RESUMO
BACKGROUND: Cognitive dysfunction (CD) is common among patients with the autoimmune disease systemic lupus erythematosus (SLE). Anti-ribosomal P autoantibodies associate with this dysfunction and have neuropathogenic effects that are mediated by cross-reacting with neuronal surface P antigen (NSPA) protein. Elucidating the function of NSPA can then reveal CD pathogenic mechanisms and treatment opportunities. In the brain, NSPA somehow contributes to glutamatergic NMDA receptor (NMDAR) activity in synaptic plasticity and memory. Here we analyze the consequences of NSPA absence in KO mice considering its structural features shared with E3 ubiquitin ligases and the crucial role of ubiquitination in synaptic plasticity. RESULTS: Electrophysiological studies revealed a decreased long-term potentiation in CA3-CA1 and medial perforant pathway-dentate gyrus (MPP-DG) hippocampal circuits, reflecting glutamatergic synaptic plasticity impairment in NSPA-KO mice. The hippocampal dentate gyrus of these mice showed a lower number of Arc-positive cells indicative of decreased synaptic activity and also showed proliferation defects of neural progenitors underlying less adult neurogenesis. All this translates into poor spatial and recognition memory when NSPA is absent. A cell-based assay demonstrated ubiquitination of NSPA as a property of RBR-type E3 ligases, while biochemical analysis of synaptic regions disclosed the tyrosine phosphatase PTPMEG as a potential substrate. Mice lacking NSPA have increased levels of PTPMEG due to its reduced ubiquitination and proteasomal degradation, which correlated with lower levels of GluN2A and GluN2B NMDAR subunits only at postsynaptic densities (PSDs), indicating selective trafficking of these proteins out of PSDs. As both GluN2A and GluN2B interact with PTPMEG, tyrosine (Tyr) dephosphorylation likely drives their endocytic removal from the PSD. Actually, immunoblot analysis showed reduced phosphorylation of the GluN2B endocytic signal Tyr1472 in NSPA-KO mice. CONCLUSIONS: NSPA contributes to hippocampal plasticity and memory processes ensuring appropriate levels of adult neurogenesis and PSD-located NMDAR. PTPMEG qualifies as NSPA ubiquitination substrate that regulates Tyr phosphorylation-dependent NMDAR stability at PSDs. The NSPA/PTPMEG pathway emerges as a new regulator of glutamatergic transmission and plasticity and may provide mechanistic clues and therapeutic opportunities for anti-P-mediated pathogenicity in SLE, a still unmet need.
Assuntos
Antígenos de Superfície/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 4/genética , Receptores de N-Metil-D-Aspartato/genética , Animais , Antígenos de Superfície/metabolismo , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Proteína Tirosina Fosfatase não Receptora Tipo 4/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , UbiquitinaçãoRESUMO
Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients. Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. ß-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expression in a wide range of CNS regions, including high expression in the choroid-plexus. Accordingly, we detected Gal-8 in human cerebrospinal fluid, suggesting a role in the CNS immune-surveillance circuit. In addition, we show that MS patients generate function-blocking anti-Gal-8 antibodies with pathogenic potential. Such antibodies block cell adhesion and Gal-8-induced Th17 apoptosis. Furthermore, circulating anti-Gal-8 antibodies associate with relapsing-remitting MS (RRMS), and not with progressive MS phenotypes, predicting clinical disability at diagnosis within the first year of follow-up. Our results reveal that Gal-8 has an immunosuppressive protective role against autoimmune CNS inflammation, modulating the balance of Th17 and Th1 polarization and their respective Tregs. Such a role can be counteracted during RRMS by anti-Gal-8 antibodies, worsening disease prognosis. Even though anti-Gal-8 antibodies are not specific for MS, our results suggest that they could be a potential early severity biomarker in RRMS.
Assuntos
Autoanticorpos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Galectinas/imunologia , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Apoptose/fisiologia , Encéfalo/imunologia , Encéfalo/metabolismo , Adesão Celular/fisiologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Galectinas/genética , Galectinas/metabolismo , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Prognóstico , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismoRESUMO
Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPµ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPµ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPµ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPµ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPµ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.
Assuntos
Endotélio Vascular/metabolismo , Rim/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Camundongos , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvß3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvß3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvß3 interaction enhances Src recruitment to integrin αvß3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvß3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvß3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvß3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Angiopoietinas/metabolismo , Permeabilidade Capilar/fisiologia , Integrina alfaVbeta3/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/deficiência , Animais , Hipóxia Celular/fisiologia , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/fisiopatologia , Humanos , Camundongos Knockout , Fosforilação/fisiologia , Retina/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismoRESUMO
Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury.
