Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Filtros adicionais











Intervalo de ano
1.
J Exp Med ; 216(9): 2038-2056, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31217193

RESUMO

Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-ß and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.

2.
Eur J Pharm Biopharm ; 142: 195-203, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31228557

RESUMO

We recently constructed a multicellular spheroid model of pancreatic tumor based on a triple co-culture of cancer cells, fibroblasts and endothelial cells and characterized by the presence of fibronectin, an important component of the tumor extracellular matrix. By combining cancer cells and stromal components, this model recreates in vitro the three-dimensional (3D) architecture of solid tumors. In this study, we used these hetero-type spheroids as a tool to assess the penetration of doxorubicin (used as a model drug) through the whole tumor mass either in a free form or loaded into polymer nanoparticles (NPs), and we investigated whether microscopy images, acquired by Confocal Laser Scanning Microscopy (CLSM) and Light Sheet Fluorescence Microscopy (LSFM), would be best to provide reliable information on this process. Results clearly demonstrated that CLSM was not suitable to accurately monitor the diffusion of small molecules such as the doxorubicin. Indeed, it only allowed to scan a layer of 100 µm depth and no information on deeper layers could be available because of a progressive loss of the fluorescence signal. On the contrary, a complete 3D tomography of the hetero-type multicellular tumor spheroids (MCTS) was obtained by LSFM and multi-view image fusion which revealed that the fluorescent molecule was able to reach the core of spheroids as large as 1 mm in diameter. However, no doxorubicin-loaded polymer nanoparticles were detected in the spheroids, highlighting the challenge of nanomedicine delivery through biological barriers. Overall, the combination of hetero-type MCTS and LSFM allowed to carry out a highly informative microscopic assessment and represents a suitable approach to precisely follow up the drug penetration in tumors. Accordingly, it could provide useful support in the preclinical investigation and optimization of nanoscale systems for drug delivery to solid tumors.

3.
Genes (Basel) ; 10(5)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091803

RESUMO

Mutations in CEP290 encoding a centrosomal protein important to cilia formation cause a spectrum of diseases, from isolated retinal dystrophies to multivisceral and sometimes embryo-lethal ciliopathies. In recent years, endogenous and/or selective non-canonical exon skipping of mutant exons have been documented in attenuated retinal disease cases. This observation led us to consider targeted exon skipping to bypass protein truncation resulting from a recurrent mutation in exon 36 (c.4723A > T, p.Lys1575*) causing isolated retinal ciliopathy. Here, we report two unrelated individuals (P1 and P2), carrying the mutation in homozygosity but affected with early-onset severe retinal dystrophy and congenital blindness, respectively. Studying skin-derived fibroblasts, we observed basal skipping and nonsense associated-altered splicing of exon 36, producing low (P1) and very low (P2) levels of CEP290 products. Consistent with a more severe disease, fibroblasts from P2 exhibited reduced ciliation compared to P1 cells displaying normally abundant cilia; both lines presented however significantly elongated cilia, suggesting altered axonemal trafficking. Antisense oligonucleotides (AONs)-mediated skipping of exon 36 increased the abundance of the premature termination codon (PTC)-free mRNA and protein, reduced axonemal length and improved cilia formation in P2 but not in P1 expressing higher levels of skipped mRNA, questioning AON-mediated exon skipping to treat patients carrying the recurrent c.4723A > T mutation.

4.
FASEB J ; : fj201800753RR, 2018 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-30303737

RESUMO

Mutations in the a disintegrin and metalloproteinase with thrombospondin motif-like 2 ( ADAMTSL2) gene are responsible for the autosomal recessive form of geleophysic dysplasia, which is characterized by short stature, short extremities, and skeletal abnormalities. However, the exact function of ADAMTSL2 is unknown. To elucidate the role of this protein in skeletal development, we generated complementary knockout (KO) mouse models with either total or chondrocyte Adamtsl2 deficiency. We observed that the Adamtsl2 KO mice displayed skeletal abnormalities reminiscent of the human phenotype. Adamtsl2 deletion affected the growth plate formation with abnormal differentiation and proliferation of chondrocytes. In addition, a TGF-ß signaling impairment in limbs lacking Adamtsl2 was demonstrated. Further investigations revealed that Adamtsl2 KO chondrocytes failed to establish a microfibrillar network composed by fibrillin1 and latent TGF-ß binding protein 1 fibrils. Chondrocyte Adamtsl2 KO mice also exhibited dwarfism. These studies uncover the function of Adamtsl2 in the maintenance of the growth plate ECM by modulating the microfibrillar network.-Delhon, L., Mahaut, C., Goudin, N., Gaudas, E., Piquand, K., Le Goff, W., Cormier-Daire, V., Le Goff, C. Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency.

