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2.
J Biol Chem ; 294(36): 13421-13433, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31337702

RESUMO

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor whose loss of function results in immunodeficiency, but its role in the central nervous system (CNS) has been unclear. Microglia are the resident immune cells of the CNS and are implicated in the pathogenesis of various neurodegenerative diseases, including multiple sclerosis (MS) and glaucoma, which affects the visual system. However, the exact roles of microglia in these diseases remain unknown. Herein, we report that DOCK8 is expressed in microglia but not in neurons or astrocytes and that its expression is increased during neuroinflammation. To define the role of DOCK8 in microglial activity, we focused on the retina, a tissue devoid of infiltrating T cells. The retina is divided into distinct layers, and in a disease model of MS/optic neuritis, DOCK8-deficient mice exhibited a clear reduction in microglial migration through these layers. Moreover, neuroinflammation severity, indicated by clinical scores, visual function, and retinal ganglion cell (RGC) death, was reduced in the DOCK8-deficient mice. Furthermore, using a glaucoma disease model, we observed impaired microglial phagocytosis of RGCs in DOCK8-deficient mice. Our data demonstrate that DOCK8 is expressed in microglia and regulates microglial activity in disease states. These findings contribute to a better understanding of the molecular pathways involved in microglial activation and implicate a role of DOCK8 in several neurological diseases.


Assuntos
Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Células Cultivadas , Feminino , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
3.
Nat Commun ; 10(1): 3106, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308374

RESUMO

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.


Assuntos
Antígeno CTLA-4/metabolismo , Proteínas de Ligação a DNA/deficiência , Fatores de Troca do Nucleotídeo Guanina/deficiência , Doenças da Imunodeficiência Primária/genética , Antígeno B7-1/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Homeostase , Humanos , Células Jurkat , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
5.
PLoS Genet ; 15(2): e1007964, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30817801

RESUMO

Transmission ratio distortion (TRD) by the mouse t-haplotype, a variant region on chromosome 17, is a well-studied model of non-Mendelian inheritance. It is characterized by the high transmission ratio (up to 99%) of the t-haplotype from t/+ males to their offspring. TRD is achieved by the exquisite ability of the responder (Tcr) to trigger non-Mendelian inheritance of homologous chromosomes. Several distorters (Tcd1-Tcd4), which act cumulatively, together promote the high transmission ratio of Tcr and the t-haplotype. Molecularly, TRD is brought about by deregulation of Rho signaling pathways via the distorter products, which impair sperm motility, and the t-sperm specific rescue of sperm motility by the responder. The t-sperm thus can reach the egg cells faster than +-sperm and fertilize them. Previously we have shown that the responder function is accomplished by a dominant negative form of sperm motility kinase (SMOKTCR), while the distorter functions are accomplished by the Rho G protein regulators TAGAP, FGD2 and NME3 proposed to function in two oppositely acting pathways. Here we identify the RAC1-specific guanine nucleotide exchange factor TIAM2 as modifier of t-haplotype TRD. Tiam2 is expressed in two isoforms, the full-length (Tiam2l) and a short transcript (Tiam2s). Tiam2s expression from the t-allele is strongly increased compared to the wild-type allele. By transgenic approaches we show that Tiam2s enhances t-haplotype transmission, while Tiam2l has the opposite effect. Our data show that a single modifier locus can encode different gene products exerting opposite effects on a trait. They also suggest that the expression ratio of the isoforms determines if the outcome is an enhancing or a suppressive effect on the trait.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Padrões de Herança , Região do Complexo-t do Genoma , Alelos , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Herança Paterna , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Motilidade Espermática/genética , Motilidade Espermática/fisiologia , Espermatogênese/genética
6.
Scand J Immunol ; 89(6): e12759, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30793341

