot study was conducted on participants in the Northern Manhattan Study (NOMAS). Three groups were selected based on their dementia status and evidence of subclinical CSVD and chosen to be similar in age, sex, and education attainment: No dementia/No CSVD (n=19), Dementia/No CSVD (n=22), and Dementia+CSVD (n=21). Dementia (any type) was diagnosed by consensus adjudication following a series of comprehensive neuropsychological assessments and a review of the medical history. CSVD was indicated by silent brain infarcts, enlarged perivascular spaces, cerebral microbleeds, and white matter hyperintensity volumes (WMHV) on MRI. Multinomial logistic regression was used to examine the difference in OPN levels across groups, adjusting for key determinants of CSVD and neurodegeneration. RESULTS: Plasma osteopontin levels were elevated in the Dementia+CSVD group (mean=70.69±39.00 ng/ml) but not in the Dementia/No CSVD group (mean=45.46±19.11 ng/ml) compared to the No dementia/No CSVD group (mean=36.43±15.72 ng/ml). Osteopontin was associated with Dementia+CSVD (Odds Ratio (OR) per ng/ml=1.06, 95%CI 1.02-1.11) after adjusting for covariates, including brain volume. OPN was strongly correlated with WMHV (Spearman's rank correlation ï²=0.46, p=0.0001), but not with other components of CSVD. CONCLUSION: In this pilot, greater levels of plasma osteopontin were associated with dementia with evidence of CSVD. This link was predominately driven by the contribution of OPN to dementia through the burden of white matter lesions.
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Screening, Brief Intervention, and Referral to Treatment (SBIRT) is a supplementary intervention that can be incorporated into the Pre-Exposure Prophylaxis (PrEP) Care Continuum, complementing initiatives and endeavors focused on Human Immunodeficiency Virus (HIV) prevention in clinical care and community-based work. Referencing the Transtheoretical Model of Change and the PrEP Awareness Continuum, this conceptual analysis highlights how SBIRT amplifies ongoing HIV prevention initiatives and presents a distinct chance to address identified gaps. SBIRT's mechanisms show promise of fit and feasibility through (a) implementing universal Screening (S), (b) administering a Brief Intervention (BI) grounded in motivational interviewing aimed at assisting individuals in recognizing the significance of PrEP in their lives, (c) providing an affirming and supportive Referral to Treatment (RT) to access clinical PrEP care, and (d) employing client-centered and destigmatized approaches. SBIRT is uniquely positioned to help address the complex challenges facing PrEP awareness and initiation efforts. Adapting the SBIRT model to integrate and amplify HIV prevention efforts merits further examination.
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Intervención en la Crisis (Psiquiatría) , Infecciones por VIH , Humanos , Estudios de Factibilidad , Cognición , Derivación y Consulta , Infecciones por VIH/diagnóstico , Infecciones por VIH/prevención & controlRESUMEN
BACKGROUND: Imbalance in tryptophan (TRP) metabolism and its neuroactive metabolites, serotonin and kynurenine (KYN), is a known pathogenic mechanism underlying neurocognitive impairment. Gut microbiota plays an important role in TRP metabolism, and the production of these neuroactive molecules affects neurocognitive function. Although both HIV infection and normal aging independently induce gut dysbiosis and influence TRP metabolism, their interactive effects on compositional/functional changes in gut microbiota and consequent alterations in TRP metabolites remain largely undetermined. METHODS: Older people living with HIV infection (PLWH, aged 50-70 years, n = 22) were enrolled in this cross-sectional pilot study. Metagenomic analysis of fecal microbiome using 16S Ribosomal ribonucleic acid gene sequencing and metabolomics analysis of plasma using mass spectrometry with a reverse-phase iquid chromatography tandem mass spectrometry were performed. Statistical analyses included the univariate linear regression and Spearman correlation analyses. RESULTS: Age-associated changes in plasma levels of key neuroactive TRP metabolites, serotonin and KYN, were seen in PLWH. Specifically, we observed age-dependent decreases in serotonin and increases in KYN and KYN-to-TRP ratio, indicative of dysfunctional TRP metabolism. Furthermore, the gut dysbiosis seen in older PLWH is characterized by a reduction of Firmicutes/Bacteroidetes ratio and butyrate-producing microbial families Lachnospiraceae and Lactobacillaceae. Of importance, correspondent with gut dysbiosis, increasing age was significantly associated with decreased plasma butyrate levels, which in turn correlated positively with serotonin and negatively with KYN/TRP ratio. CONCLUSIONS: Age-dependent gut microbial dysbiosis distinguished by a decrease in butyrogenic potential is a key pathogenic feature associated with the shift in TRP metabolism from serotonin to KYN in older PLWH.
