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Herpes simplex virus type 2 (HSV-2) coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2-infected and bystander 2D10 cells. Bulk and single-cell RNA-Seq studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2-infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms, including upregulation of MALAT1 to release epigenetic silencing.
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Infecciones por VIH , ARN Largo no Codificante , Humanos , Herpesvirus Humano 2/genética , Linfocitos T CD4-Positivos , ARN Largo no Codificante/genética , Regulación hacia Arriba , Etopósido , Infecciones por VIH/genética , Latencia del VirusRESUMEN
Knockout (KO) mouse models play critical roles in elucidating biological processes behind disease-associated or disease-resistant traits. As a presumed consequence of gene KO, mice display certain phenotypes. Based on insight into the molecular role of said gene in a biological process, it is inferred that the particular biological process causally underlies the trait. This approach has been crucial towards understanding the basis of pathological and/or advantageous traits associated with Mertk KO mice. Mertk KO mice suffer from severe, early-onset retinal degeneration. MERTK, expressed in retinal pigment epithelia, is a receptor tyrosine kinase with a critical role in phagocytosis of apoptotic cells or cellular debris. Therefore, early-onset, severe retinal degeneration was described to be a direct consequence of failed MERTK-mediated phagocytosis of photoreceptor outer segments by retinal pigment epithelia. Here, we report that the loss of Mertk alone is not sufficient for retinal degeneration. The widely used Mertk KO mouse carries multiple coincidental changes in its genome that affect the expression of a number of genes, including the Mertk paralog Tyro3. Retinal degeneration manifests only when the function of Tyro3 is concomitantly lost. Furthermore, Mertk KO mice display improved anti-tumor immunity. MERTK is expressed in macrophages. Therefore, enhanced anti-tumor immunity was inferred to result from the failure of macrophages to dispose of cancer cell corpses, resulting in a pro-inflammatory tumor microenvironment. The resistance against two syngeneic mouse tumor models observed in Mertk KO mice is not, however, phenocopied by the loss of Mertk alone. Neither Tyro3 nor macrophage phagocytosis by alternate genetic redundancy accounts for the absence of anti-tumor immunity. Collectively, our results indicate that context-dependent epistasis of independent modifier alleles determines Mertk KO traits.
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Degeneración Retiniana , Alelos , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Fagocitosis/genética , Fenotipo , Proteínas Proto-Oncogénicas/genética , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Pigmentos Retinianos , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa c-Mer/metabolismoRESUMEN
INTRODUCTION: Pre-exposure prophylaxis (PrEP) is proven to be a highly effective HIV prevention method for key populations. However, its scale-up in resource-limited settings remains suboptimal. This paper seeks to describe PrEP initiation and continuation among key populations in Cameroon. METHODOLOGY: From June 2019 through October 2020, we collected routine program data on PrEP uptake and continuation among female sex workers (FSWs) and men who have sex with men (MSM) in the Continuum of prevention, care and treatment of HIV/AIDS with Most-at-risk Populations (CHAMP) program in Cameroon. PrEP was offered to clients who tested negative for HIV and were assessed to potentially benefit from PrEP. Using survival analysis, we identified factors associated with PrEP discontinuation over time with significance set at 5%. RESULTS: Overall, 27,750 clients were sensitized for PrEP of whom 3,138 persons were eligible to start PrEP and 1,409 (45%; FSW: 691 and MSM: 718) initiated PrEP. The PrEP continuation rate was 37% at 3 months, 28% at 6 months and 19% at 12 months. PrEP discontinuation was significantly higher among FSW than MSM [adjusted hazard ratio (aHR) 1.5 (95% CI: 1.2 to 1.9)] in Yaounde [aHR 1.5 (95% CI: 1.2 to 1.9)] and Bafoussam/Bertoua [aHR 3.1 (2.2-4.5)] relative to Douala. Discontinuation was lower among those with moderate [aHR 0.3 (0.3-0.4)] or good adherence [aHR 0.4 (0.3-0.6)] compared with poor adherence (all P < 0.001). CONCLUSION: Differentiated approaches to deliver PrEP, create demand, and provide more intensive support for adherence and continuation may support scale-up of PrEP in Cameroon for equitable and prolonged impact on HIV prevention.
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Fármacos Anti-VIH , Infecciones por VIH , Profilaxis Pre-Exposición , Trabajadores Sexuales , Minorías Sexuales y de Género , Fármacos Anti-VIH/uso terapéutico , Camerún , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Homosexualidad Masculina , Humanos , Masculino , Profilaxis Pre-Exposición/métodosRESUMEN
BACKGROUND AND OBJECTIVES: Autoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. METHODS: An HEK293T cell line-modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)-was used to measure AChR autoantibody-mediated MAC formation through flow cytometry. RESULTS: Serum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58-1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10-90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, p = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset. DISCUSSION: A novel assay for evaluating AChR autoantibody-mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.
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Miastenia Gravis , Autoanticuerpos , Activación de Complemento , Células HEK293 , Humanos , Receptores ColinérgicosRESUMEN
Background: The use of information and communications technologies (ICT) in low- and middle-income countries (LMIC) has increased significantly in the last several years, particularly in health, including related areas such as preventing and responding to sexual and gender-based violence (SGBV) against women and children. While the evidence for ICT effectiveness has grown significantly in the past 5 years in other aspects of health, it has not for effectiveness of using ICT for the prevention and response to SGBV against women and children in LMIC. Objectives: The primary goal of this evidence and gap map (EGM) is to establish a baseline for the state of the evidence connected with the use of ICT for preventing and responding to SGBV against women and children in LMIC. Objectives that contribute to the achievement of this goal are: (1)identifying evidence of effectiveness for the use of ICT targeting the prevention of, and response to, SGBV against women and children in LMIC;(2)identifying key gaps in the available ICT for SGBV prevention and/or response evidence;(3)identifying research methodology issues reflected in the current evidence;(4)identifying any clusters of evidence in one or more ICT interventions suitable for systematic review;(5)identifying enabling factors associated with effective interventions using ICT for the prevention of, and response to, SGBV against women and children in LMIC; and(6)providing a structured and accessible guide to stakeholders for future investment into interventions and research using ICT for SGBV prevention and response in LMIC. Search Methods: The date of the last search from which records were evaluated, and any studies identified were incorporated into the EGM was July 11, 2021. Twenty (20) databases were searched, and identified under "Methods." Selection Criteria: We conducted systematic searches of multiple academic databases using search terms and criteria related to the use of ICT for prevention and/or response to SGBV against women and children. Although excluded, we did consider studies conducted in higher-income countries (HIC) only to provide context and contrast for the EGM discussion of the eligible studies from LMIC. Data Collection and Analysis: The EGM search process included five phases: (1) initial search of academic databases conducted by two researchers simultaneously; (2) comparison of search results, and abstract screening by two researchers collaboratively; (3) second screening by reviewing full articles of the studies identified in the first screening by two reviewers independently; (4) comparison of results of second screening; resolution of discrepancies of screening results; and (5) data extraction and analysis. Main Results: The EGM includes 10 studies published in English of which 4 were systematic, literature or scoping reviews directly addressing some aspect of the use of ICT for SGBV prevention and/or response in women and girls. The six individual studies were, or are being, conducted in LMIC (a condition for eligibility). No eligible studies addressed children as a target group, although a number of the ineligible studies reported on the use of ICT for intermediate outcomes connected with violence against children (e.g., digital parenting). Yet, such studies did not explicitly attach those intermediate outcomes to SGBV prevention or response outcomes. Countries represented among the eligible individual studies include Cambodia, Kenya, Nepal Democratic Republic of Congo (DRC), and Lebanon. Of the 10 eligible studies (individual and reviews), most focused on intimate partner violence against women (IPV). Intervention areas among the eligible studies include safety planning using decision algorithms, educational and empowerment messaging regarding norms and attitudes towards gender-based violence (GBV), multi-media radio drama for social behavior change, the collection of survivor experience to inform SGBV/GBV services, and the collection of forensic evidence connected to the perpetration of SGBV. Thirty-one studies which otherwise would have been eligible for the evidence and gap map (EGM) were conducted in HIC (identified under "Excluded Reviews"). None of the eligible studies reported results related to effectiveness of using ICT in a control setting, for the primary prevention of SGBV as an outcome, but rather reported on outcomes such as usability, secondary and tertiary prevention, feasibility, access to services and other outcomes primarily relating to the development of the interventions. Two studies identified IPV prevention as a measurable outcome within their protocols, but one of these had not yet formally published results regarding primary prevention as an outcome. The other study, while reporting on the protocol (and steps to adapt the ICT application, previously reported as effective in HIC contexts to a specific LMIC context), has not yet as of the date of writing this EGM, published outcome results related to the reduction of IPV. Of the four reviews identified as eligible, two are better characterized as either a literature review or case study rather than as traditional systematic reviews reporting on impact outcomes with methodologically rigorous protocols. Authors' Conclusions: The evidence baseline for using ICT to prevent and/or respond to SGBV against women and children in LMIC is nascent. Promising areas for future study include: (1) how ICT can contribute changing gender and social norms related to SGBV and primary prevention; (2) mobile phone applications that promote safety and security; (3) mobile technology for the collection and analysis of survivors' experience with SGBV response services; and (4) digital tools that support the collection of forensic evidence for SGBV response and secondary prevention. Most striking is the paucity of eligible studies examining the use of ICT in connection with preventing or responding to SGBV against children. In light of the exponential increase in the use of ICT by children and adolescents, even in LMIC, greater attention should be given to examining how ICT can be used during adolescence to address gender norms that lead to SGBV. While there appears to be interest in using ICT for SGBV prevention and/or response in LMIC, other than several ad hoc studies, there is little evidence of if, and how effective these interventions are. Further inquiry should be made regarding if and how interventions proven effective in HIC can be adapted to LMIC contexts.