5.
Hum Mol Genet ; 2018 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-29771326

RESUMO

CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base-pair deletion in exon 17, introducing a premature termination codon (PTC) in exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in exon 8 (c.508A>T, p.Lys170*) and exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing (NAS) alone (exon 8), or with BES (exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms.

6.
Arterioscler Thromb Vasc Biol ; 38(5): 1037-1051, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29519941

RESUMO

OBJECTIVE: Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized. APPROACH AND RESULTS: By studying a mouse model (cKO [conditional knockout]Kif5b) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKOKif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKOKif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKOKif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein. CONCLUSIONS: Our results indicate that a kinesin-1-dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

7.
Am J Hum Genet ; 102(2): 266-277, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29395073

RESUMO

Neurodegeneration with brain iron accumulation (NBIA) is a genetically heterogeneous condition characterized by progressive dystonia with iron accumulation in the basal ganglia. How NBIA-associated mutations trigger iron overload remains poorly understood. After studying fibroblast cell lines from subjects carrying both known and unreported biallelic mutations in CRAT and REPS1, we ascribe iron overload to the abnormal recycling of transferrin receptor (TfR1) and the reduction of TfR1 palmitoylation in NBIA. Moreover, we describe palmitoylation as a hitherto unreported level of post-translational TfR1 regulation. A widely used antimalarial agent, artesunate, rescued abnormal TfR1 palmitoylation in cultured fibroblasts of NBIA subjects. These observations suggest therapeutic strategies aimed at targeting impaired TfR1 recycling and palmitoylation in NBIA.

8.
Hum Mol Genet ; 27(1): 1-13, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29040558

RESUMO

Fibroblast growth factor receptor 3 (FGFR3) gain-of-function mutations cause dwarfisms, including achondroplasia (ACH) and thanatophoric dysplasia (TD). The constitutive activation of FGFR3 disrupts the normal process of skeletal growth. Bone-growth anomalies have been identified in skeletal ciliopathies, in which primary cilia (PC) function is disrupted. In human ACH and TD, the impact of FGFR3 mutations on PC in growth plate cartilage remains unknown. Here we showed that in chondrocytes from human (ACH, TD) and mouse Fgfr3Y367C/+ cartilage, the constitutively active FGFR3 perturbed PC length and the sorting and trafficking of intraflagellar transport (IFT) 20 to the PC. We demonstrated that inhibiting FGFR3 with FGFR inhibitor, PD173074, rescued both PC length and IFT20 trafficking. We also studied the impact of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) pathway. Interestingly, mTOR inhibition also rescued PC length and IFT20 trafficking. Together, we provide evidence that the growth plate defects ascribed to FGFR3-related dwarfisms are potentially due to loss of PC function, and these dwarfisms may represent a novel type of skeletal disorders with defective ciliogenesis.

9.
Am J Hum Genet ; 101(6): 1006-1012, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29198720

RESUMO

Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. It occasionally occurs in syndromic metabolic diseases and plurisystemic ciliopathies. Using exome sequencing in a multiplex family and three simplex case subjects with an atypical association of LCA with early-onset hearing loss, we identified two heterozygous mutations affecting Arg391 in ß-tubulin 4B isotype-encoding (TUBB4B). Inspection of the atomic structure of the microtubule (MT) protofilament reveals that the ß-tubulin Arg391 residue contributes to a binding pocket that interacts with α-tubulin contained in the longitudinally adjacent αß-heterodimer, consistent with a role in maintaining MT stability. Functional analysis in cultured cells overexpressing FLAG-tagged wild-type or mutant TUBB4B as well as in primary skin-derived fibroblasts showed that the mutant TUBB4B is able to fold, form αß-heterodimers, and co-assemble into the endogenous MT lattice. However, the dynamics of growing MTs were consistently altered, showing that the mutations have a significant dampening impact on normal MT growth. Our findings provide a link between sensorineural disease and anomalies in MT behavior and describe a syndromic LCA unrelated to ciliary dysfunction.


Assuntos
Amaurose Congênita de Leber/genética , Microtúbulos/genética , Tubulina (Proteína)/genética , Adulto , Sítios de Ligação/genética , Células Cultivadas , Criança , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Células Fotorreceptoras/metabolismo , Tubulina (Proteína)/metabolismo , Sequenciamento Completo do Exoma
10.
Am J Hum Genet ; 101(2): 283-290, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28757203

RESUMO

Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.