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Infecciones por VIH , Triptófano , Anciano , Estudios Transversales , Disbiosis , Humanos , Quinurenina/metabolismo , Persona de Mediana Edad , Proyectos Piloto , Espectrometría de Masas en Tándem , Triptófano/metabolismoRESUMEN
Serological assays intended for diagnosis, sero-epidemiologic assessment, and measurement of protective antibody titers upon infection or vaccination are essential for managing the SARS-CoV-2 pandemic. Serological assays measuring the antibody responses against SARS-CoV-2 antigens are readily available. However, some lack appropriate characteristics to accurately measure SARS-CoV-2 antibodies titers and neutralization. We developed an Enzyme-linked Immunosorbent Assay (ELISA) methods for measuring IgG, IgA, and IgM responses to SARS-CoV-2, Spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins. Performance characteristics of sensitivity and specificity have been defined. ELISA results show positive correlation with microneutralization and Plaque Reduction Neutralization assays with infectious SARS-CoV-2. Our ELISA was used to screen healthcare workers in Louisville, KY during the first wave of the local pandemic in the months of May and July 2020. We found a seropositive rate of approximately 1.4% and 2.3%, respectively. Our analyses demonstrate a broad immune response among individuals and suggest some non-RBD specific S IgG and IgA antibodies neutralize SARS-CoV-2.
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Anticuerpos Antivirales/sangre , COVID-19/epidemiología , SARS-CoV-2/inmunología , Área Bajo la Curva , COVID-19/sangre , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Kentucky/epidemiología , Pandemias , Fosfoproteínas/inmunología , Curva ROC , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Emerging research evidence has established the critical role of the gut-liver axis in the development of alcohol-associated liver disease (ALD). The present study employed 16S rRNA gene and whole genome shotgun (WGS) metagenomic analysis in combination with a revised microbial dataset to comprehensively detail the butyrate-producing microbial communities and the associated butyrate metabolic pathways affected by chronic ethanol feeding. Specifically, the data demonstrated that a decrease in several butyrate-producing bacterial genera belonging to distinct families within the Firmicutes phyla was a significant component of ethanol-induced dysbiosis. WGS analysis of total bacterial genomes encompassing butyrate synthesizing pathways provided the functional characteristics of the microbiome associated with butyrate synthesis. The data revealed that in control mice microbiome, the acetyl-coenzyme A (CoA) butyrate synthesizing pathway was the most prevalent and was significantly and maximally decreased by chronic ethanol feeding. Further WGS analysis i) validated the ethanol-induced decrease in the acetyl-CoA pathway by identifying the decrease in two critical genes but - (butyryl-CoA: acetate CoA transferase) and buk - (butyrate kinase) that encode the terminal condensing enzymes required for converting butyryl-CoA to butyrate and ii) detection of specific taxa of butyrate-producing bacteria containing but and buk genes. Notably, the administration of tributyrin (Tb) - a butyrate prodrug - significantly prevented ethanol-induced decrease in butyrate-producing bacteria, hepatic steatosis, inflammation, and injury. Taken together, our findings strongly suggest that the loss of butyrate-producing bacteria using the acetyl-CoA pathway is a significant pathogenic feature of ethanol-induced microbial dysbiosis and ALD and can be targeted for therapy.
