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1.
Mol Ther ; 28(2): 664-676, 2020 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-31843448

RESUMEN

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.


Asunto(s)
Anticuerpos Biespecíficos/farmacología , Distroglicanos/metabolismo , Laminina/metabolismo , Síndrome de Walker-Warburg/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/metabolismo , Modelos Animales de Enfermedad , Distroglicanos/inmunología , Expresión Génica , Humanos , Inmunohistoquímica , Inyecciones Intramusculares , Laminina/genética , Laminina/inmunología , Ratones , Ratones Noqueados , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/genética , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Síndrome de Walker-Warburg/tratamiento farmacológico , Síndrome de Walker-Warburg/etiología
2.
J Biol Chem ; 287(14): 11098-107, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22337882

RESUMEN

Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-ß and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-ß. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.


Asunto(s)
Antígeno CTLA-4/metabolismo , Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Animales , Comunicación Autocrina/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos HLA/metabolismo , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Linfocitos T Reguladores/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factor de Crecimiento Transformador beta/farmacología
3.
J Biol Chem ; 286(17): 14787-94, 2011 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-21402703

RESUMEN

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Glicoesfingolípidos/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Células Th17/citología , Animales , Diferenciación Celular , Linaje de la Célula , Citocinas/biosíntesis , Sinapsis Inmunológicas , Microdominios de Membrana , Ratones , Ratones Transgénicos
4.
Int Immunol ; 22(7): 593-603, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20497953

RESUMEN

Lipid rafts reportedly play an important role in modulating the activation of mast cells and granulocytes, the primary effector cells of airway hyperresponsiveness and asthma. Activation is mediated through resident signaling molecules whose activity, in part, may be modulated by the composition of glycosphingolipids (GSLs) in membrane rafts. In this study, we evaluated the impact of inhibiting GSL biosynthesis in mast cells and in the ovalbumin (OVA)-induced mouse model of asthma using either a small molecule inhibitor or anti-sense oligonucleotides (ASOs) directed against specific enzymes in the GSL pathway. Lowering GSL levels in mast cells through inhibition of glucosylceramide synthase (GCS) reduced phosphorylation of Syk tyrosine kinase and phospholipase C gamma 2 (PLC-gamma2) as well as cytoplasmic Ca(2+) levels. Modulating these intracellular signaling events also resulted in a significant decrease in mast cell degranulation. Primary mast cells isolated from a GM3 synthase (GM3S) knockout mouse exhibited suppressed activation-induced degranulation activity further supporting a role of GSLs in this process. In previously OVA-sensitized mice, intra-nasal administration of ASOs to GCS, GM3S or lactosylceramide synthase (LCS) significantly suppressed metacholine-induced airway hyperresponsiveness and pulmonary inflammation to a subsequent local challenge with OVA. However, administration of the ASOs into mice that had been sensitized and locally challenged with the allergen did not abate the consequent pulmonary inflammatory sequelae. These results suggest that GSLs contribute to the initiation phase of the pathogenesis of airway hyperreactivity and asthma and lowering GSL levels may offer a novel strategy to modulate these manifestations.


Asunto(s)
Asma/inmunología , Asma/fisiopatología , Glicoesfingolípidos/biosíntesis , Animales , Asma/tratamiento farmacológico , Asma/patología , Degranulación de la Célula/efectos de los fármacos , Dioxanos/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Glucosiltransferasas/antagonistas & inhibidores , Glicoesfingolípidos/inmunología , Mastocitos/citología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Peso Molecular , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Ovalbúmina/inmunología , Fosfolipasa C gamma/antagonistas & inhibidores , Fosfolipasa C gamma/inmunología , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/inmunología , Pirrolidinas/farmacología , Sialiltransferasas/inmunología , Transducción de Señal/inmunología
5.
Mol Ther ; 17(6): 954-63, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19277015

RESUMEN

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.


Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Manosafosfatos/química , Oligosacáridos/química , Ingeniería de Proteínas/métodos , alfa-Glucosidasas/metabolismo , alfa-Glucosidasas/uso terapéutico , Animales , Modelos Animales de Enfermedad , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Unión Proteica , Receptor IGF Tipo 2/metabolismo , alfa-Glucosidasas/química , alfa-Glucosidasas/genética , alfa-Glucosidasas/farmacología
6.
PLoS One ; 8(5): e63530, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23704916

RESUMEN

Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3(+) regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9-12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4-13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing ß-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.


Asunto(s)
Antígeno CTLA-4/metabolismo , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/prevención & control , Antígenos de Histocompatibilidad Clase II/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Antígenos/inmunología , Asparagina/metabolismo , Diferenciación Celular/efectos de los fármacos , Pollos , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Femenino , Glicosilación/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Factores de Tiempo
7.
PLoS One ; 7(4): e34684, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22514654

RESUMEN

Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezomib efficiently reduces AAV-specific IgG titres and moderates the cytotoxic T cell response in mice that have a pre-existing immunity to AAV2/8. Significant depletion of AAV2/8-specific IgG-producing plasma cells in secondary lymphoid organs and bone marrow was observed. However, this inhibition of the immune response by bortezomib was insufficient to allow subsequent re-infection with a recombinant AAV vector of a similar serotype. We show that this shortcoming is probably due to the combination of residual antibody levels and the inability of bortezomib to completely deplete the memory B cells that are re-activated in response to a repeated infection with a recombinant AAV vector. Taken together, the results of this study argue for the use of immunosuppressive therapies that target both plasma and memory B cells for the efficient elimination of pre-existing immunity against AAV2/8 vectors.


Asunto(s)
Ácidos Borónicos/farmacología , Dependovirus/inmunología , Vectores Genéticos/inmunología , Inmunidad/efectos de los fármacos , Inhibidores de Proteasoma , Pirazinas/farmacología , Animales , Bortezomib , Citocinas/metabolismo , Citometría de Flujo , Masculino , Ratones
8.
J Biol Chem ; 284(28): 18664-73, 2009 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-19419960

RESUMEN

Fluorescent fusion proteins are exceedingly useful for monitoring protein localization in situ or visualizing protein behavior at the single molecule level. Unfortunately, some proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA protein (RecA803 protein) to monomeric red fluorescent protein (mRFP1) to produce a functional protein (RecA-RFP) that is suitable for in vivo and in vitro analysis. In vivo, the RecA-RFP partially restores UV resistance, conjugational recombination, and SOS induction to recA(-) cells. In vitro, the purified RecA-RFP protein forms a nucleoprotein filament whose k(cat) for single-stranded DNA-dependent ATPase activity is reduced approximately 3-fold relative to wild-type protein, and which is largely inhibited by single-stranded DNA-binding protein. However, RecA protein is also a dATPase; dATP supports RecA-RFP nucleoprotein filament formation in the presence of single-stranded DNA-binding protein. Furthermore, as for the wild-type protein, the activities of RecA-RFP are further enhanced by shifting the pH to 6.2. As a consequence, RecA-RFP is proficient for DNA strand exchange with dATP or at lower pH. Finally, using single molecule visualization, RecA-RFP was seen to assemble into a continuous filament on duplex DNA, and to extend the DNA approximately 1.7-fold. Consistent with its attenuated activities, RecA-RFP nucleates onto double-stranded DNA approximately 3-fold more slowly than the wild-type protein, but still requires approximately 3 monomers to form the rate-limited nucleus needed for filament assembly. Thus, RecA-RFP reveals that its attenuated biological functions correlate with a reduced frequency of nucleoprotein filament nucleation at the single molecule level.


Asunto(s)
Escherichia coli/metabolismo , Nucleoproteínas/química , Rec A Recombinasas/metabolismo , Núcleo Celular/metabolismo , ADN/química , ADN de Cadena Simple/química , Concentración de Iones de Hidrógeno , Cinética , Proteínas Luminiscentes/química , Plásmidos/metabolismo , Unión Proteica , Recombinación Genética , Sensibilidad y Especificidad , Rayos Ultravioleta , Proteína Fluorescente Roja
9.
Mol Microbiol ; 63(1): 103-15, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17163974

RESUMEN

RecA plays a central role in recombination, DNA repair and SOS induction through forming a RecA-DNA helical filament. Biochemical observations show that at low ratios to RecA, DinI and RecX stabilize and destabilize RecA-DNA filaments, respectively, and that the C-terminal 17 residues of RecA are important for RecX function. RecA-DNA filament formation was assayed in vivo using RecA-GFP foci formation in log-phase and UV-irradiated cells. In log-phase cells, dinI mutants have fewer foci than wild type and that recX mutants have more foci than wild type. A recADelta17::gfp mutant had more foci like a recX mutant. dinI recX double mutants have the same number of foci as dinI mutants alone, suggesting that dinI is epistatic to recX. After UV treatment, the dinI, recX and dinI recX mutants differed in their ability to form foci. All three mutants had fewer foci than wild type. The dinI mutant's foci persisted longer than wild-type foci. Roles of DinI and RecX after UV treatment differed from those during log-phase growth and may reflect the different DNA substrates, population of proteins or amounts during the SOS response. These experiments give new insight into the roles of these proteins.


Asunto(s)
Proteínas Bacterianas/fisiología , ADN Bacteriano/metabolismo , ADN Bacteriano/efectos de la radiación , Proteínas de Escherichia coli/fisiología , Escherichia coli/enzimología , Escherichia coli/genética , Rec A Recombinasas/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/química , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos
10.
Mol Microbiol ; 57(4): 1074-85, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16091045

RESUMEN

RecA is important in recombination, DNA repair and repair of replication forks. It functions through the production of a protein-DNA filament. To study the localization of RecA in live Escherichia coli cells, the RecA protein was fused to the green fluorescence protein (GFP). Strains with this gene have recombination/DNA repair activities three- to tenfold below wild type (or about 1000-fold above that of a recA null mutant). RecA-GFP cells have a background of green fluorescence punctuated with up to five foci per cell. Two types of foci have been defined: 4,6-diamidino-2-phenylindole (DAPI)-sensitive foci that are bound to DNA and DAPI-insensitive foci that are DNA-less aggregates/storage structures. In log phase cells, foci were not localized to any particular region. After UV irradiation, the number of foci increased and they localized to the cell centre. This suggested colocalization with the DNA replication factory. recA, recB and recF strains showed phenotypes and distributions of foci consistent with the predicted effects of these mutations.


Asunto(s)
Proteínas de Escherichia coli/análisis , Escherichia coli/enzimología , Rec A Recombinasas/análisis , Cromosomas Bacterianos/metabolismo , Replicación del ADN , ADN Bacteriano/metabolismo , ADN Bacteriano/efectos de la radiación , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Mutación , Rec A Recombinasas/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Recombinación Genética , Rayos Ultravioleta
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