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1.
J Immunol ; 201(9): 2579-2592, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30282751

RESUMO

Low-dose IL-2 represents an immunotherapy to selectively expand regulatory T cells (Tregs) to promote tolerance in patients with autoimmunity. In this article, we show that a fusion protein (FP) of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, has greater in vivo efficacy than rIL-2 at Treg expansion and control of autoimmunity. Biochemical and functional studies support a model in which IL-2 interacts with CD25 in the context of this FP in trans to form inactive head-to-tail dimers that slowly dissociate into an active monomer. In vitro, IL-2/CD25 has low sp. act. However, in vivo IL-2/CD25 is long lived to persistently and selectively stimulate Tregs. In female NOD mice, IL-2/CD25 administration increased Tregs within the pancreas and reduced the instance of spontaneous diabetes. Thus, IL-2/CD25 represents a distinct class of IL-2 FPs with the potential for clinical development for use in autoimmunity or other disorders of an overactive immune response.


Assuntos
Diabetes Mellitus/prevenção & controle , Tolerância Imunológica/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Interleucina-2/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas Recombinantes de Fusão/imunologia
2.
J Lipid Res ; 54(1): 177-88, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23103473

RESUMO

The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr(-/-)ApoE(-/-) mice versus ApoE(-/-) mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr(-/-)ApoE(-/-) mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE(-/-) mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr(-/-) mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.


Assuntos
Técnicas de Silenciamento de Genes , Niacina/farmacologia , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apolipoproteínas E/deficiência , Colesterol/metabolismo , Interações Medicamentosas , Determinação de Ponto Final , Feminino , Humanos , Masculino , Camundongos , Niacina/uso terapêutico , Placa Aterosclerótica/genética , Receptores Imunológicos/deficiência , Receptores de LDL/deficiência , Receptores de Prostaglandina/deficiência , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo
3.
J Lipid Res ; 53(1): 51-65, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22021650

RESUMO

In an attempt to understand the applicability of various animal models to dyslipidemia in humans and to identify improved preclinical models for target discovery and validation for dyslipidemia, we measured comprehensive plasma lipid profiles in 24 models. These included five mouse strains, six other nonprimate species, and four nonhuman primate (NHP) species, and both healthy animals and animals with metabolic disorders. Dyslipidemic humans were assessed by the same measures. Plasma lipoprotein profiles, eight major plasma lipid fractions, and FA compositions within these lipid fractions were compared both qualitatively and quantitatively across the species. Given the importance of statins in decreasing plasma low-density lipoprotein cholesterol for treatment of dyslipidemia in humans, the responses of these measures to simvastatin treatment were also assessed for each species and compared with dyslipidemic humans. NHPs, followed by dog, were the models that demonstrated closest overall match to dyslipidemic humans. For the subset of the dyslipidemic population with high plasma triglyceride levels, the data also pointed to hamster and db/db mouse as representative models for practical use in target validation. Most traditional models, including rabbit, Zucker diabetic fatty rat, and the majority of mouse models, did not demonstrate overall similarity to dyslipidemic humans in this study.


Assuntos
Modelos Animais de Doenças , Dislipidemias/sangue , Lipídeos/sangue , Animais , Cricetinae , Cães , Dislipidemias/tratamento farmacológico , Ácidos Graxos/sangue , Humanos , Camundongos , Primatas , Sinvastatina/uso terapêutico , Triglicerídeos/sangue
4.
J Med Chem ; 64(1): 677-694, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33370104

RESUMO

A search for structurally diversified Tyk2 JH2 ligands from 6 (BMS-986165), a pyridazine carboxamide-derived Tyk2 JH2 ligand as a clinical Tyk2 inhibitor currently in late development for the treatment of psoriasis, began with a survey of six-membered heteroaryl groups in place of the N-methyl triazolyl moiety in 6. The X-ray co-crystal structure of an early lead (12) revealed a potential new binding pocket. Exploration of the new pocket resulted in two frontrunners for a clinical candidate. The potential hydrogen bonding interaction with Thr599 in the pocket was achieved with a tertiary amide moiety, confirmed by the X-ray co-crystal structure of 29. When the diversity search was extended to nicotinamides, a single fluorine atom addition was found to significantly enhance the permeability, which directly led to the discovery of 7 (BMS-986202) as a clinical Tyk2 inhibitor that binds to Tyk2 JH2. The preclinical studies of 7, including efficacy studies in mouse models of IL-23-driven acanthosis, anti-CD40-induced colitis, and spontaneous lupus, will also be presented.