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Butiratos/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Coenzima A Transferasas/metabolismo , Disbiosis/inducido químicamente , Etanol/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Ruminococcus/metabolismo , Animales , Modelos Animales de Enfermedad , Disbiosis/fisiopatología , Humanos , Redes y Vías Metabólicas , RatonesRESUMEN
SARS coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that causes a clinical disease called coronavirus disease 2019 (COVID-19). Although most COVID-19 cases are asymptomatic or involve mild upper respiratory tract symptoms, a significant number of patients develop severe or critical disease. Patients with severe COVID-19 commonly present with viral pneumonia that may progress to life-threatening acute respiratory distress syndrome (ARDS). Patients with COVID-19 are also predisposed to venous and arterial thromboses that are associated with a poorer prognosis. The present study identified the emergence of a low-density inflammatory neutrophil (LDN) population expressing intermediate levels of CD16 (CD16Int) in patients with COVID-19. These cells demonstrated proinflammatory gene signatures, activated platelets, spontaneously formed neutrophil extracellular traps, and enhanced phagocytic capacity and cytokine production. Strikingly, CD16Int neutrophils were also the major immune cells within the bronchoalveolar lavage fluid, exhibiting increased CXCR3 but loss of CD44 and CD38 expression. The percentage of circulating CD16Int LDNs was associated with D-dimer, ferritin, and systemic IL-6 and TNF-α levels and changed over time with altered disease status. Our data suggest that the CD16Int LDN subset contributes to COVID-19-associated coagulopathy, systemic inflammation, and ARDS. The frequency of that LDN subset in the circulation could serve as an adjunct clinical marker to monitor disease status and progression.
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Trastornos de la Coagulación Sanguínea/sangre , Trastornos de la Coagulación Sanguínea/etiología , COVID-19/sangre , COVID-19/complicaciones , Neutrófilos/inmunología , SARS-CoV-2 , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Trastornos de la Coagulación Sanguínea/inmunología , COVID-19/inmunología , Citocinas/sangre , Femenino , Proteínas Ligadas a GPI/sangre , Hospitalización , Humanos , Mediadores de Inflamación/sangre , Masculino , Persona de Mediana Edad , Neutrófilos/clasificación , Pandemias , Fagocitosis , Activación Plaquetaria , Receptores de IgG/sangre , Síndrome de Dificultad Respiratoria/sangre , Síndrome de Dificultad Respiratoria/etiología , Síndrome de Dificultad Respiratoria/inmunología , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Chronic immune activation and CD4 T cell depletion are significant pathogenic features of HIV infection. Expression of Fas ligand (FasL), a key mediator of activation-induced cell death in T cells, is elevated in people living with HIV-1 infection (PLWH). However, the epigenetic mechanisms underlying the enhanced induction of FasL expression in CD4 T lymphocytes in PLWH are not completely elucidated. Hence, the current work examined the effect of HIV infection on FasL promoter-associated histone modifications and transcriptional regulation in CD4 T lymphocytes in PLWH. METHOD: Flow cytometric analysis was performed to examine the Fas-FasL expression on total CD4 T cells and naïve/memory CD4 T cell subsets. Epigenetic FasL promoter histone modifications were investigated by chromatin immunoprecipitation-quantitative real-time polymerase chain reaction analysis using freshly isolated total CD4 T lymphocytes from HIV-1 infected and noninfected individuals. RESULTS: All naïve/memory CD4 T cell subsets from PLWH showed markedly greater frequency of FasL expression. Notably, examination of functional outcome of FasL/Fas co-expression demonstrated the preferential susceptibility of Tcm and Tem subsets to activation-induced apoptosis. Importantly, these CD4 T cells collectively demonstrated a distinct FasL promoter histone profile involving a coordinated cross-talk between histone H3 modifications leading to enhanced FasL gene expression. Specifically, levels of transcriptionally permissive histone H3K4-trimethylation (H3K4Me3) and histone H3K9-acetylation (H3K9Ac) were increased, with a concomitant decrease in the repressive H3K9-trimethylation (H3K9Me3). CONCLUSION: The present work demonstrates that epigenetic mechanisms involving promoter-histone modifications regulate transcriptional competence and FasL expression in CD4 T cells from PLWH and render them susceptible to activation-induced cell death.
