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The increased risk for acquiring secondary illnesses in people living with HIV (PLWH) has been associated with immune dysfunction. We have previously found that circulating monocytes from PLWH display a trained phenotype. Here, we evaluated the metabolic profile of these cells and found increased mitochondrial respiration and glycolysis of monocyte-derived macrophages (MDMs) from PLWH. We additionally found that cART shifted the energy metabolism of MDMs from controls toward increased utilization of mitochondrial respiration. Importantly, both downregulation of IKAROS expression and inhibition of the mTOR pathway reversed the metabolic profile of MDMs from PLWH and cART-treated control-MDMs. Altogether, this study reveals a very specific metabolic adaptation of MDMs from PLWH, which involves an IKAROS/mTOR-dependent increase of mitochondrial respiration and glycolysis. We propose that this metabolic adaptation decreases the ability of these cells to respond to environmental cues by "locking" PLWH monocytes in a pro-inflammatory and activated phenotype.
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Infecciones por VIH , Humanos , Macrófagos , Monocitos , Fenotipo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of estrogen receptor, progesterone receptor, and HER2. Our lab previously characterized miR-3189-3p as a microRNA with potent anti-cancer activity against glioblastoma. Here, we hypothesized a similar activity in TNBC cells. As miR-3189-3p is predicted to target a variety of RNA binding proteins, we further hypothesized an inhibitory effect of this miRNA on protein synthesis. METHODS: MDA-MB-231 and MDA-MB-468 cells were used to investigate the effect of miR-3189-3p on cell proliferation, migration, and invasion. TGCA database was used to analyze the expression of miR-3189-3p, c-MYC, 4EPB1, and eIF4E in breast cancer. Western blotting and RT-qPCR assays were used to assess the expression of selected proteins and RNAs after transfections. RESULTS: Although c-MYC is not a predicted gene target for miR-3189-3p, we discovered that c-MYC protein is downregulated in miRNA-treated TNBC cells. We found that the downregulation of c-MYC by miR-3189-3p occurs in both normal growth conditions and in the absence of serum. The mechanism involved the direct inhibition of eIF4EBP1 by miR-3189-3p. Additionally, we found that miR-3189-3p could negatively affect cap-independent translation mediated by internal ribosome entry sites (IRES) or by m6A. Finally, miR-3189-3p sensitized TNBC cells to doxorubicin. CONCLUSION: Overall, results indicated that miR-3189-3p exerts its anti-tumor activity through targeting translational regulatory proteins leading to an impairment in c-MYC translation, and possibly other oncogenic factors, suggesting that miR-3189-3p, alone or in combination, could be a valuable therapeutic approach against a malignancy with few treatment options.
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OBJECTIVES: To report and analyse the characteristics and performance of the first cohort of Italian radiologists completing the national mammography self-evaluation online test established by the Italian Society of Medical Radiology (SIRM). METHODS: A specifically-built dataset of 132 mammograms (24 with screen-detected cancers and 108 negative cases) was preliminarily tested on 48 radiologists to define pass thresholds (62% sensitivity and 86% specificity) and subsequently made available online to SIRM members during a 13-month timeframe between 2018 and 2019. Associations between participants' characteristics, pass rates, and diagnostic accuracy were then investigated with descriptive statistics and univariate and multivariable regression analyses. RESULTS: A total of 342 radiologists completed the test, 151/342 (44.2%) with success. All individual variables, except gender, showed a significant correlation with pass rates and diagnostic sensitivity, confirmed by univariate logistic regression, while only involvement in organised screening programs and number of mammograms read per year showed a positive association with specificity at univariate logistic regression. In the multivariable regression analysis, fewer variables remained significant: > 3000 mammograms read per year for success rate; female gender, public practice setting, and higher experience self-judgement for sensitivity; no variables were significantly associated with specificity. CONCLUSIONS: This national self-evaluation test effectively differentiated multiple aspects of mammographic reading experience, but specific breast imaging experience was shown not to strictly guarantee good diagnostic accuracy. Due to its easy use and the validity of obtained results, this test could be extended to all Italian breast radiologists, regardless of their experience, also as a Breast Unit accreditation criterion. KEY POINTS: ⢠This self-evaluation test was found to be able to differentiate various degrees of mammographic interpretation experience. ⢠Breast cancer screening readers should undergo a self-assessment test, since experience parameters alone do not guarantee diagnostic ability.
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Neoplasias de la Mama , Radiología , Neoplasias de la Mama/diagnóstico por imagen , Autoevaluación Diagnóstica , Femenino , Humanos , Mamografía , Tamizaje Masivo , Autoevaluación (Psicología) , Sensibilidad y EspecificidadRESUMEN
Neurocognitive disorders associated with HIV-1 infection affect more than half of persons living with HIV (PLWH) under retroviral therapy. Understanding the molecular mechanisms and the complex cellular network communication underlying neurological dysfunction is critical for the development of an effective therapy. As with other neurological disorders, challenges to studying HIV infection of the brain include limited access to clinical samples and proper reproducibility of the complexity of brain networks in cellular and animal models. This review focuses on cellular models used to investigate various aspects of neurological dysfunction associated with HIV infection.
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Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV-neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.
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Vesículas Extracelulares/inmunología , Inflamación/metabolismo , MicroARNs/metabolismo , Neuroglía/inmunología , Neuronas/inmunología , Sinapsis/inmunología , Animales , Células Cultivadas , Líquido Cefalorraquídeo/metabolismo , Técnicas de Cocultivo , Vesículas Extracelulares/patología , Femenino , Hipocampo/inmunología , Hipocampo/patología , Humanos , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Neuroglía/patología , Plasticidad Neuronal/fisiología , Neuronas/patología , Cultivo Primario de Células , Ratas Sprague-Dawley , Sinapsis/patologíaRESUMEN
Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , MicroARNs/genética , Animales , Secuencia de Bases , Sitios de Unión , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Glioblastoma/genética , Glioblastoma/patología , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Ratones Desnudos , Trasplante de Neoplasias , Interferencia de ARN , Factores de Empalme de ARN , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Factores de Intercambio de Guanina Nucleótido Rho/biosíntesis , Factores de Intercambio de Guanina Nucleótido Rho/genéticaRESUMEN
The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of TAU exon 10 results in tauopathies characterized by alterations in the proportion of TAU isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the TAU molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes TAU 3R up-regulation through increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with TAU RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of TAU exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.
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Exones , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Ribonucleoproteínas/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/virología , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Infecciones por VIH/genética , VIH-1/genética , Humanos , Ratones , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Ribonucleoproteínas/genética , Factores de Empalme Serina-Arginina , Regulación hacia Arriba , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Proteínas tau/genética , Quinasas DyrKRESUMEN
Human Immunodeficiency Virus (HIV)-infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age-matched, HIV-infected (HIV(+) ) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV(+) patients with cognitive impairment from those without cognitive impairment. These results justify follow-on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV(+) patients with neurocognitive impairment. J. Cell. Physiol. 231: 829-836, 2016. © 2015 Wiley Periodicals, Inc.
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Disfunción Cognitiva/sangre , Disfunción Cognitiva/complicaciones , Infecciones por VIH/sangre , Infecciones por VIH/complicaciones , MicroARNs/sangre , Adulto , Disfunción Cognitiva/genética , Demografía , Infecciones por VIH/genética , Humanos , MicroARNs/genética , Persona de Mediana Edad , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: The aim of this study was to evaluate the presence of digital lesions in very early diagnosis of SSc (VEDOSS) patients and its possible association with internal organ involvement. METHODS: One hundred and ten VEDOSS patients were investigated for the presence of digital ulcers (DUs), digital pitting scars, calcinosis, necrosis or gangrene, nailfold videocapillaroscopic abnormalities, disease-specific autoantibodies (ACA and anti-topo I) and internal organ involvement. RESULTS: Four patients reported a history of digital pitting scars, while 25 patients presented an active DU or reported a history of DUs. In particular, 16 patients presented with active DUs (14/16 also reporting a history of previous DUs), while the other 9 patients reported a history of DUs only. A statistically significant association between DUs and oesophageal manometry alteration was found in the whole DU population, as well as in the history of DU and the presence of active DU with/without a history of DU subgroups (P < 0.01, P = 0.01 and P < 0.05, respectively). DUs were observed in VEDOSS patients with internal organ involvement but not in those without organ involvement. CONCLUSION: DUs are already present in VEDOSS patients characterized by internal organ involvement, significantly correlating and associating with gastrointestinal involvement. DUs may be a sentinel sign for early organ involvement in VEDOSS patients.
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Dedos , Enfermedades Gastrointestinales/etiología , Enfermedades Pulmonares/etiología , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/diagnóstico , Úlcera/diagnóstico , Úlcera/etiología , Adulto , Calcinosis/diagnóstico , Calcinosis/patología , Estudios de Cohortes , Diagnóstico Precoz , Femenino , Humanos , Masculino , Persona de Mediana Edad , Uñas/irrigación sanguínea , Necrosis/diagnóstico , Necrosis/patología , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Factores de Riesgo , Úlcera/patologíaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and KSHV activation of mitogen-activated protein kinases (MAPKs) initiates a number of key pathogenic determinants of KS. Direct inhibition of signal transduction as a therapeutic approach presents several challenges, and a better understanding of KSHV-induced mechanisms regulating MAPK activation may facilitate the development of new treatment or prevention strategies for KS. MAPK phosphatases, including dual-specificity phosphatase-1 (DUSP1), negatively regulate signal transduction and cytokine activation through MAPK dephosphorylation or interference with effector molecule binding to MAPKs, including the extracellular signal-regulated kinase (ERK). We found that ERK-dependent latent viral gene expression, the induction of promigratory factors, and cell invasiveness following de novo infection of primary human endothelial cells are in part dependent on KSHV suppression of DUSP1 expression during de novo infection. KSHV-encoded miR-K12-11 upregulates the expression of xCT (an amino acid transporter and KSHV fusion/entry receptor), and existing data indicate a role for xCT in the regulation of 14-3-3ß, a transcriptional repressor of DUSP1. We found that miR-K12-11 induces endothelial cell secretion of promigratory factors and cell invasiveness through upregulation of xCT-dependent, 14-3-3ß-mediated suppression of DUSP1. Finally, proof-of-principle experiments revealed that pharmacologic upregulation of DUSP1 inhibits the induction of promigratory factors and cell invasiveness during de novo KSHV infection. These data reveal an indirect role for miR-K12-11 in the regulation of DUSP1 and downstream pathogenesis.
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Fosfatasa 1 de Especificidad Dual/antagonistas & inhibidores , Herpesvirus Humano 8/patogenicidad , Interacciones Huésped-Patógeno , MicroARNs/metabolismo , Línea Celular , Regulación hacia Abajo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , ARN Viral/metabolismoRESUMEN
MicroRNAs are short non-coding RNAs that modulate gene expression by translational repression. Because of their high stability in intracellular as well as extracellular environments, miRNAs have recently emerged as important biomarkers in several human diseases. However, they have not been tested in the cerebrospinal fluid (CSF) of HIV-1 positive individuals. Here, we present results of a study aimed at determining the feasibility of detecting miRNAs in the CSF of HIV-infected individuals with and without encephalitis (HIVE). We also evaluated similarities and differences between CSF and brain tissue miRNAs in the same clinical setting. We utilized a high throughput approach of miRNA detection arrays and identified differentially expressed miRNAs in the frontal cortex of three cases each of HIV+, HIVE, and HIV- controls, and CSF of 10 HIV-positive and 10 HIV-negative individuals. For the CSF samples, the group of HIV+ individuals contained nine cases of HIV-associated neurological disorders (HAND) and, among those, four had HIVE. All the HIV-negative samples had non-viral acute disseminate encephalomyelitis. A total of 66 miRNAs were found differentially regulated in HIV+ compared to HIV- groups. The greatest difference in miRNA expression was observed when four cases of HIVE were compared to five non-HIVE cases, previously normalized with the HIV-negative group. After statistical analyses, 11 miRNAs were fund significantly up-regulated in HIVE. Although more clinical samples should be examined, this work represents the first report of CSF miRNAs in HIV-infection and offers the basis for future investigation.
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Encefalitis , Infecciones por VIH , MicroARNs , Corteza Cerebral/metabolismo , Corteza Cerebral/virología , Encefalitis/líquido cefalorraquídeo , Encefalitis/complicaciones , Encefalitis/patología , Encefalitis/virología , Regulación de la Expresión Génica , Infecciones por VIH/líquido cefalorraquídeo , Infecciones por VIH/complicaciones , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Humanos , MicroARNs/líquido cefalorraquídeo , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Glioblastomas are highly aggressive brain tumors for which therapeutic options are very limited. In a quest for new anti-glioblastoma drugs, we focused on specific structural modifications to the benzoyl-phenoxy-acetamide (BPA) structure present in a common lipid-lowering drug, fenofibrate, and in our first prototype glioblastoma drug, PP1. Here, we propose extensive computational analyses to improve the selection of the most effective glioblastoma drug candidates. Initially, over 100 structural BPA variations were analyzed and their physicochemical properties, such as water solubility (- logS), calculated partition coefficient (ClogP), probability for BBB crossing (BBB_SCORE), probability for CNS penetration (CNS-MPO) and calculated cardiotoxicity (hERG), were evaluated. This integrated approach allowed us to select pyridine variants of BPA that show improved BBB penetration, water solubility, and low cardiotoxicity. Herein the top 24 compounds were synthesized and analyzed in cell culture. Six of them demonstrated glioblastoma toxicity with IC50 ranging from 0.59 to 3.24 µM. Importantly, one of the compounds, HR68, accumulated in the brain tumor tissue at 3.7 ± 0.5 µM, which exceeds its glioblastoma IC50 (1.17 µM) by over threefold.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Barrera Hematoencefálica , Cardiotoxicidad , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Neoplasias Encefálicas/tratamiento farmacológico , Simulación por Computador , Acetamidas/farmacología , Piridinas/farmacología , Agua/farmacología , Línea Celular TumoralRESUMEN
Mild Traumatic Brain Injury (mild TBI)/concussion is a common sports injury, especially common in football players. Repeated concussions are thought to lead to long-term brain damage including chronic traumatic encephalopathy (CTE). With the worldwide growing interest in studying sport-related concussion the search for biomarkers for early diagnosis and progression of neuronal injury has also became priority. MicroRNAs are short, non-coding RNAs that regulate gene expression post-transcriptionally. Due to their high stability in biological fluids, microRNAs can serve as biomarkers in a variety of diseases including pathologies of the nervous system. In this exploratory study, we have evaluated changes in the expression of selected serum miRNAs in collegiate football players obtained during a full practice and game season. We found a miRNA signature that can distinguish with good specificity and sensitivity players with concussions from non-concussed players. Furthermore, we found miRNAs associated with the acute phase (let-7c-5p, miR-16-5p, miR-181c-5p, miR-146a-5p, miR-154-5p, miR-431-5p, miR-151a-5p, miR-181d-5p, miR-487b-3p, miR-377-3p, miR-17-5p, miR-22-3p, and miR-126-5p) and those whose changes persist up to 4 months after concussion (miR-17-5p and miR-22-3p).
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The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.
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Proteína Adaptadora GRB2/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Microglía/fisiología , Dominios Homologos src , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/fisiología , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , VIH-1/metabolismo , Interacciones Huésped-Patógeno/fisiología , Humanos , Microglía/metabolismo , Microglía/patología , Microglía/virología , Datos de Secuencia Molecular , Unión Proteica , Transducción de Señal , Replicación Viral/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
Candidate genes have been identified that confer increased risk for diabetic glomerulosclerosis (DG). Mice heterozygous for the Akita (Ins2(+/C96Y)) diabetogenic mutation with a second mutation introduced at the bradykinin 2 receptor (B2R(-/-)) locus express a disease phenotype that approximates human DG. Src homology 2 domain transforming protein 1 (p66) controls mitochondrial metabolism and cellular responses to oxidative stress, aging, and apoptosis. We generated p66-null Akita mice to test whether inactivating mutations at the p66 locus will rescue kidneys of Akita mice from disease-causing mutations at the Ins2 and B2R loci. Here we show null mutations at the p66 and B2R loci interact with the Akita (Ins2(+/C96Y)) mutation, independently and in combination, inducing divergent phenotypes in the kidney. The B2R(-/-) mutation induces detrimental phenotypes, as judged by increased systemic and renal levels of oxidative stress, histology, and urine albumin excretion, whereas the p66-null mutation confers a powerful protection phenotype. To elucidate the mechanism(s) of the protection phenotype, we turned to our in vitro system. Experiments with cultured podocytes revealed previously unrecognized cross talk between p66 and the redox-sensitive transcription factor p53 that controls hyperglycemia-induced ROS metabolism, transcription of p53 target genes (angiotensinogen, angiotensin II type-1 receptor, and bax), angiotensin II generation, and apoptosis. RNA-interference targeting p66 inhibits all of the above. Finally, protein levels of p53 target genes were upregulated in kidneys of Akita mice but unchanged in p66-null Akita mice. Taken together, p66 is a potential molecular target for therapeutic intervention in DG.
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Diabetes Mellitus/genética , Insulina/genética , Mutación/genética , Fenotipo , Receptor de Bradiquinina B2/genética , Proteínas Adaptadoras de la Señalización Shc/genética , Animales , Apoptosis , Células Cultivadas , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Modelos Animales de Enfermedad , Hiperglucemia/metabolismo , Hiperglucemia/patología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Podocitos/metabolismo , Podocitos/patología , Receptor de Bradiquinina B2/deficiencia , Receptor de Bradiquinina B2/metabolismo , Proteínas Adaptadoras de la Señalización Shc/deficiencia , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Glioblastomas are the most aggressive brain tumors for which therapeutic options are limited. Current therapies against glioblastoma include surgical resection, followed by radiotherapy plus concomitant treatment and maintenance with temozolomide (TMZ), however, these standard therapies are often ineffective, and average survival time for glioblastoma patients is between 12 and 18 months. We have previously reported a strong anti-glioblastoma activity of several metabolic compounds, which were synthetized based compounds, which were synthetized based on the chemical structure of a common lipid-lowering drug, fenofibrate, and share a general molecular skeleton of benzoylphenoxyacetamide (BPA). Extensive computational analyses of phenol and naphthol moieties added to the BPA skeleton were performed in this study with the objective of selecting new BPA variants for subsequent compound preparation and anti-glioblastoma testing. Initially, 81 structural variations were considered and their physical properties such as solubility (logS), blood-brain partitioning (logBB), and probability of entering the CNS calculated by the Central Nervous System-Multiparameter Optimization (MPO-CNS) algorithm were evaluated. From this initial list, 18 compounds were further evaluated for anti-glioblastoma activity in vitro. Nine compounds demonstrated desirable glioblastoma cell toxicity in cell culture, and two of them, HR51, and HR59 demonstrated significantly improved capability of crossing the model blood-brain-barrier (BBB) composed of endothelial cells, astrocytes and pericytes.
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Neoplasias Encefálicas , Glioblastoma , Antineoplásicos Alquilantes/farmacología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/patología , Células Endoteliales/metabolismo , Glioblastoma/patología , Humanos , Temozolomida/farmacologíaRESUMEN
Inhibitor of differentiation-1 (Id-1) is a member of helix-loop-helix (HLH) family of proteins that regulate gene transcription through their inhibitory binding to basic-HLH transcription factors. Similarly to other members of this family, Id-1 is involved in the repression of cell differentiation and activation of cell growth. The dual function of Id-1, inhibition of differentiation, and stimulation of cell proliferation, might be interdependent, as cell differentiation is generally coupled with the exit from the cell cycle. Fibroblast growth factor-2 (FGF-2) has been reported to play multiple roles in different biological processes during development of the central nervous system (CNS). In addition, FGF-2 has been described to induce "neuronal-like" differentiation and trigger apoptosis in neuroblastoma SK-N-MC cells. Although regulation of Id-1 protein by several mitogenic factors is well-established, little is known about the role of FGF-2 in the regulation of Id-1. Using human neuroblastoma cell line, SK-N-MC, we found that treatment of these cells with FGF-2 resulted in early induction of both Id-1 mRNA and protein. The induction occurs within 1 h from FGF-2 treatment and is mediated by ERK1/2 pathway, which in turn stimulates expression of the early growth response-1 (Egr-1) transcription factor. We also demonstrate direct interaction of Egr-1 with Id-1 promoter in vitro and in cell culture. Finally, inhibition of Id-1 expression results in G(2) /M accumulation of FGF-2-treated cells and delayed cell death.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Neuroblastoma/metabolismo , Sitios de Unión , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Ciclo Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína 1 Inhibidora de la Diferenciación/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
In HIV patients, antiretroviral medications trigger metabolic abnormalities, including insulin resistance. In addition, the inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated in human immunodeficiency virus encephalitis (HIVE), also induces insulin resistance and inflicts neuronal damage in vitro. In differentiated PC12 cells and rat cortical neurons, high glucose (HG; 25 mM) triggers reactive oxygen species (ROS) accumulation, contributing to the retraction of neuronal processes, with only a minimal involvement of neuronal apoptosis. In the presence of TNFα, HG-treated neurons undergo massive apoptosis. Because mammalian homolog of the Forkhead family of transcription factors, Forkhead box O transcription factor 3a (FOXO3a), controls ROS metabolism, we asked whether FOXO3a could affect the fate of differentiated neurons in the paradigm of HIVE. We observed FOXO3a nuclear translocation in HG-treated neuronal cultures, accompanied by partial loss of mitochondrial potential and gradual retraction of neuronal processes. Addition of TNFα to HG-treated neurons increased expression of the FOXO-dependent proapoptotic gene Bim, which resulted in extensive apoptotic death. Insulin-like growth factor-I (IGF-I) significantly lowered intracellular ROS, which was accompanied by IGF-I-mediated FOXO3a nuclear export and decrease in its transcriptional activity. The clinical relevance of these findings is supported by detection of nuclear FOXO3a in TUNEL-positive cortical neurons from HIVE, especially in brain areas characterized by elevated TNFα.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Factores de Transcripción Forkhead/metabolismo , Resistencia a la Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Neuronas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Western Blotting , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box O3 , Glucosa/metabolismo , Etiquetado Corte-Fin in Situ , Potencial de la Membrana Mitocondrial/fisiología , Proteínas de la Membrana/metabolismo , Neuronas/patología , Células PC12 , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
MicroRNA-mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir-146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir-146a, the chemokine CCL8/MCP-2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5-mediated HIV-1 entry and replication. In the present study, we have analyzed changes in the expression of mir-146a in primary human fetal microglial cells upon infection with HIV-1 and found increased expression of mir-146a. We further show that CCL8/MCP-2 is a target for mir-146a in HIV-1 infected microglia, as overexpression of mir-146a prevented HIV-induced secretion of MCP-2 chemokine. The clinical relevance of our findings was evaluated in HIV-encephalitis (HIVE) brain samples in which decreased levels of MCP-2 and increased levels of mir-146a were observed, suggesting a role for mir-146a in the maintenance of HIV-mediated chronic inflammation of the brain.
Asunto(s)
Quimiocina CCL8/antagonistas & inhibidores , Infecciones por VIH/etiología , VIH-1/patogenicidad , MicroARNs/genética , Microglía/virología , Células Cultivadas , Encefalitis Viral/patología , Regulación de la Expresión Génica/inmunología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Humanos , Inflamación/virologíaRESUMEN
Persons living with HIV (PLWH) are at higher risk of developing secondary illnesses than their uninfected counterparts, suggestive of a dysfunctional immune system in these individuals. Upon exposure to pathogens, monocytes undergo epigenetic remodeling that results in either a trained or a tolerant phenotype, characterized by hyper-responsiveness or hypo-responsiveness to secondary stimuli, respectively. We utilized CD14+ monocytes from virally suppressed PLWH and healthy controls for in vitro analysis following polarization of these cells toward a pro-inflammatory monocyte-derived macrophage (MDM) phenotype. We found that in PLWH-derived MDMs, pro-inflammatory signals (TNFA, IL6, IL1B, miR-155-5p, and IDO1) dominate over negative feedback signals (NCOR2, GSN, MSC, BIN1, and miR-146a-5p), favoring an abnormally trained phenotype. The mechanism of this reduction in negative feedback involves the attenuated expression of IKZF1, a transcription factor required for de novo synthesis of RELA during LPS-induced inflammatory responses. Furthermore, restoring IKZF1 expression in PLWH-MDMs partially reinstated expression of negative regulators of inflammation and lowered the expression of pro-inflammatory cytokines. Overall, this mechanism may provide a link between dysfunctional immune responses and susceptibility to co-morbidities in PLWH with low or undetectable viral load.