Assuntos
Ciclopropanos/farmacologia , Descoberta de Drogas , Oxazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , Animais , Catálise , Cristalografia por Raios X , Ciclopropanos/química , Humanos , Camundongos , Oxazóis/química , Ligação Proteica , Inibidores de Proteínas Quinases/química , Psoríase/tratamento farmacológico , Relação Estrutura-Atividade , TYK2 Quinase/metabolismo
5.
Dev Comp Immunol ; 30(9): 772-82, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16364438

RESUMO

The glycans associated with mammalian major histocompatibility complex (MHC) class II molecules have been studied extensively. Co-translational and post-translational addition of sugar molecules to proteins confers many structural and modulatory functions. In the present study we characterized the glycans associated with MHC class II molecules in the channel catfish to compare glycosylation patterns in a teleost to those known to occur in mammals. This study made use of enzymatic methods and two-dimensional (2D) gel electrophoresis to characterize the N-linked sugars. Unlike mammalian T cells which expressed complex N-linked sugars, channel catfish derived 28S T cells were found to express high-mannose/hybrid N-glycans on class II molecules. However studies with Endoglycosidase H in conjunction with cell surface labeling on peripheral blood leukocytes revealed that catfish possess the machinery to modify the intermediate high-mannose sugars to complex type sugars. Nonetheless, the majority of the class II cell surface glycoproteins were of the high-mannose type. Resolution of catfish MHC class II molecules by 2D gel analyses revealed multiple bands for class II beta chains whereas class II alpha chains focused as a single spot. Glycosylation in the channel catfish, a premier model system for studying the immune system of teleosts, has significant differences from the glycosylation patterns characterized in mammalian systems, likely with functional implications.


Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Ictaluridae/imunologia , Polissacarídeos/imunologia , Animais , Western Blotting/veterinária , Linhagem Celular , Eletroforese em Gel Bidimensional/veterinária , Glicosídeo Hidrolases/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Polissacarídeos/química
6.
Mol Ther Nucleic Acids ; 5(8): e342, 2016 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-27483025

RESUMO

Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85-95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes.

7.
Blood Coagul Fibrinolysis ; 26(8): 893-902, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26192114

RESUMO

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 µmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.


Assuntos
Endodesoxirribonucleases/genética , Fator XII/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Terapia Trombolítica/métodos , Trombose/terapia , Animais , Derivação Arteriovenosa Cirúrgica , Cloretos/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ácido Elágico/farmacologia , Endodesoxirribonucleases/metabolismo , Fator XII/genética , Fator XII/metabolismo , Compostos Férricos/farmacologia , Técnicas de Inativação de Genes , Hemorragia/prevenção & controle , Humanos , Fígado/metabolismo , Masculino , Tempo de Tromboplastina Parcial , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Trombina/metabolismo , Trombose/induzido quimicamente , Trombose/genética , Trombose/patologia , Dedos de Zinco/genética
8.
Mol Ther Nucleic Acids ; 4: e224, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25625614

RESUMO

The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.

9.
Thromb Haemost ; 113(6): 1300-11, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25790442

RESUMO

Haemophilia A and B are characterised by a life-long bleeding predisposition, and several lines of evidence suggest that risks of atherothrombotic events may also be reduced. Establishing a direct correlation between coagulation factor levels, thrombotic risks and bleeding propensity has long been hampered by an inability to selectively and specifically inhibit coagulation factor levels. Here, the exquisite selectivity of gene silencing combined with a gene knockout (KO) approach was used to define the relative contribution of factor IX (fIX) to thrombosis and primary haemostasis in the rat. Using a lipid nanoparticle (LNP) formulation, we successfully delivered fIX siRNAs to the liver by intravenous administration. The knockdown (KD) of target gene mRNA was achieved rapidly (within 24 hour post-siRNA dosing), sustained (maintained for at least 7 days post dosing) and not associated with changes in mRNA expression levels of other coagulation factors. We found that intermediate levels of liver fIX mRNA silencing (60-95 %) translating into a 50-99 % reduction of plasma fIX activity provided protection from thrombosis without prolonging the cuticle bleeding time. Over 99 % inhibition of fIX activity was required to observe increase in bleeding, a phenotype confirmed in fIX KO rats. These data provide substantial evidence of a participation of fIX in the mechanisms regulating thrombosis prior to those regulating primary haemostasis, therefore highlighting the potential of fIX as a therapeutic target. In addition, hepatic mRNA silencing using LNP-encapsulated siRNAs may represent a promising novel approach for the chronic treatment and prevention of coagulation-dependent thrombotic disorders in humans.


Assuntos
Fator IX/genética , Hemofilia B/genética , Hemorragia/genética , Fígado/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Trombose/prevenção & controle , Animais , Linhagem Celular , Cloretos , Modelos Animais de Doenças , Fator IX/metabolismo , Compostos Férricos , Regulação da Expressão Gênica , Genótipo , Hemofilia B/sangue , Hemorragia/sangue , Hemostasia/genética , Masculino , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Trombose/sangue , Trombose/induzido quimicamente , Trombose/genética , Fatores de Tempo , Transfecção
10.
J Exp Med ; 207(8): 1775-89, 2010 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-20624888

RESUMO

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G(alphaq) subunit of trimeric G proteins (Gnaq(-/-) mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq(-/-) B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq(-/-) chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq(-/-) B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.


Assuntos
Autoimunidade/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Tolerância Imunológica , Anemia/sangue , Anemia/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Complexo Antígeno-Anticorpo/metabolismo , Artrite/imunologia , Artrite/patologia , Autoantígenos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/mortalidade , Doenças Autoimunes/patologia , Autoimunidade/genética , Fator Ativador de Células B/imunologia , Fator Ativador de Células B/farmacologia , Subpopulações de Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/transplante , Diferenciação Celular/genética , Movimento Celular/genética , Movimento Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Homeostase/imunologia , Rim/metabolismo , Rim/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quimera por Radiação/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T/citologia
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