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Linfocitos T CD4-Positivos/inmunología , Muerte Celular , Epigénesis Genética , Proteína Ligando Fas/metabolismo , Infecciones por VIH/inmunología , Adulto , Proteína Ligando Fas/genética , Femenino , Regulación de la Expresión Génica , VIH-1/fisiología , Histonas/metabolismo , Humanos , Activación de Linfocitos , Masculino , Metilación , Persona de Mediana Edad , Factores de TranscripciónRESUMEN
Recent reports suggest that arylamine N-acetyltransferases (NAT1 and/or NAT2) serve important roles in regulation of energy utility and insulin sensitivity. We investigated the interaction between diet (control vs. high-fat diet) and acetylator phenotype (rapid vs. slow) using previously established congenic rat lines (in F344 background) that exhibit rapid or slow Nat2 (orthologous to human NAT1) acetylator genotypes. Male and female rats of each genotype were fed control or high-fat (Western-style) diet for 26 weeks. We then examined diet- and acetylator genotype-dependent changes in body and liver weights, systemic glucose tolerance, insulin sensitivity, and plasma lipid profile. Male and female rats on the high fat diet weighed approximately 10% more than rats on the control diet and the percentage liver to body weight was consistently higher in rapid than slow acetylator rats. Rapid acetylator rats were more prone to develop dyslipidemia overall (i.e., higher triglyceride; higher LDL; and lower HDL), compared to slow acetylator rats. Total cholesterol (TC)-to-HDL ratios were significantly higher and HDL-to-LDL ratios were significantly lower in rapid acetylator rats. Our data suggest that rats with rapid systemic Nat2 (NAT1 in humans) genotype exhibited higher dyslipidemia conferring risk for metabolic syndrome and cardiovascular dysfunction.
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Ethanol-mediated down-regulation of carnitine palmitoyltransferase-1 (CPT-1A) gene expression plays a major role in the development of hepatic steatosis; however, the underlying mechanisms are not completely elucidated. Tributyrin, a butyrate prodrug that can inhibit histone deacetylase (HDAC) activity, attenuates hepatic steatosis and injury. The present study examined the beneficial effect of tributyrin/butyrate in attenuating ethanol-induced pathogenic epigenetic mechanisms affecting CPT-1A promoter-histone modifications and gene expression and hepatic steatosis/injury. METHODS: Mice were fed a liquid Lieber-DeCarli diet (Research Diet Inc, New Brunswick, NJ) with or without ethanol for 4 weeks. In a subset of mice, tributyrin (2 g/kg) was administered orally by gavage. Primary rat hepatocytes were treated with 50 mmol/L ethanol and/or 2 mmol/L butyrate. Gene expression and epigenetic modifications at the CPT-1A promoter were analyzed by chromatin immunoprecipitation analysis. RESULTS: In vivo, ethanol induced hepatic CPT-1A promoter histone H3K9 deacetylation, which is indicative of a repressive chromatin state, and decreased CPT-1A gene expression. Our data identified HDAC1 as the predominant HDAC causing CPT-1A promoter histone H3K9 deacetylation and epigenetic down-regulation of gene expression. Significantly, Specificity Protein 1 (SP1) and Hepatocyte Nuclear Factor 4 Alpha (HNF4α) participated in the recruitment of HDAC1 to the proximal and distal regions of CPT-1A promoter, respectively, and mediated transcriptional repression. Importantly, butyrate, a dietary HDAC inhibitor, attenuated ethanol-induced recruitment of HDAC1 and facilitated p300-HAT binding by enabling SP1/p300 interaction at the proximal region and HNF4α/peroxisomal proliferator-activated receptor-γ coactivator-1α/p300 interactions at the distal region, leading to promoter histone acetylation and enhanced CPT-1A transcription. CONCLUSIONS: This study identifies HDAC1-mediated repressive epigenetic mechanisms that underlie an ethanol-mediated decrease in CPT-1A expression. Importantly, tributyrin/butyrate inhibits HDAC1, rescues CPT-1A expression, and attenuates ethanol-mediated hepatic steatosis and injury, suggesting its potential use in therapeutic strategies for alcoholic liver disease.
