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1.
Cell ; 183(5): 1264-1281.e20, 2020 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-33091337

RESUMEN

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.


Asunto(s)
Subtipos Serológicos HLA-DR/inmunología , Esclerosis Múltiple/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Alelos , Antígenos/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Reacciones Cruzadas/inmunología , Femenino , Humanos , Memoria Inmunológica , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Péptidos/inmunología , Proteoma/metabolismo , Adulto Joven
2.
Cell ; 175(1): 85-100.e23, 2018 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-30173916

RESUMEN

Multiple sclerosis is an autoimmune disease that is caused by the interplay of genetic, particularly the HLA-DR15 haplotype, and environmental risk factors. How these etiologic factors contribute to generating an autoreactive CD4+ T cell repertoire is not clear. Here, we demonstrate that self-reactivity, defined as "autoproliferation" of peripheral Th1 cells, is elevated in patients carrying the HLA-DR15 haplotype. Autoproliferation is mediated by memory B cells in a HLA-DR-dependent manner. Depletion of B cells in vitro and therapeutically in vivo by anti-CD20 effectively reduces T cell autoproliferation. T cell receptor deep sequencing showed that in vitro autoproliferating T cells are enriched for brain-homing T cells. Using an unbiased epitope discovery approach, we identified RASGRP2 as target autoantigen that is expressed in the brain and B cells. These findings will be instrumental to address important questions regarding pathogenic B-T cell interactions in multiple sclerosis and possibly also to develop novel therapies.


Asunto(s)
Linfocitos B/patología , Subtipos Serológicos HLA-DR/inmunología , Esclerosis Múltiple/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/fisiopatología , Linfocitos B/metabolismo , Encéfalo/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Subtipos Serológicos HLA-DR/genética , Humanos , Esclerosis Múltiple/genética , Esclerosis Múltiple/fisiopatología , Receptores de Antígenos de Linfocitos T , Células TH1/fisiología
3.
Nature ; 617(7962): 807-817, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37198490

RESUMEN

Microbial organisms have key roles in numerous physiological processes in the human body and have recently been shown to modify the response to immune checkpoint inhibitors1,2. Here we aim to address the role of microbial organisms and their potential role in immune reactivity against glioblastoma. We demonstrate that HLA molecules of both glioblastoma tissues and tumour cell lines present bacteria-specific peptides. This finding prompted us to examine whether tumour-infiltrating lymphocytes (TILs) recognize tumour-derived bacterial peptides. Bacterial peptides eluted from HLA class II molecules are recognized by TILs, albeit very weakly. Using an unbiased antigen discovery approach to probe the specificity of a TIL CD4+ T cell clone, we show that it recognizes a broad spectrum of peptides from pathogenic bacteria, commensal gut microbiota and also glioblastoma-related tumour antigens. These peptides were also strongly stimulatory for bulk TILs and peripheral blood memory cells, which then respond to tumour-derived target peptides. Our data hint at how bacterial pathogens and bacterial gut microbiota can be involved in specific immune recognition of tumour antigens. The unbiased identification of microbial target antigens for TILs holds promise for future personalized tumour vaccination approaches.


Asunto(s)
Antígenos de Neoplasias , Bacterias , Proteínas Bacterianas , Glioblastoma , Linfocitos Infiltrantes de Tumor , Fragmentos de Péptidos , Humanos , Antígenos de Neoplasias/inmunología , Proteínas Bacterianas/inmunología , Vacunas contra el Cáncer/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Línea Celular Tumoral , Microbioma Gastrointestinal/inmunología , Glioblastoma/inmunología , Glioblastoma/patología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos HLA/inmunología , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Fragmentos de Péptidos/inmunología , Simbiosis , Bacterias/inmunología , Bacterias/patogenicidad
4.
Magn Reson Med ; 89(2): 665-677, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36253953

RESUMEN

PURPOSE: To explore the properties of short-T2 signals in human brain, investigate the impact of various experimental procedures on these properties and evaluate the performance of three-component analysis. METHODS: Eight samples of non-pathological human brain tissue were subjected to different combinations of experimental procedures including D2 O exchange and frozen storage. Short-T2 imaging techniques were employed to acquire multi-TE (33-2067 µs) data, to which a three-component complex model was fitted in two steps to recover the properties of the underlying signal components and produce amplitude maps of each component. For validation of the component amplitude maps, the samples underwent immunohistochemical myelin staining. RESULTS: The signal component representing the myelin bilayer exhibited super-exponential decay with T2,min of 5.48 µs and a chemical shift of 1.07 ppm, and its amplitude could be successfully mapped in both white and gray matter in all samples. These myelin maps corresponded well to myelin-stained tissue sections. Gray matter signals exhibited somewhat different components than white matter signals, but both tissue types were well represented by the signal model. Frozen tissue storage did not alter the signal components but influenced component amplitudes. D2 O exchange was necessary to characterize the non-aqueous signal components, but component amplitude mapping could be reliably performed also in the presence of H2 O signals. CONCLUSIONS: The myelin mapping approach explored here produced reasonable and stable results for all samples. The extensive tissue and methodological investigations performed in this work form a basis for signal interpretation in future studies both ex vivo and in vivo.


Asunto(s)
Vaina de Mielina , Sustancia Blanca , Humanos , Vaina de Mielina/química , Imagen por Resonancia Magnética/métodos , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Sustancia Gris/diagnóstico por imagen , Sustancia Blanca/diagnóstico por imagen
5.
Proc Natl Acad Sci U S A ; 116(19): 9671-9676, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-31004050

RESUMEN

Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor's downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19+ cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors.


Asunto(s)
Linfocitos B , Sistema de Señalización de MAP Quinasas/genética , Esclerosis Múltiple , Fosfoproteínas , Polimorfismo de Nucleótido Simple , Proteómica , Linfocitos B/metabolismo , Linfocitos B/patología , Proliferación Celular , Supervivencia Celular , Femenino , Humanos , Masculino , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo
6.
J Sleep Res ; 28(3): e12721, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-29961995

RESUMEN

Fatigue in multiple sclerosis is a very common and cumbersome symptom, but its aetiology is poorly understood. Proteomics is increasingly implemented in multiple sclerosis research, but has not yet been used to study the neurobiological basis of fatigue in multiple sclerosis. To identify potential cerebrospinal fluid biomarkers of fatigue in multiple sclerosis, we collected cerebrospinal fluid of 20 patients with multiple sclerosis with fatigue (MS+), 20 patients with multiple sclerosis without fatigue (MS-), and 20 control subjects without multiple sclerosis and without fatigue (HC). We used a shotgun proteomics approach and label-free quantitative proteomics to analyse the protein content in cerebrospinal fluid. Selected proteins with differential abundance were further validated by immunoblotting. Out of 591 detected cerebrospinal fluid proteins, the abundance of nine proteins differed between the three groups, and seven additional proteins differed between MS+ and MS- patients. Using immunoblot or slot-blot techniques, we confirmed decreased levels of protein kinase C-binding protein NELL2, neural cell adhesion molecule L1-like protein, and reelin in MS+ patients. In conclusion, cerebrospinal fluid proteomics may provide insight into the neurobiological basis of fatigue in multiple sclerosis. The proteins identified to be decreased in MS+ are involved in synaptic plasticity and energy homeostasis, and thus appear as plausible biomarkers of this common symptom.


Asunto(s)
Biomarcadores/metabolismo , Proteínas del Líquido Cefalorraquídeo/metabolismo , Fatiga/etiología , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/diagnóstico , Proteómica/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Proteína Reelina
7.
J Neurovirol ; 21(6): 614-22, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25740538

RESUMEN

The high prevalence of asymptomatic JC polyomavirus (JCV) infection in the general population indicates coexistence with the human host and efficient immune control in healthy individuals. For unknown reasons, kidney-resident archetypic JCV strains can turn into neurotropic JCV strains which in hereditary or acquired states of immunodeficiency cause opportunistic infection and cytolytic destruction of glial cells or granule cell neurons resulting in progressive multifocal demyelination in the central nervous system (CNS) or cerebellar atrophy, respectively. Immunomodulatory or immunosuppressive therapies with specific monoclonal antibodies including natalizumab, efalizumab, and rituximab have increased the risk of progressive multifocal leukoencephalopathy (PML) among treated patients, highlighting that symptomatic JCV infection of the CNS is associated with disturbances of adaptive immunity affecting B cells, antibodies, and CD4(+) and/or CD8(+) T cells. To date, no specific therapy to overcome PML is available and the only way to eliminate the virus from the CNS is to reconstitute global immune function. However, since the identification of JCV as the causative agent of PML 40 years ago, it is still not fully understood which components of the immune system prevent the development of PML and which immune mechanisms are involved in eliminating the virus from the CNS. This review gives an update about adaptive JCV-specific immune responses.


Asunto(s)
Virus JC/inmunología , Leucoencefalopatía Multifocal Progresiva/inmunología , Leucoencefalopatía Multifocal Progresiva/virología , Humanos
8.
Neurol Neuroimmunol Neuroinflamm ; 11(4): e200251, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38838284

RESUMEN

BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is considered a prototypic autoimmune disease of the CNS. It is the leading cause of chronic neurologic disability in young adults. Proinflammatory B cells and autoreactive T cells both play important roles in its pathogenesis. We aimed to study alterations of regulatory T cells (Tregs), which likely also contribute to the disease, but their involvement is less clear. METHODS: By combining multiple experimental approaches, we examined the Treg compartments in 41 patients with relapsing-remitting MS and 17 healthy donors. RESULTS: Patients with MS showed a reduced frequency of CD4+ T cells and Foxp3+ Tregs and age-dependent alterations of Treg subsets. Treg suppressive function was compromised in patients, who were treated with natalizumab, while it was unaffected in untreated and anti-CD20-treated patients. The changes in natalizumab-treated patients included increased proinflammatory cytokines and an altered transcriptome in thymus-derived (t)-Tregs, but not in peripheral (p)-Tregs. DISCUSSION: Treg dysfunction in patients with MS might be related to an altered transcriptome of t-Tregs and a proinflammatory environment. Our findings contribute to a better understanding of Tregs and their subtypes in MS.


Asunto(s)
Esclerosis Múltiple Recurrente-Remitente , Natalizumab , Linfocitos T Reguladores , Humanos , Linfocitos T Reguladores/inmunología , Adulto , Femenino , Masculino , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Natalizumab/farmacología , Persona de Mediana Edad , Timo/inmunología , Factores Inmunológicos/farmacología , Adulto Joven
9.
J Cell Sci ; 124(Pt 20): 3414-27, 2011 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-22010197

RESUMEN

The Scar/Wave complex (SWC) generates lamellipodia through Arp2/3-dependent polymerisation of branched actin networks. In order to identify new SWC regulators, we conducted a screen in Drosophila cells combining proteomics with functional genomics. This screen identified Clathrin heavy chain (CHC) as a protein that binds to the SWC and whose depletion affects lamellipodium formation. This role of CHC in lamellipodium formation can be uncoupled from its role in membrane trafficking by several experimental approaches. Furthermore, CHC is detected in lamellipodia in the absence of the adaptor and accessory proteins of endocytosis. We found that CHC overexpression decreased membrane recruitment of the SWC, resulting in reduced velocity of protrusions and reduced cell migration. By contrast, when CHC was targeted to the membrane by fusion to a myristoylation sequence, we observed an increase in membrane recruitment of the SWC, protrusion velocity and cell migration. Together these data suggest that, in addition to its classical role in membrane trafficking, CHC brings the SWC to the plasma membrane, thereby controlling lamellipodium formation.


Asunto(s)
Clatrina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Microfilamentos/metabolismo , Seudópodos/metabolismo , Animales , Movimiento Celular/genética , Extensiones de la Superficie Celular/metabolismo , Extensiones de la Superficie Celular/patología , Clatrina/genética , Drosophila , Proteínas de Drosophila/genética , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , Unión Proteica/genética , Transporte de Proteínas/genética , Proteómica , Seudópodos/patología , Eliminación de Secuencia/genética , Transgenes/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo
10.
J Immunol ; 187(10): 5299-309, 2011 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-22013110

RESUMEN

The C-type lectin receptor dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is an important player in the recognition of pathogens by dendritic cells. A plethora of pathogens including viruses, bacteria, parasites, and fungi are recognized by DC-SIGN through both mannose and fucose-containing glycans expressed on the pathogen surface. In this study, we identified semen clusterin as a novel DC-SIGN ligand. Semen clusterin, but not serum clusterin, expresses an extreme abundance of fucose-containing blood-type Ags such as Le(x) and Le(y), which are both excellent DC-SIGN ligands. These motifs enable semen clusterin to bind DC-SIGN with very high affinity (K(d) 76 nM) and abrogate the binding of HIV-1 to DC-SIGN. Depletion of clusterin from semen samples, however, did not completely prevent the ability of semen to inhibit the capture of HIV-1 by DC-SIGN, supporting that besides clusterin, semen contains other DC-SIGN ligands. Further studies are needed to characterize these ligands and define their contribution to the DC-SIGN-blocking activity mediated by semen. Clusterin is an enigmatic protein involved in a variety of physiologic and pathologic processes including inflammation, atherosclerosis, and cancer. Our results uncover an unexpected heterogeneity in the glycosylation pattern of clusterin and suggest that the expression of high concentrations of fucose-containing glycans enables semen clusterin to display a unique set of biological functions that might affect the early course of sexually transmitted infectious diseases.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Clusterina/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Semen/inmunología , Semen/metabolismo , Adulto , Antivirales/sangre , Antivirales/metabolismo , Moléculas de Adhesión Celular/sangre , Clusterina/sangre , Células Dendríticas/virología , Fucosa/metabolismo , Glicosilación , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Lectinas Tipo C/sangre , Ligandos , Masculino , Manosa/metabolismo , Persona de Mediana Edad , Unión Proteica/inmunología , Receptores de Superficie Celular/sangre , Proteínas Recombinantes/sangre , Proteínas Recombinantes/metabolismo , Semen/virología
11.
Sci Adv ; 9(32): eadi0611, 2023 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-37566661

RESUMEN

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by loss of myelin (demyelination) and, to a certain extent, subsequent myelin repair (remyelination). To better understand the pathomechanisms underlying de- and remyelination and to monitor the efficacy of treatments aimed at regenerating myelin, techniques offering noninvasive visualizations of myelin are warranted. Magnetic resonance (MR) imaging has long been at the forefront of efforts to visualize myelin, but it has only recently become feasible to access the rapidly decaying resonance signals stemming from the myelin lipid-protein bilayer itself. Here, we show that direct MR mapping of the bilayer yields highly specific myelin maps in brain tissue from patients with MS. Furthermore, examination of the bilayer signal behavior is found to reveal pathological alterations in normal-appearing white and gray matter. These results indicate promise for in vivo implementations of the myelin bilayer mapping technique, with prospective applications in basic research, diagnostics, disease monitoring, and drug development.


Asunto(s)
Esclerosis Múltiple , Vaina de Mielina , Humanos , Vaina de Mielina/patología , Esclerosis Múltiple/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Espectroscopía de Resonancia Magnética
12.
Brain Commun ; 5(6): fcad307, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38025281

RESUMEN

Magnetic resonance imaging (MRI) has limitations in identifying underlying tissue pathology, which is relevant for neurological diseases such as multiple sclerosis, stroke or brain tumours. However, there are no standardized methods for correlating MRI features with histopathology. Thus, here we aimed to develop and validate a tool that can facilitate the correlation of brain MRI features to corresponding histopathology. For this, we designed the Brainbox, a waterproof and MRI-compatible 3D printed container with an integrated 3D coordinate system. We used the Brainbox to acquire post-mortem ex vivo MRI of eight human brains, fresh and formalin-fixed, and correlated focal imaging features to histopathology using the built-in 3D coordinate system. With its built-in 3D coordinate system, the Brainbox allowed correlation of MRI features to corresponding tissue substrates. The Brainbox was used to correlate different MR image features of interest to the respective tissue substrate, including normal anatomical structures such as the hippocampus or perivascular spaces, as well as a lacunar stroke. Brain volume decreased upon fixation by 7% (P = 0.01). The Brainbox enabled degassing of specimens before scanning, reducing susceptibility artefacts and minimizing bulk motion during scanning. In conclusion, our proof-of-principle experiments demonstrate the usability of the Brainbox, which can contribute to improving the specificity of MRI and the standardization of the correlation between post-mortem ex vivo human brain MRI and histopathology. Brainboxes are available upon request from our institution.

13.
Artículo en Inglés | MEDLINE | ID: mdl-33649179

RESUMEN

OBJECTIVE: CNS damage can increase the susceptibility of the blood-brain barrier (BBB) to changes induced by systemic inflammation. The aim of this study is to better understand BBB permeability in patients with MS and to examine whether compromised BBB integrity in some of these patients is associated with CNS damage and systemic inflammation. METHODS: Routine CSF measurements of 121 patients with MS were analyzed including number and type of infiltrating cells, total protein, lactate, and oligoclonal bands, as well as intrathecal production of immunoglobulins and CSF/serum quotients for albumin, immunoglobulins, and glucose. In addition, in a subcohort of these patients, we performed ex vivo immunophenotyping of CSF-infiltrating and paired circulating lymphocytes using a panel of 13 monoclonal antibodies, we quantified intrathecal neurofilament light chain (NF-L) and chitinase 3-like 1 (CHI3L1), and we performed intrathecal lipidomic analysis. RESULTS: Patients with MS with abnormal high levels of albumin in the CSF showed a distinct CSF cell infiltrate and markers of CNS damage such as increased intrathecal levels of NF-L and CHI3L1 as well as a distinct CSF lipidomic profile. In addition, these patients showed higher numbers of circulating proinflammatory Th1 and Th1* cells compatible with systemic inflammation. Of interest, the abnormally high levels of albumin in the CSF of those patients were preserved over time. CONCLUSIONS: Our results support the hypothesis that CNS damage may increase BBB vulnerability to systemic inflammation in a subset of patients and thus contribute to disease heterogeneity.


Asunto(s)
Albúminas/líquido cefalorraquídeo , Lesiones Encefálicas/líquido cefalorraquídeo , Inflamación/líquido cefalorraquídeo , Esclerosis Múltiple/líquido cefalorraquídeo , Adulto , Biomarcadores/líquido cefalorraquídeo , Barrera Hematoencefálica/metabolismo , Lesiones Encefálicas/inmunología , Femenino , Humanos , Inmunoglobulina G/líquido cefalorraquídeo , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología
14.
Genome Med ; 13(1): 117, 2021 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-34271980

RESUMEN

BACKGROUND: Multiple sclerosis (MS) is a major health problem, leading to a significant disability and patient suffering. Although chronic activation of the immune system is a hallmark of the disease, its pathogenesis is poorly understood, while current treatments only ameliorate the disease and may produce severe side effects. METHODS: Here, we applied a network-based modeling approach based on phosphoproteomic data to uncover the differential activation in signaling wiring between healthy donors, untreated patients, and those under different treatments. Based in the patient-specific networks, we aimed to create a new approach to identify drug combinations that revert signaling to a healthy-like state. We performed ex vivo multiplexed phosphoproteomic assays upon perturbations with multiple drugs and ligands in primary immune cells from 169 subjects (MS patients, n=129 and matched healthy controls, n=40). Patients were either untreated or treated with fingolimod, natalizumab, interferon-ß, glatiramer acetate, or the experimental therapy epigallocatechin gallate (EGCG). We generated for each donor a dynamic logic model by fitting a bespoke literature-derived network of MS-related pathways to the perturbation data. Last, we developed an approach based on network topology to identify deregulated interactions whose activity could be reverted to a "healthy-like" status by combination therapy. The experimental autoimmune encephalomyelitis (EAE) mouse model of MS was used to validate the prediction of combination therapies. RESULTS: Analysis of the models uncovered features of healthy-, disease-, and drug-specific signaling networks. We predicted several combinations with approved MS drugs that could revert signaling to a healthy-like state. Specifically, TGF-ß activated kinase 1 (TAK1) kinase, involved in Transforming growth factor ß-1 proprotein (TGF-ß), Toll-like receptor, B cell receptor, and response to inflammation pathways, was found to be highly deregulated and co-druggable with all MS drugs studied. One of these predicted combinations, fingolimod with a TAK1 inhibitor, was validated in an animal model of MS. CONCLUSIONS: Our approach based on donor-specific signaling networks enables prediction of targets for combination therapy for MS and other complex diseases.


Asunto(s)
Sistema Inmunológico/metabolismo , Modelos Biológicos , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/terapia , Transducción de Señal , Adulto , Algoritmos , Biomarcadores , Estudios de Casos y Controles , Terapia Combinada/métodos , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Humanos , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/inmunología , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/etiología , Fosfoproteínas/metabolismo , Pronóstico , Proteoma , Proteómica/métodos , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento
15.
Front Immunol ; 10: 540, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31024521

RESUMEN

Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification (PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4+ T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples.


Asunto(s)
Encéfalo/inmunología , Esclerosis Múltiple/inmunología , Proteína Básica de Mielina/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Líquido Cefalorraquídeo/inmunología , Citrulinación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Adulto Joven
16.
Sci Transl Med ; 10(462)2018 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-30305453

RESUMEN

Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system that develops in genetically susceptible individuals and likely requires environmental triggers. The autoantigens and molecular mimics triggering the autoimmune response in multiple sclerosis remain incompletely understood. By using a brain-infiltrating CD4+ T cell clone that is clonally expanded in multiple sclerosis brain lesions and a systematic approach for the identification of its target antigens, positional scanning peptide libraries in combination with biometrical analysis, we have identified guanosine diphosphate (GDP)-l-fucose synthase as an autoantigen that is recognized by cerebrospinal fluid-infiltrating CD4+ T cells from HLA-DRB3*-positive patients. Significant associations were found between reactivity to GDP-l-fucose synthase peptides and DRB3*02:02 expression, along with reactivity against an immunodominant myelin basic protein peptide. These results, coupled with the cross-recognition of homologous peptides from gut microbiota, suggest a possible role of this antigen as an inducer or driver of pathogenic autoimmune responses in multiple sclerosis.


Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Fucosa/metabolismo , Glucosiltransferasas/metabolismo , Cadenas HLA-DRB3/metabolismo , Esclerosis Múltiple/inmunología , Alelos , Secuencia de Aminoácidos , Encéfalo/metabolismo , Células Clonales , Humanos , Esclerosis Múltiple/líquido cefalorraquídeo , Proteína Básica de Mielina/metabolismo , Péptidos/química , Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
17.
Int J Parasitol ; 37(11): 1187-99, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17481635

RESUMEN

During the infectious cycle, protozoan parasites undergo various developmental transitions and switch virulence factors in response to extracellular signals in insect vectors and human hosts. Despite the importance of environmental sensing in parasite pathogenicity, little is known about the pathways that transduce extracellular signals into stage-specific gene expression. Here, we used a transgenic approach to gain insight into localisation and activity of three green fluorescence protein (GFP)-tagged Leishmania major mitogen-activated protein kinases, LmaMPK4, 7 and 10. The GFP-LmaMPKs in both L. major and Leishmania donovani transgenic lines showed predominant cytoplasmic localisation and the over-expression had no effect on promastigote morphology, growth and the ability to differentiate into stationary-phase metacyclics for L. major and axenic amastigotes for L. donovani. We isolated the GFP-tagged MPKs from parasite extracts and tested their phosphotransferase activity across various culture conditions. For all three GFP-LmaMPKs, kinase activity was low or absent in promastigote extracts but significantly increased in L. major promastigotes after exposure to pH 5.5 and 34 degrees C, and in axenic L. donovani amastigotes. Enhanced activity correlated with increased GFP-LmaMPK phosphorylation as judged by phospho-specific fluorescent staining of the immuno-precipitated kinases. We could extend these findings to the endogenous LmaMPK10, which accumulated in the phospho-protein fraction of axenic amastigotes but not promastigotes, and thus follows the stage-specific phosphorylation profile of episomally expressed GFP-LmaMPK10. These results provide evidence for the functional conservation of Leishmania MAP kinases in parasite environmental sensing and underscore the potential of transgenic approaches to gain insight into signaling events during the Leishmania life cycle.


Asunto(s)
Leishmania major/enzimología , Leishmaniasis/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Activación Enzimática , Expresión Génica , Genes Protozoarios , Proteínas Fluorescentes Verdes/genética , Interacciones Huésped-Parásitos , Calor , Concentración de Iones de Hidrógeno , Inmunoprecipitación , Leishmania major/genética , Microscopía Confocal , Proteínas Quinasas Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Parasitología/métodos , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia , Transgenes
18.
Mol Cell Biol ; 24(14): 6379-92, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15226438

RESUMEN

We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.


Asunto(s)
División Celular/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/fisiología , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Supervivencia Celular , Cromosomas Fúngicos , Humanos , Sustancias Macromoleculares , Mutación , Sistemas de Lectura Abierta , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura , Proteína de Unión al GTP ran/genética
19.
Int J Parasitol ; 36(13): 1399-407, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16959255

RESUMEN

Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3-10 and 4-7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7', the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.


Asunto(s)
Eimeria tenella/química , Proteínas Protozoarias/análisis , Animales , Eimeria tenella/ultraestructura , Electroforesis en Gel Bidimensional/métodos , Espectrometría de Masas/métodos , Proteoma , Proteómica/métodos , Proteínas Protozoarias/aislamiento & purificación , Esporozoítos/química
20.
Lab Chip ; 5(9): 935-42, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16100577

RESUMEN

The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.


Asunto(s)
Magnetismo , Microcomputadores , Técnicas Analíticas Microfluídicas/métodos , Proteínas/química , Tripsina/química , Dimetilpolisiloxanos/química , Hormona de Crecimiento Humana/química , Hormona de Crecimiento Humana/metabolismo , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Nylons/química , Porosidad , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Tripsina/metabolismo
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