Assuntos
Aciltransferases/genética , Atrofia/patologia , Encefalopatias/genética , Encéfalo/patologia , Lipoilação/genética , Mitocôndrias/metabolismo , Aminoácidos/metabolismo , Encéfalo/diagnóstico por imagem , Encefalopatias/patologia , Mapeamento Encefálico/métodos , Células Cultivadas , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Glicina/sangue , Humanos , Recém-Nascido , Imagem por Ressonância Magnética , Mitocôndrias/genética , Consumo de Oxigênio/genética , Ligação Proteica/genética , Ácido Tióctico/metabolismo
12.
J Cell Biol ; 215(2): 203-216, 2016 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-27810912

RESUMO

Cross-linking of mast cell (MC) IgE receptors (FcεRI) triggers degranulation of secretory granules (SGs) and the release of many allergic and inflammatory mediators. Although degranulation depends crucially on microtubule dynamics, the molecular machinery that couples SGs to microtubule-dependent transport is poorly understood. In this study, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in hematopoietic cells are less sensitive to IgE-mediated, passive, systemic anaphylaxis. After IgE-induced stimulation, bone marrow-derived MCs from Kif5b knockout mice exhibited a marked reduction in SG translocation toward the secretion site. In contrast, a lack of Kif5b did not affect cytokine secretion, early FcεRI-initiated signaling pathways, or microtubule reorganization upon FcεRI stimulation. We identified Slp3 as the critical effector linking kinesin-1 to Rab27b-associated SGs. Kinesin-1 recruitment to the Slp3/Rab27b effector complex was independent of microtubule reorganization but occurred only upon stimulation requiring phosphatidylinositol 3-kinase (PI3K) activity. Our findings demonstrate that PI3K-dependent formation of a kinesin-1/Slp3/Rab27b complex is critical for the microtubule-dependent movement of SGs required for MC degranulation.


Assuntos
Degranulação Celular , Cinesina/metabolismo , Mastócitos/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Membrana Celular/metabolismo , Citocinas/metabolismo , Ativação Enzimática , Camundongos Knockout , Microscopia de Vídeo , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Receptores de IgE/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismo
13.
Infect Immun ; 84(10): 3017-23, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27481255

RESUMO

Meningococcal septic shock is associated with profound vasoplegia, early and severe myocardial dysfunction, and extended skin necrosis responsible for a specific clinical entity designated purpura fulminans (PF). PF represents 90% of fatal meningococcal infections. One characteristic of meningococcal PF is the myocardial dysfunction that occurs in the early phase of sepsis. Furthermore, hemodynamic studies have shown that the prognosis of meningococcal sepsis is directly related to the degree of impairment of cardiac contractility during the initial phase of the disease. To gain insight into a potential interaction of Neisseria meningitidis with the myocardial microvasculature, we modified a previously described humanized mouse model by grafting human myocardial tissue to SCID mice. We then infected the grafted mice with N. meningitides Using the humanized SCID mouse model, we demonstrated that N. meningitidis targets the human myocardial tissue vasculature, leading to the formation of blood thrombi, infectious vasculitis, and vascular leakage. These results suggest a novel mechanism of myocardial injury in the course of severe N. meningitidis sepsis that is likely to participate in primary myocardial dysfunction.


Assuntos
Coração/microbiologia , Infecções Meningocócicas/microbiologia , Microvasos/microbiologia , Animais , Bacteriemia/microbiologia , Modelos Animais de Doenças , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Feminino , Humanos , Infecções Meningocócicas/patologia , Camundongos SCID , Miocárdio , Neisseria meningitidis , Choque Séptico/sangue , Vasculite/patologia , Trombose Venosa/patologia
14.
PLoS One ; 11(6): e0157822, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27341421

RESUMO

Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

15.
J Exp Med ; 212(5): 619-31, 2015 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-25918342

RESUMO

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA- and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC-deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.


Assuntos
Candida albicans/imunologia , Candidíase Mucocutânea Crônica/imunologia , Homozigoto , Receptores de Interleucina/deficiência , Dermatopatias Genéticas/imunologia , Adulto , Candidíase Mucocutânea Crônica/genética , Candidíase Mucocutânea Crônica/patologia , Criança , Feminino , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Masculino , Dermatopatias Genéticas/genética , Dermatopatias Genéticas/patologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/imunologia
16.
Traffic ; 16(2): 191-203, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25425525

RESUMO

Chediak-Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome-like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling-late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal-sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13-4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin-containing vesicles into secretory cytotoxic granules.