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Carnitina O-Palmitoiltransferasa/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Hígado Graso Alcohólico/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Triglicéridos/farmacología , Acetilación/efectos de los fármacos , Administración Oral , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Represión Epigenética/efectos de los fármacos , Etanol/toxicidad , Hígado Graso Alcohólico/diagnóstico , Hígado Graso Alcohólico/patología , Hepatocitos , Inhibidores de Histona Desacetilasas/uso terapéutico , Histonas/metabolismo , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/patología , Pruebas de Función Hepática , Masculino , Ratones , Cultivo Primario de Células , Regiones Promotoras Genéticas/genética , Triglicéridos/uso terapéuticoRESUMEN
Leukemias bearing mixed lineage leukemia (MLL) rearrangement (MLL-R) resulting in expression of oncogenic MLL fusion proteins (MLL-FPs) represent an especially aggressive disease subtype with the worst overall prognoses and chemotherapeutic response. MLL-R leukemias are uniquely dependent on the epigenetic function of the H3K79 methyltransferase DOT1L, which is misdirected by MLL-FPs activating gene expression, driving transformation and leukemogenesis. Given the functional necessity of these leukemias to maintain adequate methylation potential allowing aberrant activating histone methylation to proceed, driving leukemic gene expression, we investigated perturbation of methionine (Met)/S-adenosylmethionine (SAM) metabolism as a novel therapeutic paradigm for MLL-R leukemia. Disruption of Met/SAM metabolism, by either methionine deprivation or pharmacologic inhibition of downstream metabolism, reduced overall cellular methylation potential, reduced relative cell numbers, and induced apoptosis selectively in established MLL-AF4 cell lines or MLL-AF6-expressing patient blasts but not in BCR-ABL-driven K562 cells. Global histone methylation dynamics were altered, with a profound loss of requisite H3K79 methylation, indicating inhibition of DOT1L function. Relative occupancy of the repressive H3K27me3 modification was increased at the DOT1L promoter in MLL-R cells, and DOT1L mRNA and protein expression was reduced. Finally, pharmacologic inhibition of Met/SAM metabolism significantly prolonged survival in an advanced, clinically relevant patient-derived MLL-R leukemia xenograft model, in combination with cytotoxic induction chemotherapy. Our findings provide support for further investigation into the development of highly specific allosteric inhibitors of enzymatic mediators of Met/SAM metabolism or dietary manipulation of methionine levels. Such inhibitors may lead to enhanced treatment outcomes for MLL-R leukemia, along with cytotoxic chemotherapy or DOT1L inhibitors.
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Leucemia Bifenotípica Aguda/metabolismo , Metionina/metabolismo , S-Adenosilmetionina/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Leucemia/metabolismo , Metionina/genética , Metionina/uso terapéutico , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/uso terapéutico , Procesamiento Proteico-Postraduccional , S-Adenosilmetionina/uso terapéuticoRESUMEN
Zidovudine (AZT) remains the mainstay of antiretroviral therapy against HIV in resource-poor countries; however, its use is frequently associated with hepatotoxicity. Not all HIV patients on AZT develop hepatotoxicity, and the determining factors are unclear. Alcohol consumption and cigarette smoking are known risk factors for HIV hepatotoxicity, and both are significant sources of acrolein, a highly reactive and toxic aldehyde. This study examines the potential hepatotoxic interactions between acrolein and AZT. Our data demonstrate that acrolein markedly enhanced AZT-induced transcriptionally permissive histone modifications (H3K9Ac and H3K9Me3) allowing the recruitment of transcription factor NF-kB and RNA polymerase II at the FasL gene promoter, resulting in FasL upregulation and apoptosis in hepatocytes. Notably, the acrolein scavenger, hydralazine prevented these promoter-associated epigenetic changes and inhibited FasL upregulation and apoptosis induced by the combination of AZT and acrolein, as well as AZT alone. Our data strongly suggest that acrolein enhancement of promoter histone modifications and FasL upregulation are major pathogenic mechanisms driving AZT-induced hepatotoxicity. Moreover, these data also indicate the therapeutic potential of hydralazine in mitigating AZT hepatotoxicity.
