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1.
J Biol Chem ; 298(12): 102625, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36306823

RESUMO

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the nonreducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary cells to produce a highly soluble and fully active recombinant human sulfamidase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of 9 days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease-specific HS-NRE were dramatically reduced, reaching a nadir 2 weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuroimmune response lasted through the 56 days tested. Furthermore, seven weekly 148 µg doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuroimmune response when delivered ICV.


Assuntos
Encefalopatias , Mucopolissacaridose III , Cricetinae , Animais , Humanos , Camundongos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/metabolismo , Células CHO , Projetos Piloto , Cricetulus , Hidrolases/metabolismo , Encéfalo/metabolismo , Heparitina Sulfato/metabolismo , Encefalopatias/metabolismo , Lisossomos/metabolismo , Modelos Animais de Doenças
2.
J Immunol ; 204(6): 1592-1597, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31988179

RESUMO

IL-33 and its receptor ST2 are contributing factors to airway inflammation and asthma exacerbation. The IL-33/ST2 signaling pathway is involved in both the onset and the acute exacerbations of asthma. In this study, we address the role of endogenous IL-33 and its autoamplification of the IL-33/ST2 pathway in Ag-dependent and Ag-independent asthma-like models. Wild-type, IL-33 knockout, ST2 knockout mice were either intratracheally administrated with 500 ng of rIL-33 per day for four consecutive days or were sensitized and challenged with OVA over 21 d. In wild-type mice, IL-33 or OVA induced similar airway hyperresponsiveness and eosinophilic airway inflammation. IL-33 induced its own mRNA and ST2L mRNA expression in the lung. IL-33 autoamplified itself and ST2 protein expression in airway epithelial cells. OVA also induced IL-33 and ST2 protein expression. In IL-33 knockout mice, the IL-33- and OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation were both significantly attenuated, whereas IL-33-induced ST2L mRNA expression was preserved, although no autoamplification of IL-33/ST2 pathway was observed. In ST2 knockout mice, IL-33 and OVA induced airway hyperresponsiveness and eosinophilic airway inflammation were both completely diminished, and no IL-33/ST2 autoamplification was observed. These results suggest that endogenous IL-33 and its autoamplification of IL-33/ST2 pathway play an important role in the induction of asthma-like phenotype. Thus an intact IL-33/ST2 pathway is necessary for both Ag-dependent and Ag-independent asthma-like mouse models.


Assuntos
Asma/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Asma/sangue , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/administração & dosagem , Interleucina-33/genética , Camundongos , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Transdução de Sinais/genética
3.
J Immunol ; 200(12): 4036-4043, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29728508

RESUMO

IL-33 and its receptor ST2 play important roles in airway inflammation and contribute to asthma onset and exacerbation. The IL-33/ST2 signaling pathway recruits adapter protein myeloid differentiation primary response 88 (MyD88) to transduce intracellular signaling. MyD88 forms a complex with IL-R-associated kinases (IRAKs), IRAK4 and IRAK2, called the Myddosome (MyD88-IRAK4-IRAK2). The myddosome subsequently activates downstream NF-κB and MAPKs p38 and JNK. We established an asthma-like mouse model by intratracheal administration of IL-33. The IL-33 model has a very similar phenotype compared with the OVA-induced mouse asthma model. The importance of MyD88 in the IL-33/ST2 signaling transduction was demonstrated by the MyD88 knockout mice, which were protected from the IL-33-induced asthma. We synthesized small molecule mimetics of the α-helical domain of IRAK2 with drug-like characteristics based on the recent advances in the designing of α-helix compounds. The mimetics can competitively interfere in the protein-protein interaction between IRAK2 and IRAK4, leading to disruption of Myddosome formation. A series of small molecules were screened using an NF-κB promoter assay in vitro. The lead compound, 7004, was further studied in the IL-33-induced and OVA-induced asthma mouse models in vivo. Compound 7004 can inhibit the IL-33-induced NF-κB activity, disrupt Myddosome formation, and attenuate the proinflammatory effects in asthma-like models. Our data indicate that the Myddosome may represent a novel intracellular therapeutic target for diseases in which IL-33/ST2 plays important roles, such as asthma and other inflammatory diseases.


Assuntos
Asma/tratamento farmacológico , Inflamação/tratamento farmacológico , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Asma/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fenótipo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
4.
J Vis Exp ; (86)2014 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-24747695

RESUMO

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.


Assuntos
Intubação Intratraqueal/métodos , Intubação Intratraqueal/veterinária , Laringoscopia/métodos , Laringoscopia/veterinária , Otoscópios/veterinária , Animais , Intubação Intratraqueal/instrumentação , Laringoscopia/instrumentação , Camundongos , Modelos Animais
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