RESUMO
Multiple sulfatase deficiency (MSD) is a severe, lysosomal storage disorder caused by pathogenic variants in the gene SUMF1, encoding the sulfatase modifying factor formylglycine-generating enzyme. Patients with MSD exhibit functional deficiencies in all cellular sulfatases. The inability of sulfatases to break down their substrates leads to progressive and multi-systemic complications in patients, similar to those seen in single-sulfatase disorders such as metachromatic leukodystrophy and mucopolysaccharidoses IIIA. Here, we aimed to determine if hematopoietic stem cell transplant with ex vivo SUMF1 lentiviral gene therapy could improve outcomes in a clinically relevant mouse model of MSD. We first tested our approach in MSD patient-derived cells and found that our SUMF1 lentiviral vector improved protein expression, sulfatase activities, and glycosaminoglycan accumulation. In vivo, we found that our gene therapy approach rescued biochemical deficits, including sulfatase activity and glycosaminoglycan accumulation, in affected organs of MSD mice treated post-symptom onset. In addition, treated mice demonstrated improved neuroinflammation and neurocognitive function. Together, these findings suggest that SUMF1 HSCT-GT can improve both biochemical and functional disease markers in the MSD mouse.
RESUMO
Type 1 diabetes (T1D) in both humans and NOD mice is caused by T cell-mediated autoimmune destruction of pancreatic ß cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control these pathogenic effectors have both been implicated in T1D etiology. Due to the expression of MHC class I molecules on ß cells, CD8 T cells represent the ultimate effector population mediating T1D. Developing autoreactive CD8 T cells normally undergo extensive thymic negative selection, but this process is impaired in NOD mice and also likely T1D patients. Previous studies identified an allelic variant of Nfkbid, a NF-κB signal modulator, as a gene strongly contributing to defective thymic deletion of autoreactive CD8 T cells in NOD mice. These previous studies found ablation of Nfkbid in NOD mice using the clustered regularly interspaced short palindromic repeats system resulted in greater thymic deletion of pathogenic CD8 AI4 and NY8.3 TCR transgenic T cells but an unexpected acceleration of T1D onset. This acceleration was associated with reductions in the frequency of peripheral Tregs. In this article, we report transgenic overexpression of Nfkbid in NOD mice also paradoxically results in enhanced thymic deletion of autoreactive CD8 AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs, in part contributing to the induction of complete T1D resistance. Thus, future identification of a pharmaceutical means to enhance Nfkbid expression might ultimately provide an effective T1D intervention approach.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Animais , Linfócitos T CD8-Positivos , Diabetes Mellitus Experimental/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Linfócitos T ReguladoresRESUMO
Multiple sulfatase deficiency (MSD) is an ultrarare lysosomal storage disorder due to deficiency of all known sulfatases. MSD is caused by mutations in the Sulfatase Modifying Factor 1 (SUMF1) gene encoding the enzyme responsible for the post-translational modification and activation of all sulfatases. Most MSD patients carry hypomorph SUMF1 variants resulting in variable degrees of residual sulfatase activities. In contrast, Sumf1 null mice with complete deficiency in all sulfatase enzyme activities, have very short lifespan with significant pre-wean lethality, owing to a challenging preclinical model. To overcome this limitation, we genetically engineered and characterized in mice two commonly identified patient-based SUMF1 pathogenic variants, namely p.Ser153Pro and p.Ala277Val. These pathogenic missense variants correspond to variants detected in patients with attenuated MSD presenting with partial-enzyme deficiency and relatively less severe disease. These novel MSD mouse models have a longer lifespan and show biochemical and pathological abnormalities observed in humans. In conclusion, mice harboring the p.Ser153Pro or the p.Ala277Val variant mimic the attenuated MSD and are attractive preclinical models for investigation of pathogenesis and treatments for MSD.
Assuntos
Doenças por Armazenamento dos Lisossomos , Doença da Deficiência de Múltiplas Sulfatases , Humanos , Animais , Camundongos , Doença da Deficiência de Múltiplas Sulfatases/genética , Mutação , Sulfatases , Mutação de Sentido Incorreto , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genéticaRESUMO
In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T cells recognizing pancreatic ß cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-Kd and/or H2-Db class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8+ T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene(s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated Nfkbid expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T cells. These results indicated that allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8+ T cells. However, although enhancing thymic deletion of pathogenic CD8+ T cells, ablating Nfkbid expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cromossomos Humanos Par 7/genética , Diabetes Mellitus Tipo 1/imunologia , Proteínas I-kappa B/genética , Timo/imunologia , Alelos , Animais , Autoantígenos/imunologia , Diferenciação Celular , Células Cultivadas , Deleção Clonal , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Proteínas I-kappa B/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Polimorfismo GenéticoRESUMO
NOD-scid.Il2rg(null) (NSG) mice are currently being used as recipients to screen for pathogenic autoreactive T cells in type 1 diabetes (T1D) patients. We questioned whether the restriction of IL-2R γ-chain (Il-2rγ)-dependent cytokine signaling only to donor cells in NSG recipients differently influenced the activities of transferred diabetogenic T cells when they were introduced as a monoclonal/oligoclonal population versus being part of a polyclonal repertoire. Unexpectedly, a significantly decreased T1D transfer by splenocytes from prediabetic NOD donors was observed in Il-2rγ(null)-NSG versus Il-2rγ-intact standard NOD-scid recipients. In contrast, NOD-derived monoclonal/oligoclonal TCR transgenic ß cell-autoreactive T cells in either the CD8 (AI4, NY8.3) or CD4 (BDC2.5) compartments transferred disease significantly more rapidly to NSG than to NOD-scid recipients. The reduced diabetes transfer efficiency by polyclonal T cells in NSG recipients was associated with enhanced activation of regulatory T cells (Tregs) mediated by NSG myeloid APC. This enhanced suppressor activity was associated with higher levels of Treg GITR expression in the presence of NSG than NOD-scid APC. These collective results indicate NSG recipients might be efficiently employed to test the activity of T1D patient-derived ß cell-autoreactive T cell clones and lines, but, when screening for pathogenic effectors within polyclonal populations, Tregs should be removed from the transfer inoculum to avoid false-negative results.
Assuntos
Transferência Adotiva , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Receptores de Interleucina-2/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Proteína Relacionada a TNFR Induzida por Glucocorticoide/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Interleucina-2/genética , Transdução de Sinais/imunologia , Baço/citologia , Baço/imunologia , Linfócitos T Reguladores/transplanteRESUMO
Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. In this study, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target Ag of islet-infiltrating B cells. PRPH autoreactive B cells recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. Autoantibodies to this epitope were also detected in the disease-resistant NOR and C57BL/6 strains. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cells during disease progression. Before extended insulitis was established, anti-PRPH B cells preferentially accumulated in the peritoneum. Anti-PRPH B cells were likewise detected in C57BL/6 mice, albeit at lower frequencies. As disease unfolded in NOD mice, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not prediabetic mice. Isotype-switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes is established. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis.
Assuntos
Linfócitos B/imunologia , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Periferinas/imunologia , Sequência de Aminoácidos , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Western Blotting , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Progressão da Doença , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Feminino , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Microscopia de Fluorescência , Dados de Sequência Molecular , Periferinas/genética , Periferinas/metabolismo , Peritônio/imunologia , Peritônio/metabolismo , Isoformas de Proteínas/imunologia , Baço/imunologia , Baço/metabolismoRESUMO
The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag-specific CD4(+) T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4(+) T-cell epitopes, the BDC-2.5 T-cell 2.5 mi mimotope and glutamic acid decarboxylase (GAD) 286-300, affected diabetogenic CD4(+) T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB-2.5 mi activated 2.5 mi(+) T cells and following intragastric delivery generated Ag-specific Foxp3(+) Treg and Th2 cells. While 2.5 mi(+) and GAD-specific T cells were tolerized in diabetes-resistant NODxB6.Foxp3(EGFP) F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB-fused epitopes. In contrast to NODxB6.Foxp3(EGFP) F1 mice, oral treatment in NOD mice lead to strong 2.5 mi(+) T-cell activation and the sequestration of these cells to the effector-memory pool. Oral treatment of NOD mice with CTB-2.5 mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Toxina da Cólera/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Administração Oral , Animais , Linfócitos T CD4-Positivos/imunologia , Toxina da Cólera/administração & dosagem , Vacinas contra Cólera/imunologia , Glutamato Descarboxilase/imunologia , Tolerância Imunológica/imunologia , Interferon gama/biossíntese , Interleucina-4/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Fragmentos de Peptídeos/administração & dosagemRESUMO
The mRNA transcript of the human STMN2 gene, encoding for stathmin-2 protein (also called SCG10), is profoundly impacted by TAR DNA-binding protein 43 (TDP-43) loss of function. The latter is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Using a combination of approaches, including transient antisense oligonucleotide-mediated suppression, sustained shRNA-induced depletion in aging mice, and germline deletion, we show that stathmin-2 has an important role in the establishment and maintenance of neurofilament-dependent axoplasmic organization that is critical for preserving the caliber and conduction velocity of myelinated large-diameter axons. Persistent stathmin-2 loss in adult mice results in pathologies found in ALS, including reduced interneurofilament spacing, axonal caliber collapse that drives tearing within outer myelin layers, diminished conduction velocity, progressive motor and sensory deficits, and muscle denervation. These findings reinforce restoration of stathmin-2 as an attractive therapeutic approach for ALS and other TDP-43-dependent neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica , Animais , Camundongos , Esclerose Lateral Amiotrófica/metabolismo , Axônios/fisiologia , Denervação , Proteínas de Ligação a DNA/genética , Filamentos Intermediários/metabolismo , Filamentos Intermediários/patologia , Neurônios Motores/metabolismo , Estatmina/genética , Estatmina/metabolismoRESUMO
CD4 T cells are crucial effectors in the pathology of type 1 diabetes (T1D). Successful therapeutic interventions for prevention and cure of T1D in humans are still elusive. Recent research efforts have focused on the manipulation of T cells by treatment with DNA. In this paper, we studied the effects of a DNA treatment strategy designed to target antigenic peptides to the lysosomal compartment on a monospecific T cell population termed 2.5mi(+) T cells that shares reactivity with the diabetogenic T cell clone BDC-2.5 in the NOD mouse. MHC class II tetramer analysis showed that repeated administrations were necessary to expand 2.5mi(+) T cells in vivo. This expansion was independent of Ag presentation by B cells. A single peptide epitope was sufficient to induce protection against T1D, which was not due to Ag-specific T cell anergy. Typical Th2 cytokines such as IL-10 or IL-4 were undetectable in 2.5mi(+) T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi(+) T cell population produced IFN-γ similar to 2.5mi(+) T cells from naive mice. Protection against T1D by DNA treatment was completely lost in NOD.CD28(-/-) mice which are largely deficient of natural regulatory T cells (Treg). Although Ag-specific Foxp3(+) Treg did not expand in response to DNA treatment, diabetes onset was delayed in Treg-reconstituted and DNA-treated NOD.SCID mice. These observations provide evidence for a Treg-mediated protective mechanism that is independent of the expansion or de novo generation of Ag-specific Treg.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Sistemas de Liberação de Medicamentos/métodos , Tolerância Imunológica , Lisossomos/imunologia , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/metabolismo , Vacinas de DNA/agonistas , Vacinas de DNA/imunologia , Transferência Adotiva/métodos , Animais , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Relação Dose-Resposta Imunológica , Feminino , Tolerância Imunológica/genética , Lisossomos/genética , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Fragmentos de Peptídeos/administração & dosagem , Baço/citologia , Baço/imunologia , Baço/transplante , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span. There is currently no treatment for CMT4J. Here, we present the results of preclinical studies testing a gene-therapy approach to restoring FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded adeno-associated virus serotype 9 (AAV9) to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at P1 or P4, mice survived at least 1 year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When mice were treated at P7 or P11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.
Assuntos
Doença de Charcot-Marie-Tooth/terapia , Dependovirus , Flavoproteínas/biossíntese , Longevidade , Fosfatases de Fosfoinositídeos/biossíntese , Transdução Genética , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Modelos Animais de Doenças , Feminino , Flavoproteínas/genética , Masculino , Camundongos , Camundongos Knockout , Fosfatases de Fosfoinositídeos/genéticaRESUMO
During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Glicoproteínas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Translocação Genética/fisiologia , Animais , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Glicoproteínas/genética , GlicosilaçãoRESUMO
Type 1 diabetes can be overcome by regulatory T cells (Treg) in NOD mice yet an efficient method to generate and maintain antigen-specific Treg is difficult to come by. Here, we devised a combination therapy of peptide/MHC tetramers and IL-2/anti-IL-2 monoclonal antibody complexes to generate antigen-specific Treg and maintain them over extended time periods. We first optimized treatment protocols conceived to obtain an improved islet-specific Treg/effector T cell ratio that led to the in vivo expansion and activation of these Treg as well as to an improved suppressor function. Optimized protocols were applied to treatment for testing diabetes prevention in NOD mice as well as in an accelerated T cell transfer model of T1D. The combined treatment led to robust protection against diabetes, and in the NOD model, to a close to complete prevention of insulitis. Treatment was accompanied with increased secretion of IL-10, detectable in total splenocytes and in Foxp3- CD4 T cells. Our data suggest that a dual protection mechanism takes place by the collaboration of Foxp3+ and Foxp3- regulatory cells. We conclude that antigen-specific Treg are an important target to improve current clinical interventions against this disease.
Assuntos
Anticorpos Monoclonais/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Antígenos de Histocompatibilidade/química , Interleucina-2/imunologia , Peptídeos/química , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Diabetes Mellitus Tipo 1/tratamento farmacológico , Feminino , Camundongos , Peptídeos/farmacologia , Fenótipo , Multimerização Proteica , Estrutura Quaternária de Proteína , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
The aim of this study was to detect toxic metabolites from fungi contaminating food and medicinal herbs by applying the toxicity assay to Artemia salina. According to toxicity percentages, the extracts were classified as nontoxic (NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified as T and HT were assayed for mycotoxins. Only 6 out of 71 strains were found to be T (8.5%) for A. salina. Penicillium brevicompactum Dierckx, isolated from sausages, was found to be HT, mainly due to the presence of ochratoxin A and two other unidentified metabolites.
Assuntos
Artemia/efeitos dos fármacos , Bioensaio/métodos , Contaminação de Medicamentos , Contaminação de Alimentos , Fungos/isolamento & purificação , Micotoxinas/análise , Plantas Medicinais/microbiologia , Toxicologia/métodos , Animais , Baccharis/microbiologia , Análise de Alimentos/métodos , Microbiologia de Alimentos , Fungos/metabolismo , Ilex paraguariensis/microbiologia , Lippia/microbiologia , Malva/microbiologia , Produtos da Carne/microbiologia , Mentha piperita/microbiologia , Fungos Mitospóricos/isolamento & purificação , Fungos Mitospóricos/metabolismo , Micotoxinas/farmacologia , Glycine max/microbiologia , Zea mays/microbiologiaRESUMO
El objetivo fue aplicar un ensayo de toxicidad sobre Artemia salina para ladetección de metabolitos fúngicos tóxicos, obtenidos a partir de hongoscontaminantes de hierbas medicinales y alimentos.Los extractos fúngicos fueron clasificados, según el porcentaje de mortalidadhallado, como: no tóxico (NT), levemente tóxico (LT), tóxico (T) y muy tóxico(MT). En los tipos T y MT se investigó la presencia de micotoxinas.Seis extractos resultaron de tipo T (8,5%). Penicillium brevicompactumDierckx, aislado de un embutido, fue el único MT, debido principalmente a lapresencia de ocratoxina A y de otros dos metabolitos no identificados(AU)
The aim of this study was to detect toxic metabolites from fungi contaminatingfood and medicinal herbs by applying the toxicity assay to Artemia salina.According to toxicity percentages, the extracts were classified as nontoxic(NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified asT and HT were assayed for mycotoxins.Only 6 out of 71 strains were found to be T (8.5%) for A. salina.Penicillium brevicompactum Dierckx, isolated from sausages, was found tobe HT, mainly due to the presence of ochratoxin A and two other unidentifiedmetabolites(AU)