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Acroleína/toxicidad , Fármacos Anti-VIH/toxicidad , Epigénesis Genética/efectos de los fármacos , Proteína Ligando Fas/genética , Hepatocitos/efectos de los fármacos , Zidovudina/toxicidad , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fragmentación del ADN , Células Hep G2 , Hepatocitos/metabolismo , Histonas/genética , Humanos , Hidralazina/farmacología , ARN Polimerasa II/genética , RatasRESUMEN
BACKGROUND & AIMS: Alcoholic liver disease (ALD) remains a major cause of morbidity and mortality, with no Food and Drug Administration-approved therapy. Chronic alcohol consumption causes a pro-oxidant environment and increases hepatic lipid peroxidation, with acrolein being the most reactive/toxic by-product. This study investigated the pathogenic role of acrolein in hepatic endoplasmic reticulum (ER) stress, steatosis, and injury in experimental ALD, and tested acrolein elimination/scavenging (using hydralazine) as a potential therapy in ALD. METHODS: In vitro (rat hepatoma H4IIEC cells) and in vivo (chronic+binge alcohol feeding in C57Bl/6 mice) models were used to examine alcohol-induced acrolein accumulation and consequent hepatic ER stress, apoptosis, and injury. In addition, the potential protective effects of the acrolein scavenger, hydralazine, were examined both in vitro and in vivo. RESULTS: Alcohol consumption/metabolism resulted in hepatic accumulation of acrolein-protein adducts, by up-regulation of cytochrome P4502E1 and alcohol dehydrogenase, and down-regulation of glutathione-s-transferase-P, which metabolizes/detoxifies acrolein. Alcohol-induced acrolein adduct accumulation led to hepatic ER stress, proapoptotic signaling, steatosis, apoptosis, and liver injury; however, ER-protective/adaptive responses were not induced. Notably, direct exposure to acrolein in vitro mimicked the in vivo effects of alcohol, indicating that acrolein mediates the adverse effects of alcohol. Importantly, hydralazine, a known acrolein scavenger, protected against alcohol-induced ER stress and liver injury, both in vitro and in mice. CONCLUSIONS: Our study shows the following: (1) alcohol consumption triggers pathologic ER stress without ER adaptation/protection; (2) alcohol-induced acrolein is a potential therapeutic target and pathogenic mediator of hepatic ER stress, cell death, and injury; and (3) removal/clearance of acrolein by scavengers may have therapeutic potential in ALD.
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Dysregulated cytokine metabolism plays a critical role in the pathogenesis of many forms of liver disease, including alcoholic and non-alcoholic liver disease. In this study we examined the efficacy of Misoprostol in modulating LPS-inducible TNFα and IL-10 expression in healthy human subjects and evaluated molecular mechanisms for Misoprostol modulation of cytokines in vitro. Healthy subjects were given 14day courses of Misoprostol at doses of 100, 200, and 300µg four times a day, in random order. Baseline and LPS-inducible cytokine levels were examined ex vivo in whole blood at the beginning and the end of the study. Additionally, in vitro studies were performed using primary human PBMCs and the murine macrophage cell line, RAW 264.7, to investigate underlying mechanisms of misoprostol on cytokine production. Administration of Misoprostol reduced LPS inducible TNF production by 29%, while increasing IL-10 production by 79% in human subjects with no significant dose effect on ex vivo cytokine activity; In vitro, the effect of Misoprostol was largely mediated by increased cAMP levels and consequent changes in CRE and NFκB activity, which are critical for regulating IL-10 and TNF expression. Additionally, chromatin immunoprecipitation (ChIP) studies demonstrated that Misoprostol treatment led to changes in transcription factor and RNA Polymerase II binding, resulting in changes in mRNA levels. In summary, Misoprostol was effective at beneficially modulating TNF and IL-10 levels both in vivo and in vitro; these studies suggest a potential rationale for Misoprostol use in ALD, NASH and other liver diseases where inflammation plays an etiologic role.