Assuntos
Injúria Renal Aguda/metabolismo , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Proteínas dos Microfilamentos/genética , Podócitos/metabolismo , Actinas/metabolismo , Injúria Renal Aguda/induzido quimicamente , Animais , Células Cultivadas , Modelos Animais de Doenças , Forminas , Heparina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica de Transmissão , Fenótipo , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/patologia , Podócitos/ultraestrutura , Mutação Puntual , Protaminas/toxicidade , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Improved surgical and medical therapy have prolonged survival in patients with congenital heart disease (CHD) such that general medical conditions like coronary artery disease (CAD) are now the main determinants of mortality. A summary of the association of CAD with CHD, as well as a discussion of the radiologic evaluation of the coronary arteries in adults with CHD is described herein. Cross sectional imaging to evaluate CAD in adults with CHD should follow the same appropriateness criteria as gender and aged matched patients without CHD. Coronary CT imaging may be particularly valuable in evaluating the coronary arteries in this patient population as invasive coronary angiography may prove challenging secondary to complicated or unconventional anatomy of the coronary arteries. Further, typical methods for evaluating CAD such as stress or echocardiography may be impractical in adults with CHD. Finally, delineating the anatomic relationship of the coronary arteries and their relationship with the sternum, chest wall, conduits, grafts, and valves is highly recommended in patients with CHD prior to reintervention to avoid iatrogenic complications.
Assuntos
Angiografia Coronária/métodos , Doença da Artéria Coronariana/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Cardiopatias Congênitas/complicações , Tomografia Computadorizada por Raios X , Adulto , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/terapia , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/terapia , Humanos , Valor Preditivo dos Testes , PrognósticoRESUMO
Kir7.1 is an inwardly rectifying K+ channel of the Kir superfamily encoded by the kcnj13 gene. Kir7.1 is present in epithelial tissues where it colocalizes with the Na+/K+-pump probably serving to recycle K+ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree.
Assuntos
Fissura Palatina/patologia , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Animais , Animais Recém-Nascidos , Peso Corporal , Fissura Palatina/embriologia , Desenvolvimento Embrionário , Epitélio/metabolismo , Epitélio/patologia , Pulmão/anormalidades , Pulmão/embriologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Análise de SobrevidaRESUMO
Secreted frizzled-related protein 4 (SFRP4) is an extracellular regulator of the wingless-type mouse mammary tumor virus integration site family (WNT) pathway. SFRP4 has been implicated in adipocyte dysfunction, obesity, insulin resistance, and impaired insulin secretion in patients with type 2 diabetes. However, the exact role of SFRP4 in regulating whole-body metabolism and glucose homeostasis is unknown. We show here that male Sfrp4(-/-) mice have increased spine length and gain more weight when fed a high-fat diet. The body composition and body mass per spine length of diet-induced obese Sfrp4(-/-) mice is similar to wild-type littermates, suggesting that the increase in body weight can be accounted for by their longer body size. The diet-induced obese Sfrp4(-/-) mice have reduced energy expenditure, food intake, and bone mineral density. Sfrp4(-/-) mice have normal glucose and insulin tolerance and ß-cell mass. Diet-induced obese Sfrp4(-/-) and control mice show similar impairments of glucose tolerance and a 5-fold compensatory expansion of their ß-cell mass. In summary, our data suggest that loss of SFRP4 alters body length and bone mineral density as well as energy expenditure and food intake. However, SFRP4 does not control glucose homeostasis and ß-cell mass in mice.
Assuntos
Tamanho Corporal/genética , Densidade Óssea/genética , Dieta Hiperlipídica , Ingestão de Alimentos/genética , Metabolismo Energético/genética , Células Secretoras de Insulina/metabolismo , Obesidade , Proteínas Proto-Oncogênicas/genética , Animais , Glicemia/metabolismo , Composição Corporal/genética , Comportamento Alimentar , Técnicas de Introdução de Genes , Teste de Tolerância a Glucose , Células HEK293 , Homeostase/genética , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Via de Sinalização Wnt , Microtomografia por Raio-XRESUMO
Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia.
Assuntos
Angiopoietinas/imunologia , Anticorpos Monoclonais/imunologia , Dislipidemias/sangue , Lipídeos/sangue , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/metabolismo , Animais , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Humanos , Lipase/sangue , Lipase Lipoproteica/sangue , Macaca fascicularis , Masculino , Camundongos , Ratos , Triglicerídeos/sangueRESUMO
Antagonizing glucagon action represents an attractive therapeutic option for reducing hepatic glucose production in settings of hyperglycemia where glucagon excess plays a key pathophysiological role. We therefore generated REGN1193, a fully human monoclonal antibody that binds and inhibits glucagon receptor (GCGR) signaling in vitro. REGN1193 administration to diabetic ob/ob and diet-induced obese mice lowered blood glucose to levels observed in GCGR-deficient mice. In diet-induced obese mice, REGN1193 reduced food intake, adipose tissue mass, and body weight. REGN1193 increased circulating levels of glucagon and glucagon-like peptide 1 and was associated with reversible expansion of pancreatic α-cell area. Hyperglucagonemia and α-cell hyperplasia was observed in fibroblast growth factor 21-deficient mice treated with REGN1193. Single administration of REGN1193 to diabetic cynomolgus monkeys normalized fasting blood glucose and glucose tolerance and increased circulating levels of glucagon and amino acids. Finally, administration of REGN1193 for 8 weeks to normoglycemic cynomolgus monkeys did not cause hypoglycemia or increase pancreatic α-cell area. In summary, the GCGR-blocking antibody REGN1193 normalizes blood glucose in diabetic mice and monkeys but does not produce hypoglycemia in normoglycemic monkeys. Thus, REGN1193 provides a potential therapeutic modality for diabetes mellitus and acute hyperglycemic conditions.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Receptores de Glucagon/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Glicemia/metabolismo , Diabetes Mellitus Experimental/complicações , Feminino , Fatores de Crescimento de Fibroblastos/genética , Humanos , Hipoglicemiantes/farmacologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/patologia , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/genéticaRESUMO
In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a lacZ reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.
Assuntos
RNA Longo não Codificante/genética , Transcrição Gênica/genética , Transcriptoma/genética , Alelos , Animais , Desenvolvimento Embrionário/genética , Feminino , Genes Reporter/genética , Masculino , Mamíferos/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , FenótipoRESUMO
G protein-coupled receptor 17 (GPR17) was recently reported to be a Foxo1 target in agouti-related peptide (AGRP) neurons. Intracerebroventricular injection of GPR17 agonists induced food intake, whereas administration of an antagonist to the receptor reduced feeding. These data lead to the conclusion that pharmacological modulation of GPR17 has therapeutic potential to treat obesity. Here we report that mice deficient in Gpr17 (Gpr17(-/-)) have similar food intake and body weight compared with their wild-type littermates. Gpr17(-/-) mice have normal hypothalamic Agrp mRNA expression, AGRP plasma levels, and metabolic rate. GPR17 deficiency in mice did not affect glucose homeostasis or prevent fat-induced insulin resistance. These data do not support a role for GPR17 in the control of food intake, body weight, or glycemic control.
Assuntos
Ingestão de Alimentos/genética , Glucose/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G/genética , Proteína Relacionada com Agouti/metabolismo , Análise de Variância , Animais , Sequência de Bases , Composição Corporal/efeitos dos fármacos , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neurônios/metabolismo , Análise de Sequência de RNA , Fatores de Tempo , Microtomografia por Raio-XRESUMO
Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4(+) T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.
Assuntos
Genes Dominantes/genética , Genes Virais/genética , HIV-1/genética , HIV-1/imunologia , Mutação/genética , Linfócitos T Citotóxicos/imunologia , Latência Viral/imunologia , Doença Aguda/terapia , Animais , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Doença Crônica/tratamento farmacológico , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Humanos , Masculino , Camundongos , RNA Viral/sangue , Carga Viral/efeitos dos fármacos , Latência Viral/genética , Replicação Viral/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Known examples of male to female sex reversal in mice are caused by either strain incompatibilities or mutations in genes required for male sex determination. The resultant XY females are often sterile or exhibit very poor fertility. We describe here embryonic stem (ES) cell growth conditions that promote the production of healthy, anatomically normal fertile and fecund female F0 generation mice completely derived from gene-targeted XY male ES cells. The sex reversal is a transient trait that is not transmitted to the F1 progeny. Growth media with low osmolality and reduced sodium bicarbonate, maintained throughout the gene targeting process, enhance the yield of XY females. As a practical application of the induced sex reversal, we demonstrate the generation of homozygous mutant mice ready for phenotypic studies by the breeding of F0 XY females with their isogenic XY male clonal siblings, thereby eliminating one generation of breeding and the associated costs.