RESUMO

DOCK8 immunodeficiency syndrome (DIDS) is a combined immunodeficiency characterized by recurrent viral infections, severe atopy and early onset malignancy. Immunological abnormalities include lymphopenia, CD8+ T-cell cytoskeleton dysfunction, defective B cell memory and variable serum immunoglobulin levels. Here, we analyse the B cell receptor repertoire (BCR) characteristics and antibody avidity of four DIDS patients, attempt to understand the dysregulated humoral immunity in DIDS patients with a normal antibody titre and suggest a scientific basis for intravenous immunoglobulin (IVIG) replacement therapy for these patients. We analysed BCR characteristics, including somatic hypermutation (SHM) frequency, using deep sequencing of multiplex PCR products derived from BCR heavy chain CDR3 regions from DIDS patients and controls. The antibody avidity of human tetanus and hemophilus influenza B antibodies was determined by ELISA using thiocyanate elution. IVIG replacement treatment and infection conditions were investigated retrospectively. We found skewing of the BCR repertoire and decreased antibody avidity in patients with DIDS. DIDS patients had fewer negatively charged amino acids than healthy controls. The SHM frequency of the IGHV3 gene was lower in patients with DIDS. Patients received regular IVIG therapy, resulting in fewer and less severe infections. We conclude that although IgG levels are normal in most DIDS patients, IVIG replacement therapy is still necessary.


Assuntos
Afinidade de Anticorpos/imunologia , Linfócitos B/imunologia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/terapia , Imunoglobulinas Intravenosas/uso terapêutico , Receptores de Antígenos de Linfócitos B/imunologia , Adolescente , Anticorpos Antibacterianos/imunologia , Linfócitos T CD8-Positivos/imunologia , Pré-Escolar , Clostridium tetani/imunologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Haemophilus influenzae tipo b/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/sangue , Memória Imunológica/imunologia , Masculino , Receptores de Antígenos de Linfócitos B/genética
7.
Neurobiol Dis ; 124: 218-229, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30468864

RESUMO

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Junção Neuromuscular/patologia , Nervo Isquiático/ultraestrutura , Degenerações Espinocerebelares/genética , Degenerações Espinocerebelares/patologia , Animais , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/deficiência , Humanos , Camundongos Transgênicos , Músculo Esquelético/inervação , Músculo Esquelético/ultraestrutura , Junção Neuromuscular/metabolismo , Proteômica , Nervo Isquiático/metabolismo , Degenerações Espinocerebelares/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
8.
FASEB J ; 33(3): 4547-4558, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30592629

RESUMO

Endoplasmic reticulum (ER)-Golgi vesicle trafficking plays a pivotal role in the conventional secretory pathway of many cytokines; however, the precise release mechanism of a major inflammasome mediator, IL-1ß, is not thought to follow the conventional ER-Golgi route and remains elusive. Here, we found that perturbation of ER-Golgi trafficking by brefeldin A (BFA) treatment attenuated nucleotide-binding oligomerization domain-like receptor family, pyrin-domain-containing 3 (NLRP3) inflammasome activation in mouse bone marrow-derived macrophages (BMDMs). BFA treatment inhibited NLRP3-mediated inflammasome assembly and caspase-1 activation but did not block IL-1ß secretion from BMDMs following BFA administration after NLRP3 inflammasome activation. Consistently, short-hairpin RNA-dependent knockdown of BFA-inhibited guanine nucleotide-exchange protein 1 (BIG1), a molecular target of BFA and an initiator of Golgi-specific vesicle trafficking, abolished NLRP3-dependent apoptosis-associated speck-like protein containing a caspase-recruitment domain oligomerization and caspase-1 activation in BMDMs. Similarly, knockdown of Golgi-specific BFA-resistance guanine nucleotide exchange factor 1, another target of BFA, clearly attenuated NLRP3-mediated caspase-1 activation in BMDMs. Mechanistically, inhibition of BIG1-mediated vesicle trafficking did not impair NLRP3-activating signal 2-promoted events, such as potassium efflux and mitochondrial rearrangement, but caused significant impairment of signal 1-triggered priming steps, including NF-κB-mediated pathways. These data suggest that BFA-targeted vesicle trafficking at the Golgi contributes to activation of the NLRP3 inflammasome signaling.-Hong, S., Hwang, I., Gim, E., Yang, J., Park, S., Yoon, S.-H., Lee, W.-W., Yu, J.-W. Brefeldin A-sensitive ER-Golgi vesicle trafficking contributes to NLRP3-dependent caspase-1 activation.


Assuntos
Brefeldina A/farmacologia , Caspase 1/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Inflamassomos/fisiologia , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Transporte Proteico/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Animais , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/biossíntese , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Potássio/metabolismo , Organismos Livres de Patógenos Específicos , Células THP-1
9.
J Physiol Sci ; 69(2): 175-184, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30084082

RESUMO

Vascular smooth muscle cell (VSMC) migration and the subsequent intimal thickening play roles in vascular restenosis. We previously reported that an exchange protein activated by cAMP 1 (Epac1) promotes platelet-derived growth factor (PDGF)-induced VSMC migration and intimal thickening. Because basic fibroblast growth factor (bFGF) also plays a pivotal role in restenosis, we examined whether Epac1 was involved in bFGF-mediated VSMC migration. bFGF-induced lamellipodia formation and migration were significantly decreased in VSMCs obtained from Epac1-/- mice compared to those in Epac1+/+-VSMCs. The bFGF-induced phosphorylation of Akt and glycogen synthase kinase 3ß (GSK3ß), which play a role in bFGF-induced cell migration, was attenuated in Epac1-/--VSMCs. Intimal thickening induced by the insertion of a large wire was attenuated in Epac1-/- mice, and was accompanied by the decreased phosphorylation of GSK3ß. These data suggest that Epac1 deficiency attenuates bFGF-induced VSMC migration, possibly via Akt/GSK3ß pathways.


Assuntos
Movimento Celular/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Animais , Células Cultivadas , Reestenose Coronária/metabolismo , Reestenose Coronária/fisiopatologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia
10.
J Cell Biol ; 218(1): 350-379, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30523041

RESUMO

Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.


Assuntos
Cones de Crescimento/ultraestrutura , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imagem Molecular/normas , Neurônios/ultraestrutura , Software , Imagem com Lapso de Tempo/normas , Proteínas rho de Ligação ao GTP/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Forma Celular/genética , Regulação da Expressão Gênica , Heterogeneidade Genética , Cones de Crescimento/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Imagem Molecular/métodos , Neurônios/metabolismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Fatores de Troca de Nucleotídeo Guanina Rho/deficiência , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais , Imagem com Lapso de Tempo/métodos , Proteína cdc42 de Ligação ao GTP/deficiência , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/deficiência
11.
J Biol Chem ; 294(4): 1218-1229, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30518550

RESUMO

Autophagy is critical for maintaining cellular function via clearance of excess nutrients and damaged organelles. In pancreatic ß-cells, it helps counter the endoplasmic reticulum (ER) stress that impairs insulin secretory capacity during Type 2 diabetes. Chronic exposure of ß-cells to saturated fatty acids (FAs) such as palmitate stimulates ER stress and modulates autophagy, but the effects of unsaturated FAs such as oleate, which are also elevated during obesity, are less well understood. We therefore treated MIN6 cells and mouse islets for 8-48 h with either palmitate or oleate, and then monitored autophagic flux, signaling pathways, lysosomal biology, and phospholipid profiles. Compared with palmitate, oleate more effectively stimulated both autophagic flux and clearance of autophagosomes. The flux stimulation occurred independently of ER stress, nutrient-sensing (mTOR) and signaling pathways (protein kinases A, C, and D). Instead the mechanism involved the exchange factor directly activated by cAMP 2 (EPAC2). Oleate reduced cellular cAMP, and its effects on autophagic flux were reproduced or inhibited, respectively, by Epac2 knockdown or activation. Oleate also increased lysosomal acidity and increased phospholipid saturation, consistent with improved autophagosomal fusion with lysosomes. We conclude that a potent stimulation of autophagy might help explain the known benefits of unsaturated FAs in countering the toxicity of saturated FAs in ß-cells during obesity and lipid loading.


Assuntos
Apoptose/efeitos dos fármacos , AMP Cíclico/antagonistas & inibidores , Células Secretoras de Insulina/efeitos dos fármacos , Ácido Oleico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas
12.
Allergy ; 74(2): 370-379, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30252138

RESUMO

BACKGROUND: Hyper-IgE syndromes (HIES) are a clinically overlapping, heterogeneous group of inborn errors of immunity characterized by elevated serum IgE level, eosinophilia, atopy, and immune dysregulation. Deficiency of DOCK8 protein is potentially a life-threatening autosomal recessive HIES and only curable with bone marrow transplantation. Hence, the diagnosis of DOCK8 deficiency is critical and should be sought at an early stage to initiate definitive therapy. METHODS: Serum samples from patients with DOCK8 deficiency and atopic dermatitis were profiled on a cytokine/chemokine panel for potential differential expression. RESULTS: CXCL10 and TNF-A were upregulated in DOCK8 patients when compared to AD, possibly contributing toward increased susceptibility to infections and cancer. In contrast, epidermal growth factor (EGF) was significantly downregulated in a subgroup of DOCK8-deficient and AD patients, while IL-31 expression was comparable between both DOCK8-deficient and AD cohorts, possibly contributing toward pruritus seen in both groups. CONCLUSION: This comprehensive cytokine profile in HIES patients reveals distinctive biomarkers that differentiate between the DOCK8-deficient and AD patients. The unique expression profile of various inflammatory cytokines in patients with DOCK8 deficiency vs atopic dermatitis likely reflects disease-specific perturbations in multiple cellular processes and pathways leading to a predisposition to infections and allergies seen in these patients. These data agree with the role for EGF replacement therapy in EGF-deficient individuals with AD as well as DOCK8 deficiency through a potential shared pathway. In addition, these novel biomarkers may be potentially useful in distinguishing DOCK8 deficiency from AD allowing early-targeted treatment options.


Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Dermatite Atópica/etiologia , Dermatite Atópica/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Adolescente , Adulto , Biomarcadores , Linhagem Celular , Criança , Dermatite Atópica/diagnóstico , Suscetibilidade a Doenças , Feminino , Humanos , Imunoglobulina E/imunologia , Masculino , Mutação , Curva ROC , Adulto Jovem
13.
Mol Vis ; 24: 727-732, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30581279

RESUMO

Purpose: Inflammation is a key component of retinal disease. We previously reported that exchange protein for cAMP 1 (Epac1) reduced inflammatory mediators, including total levels of high mobility group box 1 (HMGB1) in retinal endothelial cells (RECs) and the mouse retina. The goal of this study was to determine intermediate pathways that allow Epac1 to reduce HMGB1, which could lead to novel targets for therapeutics. Methods: We used endothelial cell-specific conditional knockout mice for Epac1 and RECs to investigate whether Epac1 requires activation of insulin like growth factor binding protein 3 (IGFBP-3) and sirtuin 1 (SIRT1) to reduce acetylated HMGB1 levels with immunoprecipitation, western blot, and enzyme-linked immunosorbent assay (ELISA). Results: Data showed that high glucose reduced IGFBP-3 and SIRT1 levels, and increased acetylation of HMGB1 in RECs. An Epac1 agonist reduced acetylated HMGB1 levels in high glucose. The Epac1 agonist could not reduce HMGB1 or SIRT1 levels when IGFBP-3 siRNA was used. The agonist also could not reduce HMGB1 when SIRT1 siRNA was used. The mouse retina showed that loss of Epac1 increases acetylated HMGB1 levels and reduces IGFBP-3 and SIRT1 levels. Conclusions: Taken together, the data suggest that Epac1 activates IGFBP-3 to increase SIRT1, leading to a significant reduction in acetylated HMGB1. These findings provide novel therapeutic targets for reducing key inflammatory cascades in the retina.


Assuntos
Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Proteína HMGB1/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Vasos Retinianos/metabolismo , Sirtuína 1/genética , Acetilação/efeitos dos fármacos , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Glucose/farmacologia , Fatores de Troca do Nucleotídeo Guanina/agonistas , Fatores de Troca do Nucleotídeo Guanina/deficiência , Proteína HMGB1/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/antagonistas & inibidores , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Retina/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/efeitos dos fármacos , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismo , Tionucleotídeos/farmacologia
14.
J Pediatric Infect Dis Soc ; 7(suppl_2): S79-S82, 2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30590619

RESUMO

Hematopoietic stem cell transplantation (HSCT) has been the standard of care for infants with severe combined immunodeficiency (SCID) for several decades due to the dismal prognosis early in life without immune reconstitution. In recent years, as HSCT conditioning regimens and supportive care have greatly improved, HSCT is gaining in acceptance for more non-SCID primary immunodeficiencies (PIDs) and outside the early childhood period. In addition, potential donor options for non-SCID PIDs are expanding with increasing success for haploidentical donor transplants. In this brief report of a presentation at the PIDS-St. Jude 2018 conference, PIDs for which transplants are increasingly performed outside of early childhood will be discussed.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Síndromes de Imunodeficiência/terapia , Criança , Deficiência de GATA2/terapia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Humanos , Lactente , Imunodeficiência Combinada Severa/terapia
15.
Nat Med ; 24(12): 1815-1821, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30397357

RESUMO

Human microbiome studies have revealed the intricate interplay of host immunity and bacterial communities to achieve homeostatic balance. Healthy skin microbial communities are dominated by bacteria with low viral representation1-3, mainly bacteriophage. Specific eukaryotic viruses have been implicated in both common and rare skin diseases, but cataloging skin viral communities has been limited. Alterations in host immunity provide an opportunity to expand our understanding of microbial-host interactions. Primary immunodeficient patients manifest with various viral, bacterial, fungal, and parasitic infections, including skin infections4. Dedicator of cytokinesis 8 (DOCK8) deficiency is a rare primary human immunodeficiency characterized by recurrent cutaneous and systemic infections, as well as atopy and cancer susceptibility5. DOCK8, encoding a guanine nucleotide exchange factor highly expressed in lymphocytes, regulates actin cytoskeleton, which is critical for migration through collagen-dense tissues such as skin6. Analyzing deep metagenomic sequencing data from DOCK8-deficient skin samples demonstrated a notable increase in eukaryotic viral representation and diversity compared with healthy volunteers. De novo assembly approaches identified hundreds of novel human papillomavirus genomes, illuminating microbial dark matter. Expansion of the skin virome in DOCK8-deficient patients underscores the importance of immune surveillance in controlling eukaryotic viral colonization and infection.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/virologia , Dermatopatias/virologia , Pele/virologia , Adolescente , Bacteriófagos/genética , Criança , Feminino , Genoma Viral/genética , Fatores de Troca do Nucleotídeo Guanina/deficiência , Voluntários Saudáveis , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade/genética , Síndromes de Imunodeficiência/microbiologia , Síndromes de Imunodeficiência/patologia , Linfócitos/virologia , Masculino , Metagenoma/genética , Metagenoma/imunologia , Microbiota/genética , Papillomaviridae/isolamento & purificação , Papillomaviridae/patogenicidade , Pele/microbiologia , Dermatopatias/genética , Dermatopatias/microbiologia , Dermatopatias/patologia
16.
PLoS One ; 13(9): e0204346, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30235337

RESUMO

We had previously reported that exchange protein for cAMP 1 (Epac1) reduced inflammatory mediators in the retina of mice and in retinal endothelial cells (REC). Since ischemia can induce retinal damage potentially through activation of inflammatory cascades, we hypothesized that Epac1 would protect the retina against neuronal and vascular damage after exposure to ischemia/reperfusion (I/R). We used Epac1 floxed and endothelial cell specific Epac1 knockout mice for this work. We exposed them to ischemia for 90 minutes followed by reperfusion. One day after I/R, some mice were used for fluorescein angiography imaging or Evan's blue measurements of permeability. Mice were sacrificed at 2 days for neuronal measurements and at 10 days for measurements of degenerate capillaries. Data show increased leakage in the Epac1 Cre-Lox (Epac1 EC-KO) mice exposed to I/R when compared to Epac1 floxed mice with the same treatment. I/R also increased numbers of degenerate capillaries and cell loss in all retinal layers of Epac1 EC-KO mice. Retinal thickness was reduced more significantly in the Epac1 EC-KO mice compared to Epac1 floxed mice after I/R. Taken together, the data suggest that Epac1 is protective against both neuronal and vascular damage to the retina after exposure to I/R.


Assuntos
Vasos Sanguíneos/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neurônios/patologia , Traumatismo por Reperfusão/prevenção & controle , Retina/patologia , Animais , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Traumatismo por Reperfusão/patologia , Retina/fisiopatologia
17.
Transfusion ; 58(9): 2122-2127, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30179262

RESUMO

BACKGROUND: After hematopoietic stem cell transplantation (HSCT) autoimmune hemolytic anemia (AIHA) is a known and fairly common complication. It is often refractory to conventional therapies including corticosteroids, intravenous immunoglobulin, splenectomy, and the more recently described use of monoclonal antibodies. The high morbidity associated with these severe persistent cases elucidates the gaps in alternative therapies available for treatment. STUDY DESIGN AND METHODS: We described the successful use of abatacept for severe refractory AIHA after HSCT in three patients. RESULTS: Three pediatric patients with refractory AIHA after allogeneic stem cell transplantation were observed to be unresponsive to multitude immunosuppressive therapies, resulting in persistent transfusion dependency. Treatment with abatacept, a fusion protein that inhibits T-cell activation by binding to CD80/CD86 on antigen-presenting cells (APCs), thus blocking the required CD28 interaction between APCs and T cells, resulted in the resolution of hemolysis. CONCLUSION: Abatacept may provide significant clinical benefit in the management of AIHA after HSCT.


Assuntos
Abatacepte/uso terapêutico , Anemia Hemolítica Autoimune/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunossupressores/uso terapêutico , Adolescente , Anemia Hemolítica Autoimune/etiologia , Anemia Falciforme/terapia , Bacteriemia/complicações , Tipagem e Reações Cruzadas Sanguíneas , Criança , Pré-Escolar , Resistência a Medicamentos , Substituição de Medicamentos , Feminino , Fatores de Troca do Nucleotídeo Guanina/deficiência , Humanos , Síndrome de Job/complicações , Linfo-Histiocitose Hemofagocítica/terapia , Masculino , Staphylococcus aureus Resistente à Meticilina , Pneumonia por Pneumocystis/complicações , Indução de Remissão , Estudos Retrospectivos , Infecções Estafilocócicas/complicações , Viroses/complicações
18.
Mamm Genome ; 29(7-8): 603-617, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30094507

RESUMO

Spectacular progress has been made in the characterization of human hyper-IgE syndrome (HIES) over the last 50 years. HIES is a primary immunodeficiency defined as an association of atopy in a context of very high serum IgE levels, characteristic bacterial and fungal diseases, low-level clinical and biological inflammation, and various non-hematopoietic developmental manifestations. Somewhat arbitrarily, three disorders were successively put forward as the underlying cause of HIES: autosomal dominant (AD) STAT3 deficiency, the only disorder corresponding to the original definition of HIES, and autosomal recessive (AR) DOCK8 and PGM3 deficiencies, in which atopy and high serum IgE levels occur in a context of manifestations not seen in patients with typical HIES. Indeed, these three disorders disrupt different molecular pathways, affect different cell types, and underlie different clinical phenotypes. Surprisingly, several other inherited inborn errors of immunity in which serum IgE levels are high, sometimes almost as high as those in HIES patients, are not considered to belong to the HIES group of diseases. Studies of HIES have been further complicated by the lack of a high serum IgE phenotype in all mouse models of the disease other than two Stat3 mutant strains. The study of infections in mutant mice has helped elucidate only some forms of HIES and infection. Mouse models of these conditions have also been used to study non-hematopoietic phenotypes for STAT3 deficiency, tissue-specific immunity for DOCK8 deficiency, and cell lineage maturation for PGM3 deficiency. We review here the history of the field of HIES since the first clinical description of this condition in 1966, together with the three disorders commonly referred to as HIES, focusing, in particular, on their mouse models. We propose the restriction of the term "HIES" to patients with an AD STAT3-deficiency phenotype, including the most recently described AR ZNF341 deficiency, thus excluding AR DOCK8 and PGM3 deficiencies from the definition of this disease.


Assuntos
Suscetibilidade a Doenças , Síndrome de Job/etiologia , Síndrome de Job/metabolismo , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Biomarcadores , Modelos Animais de Doenças , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina/deficiência , Humanos , Imunoglobulina E/imunologia , Síndrome de Job/diagnóstico , Fenótipo , Fosfoglucomutase/deficiência , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
19.
PLoS One ; 13(7): e0200935, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30048476

RESUMO

Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.


Assuntos
Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Hipocampo/citologia , Transdução de Sinais/genética , Animais , Corticosterona/sangue , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Psicológico/genética , Estresse Psicológico/patologia
20.
Circulation ; 138(21): 2413-2433, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-29921611

RESUMO

BACKGROUND: Thoracic aortic aneurysm (TAA) and dissection are fatal diseases that cause aortic rupture and sudden death. The small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Previous studies revealed that reduced force generation in aortic smooth muscle cells (AoSMCs) causes TAA and thoracic aortic dissection. METHODS: To examine the role of SmgGDS in TAA formation, we used an angiotensin II (1000 ng·min-1·kg-1, 4 weeks)-induced TAA model. RESULTS: We found that 33% of Apoe-/- SmgGDS+/- mice died suddenly as a result of TAA rupture, whereas there was no TAA rupture in Apoe-/- control mice. In contrast, there was no significant difference in the ratio of abdominal aortic aneurysm rupture between the 2 genotypes. We performed ultrasound imaging every week to follow up the serial changes in aortic diameters. The diameter of the ascending aorta progressively increased in Apoe-/- SmgGDS+/- mice compared with Apoe-/- mice, whereas that of the abdominal aorta remained comparable between the 2 genotypes. Histological analysis of Apoe-/- SmgGDS+/- mice showed dissections of major thoracic aorta in the early phase of angiotensin II infusion (day 3 to 5) and more severe elastin degradation compared with Apoe-/- mice. Mechanistically, Apoe-/- SmgGDS+/- mice showed significantly higher levels of oxidative stress, matrix metalloproteinases, and inflammatory cell migration in the ascending aorta compared with Apoe-/- mice. For mechanistic analyses, we primary cultured AoSMCs from the 2 genotypes. After angiotensin II (100 nmol/L) treatment for 24 hours, Apoe-/- SmgGDS+/- AoSMCs showed significantly increased matrix metalloproteinase activity and oxidative stress levels compared with Apoe-/- AoSMCs. In addition, SmgGDS deficiency increased cytokines/chemokines and growth factors in AoSMCs. Moreover, expressions of fibrillin-1 ( FBN1), α-smooth muscle actin ( ACTA2), myosin-11 ( MYH11), MYLLK, and PRKG1, which are force generation genes, were significantly reduced in Apoe-/- SmgGDS+/- AoSMCs compared with Apoe-/- AoSMCs. A similar tendency was noted in AoSMCs from patients with TAA compared with those from control subjects. Finally, local delivery of the SmgGDS gene construct reversed the dilation of the ascending aorta in Apoe-/- SmgGDS+/- mice. CONCLUSIONS: These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.


Assuntos
Aorta/metabolismo , Aneurisma da Aorta Torácica/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Actinas/genética , Actinas/metabolismo , Angiotensina II/administração & dosagem , Angiotensina II/efeitos adversos , Animais , Aorta/citologia , Aorta/patologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/prevenção & controle , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrilina-1/genética , Fibrilina-1/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína rhoA de Ligação ao GTP/genética
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