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AMP Cíclico/metabolismo , Citocinas/metabolismo , Misoprostol/farmacología , Transducción de Señal/efectos de los fármacos , Dolor Abdominal/inducido químicamente , Animales , Antiulcerosos/efectos adversos , Antiulcerosos/farmacología , Línea Celular , Células Cultivadas , Citocinas/sangre , Citocinas/genética , Diarrea/inducido químicamente , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Hepatopatías/tratamiento farmacológico , Hepatopatías/genética , Hepatopatías/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Misoprostol/efectos adversos , Náusea/inducido químicamente , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Acrolein, a highly reactive unsaturated aldehyde, is a ubiquitous environmental pollutant and its potential as a serious environmental health threat is beginning to be recognized. Humans are exposed to acrolein per oral (food and water), respiratory (cigarette smoke, automobile exhaust, and biocide use) and dermal routes, in addition to endogenous generation (metabolism and lipid peroxidation). Acrolein has been suggested to play a role in several disease states including spinal cord injury, multiple sclerosis, Alzheimer's disease, cardiovascular disease, diabetes mellitus, and neuro-, hepato-, and nephro-toxicity. On the cellular level, acrolein exposure has diverse toxic effects, including DNA and protein adduction, oxidative stress, mitochondrial disruption, membrane damage, endoplasmic reticulum stress, and immune dysfunction. This review addresses our current understanding of each pathogenic mechanism of acrolein toxicity, with emphasis on the known and anticipated contribution to clinical disease, and potential therapies.
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Acroleína/toxicidad , Enfermedad/etiología , Exposición a Riesgos Ambientales/efectos adversos , Contaminantes Ambientales/toxicidad , Estrés Oxidativo/efectos de los fármacos , Acroleína/metabolismo , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Citocinas/inmunología , Aductos de ADN/metabolismo , Contaminantes Ambientales/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Activation-induced Fas ligand (FasL) mRNA expression in CD4+ T cells is mainly controlled at transcriptional initiation. To elucidate the epigenetic mechanisms regulating physiologic and pathologic FasL transcription, TCR stimulation-responsive promoter histone modifications in normal and alcohol-exposed primary human CD4+ T cells were examined. TCR stimulation of normal and alcohol-exposed cells led to discernible changes in promoter histone H3 lysine trimethylation, as documented by an increase in the levels of transcriptionally permissive histone 3 lysine 4 trimethylation and a concomitant decrease in the repressive histone 3 lysine 9 trimethylation. Moreover, acetylation of histone 3 lysine 9 (H3K9), a critical feature of the active promoter state that is opposed by histone 3 lysine 9 trimethylation, was significantly increased and was essentially mediated by the p300-histone acetyltransferase. Notably, the degree of these coordinated histone modifications and subsequent recruitment of transcription factors and RNA polymerase II were significantly enhanced in alcohol-exposed CD4+ T cells and were commensurate with the pathologic increase in the levels of FasL mRNA. The clinical relevance of these findings is further supported by CD4+ T cells obtained from individuals with a history of heavy alcohol consumption, which demonstrate significantly greater p300-dependent H3K9 acetylation and FasL expression. Overall, these data show that, in human CD4+ T cells, TCR stimulation induces a distinct promoter histone profile involving a coordinated cross-talk between histone 3 lysine 4 and H3K9 methylation and acetylation that dictates the transcriptional activation of FasL under physiologic, as well as pathologic, conditions of alcohol exposure.