Assuntos
Transtornos do Desenvolvimento Sexual/genética , Fertilidade/genética , Disgenesia Gonadal 46 XY/genética , Processos de Determinação Sexual , Animais , Células-Tronco Embrionárias/citologia , Feminino , Marcação de Genes , Masculino , Camundongos , Microinjeções , MutaçãoRESUMO
Intraluminal procedures for the gastrointestinal tract range from simple intubation for feeding or bowel decompression to endoscopic procedures including stenting and pancreatobiliary ductal catheterization. Each of these procedures and interventions carries a risk of iatrogenic injury, including bleeding, perforation, infection, adhesions, and obstruction. An understanding of how anatomy and function may predispose to injury, and the distinct patterns of injury, can help the radiologist identify and characterize iatrogenic injury rapidly at computed tomography (CT) imaging. Furthermore, selective use of intravenous or oral CT contrast material can help reveal injury and triage clinical management.
Assuntos
Endoscopia Gastrointestinal/efeitos adversos , Doença Iatrogênica , Intubação Gastrointestinal/efeitos adversos , Stents/efeitos adversos , Tomografia Computadorizada por Raios X/métodos , Trato Gastrointestinal Superior/diagnóstico por imagem , Humanos , Trato Gastrointestinal Superior/lesõesRESUMO
Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.
Assuntos
Formação de Anticorpos , Genes de Imunoglobulinas , Alelos , Animais , Linfócitos B/imunologia , Citometria de Fluxo , Humanos , Camundongos , MutaçãoRESUMO
Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.
Assuntos
Genes de Imunoglobulinas , Animais , Cromossomos Artificiais Bacterianos , Células-Tronco Embrionárias/imunologia , Recombinação Homóloga , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , TransgenesRESUMO
Nonalcoholic fatty liver disease is the most common chronic liver disorder in developed countries. Its pathogenesis is poorly understood, and therapeutic options are limited. Here, we show that SIRT7, an NAD(+)-dependent H3K18Ac deacetylase, functions at chromatin to suppress ER stress and prevent the development of fatty liver disease. SIRT7 is induced upon ER stress and is stabilized at the promoters of ribosomal proteins through its interaction with the transcription factor Myc to silence gene expression and to relieve ER stress. SIRT7-deficient mice develop chronic hepatosteatosis resembling human fatty liver disease. Myc inactivation or pharmacological suppression of ER stress alleviates fatty liver caused by SIRT7 deficiency. Importantly, SIRT7 suppresses ER stress and reverts the fatty liver disease in diet-induced obese mice. Our study identifies SIRT7 as a cofactor of Myc for transcriptional repression and delineates a druggable regulatory branch of the ER stress response that prevents and reverts fatty liver disease.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Fígado Gorduroso/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuínas/metabolismo , Animais , Feminino , Genes myc , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Sirtuínas/deficiência , Sirtuínas/genética , TransfecçãoRESUMO
Angiopoietin-like protein (ANGPTL)8 (alternatively called TD26, RIFL, Lipasin, and Betatrophin) is a newly recognized ANGPTL family member that has been implicated in both triglyceride (TG) and glucose metabolism. Hepatic overexpression of ANGPTL8 causes hypertriglyceridemia and increased insulin secretion. Here we examined the effects of inactivating Angptl8 on TG and glucose metabolism in mice. Angptl8 knockout (Angptl8(-/-)) mice gained weight more slowly than wild-type littermates due to a selective reduction in adipose tissue accretion. Plasma levels of TGs of the Angptl8(-/-) mice were similar to wild-type animals in the fasted state but paradoxically decreased after refeeding. The lower TG levels were associated with both a reduction in very low density lipoprotein secretion and an increase in lipoprotein lipase (LPL) activity. Despite the increase in LPL activity, the uptake of very low density lipoprotein-TG is markedly reduced in adipose tissue but preserved in hearts of fed Angptl8(-/-) mice. Taken together, these data indicate that ANGPTL8 plays a key role in the metabolic transition between fasting and refeeding; it is required to direct fatty acids to adipose tissue for storage in the fed state. Finally, glucose and insulin tolerance testing revealed no alterations in glucose homeostasis in mice fed either a chow or high fat diet. Thus, although absence of ANGPTL8 profoundly disrupts TG metabolism, we found no evidence that it is required for maintenance of glucose homeostasis.
Assuntos
Dislipidemias/metabolismo , Glucose/metabolismo , Homeostase/fisiologia , Hormônios Peptídicos/deficiência , Triglicerídeos/metabolismo , Tecido Adiposo/metabolismo , Proteína 8 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Transporte Biológico/fisiologia , Calorimetria Indireta , VLDL-Colesterol/sangue , Dislipidemias/tratamento farmacológico , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônios Peptídicos/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Triglicerídeos/sangueRESUMO
Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.