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TUG tethering proteins bind and sequester GLUT4 glucose transporters intracellularly, and insulin stimulates TUG cleavage to translocate GLUT4 to the cell surface and increase glucose uptake. This effect of insulin is independent of phosphatidylinositol 3-kinase, and its physiological relevance remains uncertain. Here we show that this TUG cleavage pathway regulates both insulin-stimulated glucose uptake in muscle and organism-level energy expenditure. Using mice with muscle-specific Tug (Aspscr1)-knockout and muscle-specific constitutive TUG cleavage, we show that, after GLUT4 release, the TUG C-terminal cleavage product enters the nucleus, binds peroxisome proliferator-activated receptor (PPAR)γ and its coactivator PGC-1α and regulates gene expression to promote lipid oxidation and thermogenesis. This pathway acts in muscle and adipose cells to upregulate sarcolipin and uncoupling protein 1 (UCP1), respectively. The PPARγ2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The ATE1 arginyltransferase, which mediates a specific protein degradation pathway and controls thermogenesis, regulates the stability of the TUG product. We conclude that insulin-stimulated TUG cleavage coordinates whole-body energy expenditure with glucose uptake, that this mechanism might contribute to the thermic effect of food and that its attenuation could promote obesity.
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Metabolismo Energético , Glucosa/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células 3T3-L1 , Aminoaciltransferasas/metabolismo , Animales , Ratones , Ratones Noqueados , Oxidación-Reducción , PPAR gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteolisis , TermogénesisRESUMEN
Endogenous retroviruses (ERVs) are genomic sequences that originated from retroviruses and are present in most eukaryotic genomes. Both beneficial and detrimental functions are attributed to ERVs, but whether ERVs contribute to antiviral immunity is not well understood. Here, we used herpes simplex virus type 2 (HSV-2) infection as a model and found that Toll-like receptor 7 (Tlr7-/-) deficient mice that have high systemic levels of infectious ERVs are protected from intravaginal HSV-2 infection and disease, compared to wildtype C57BL/6 mice. We deleted the endogenous ecotropic murine leukemia virus (Emv2) locus on the Tlr7-/- background (Emv2-/-Tlr7-/-) and found that Emv2-/-Tlr7-/- mice lose protection against HSV-2 infection. Intravaginal application of purified ERVs from Tlr7-/- mice prior to HSV-2 infection delays disease in both wildtype and highly susceptible interferon-alpha receptor-deficient (Ifnar1-/-) mice. However, intravaginal ERV treatment did not protect Emv2-/-Tlr7-/- mice from HSV-2 disease, suggesting that the protective mechanism mediated by exogenous ERV treatment may differ from that of constitutively and systemically expressed ERVs in Tlr7-/- mice. We did not observe enhanced type I interferon (IFN-I) signaling in the vaginal tissues from Tlr7-/- mice, and instead found enrichment in genes associated with extracellular matrix organization. Together, our results revealed that constitutive and/or systemic expression of ERVs protect mice against vaginal HSV-2 infection and delay disease.
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Retrovirus Endógenos/inmunología , Herpes Genital/inmunología , Herpes Genital/prevención & control , Herpesvirus Humano 2/inmunología , Enfermedades Vaginales/inmunología , Enfermedades Vaginales/prevención & control , Animales , Retrovirus Endógenos/genética , Femenino , Herpes Genital/genética , Herpesvirus Humano 2/genética , Ratones , Ratones Noqueados , Enfermedades Vaginales/genéticaRESUMEN
This is the protocol for the development of a Campbell Collaboration evidence and gaps map (EGM). The primary objective of this evidence and gap map (EGM) is to answer the following question: (1) What is the evidence connected with the use of information and communications technologies (ICT) for preventing and responding to sexual and gender-based violence (SGBV) against women and children in lower- and middle-income countries (LMIC)? (a) the EGM will provide a structured and accessible contextual framework for research to stakeholders and policymakers in SGBV and ICT; (b) the EGM will identify gaps in the available ICT and SGBV evidence; (c) the EGM will identify clusters of evidence suitable for systematic review; and (d) the EGM will look for and build connections between related areas of research in ICT and SGBV. As part of identifying the evidence connected with the use of ICT for preventing and responding to SGBV we seek to answer the following questions based upon the available evidence: (a)Does the use of ICT prevent SGBV against women and children in LMIC?(b)How effective is ICT at improving access to quality services for SGBV survivors in LMIC?(c)Does the use of ICT contribute to effectively achieving intermediate outcomes that lead to the prevention of SGBV against women and children, and/or improving access for SGBV survivors to response services in LMIC?(d)What are the enabling factors associated with the implementation of ICT and SGBV interventions?
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The receptor for advanced glycation endproducts (RAGE) is expressed in T cells after activation with antigen and is constitutively expressed in T cells from patients at-risk for and with type 1 diabetes mellitus (T1D). RAGE expression was associated with an activated T cell phenotype, leading us to examine whether RAGE is involved in T cell signaling. In primary CD4+ and CD8+ T cells from patients with T1D or healthy control subjects, RAGE- cells showed reduced phosphorylation of Erk. To study T cell receptor signaling in RAGE+ or-T cells, we compared signaling in RAGE+/+ Jurkat cells, Jurkat cells with RAGE eliminated by CRISPR/Cas9, or silenced with siRNA. In RAGE KO Jurkat cells, there was reduced phosphorylation of Zap70, Erk and MEK, but not Lck or CD3ξ. RAGE KO cells produced less IL-2 when activated with anti-CD3 +/- anti-CD28. Stimulation with PMA restored signaling and (with ionomycin) IL-2 production. Silencing RAGE with siRNA also decreased signaling. Our studies show that RAGE expression in human T cells is associated with an activated signaling cascade. These findings suggest a link between inflammatory products that are found in patients with diabetes, other autoimmune diseases, and inflammation that may enhance T cell reactivity.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de Señal , Adolescente , Adulto , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Niño , Femenino , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Inflamación/metabolismo , Células Jurkat , Masculino , Adulto JovenRESUMEN
Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.
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Adenosina Trifosfato/metabolismo , Glucosa/metabolismo , Guanosina Trifosfato/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Succinato-CoA Ligasas/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/genética , Ciclo del Ácido Cítrico/genética , Homeostasis , Humanos , Secreción de Insulina/genética , Secreción de Insulina/fisiología , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Fosforilación Oxidativa , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Regulación hacia ArribaRESUMEN
Breast cancer-associated gene 3 (BCA3) is a recently identified adaptor protein whose functions are still being defined. BCA3 has been reported to be an important regulator of nuclear factor-κB (NF-κB) signaling. It has also been reported to interact with the small GTPase, Rac1. Consistent with that observation, in the present study, BCA3 was found to interact with nuclear Rac1 in 293 cells and influence NF-κB signaling. Additional experiments revealed that depending on cell type, BCA3 augmented, attenuated or had no effect on NF-κB signaling in vitro. Since canonical NF-κB signaling is a critical downstream target from activated receptor activator of nuclear factor κB (RANK) that is required for mature osteoclast formation and function, BCA3 was selectively overexpressed in osteoclasts in vivo using the cathepsin K promoter and the response to exogenous receptor activator of nuclear factor κB ligand (RANKL) administration was examined. Despite its ability to augment NF-κB signaling in other cells, transgenic animals injected with high-dose RANKL had the same hypercalcemic response as their wildtype littermates. Furthermore, the degree of bone loss induced by a 2-week infusion of low-dose RANKL was the same in both groups. Combined with earlier studies, the data from our study data indicate that BCA3 can affect NF-κB signaling and that BCA3 plays a cell-type dependent role in this process. The significance of the BCA3/NF-κB interaction in vivo in bone remains to be determined.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Resorción Ósea/genética , FN-kappa B/metabolismo , Neuropéptidos/metabolismo , Osteoclastos/efectos de los fármacos , Ligando RANK/administración & dosificación , Proteína de Unión al GTP rac1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Resorción Ósea/inducido químicamente , Resorción Ósea/metabolismo , Resorción Ósea/patología , Catepsina K/genética , Catepsina K/metabolismo , Línea Celular , Femenino , Fémur/efectos de los fármacos , Fémur/metabolismo , Fémur/patología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/genética , Neuropéptidos/genética , Especificidad de Órganos , Osteoclastos/citología , Osteoclastos/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal , Tibia/efectos de los fármacos , Tibia/metabolismo , Tibia/patología , Proteína de Unión al GTP rac1/genéticaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance.
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Grasas de la Dieta/efectos adversos , Resistencia a la Insulina , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Grasas de la Dieta/farmacología , Glucógeno/biosíntesis , Glucógeno/genética , Hígado/patología , Ratones , Ratones Mutantes , Mutación Missense , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/genética , Fosforilación , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismo , Receptor de Insulina/genéticaRESUMEN
CEACAM1 promotes insulin extraction, an event that occurs mainly in liver. Phenocopying global Ceacam1 null mice (Cc1(-/-) ), C57/BL6J mice fed a high-fat (HF) diet exhibited reduced hepatic CEACAM1 levels and impaired insulin clearance, followed by hyperinsulinemia, insulin resistance, and visceral obesity. Conversely, forced liver-specific expression of CEACAM1 protected insulin sensitivity and energy expenditure, and limited gain in total fat mass by HF diet in L-CC1 mice. Because CEACAM1 protein is barely detectable in white adipose tissue (WAT), we herein investigated whether hepatic CEACAM1-dependent insulin clearance pathways regulate adipose tissue biology in response to dietary fat. While HF diet caused a similar body weight gain in L-CC1, this effect was delayed and less intense relative to wild-type (WT) mice. Histological examination revealed less expansion of adipocytes in L-CC1 than WT by HF intake. Immunofluorescence analysis demonstrated a more limited recruitment of crown-like structures, and qRT-PCR analysis showed no significant rise in TNFα mRNA levels in response to HF intake in L-CC1 than WT mice. Unlike WT, HF diet did not activate TGF-ß in WAT of L-CC1 mice, as assessed by Western analysis of Smad2/3 phosphorylation. Consistently, HF diet caused relatively less collagen deposition in L-CC1 than WT mice, as shown by Trichrome staining. Coupled with reduced lipid redistribution from liver to visceral fat, lower inflammation and fibrosis could contribute to protected energy expenditure against HF diet in L-CC1 mice. The data underscore the important role of hepatic insulin clearance in the regulation of adipose tissue inflammation and fibrosis.
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Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates insulin sensitivity by promoting hepatic insulin clearance. Liver-specific inactivation or global null-mutation of Ceacam1 impairs hepatic insulin extraction to cause chronic hyperinsulinemia, resulting in insulin resistance and visceral obesity. In this study we investigated whether diet-induced insulin resistance implicates changes in hepatic CEACAM1. We report that feeding C57/BL6J mice a high-fat diet reduced hepatic CEACAM1 levels by >50% beginning at 21 days, causing hyperinsulinemia, insulin resistance, and elevation in hepatic triacylglycerol content. Conversely, liver-specific inducible CEACAM1 expression prevented hyperinsulinemia and markedly limited insulin resistance and hepatic lipid accumulation that were induced by prolonged high-fat intake. This was partly mediated by increased hepatic ß-fatty acid oxidation and energy expenditure. The data demonstrate that the high-fat diet reduced hepatic CEACAM1 expression and that overexpressing CEACAM1 in liver curtailed diet-induced metabolic abnormalities by protecting hepatic insulin clearance.
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Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Dieta Alta en Grasa , Resistencia a la Insulina/genética , Hígado/metabolismo , Animales , Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Metabolismo Energético/fisiología , Ácidos Grasos/metabolismo , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Insulina/sangre , Ratones , Ratones TransgénicosRESUMEN
The MYC oncogene is frequently mutated and overexpressed in human renal cell carcinoma (RCC). However, there have been no studies on the causative role of MYC or any other oncogene in the initiation or maintenance of kidney tumorigenesis. Here, we show through a conditional transgenic mouse model that the MYC oncogene, but not the RAS oncogene, initiates and maintains RCC. Desorption electrospray ionization-mass-spectrometric imaging was used to obtain chemical maps of metabolites and lipids in the mouse RCC samples. Gene expression analysis revealed that the mouse tumors mimicked human RCC. The data suggested that MYC-induced RCC up-regulated the glutaminolytic pathway instead of the glycolytic pathway. The pharmacologic inhibition of glutamine metabolism with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide impeded MYC-mediated RCC tumor progression. Our studies demonstrate that MYC overexpression causes RCC and points to the inhibition of glutamine metabolism as a potential therapeutic approach for the treatment of this disease.
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Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Genes myc , Glutamina/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Animales , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Genes ras , Glutaminasa/antagonistas & inhibidores , Glutaminasa/metabolismo , Humanos , Neoplasias Renales/patología , Metabolismo de los Lípidos , Ratones , Ratones SCID , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Sulfuros/farmacología , Tiadiazoles/farmacología , Regulación hacia ArribaRESUMEN
Mucus production by goblet cells of the large intestine serves as a crucial antimicrobial protective mechanism at the interface between the eukaryotic and prokaryotic cells of the mammalian intestinal ecosystem. However, the regulatory pathways involved in goblet cell-induced mucus secretion remain largely unknown. Here, we demonstrate that the NLRP6 inflammasome, a recently described regulator of colonic microbiota composition and biogeographical distribution, is a critical orchestrator of goblet cell mucin granule exocytosis. NLRP6 deficiency leads to defective autophagy in goblet cells and abrogated mucus secretion into the large intestinal lumen. Consequently, NLRP6 inflammasome-deficient mice are unable to clear enteric pathogens from the mucosal surface, rendering them highly susceptible to persistent infection. This study identifies an innate immune regulatory pathway governing goblet cell mucus secretion, linking nonhematopoietic inflammasome signaling to autophagy and highlighting the goblet cell as a critical innate immune player in the control of intestinal host-microbial mutualism. PAPERCLIP:
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Colon/inmunología , Células Caliciformes/inmunología , Inflamasomas/inmunología , Mucosa Intestinal/inmunología , Receptores de Superficie Celular/inmunología , Animales , Autofagia , Colitis/inmunología , Colitis/microbiología , Colon/microbiología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Caliciformes/citología , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Moco/metabolismoRESUMEN
This systematic review assesses the published literature to describe the landscape of mobile health technology (mHealth) for HIV/AIDS and the evidence supporting the use of these tools to address the HIV prevention, care, and treatment cascade. The speed of innovation, broad range of initiatives and tools, and heterogeneity in reporting have made it difficult to uncover and synthesize knowledge on how mHealth tools might be effective in addressing the HIV pandemic. To do address this gap, a team of reviewers collected literature on the use of mobile technology for HIV/AIDS among health, engineering, and social science literature databases and analyzed a final set of 62 articles. Articles were systematically coded, assessed for scientific rigor, and sorted for HIV programmatic relevance. The review revealed evidence that mHealth tools support HIV programmatic priorities, including: linkage to care, retention in care, and adherence to antiretroviral treatment. In terms of technical features, mHealth tools facilitate alerts and reminders, data collection, direct voice communication, educational messaging, information on demand, and more. Studies were mostly descriptive with a growing number of quasi-experimental and experimental designs. There was a lack of evidence around the use of mHealth tools to address the needs of key populations, including pregnant mothers, sex workers, users of injection drugs, and men who have sex with men. The science and practice of mHealth for HIV are evolving rapidly, but still in their early stages. Small-scale efforts, pilot projects, and preliminary descriptive studies are advancing and there is a promising trend toward implementing mHealth innovation that is feasible and acceptable within low-resource settings, positive program outcomes, operational improvements, and rigorous study design.
RESUMEN
Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG (tether containing a ubiquitin regulatory X (UBX) domain for glucose transporter 4 (GLUT4)). Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST (PDZ domain protein interacting specifically with TC10), which transmits an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations compared with controls. Whole-body glucose turnover was increased during fasting but not during hyperinsulinemic clamp studies. In muscles with the greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamp studies. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure were increased by 12-13%. After 3 weeks on a high fat diet, the decreased fasting plasma glucose in transgenic mice compared with controls was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that the effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice.
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Proteínas Portadoras/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Músculo Esquelético/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras Transductoras de Señales , Animales , Glucemia/metabolismo , Dióxido de Carbono/metabolismo , Proteínas Portadoras/genética , Desoxiglucosa/metabolismo , Ayuno/sangre , Femenino , Glucógeno/metabolismo , Proteínas de la Matriz de Golgi , Hipoglucemiantes/sangre , Hipoglucemiantes/farmacología , Immunoblotting , Insulina/sangre , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacosRESUMEN
Transplantation of human hematopoietic stem cells into severely immunocompromised newborn mice allows the development of a human hematopoietic and immune system in vivo. NOD/scid/γ(c)(-/-) (NSG) and BALB/c Rag2(-/-)γ(c)(-/-) mice are the most commonly used mouse strains for this purpose and a number of studies have demonstrated the high value of these model systems in areas spanning from basic to translational research. However, limited cross-reactivity of many murine cytokines on human cells and residual host immune function against the xenogeneic grafts results in defective development and maintenance of human cells in vivo. Whereas NSG mice have higher levels of absolute human engraftment than similar mice on a BALB/c background, they have a shorter lifespan and NOD ES cells are unsuitable for the complex genetic engineering that is required to improve human hematopoiesis and immune responses by transgenesis or knockin of human genes. We have generated mice that faithfully express a transgene of human signal regulatory protein alpha (SIRPa), a receptor that negatively regulates phagocytosis, in Rag2(-/-)γ(c)(-/-) mice on a mixed 129/BALB/c background, which can easily be genetically engineered. These mice allow significantly increased engraftment and maintenance of human hematopoietic cells reaching levels comparable to NSG mice. Furthermore, we found improved functionality of the human immune system in these mice. In summary, hSIRPa-transgenic Rag2(-/-)γ(c)(-/-) mice represent a unique mouse strain supporting high levels of human cell engraftment, which can easily be genetically manipulated.
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Antígenos de Diferenciación/metabolismo , Proteínas de Unión al ADN/deficiencia , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Receptores Inmunológicos/metabolismo , Transgenes/genética , Animales , Antígenos de Diferenciación/genética , Células de la Médula Ósea/patología , Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Epítopos/inmunología , Humanos , Inmunidad Humoral/inmunología , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Receptores Inmunológicos/genéticaRESUMEN
OBJECTIVE: Insulin resistance is a major characteristic of type 2 diabetes and is causally associated with obesity. Inflammation plays an important role in obesity-associated insulin resistance, but the underlying mechanism remains unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine with lower circulating levels in obese subjects, and acute treatment with IL-10 prevents lipid-induced insulin resistance. We examined the role of IL-10 in glucose homeostasis using transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10). RESEARCH DESIGN AND METHODS: MCK-IL10 and wild-type mice were fed a high-fat diet (HFD) for 3 weeks, and insulin sensitivity was determined using hyperinsulinemic-euglycemic clamps in conscious mice. Biochemical and molecular analyses were performed in muscle to assess glucose metabolism, insulin signaling, and inflammatory responses. RESULTS: MCK-IL10 mice developed with no obvious anomaly and showed increased whole-body insulin sensitivity. After 3 weeks of HFD, MCK-IL10 mice developed comparable obesity to wild-type littermates but remained insulin sensitive in skeletal muscle. This was mostly due to significant increases in glucose metabolism, insulin receptor substrate-1, and Akt activity in muscle. HFD increased macrophage-specific CD68 and F4/80 levels in wild-type muscle that was associated with marked increases in tumor necrosis factor-alpha, IL-6, and C-C motif chemokine receptor-2 levels. In contrast, MCK-IL10 mice were protected from diet-induced inflammatory response in muscle. CONCLUSIONS: These results demonstrate that IL-10 increases insulin sensitivity and protects skeletal muscle from obesity-associated macrophage infiltration, increases in inflammatory cytokines, and their deleterious effects on insulin signaling and glucose metabolism. Our findings provide novel insights into the role of anti-inflammatory cytokine in the treatment of type 2 diabetes.