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Consumo de Bebidas Alcohólicas/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteína Ligando Fas/inmunología , Regulación de la Expresión Génica/inmunología , Histonas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Consumo de Bebidas Alcohólicas/efectos adversos , Consumo de Bebidas Alcohólicas/patología , Linfocitos T CD4-Positivos/patología , Femenino , Humanos , Masculino , Metilación , Factores de Transcripción p300-CBP/inmunologíaRESUMEN
The role of host response-related factors in the fast progression of liver disease in individuals co-infected with HIV and HCV viruses remains poorly understood. This study compared patterns of cytokines, caspase-1 activation, endotoxin exposure in plasma as well as interferon signaling in peripheral blood mononuclear cells from HIV/HCV co-infected (HIV(+)/HCV(+)), HCV mono-infected (HIV(-)/HCV(+)), HIV mono-infected (HIV(+)/HCV(-)) female patients and HIV- and HCV-uninfected women (HIV(-)/HCV(-)) who had enrolled in the Women's Interagency HIV Study (WIHS). HIV(+)/HCV(+) women had higher plasma levels of pro-inflammatory cytokines as well as caspase-1 compared with other groups. Both HIV(+)/HCV(+) and HIV(+)/HCV(-) women had significantly higher sCD14 levels compared with other groups. Peripheral blood mononuclear cells from HCV mono-infected patients had reduced levels of phosphorylation of STAT1 compared with other groups as well as lower basal levels of expression of the IFN-stimulated genes, OAS1, ISG15, and USP18 (UBP43). Basal expression of USP18, a functional antagonist of ISG15, as well as USP18/ISG15 ratios were increased in the HIV(+)/HCV(+) group compared with HIV(-)/HCV(+) and HIV(+)/HCV(-) groups. A more pronounced systemic inflammatory profile as well as increased expression ratios of USP18 to ISG15 may contribute to the more rapid progression of liver disease in HIV(+)/HCV(+) individuals.
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Coinfección/inmunología , Endopeptidasas/metabolismo , Infecciones por VIH/inmunología , VIH/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Leucocitos Mononucleares/inmunología , 2',5'-Oligoadenilato Sintetasa/metabolismo , Adulto , Caspasa 1/sangre , Estudios de Cohortes , Coinfección/complicaciones , Citocinas/sangre , Citocinas/metabolismo , Femenino , Estudios de Seguimiento , Infecciones por VIH/complicaciones , Hepatitis C/complicaciones , Humanos , Leucocitos Mononucleares/virología , Persona de Mediana Edad , Estudios Prospectivos , Ubiquitina Tiolesterasa , Ubiquitinas/metabolismoRESUMEN
BACKGROUND: Recently, we have demonstrated that acute alcohol exposure due to binge drinking leads to hepatic steatosis with the deregulation of hepatic histone deacetylase (HDAC) expression. Various class I, II, and IV HDACs were down-regulated, whereas expression of HDAC3 was solely up-regulated. Hence, in the present work, we specifically examined the mechanistic role of HDAC3 in the development of hepatic steatosis occurring in response to binge alcohol administration. METHODS: C57BL/6 mice were gavaged 3 times with ethanol (EtOH) at a dose of 4.5 g/kg. HDAC inhibitor, Trichostatin A (TSA) was simultaneously injected intraperitoneally at a dose of 1 mg/kg. Hepatic steatosis, injury, expression of HDAC3 and carnitine palmitoyltransferase 1α (CPT1α) were evaluated. HDAC3 and histone H3 acetylation levels at the Cpt1α promoter were analyzed by chromatin immunoprecipitation (ChIP). RESULTS: The binge EtOH-mediated increase in HDAC3 was prevented by simultaneous administration of HDAC inhibitor, TSA, which markedly attenuated hepatic steatosis and injury. Importantly, HDAC3 inhibition was able to normalize the down-regulation of Cpt1α expression. Causal role of HDAC3 in the transcriptional repression of Cpt1α was demonstrated by increased HDAC3 binding at the thyroid receptor element site in the Cpt1α distal promoter region. Further, a resultant decrease in the transcriptionally permissive histone H3 lysine 9 acetylation in the proximal promoter region near the transcriptional start site was observed. Notably, TSA treatment reduced HDAC3 binding and increased H3K9 acetylation at Cpt1α promoter leading to increased Cpt1α expression. These molecular events resulted in attenuation of binge alcohol-induced hepatic steatosis. CONCLUSIONS: These findings provide insights into potential epigenetic mechanisms underlying transcriptional regulation of Cpt1α in the hepatic steatosis occurring in response to binge EtOH administration.