Assuntos
Síndrome de Chediak-Higashi/genética , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Exocitose , Humanos , Sinapses Imunológicas/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Transporte Proteico , Via Secretória , Linfócitos T/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP
17.
J Clin Invest ; 124(12): 5516-20, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25401470

RESUMO

Innate immunity to viral infection involves induction of the type I IFN response; however, dysfunctional regulation of this pathway leads to inappropriate inflammation. Here, we evaluated a nonconsanguineous family of mixed European descent, with 4 members affected by systemic inflammatory and autoimmune conditions, including lupus, with variable clinical expression. We identified a germline dominant gain-of-function mutation in TMEM173, which encodes stimulator of type I IFN gene (STING), in the affected individuals. STING is a key signaling molecule in cytosolic DNA-sensing pathways, and STING activation normally requires dimerization, which is induced by 2'3' cyclic GMP-AMP (cGAMP) produced by the cGAMP synthase in response to cytosolic DNA. Structural modeling supported constitutive activation of the mutant STING protein based on stabilized dimerization. In agreement with the model predictions, we found that the STING mutant spontaneously localizes in the Golgi of patient fibroblasts and is constitutively active in the absence of exogenous 2'3'-cGAMP in vitro. Accordingly, we observed elevated serum IFN activity and a type I IFN signature in peripheral blood from affected family members. These findings highlight the key role of STING in activating both the innate and adaptive immune responses and implicate aberrant STING activation in features of human lupus.


Assuntos
Doenças Genéticas Inatas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Mutação , Multimerização Proteica/imunologia , Transdução de Sinais/imunologia , Imunidade Adaptativa/genética , Adulto , Idoso , Pré-Escolar , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Humanos , Imunidade Inata/genética , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Proteínas de Membrana , Nucleotídeos Cíclicos/genética , Nucleotídeos Cíclicos/imunologia , Multimerização Proteica/genética , Transdução de Sinais/genética , Síndrome
18.
PLoS One ; 8(9): e70292, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086246

RESUMO

Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2ß1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.


Assuntos
Basófilos/citologia , Células da Medula Óssea/citologia , Movimento Celular , Matriz Extracelular , Animais , Basófilos/metabolismo , Células da Medula Óssea/metabolismo , Adesão Celular , Células Cultivadas , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
19.
PLoS One ; 8(5): e62292, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23667464

RESUMO

BACKGROUND: Intestinal atresia is a rare congenital disorder with an incidence of 3/10,000 birth. About one-third of patients have severe intestinal dysfunction after surgical repair. We examined whether prenatal gastrointestinal obstruction might effect on the myenteric plexus and account for subsequent functional disorders. METHODOLOGY/PRINCIPAL FINDINGS: We studied a rat model of surgically induced antenatal atresia, comparing intestinal samples from both sides of the obstruction and with healthy rat pups controls. Whole-mount preparations of the myenteric plexus were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (nNOS). Quantitative reverse transcription PCR was used to analyze mRNAs for inflammatory markers. Functional motility and permeability analyses were performed in vitro. Phenotypic studies were also performed in 8 newborns with intestinal atresia. In the experimental model, the proportion of nNOS-immunoreactive neurons was similar in proximal and distal segments (6.7±4.6% vs 5.6±4.2%, p = 0.25), but proximal segments contained a higher proportion of ChAT-immunoreactive neurons (13.2±6.2% vs 7.5±4.3%, p = 0.005). Phenotypic changes were associated with a 100-fold lower concentration-dependent contractile response to carbachol and a 1.6-fold higher EFS-induced contractile response in proximal compared to distal segments. Transcellular (p = 0.002) but not paracellular permeability was increased. Comparison with controls showed that modifications involved not only proximal but also distal segments. Phenotypic studies in human atresia confirmed the changes in ChAT expression. CONCLUSION: Experimental atresia in fetal rat induces differential myenteric plexus phenotypical as well as functional changes (motility and permeability) between the two sides of the obstruction. Delineating these changes might help to identify markers predictive of motility dysfunction and to define guidelines for post-surgical care.


Assuntos
Modelos Animais de Doenças , Doenças Fetais/fisiopatologia , Motilidade Gastrointestinal/fisiologia , Atresia Intestinal/fisiopatologia , Plexo Mientérico/patologia , Animais , Área Sob a Curva , Atropina , Colina O-Acetiltransferase/metabolismo , Peroxidase do Rábano Silvestre , Humanos , Recém-Nascido , Manitol , NG-Nitroarginina Metil Éster , Neurônios/citologia , Óxido Nítrico Sintase/metabolismo , Permeabilidade , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas
20.
Blood ; 119(17): 3879-89, 2012 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-22308290

RESUMO

Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1-dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Cinesina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Sinapses/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas rab de Ligação ao GTP/metabolismo , Western Blotting , Células Cultivadas , Grânulos Citoplasmáticos/imunologia , Imunofluorescência , Humanos , Cinesina/antagonistas & inibidores , Cinesina/genética , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA