RESUMO
Human T cell antigen receptors (TCRs) pair in millions of combinations to create complex and unique T cell repertoires for each person. Through the use of tetramers to analyze TCRs reactive to the antigen-presenting molecule CD1b, we detected T cells with highly stereotyped TCR α-chains present among genetically unrelated patients with tuberculosis. The germline-encoded, mycolyl lipid-reactive (GEM) TCRs had an α-chain bearing the variable (V) region TRAV1-2 rearranged to the joining (J) region TRAJ9 with few nontemplated (N)-region additions. Analysis of TCRs by high-throughput sequencing, binding and crystallography showed linkage of TCRα sequence motifs to high-affinity recognition of antigen. Thus, the CD1-reactive TCR repertoire is composed of at least two compartments: high-affinity GEM TCRs, and more-diverse TCRs with low affinity for CD1b-lipid complexes. We found high interdonor conservation of TCRs that probably resulted from selection by a nonpolymorphic antigen-presenting molecule and an immunodominant antigen.
Assuntos
Antígenos CD1/imunologia , Infecções por Mycobacterium/imunologia , Mycobacterium/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Sequência de Bases , Células Clonais , Cristalografia por Raios X , Citometria de Fluxo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Infecções por Mycobacterium/microbiologia , RNA/química , RNA/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Subpopulações de Linfócitos T/citologia , Linfócitos T/citologiaRESUMO
OBJECTIVES: Vasculopathy is an important hallmark of systemic chronic inflammatory connective tissue diseases (CICTD) and is associated with increased cardiovascular risk. We investigated disease-specific biomarker profiles associated with endothelial dysfunction, angiogenic homeostasis and (tissue) inflammation, and their relation to disease activity in rare CICTD. METHODS: A total of 38 serum proteins associated with endothelial (dys)function and inflammation were measured by multiplex-immunoassay in treatment-naive patients with localized scleroderma (LoS, 30), eosinophilic fasciitis (EF, 8) or (juvenile) dermatomyositis (34), 119 (follow-up) samples during treatment, and 65 controls. Data were analysed by unsupervised clustering, Spearman correlations, non-parametric t test and ANOVA. RESULTS: The systemic CICTD, EF and dermatomyositis, had distinct biomarker profiles, with 'signature' markers galectin-9 (dermatomyositis) and CCL4, CCL18, CXCL9, fetuin, fibronectin, galectin-1 and TSP-1 (EF). In LoS, CCL18, CXCL9 and CXCL10 were subtly increased. Furthermore, dermatomyositis and EF shared upregulation of markers related to interferon (CCL2, CXCL10), endothelial activation (VCAM-1), inhibition of angiogenesis (angiopoietin-2, sVEGFR-1) and inflammation/leucocyte chemo-attraction (CCL19, CXCL13, IL-18, YKL-40), as well as disturbance of the Angiopoietin-Tie receptor system and VEGF-VEGFR system. These profiles were related to disease activity, and largely normalized during treatment. However, a subgroup of CICTD patients showed continued elevation of CXCL10, CXCL13, galectin-9, IL-18, TNFR2, VCAM-1, and/or YKL-40 during clinically inactive disease, possibly indicating subclinical interferon-driven inflammation and/or endothelial dysfunction. CONCLUSION: CICTD-specific biomarker profiles revealed an anti-angiogenic, interferon-driven environment during active disease, with incomplete normalization under treatment. This warrants further investigation into monitoring of vascular biomarkers during clinical follow-up, or targeted interventions to minimize cardiovascular risk in the long term.
Assuntos
Biomarcadores/sangue , Dermatomiosite , Endotélio Vascular/imunologia , Eosinofilia , Fasciite , Esclerodermia Localizada , Autoimunidade , Quimiocina CXCL10/sangue , Quimiocina CXCL13/sangue , Dermatomiosite/sangue , Dermatomiosite/diagnóstico , Eosinofilia/sangue , Eosinofilia/diagnóstico , Fasciite/sangue , Fasciite/diagnóstico , Feminino , Galectinas/sangue , Fatores de Risco de Doenças Cardíacas , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização Imunológica/métodos , Países Baixos , Gravidade do Paciente , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Esclerodermia Localizada/sangue , Esclerodermia Localizada/diagnóstico , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
BACKGROUND: Short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS) and CpG-ODN affect intestinal epithelial cells (IEC). Epithelial IL1α may contribute to allergic sensitization via autocrine mediator release affecting dendritic cells (DC). We studied whether IL1α contributes to Th2-associated mediator release by activated IEC and IEC/DC cocultures and possible modulation by scGOS/lcFOS±CpG-ODN. METHODS: Solid phase or transwell cultured IEC were preincubated with IL1α and/or IFNγ/TNFα for 6 h. The transwell IEC were also apically exposed to scGOS/lcFOS±CpG-ODN for 6 h, washed, and re-exposed, while cocultured with immature moDC (ccDC) for 48 h. These ccDC were subsequently added to allogeneic naïve T cells (MLR). IEC- and/or DC-derived mediators and T cell cytokines were measured. RESULTS: IL1α tended to enhance IL25 and enhanced IL33 and CCL20 release by IEC, while IL1α or TNFα or IFNγ enhanced CCL22. These were all further increased upon combined exposure of IFNγ/TNFα±IL1α coinciding with increased IL33 secretion in the solid phase culture. In the transwell, IL25 and IL33 remained under detection, while CCL20 and CCL22 were induced by IL1α or IFNγ/TNFα, respectively, and a synergistic increase was observed upon combined exposure of IFNγ/TNFα and IL1α. Furthermore, IFNγ was found to enhance galectin-9 secretion, which was more pronounced in IFNγ/TNFα±IL1α-exposed IEC and coincided with TGFß increase. Epithelial CpG-ODN exposure further increased CCL20, while reducing CCL22 release by IFNγ/TNFα/IL1α-activated IEC; however, scGOS/lcFOS suppressed both. Combined scGOS/lcFOS and CpG-ODN reduced CCL22, while CCL20 and regulatory galectin-9 and TGFß remained high in the supernatant of IFNγ/TNFα/IL1α-activated IEC and the following IEC/DC coculture. ccDC of scGOS/lcFOS- and CpG-ODN-exposed IFNγ/TNFα/IL1α-activated IEC increased IFNγ, IL10, TGFß, and galectin-9 secretion in the MLR compared to ccDC exposed to control-activated IEC. CONCLUSION: IL1α enhanced CCL20 and Th2-associated CCL22 release by IFNγ/TNFα-activated IEC. Combined scGOS/lcFOS and CpG-ODN exposure suppressed CCL22, while maintaining high CCL20, TGFß, and galectin-9 concentrations. In addition, ccDC derived from this IEC/DC coculture enhanced Th1 and regulatory mediator secretion mimicking known in vivo effects.
Assuntos
Galectinas/metabolismo , Oligossacarídeos/farmacologia , Células Th1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Células HT29 , Humanos , Teste de Cultura Mista de Linfócitos , Células Th1/efeitos dos fármacosRESUMO
OBJECTIVE: The interferon (IFN) signature is related to disease activity and vascular disease in systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and represents a promising therapeutic target. Quantification of the IFN signature is currently performed by gene expression analysis, limiting its current applicability in clinical practice. Therefore, the objective of this study was to establish an easy to measure biomarker for the IFN signature. METHODS: Serum levels of galectin-9, CXCL-10 (IP-10) and tumour necrosis factor receptor type II (TNF-RII) were measured in patients with SLE, SLE+APS and primary APS (PAPS) and healthy controls (n=148) after an initial screening of serum analytes in a smaller cohort (n=43). Analytes were correlated to measures of disease activity and the IFN signature. The performance of galectin-9, CXCL-10 and TNF-RII as biomarkers to detect the IFN signature was assessed by receiver operating characteristic curves. RESULTS: Galectin-9, CXCL-10 and TNF-RII were elevated in patients with SLE, SLE+APS and PAPS (p<0.05) and correlated with disease activity and tissue factor expression. Galectin-9 correlated stronger than CXCL-10 or TNF-RII with the IFN score (r=0.70, p<0.001) and was superior to CXCL-10 or TNF-RII in detecting the IFN signature (area under the curve (AUC) 0.86). Importantly, in patients with SLE(±APS), galectin-9 was also superior to anti-dsDNA antibody (AUC 0.70), or complement C3 (AUC 0.70) and C4 (AUC 0.78) levels in detecting the IFN signature. CONCLUSION: Galectin-9 is a novel, easy to measure hence clinically applicable biomarker to detect the IFN signature in patients with systemic autoimmune diseases such as SLE and APS.
Assuntos
Síndrome Antifosfolipídica/sangue , Biomarcadores/sangue , Galectinas/sangue , Interferons/análise , Lúpus Eritematoso Sistêmico/sangue , Adulto , Síndrome Antifosfolipídica/imunologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89-107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.
Assuntos
Anticoagulantes/farmacologia , Voluntários Saudáveis , Imunoproteínas/metabolismo , Adulto , Ácido Edético/farmacologia , Feminino , Congelamento , Heparina/farmacologia , Humanos , Imunoensaio , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Estabilidade Proteica , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
We aimed to study serum cytokine levels in 11 electrical status epilepticus in sleep (ESES) patients and 20 healthy control children. Patients showed significantly higher levels of interleukin (IL)-1α, IL-6, IL-10, chemokine (C-C motif) ligand (CCL)2 and chemokine (C-X-C motif) ligand (CXCL)8/IL-8 than controls, while macrophage migration inhibitory factor (MIF) and CCL3 were significantly lower. Follow-up analyses in five patients revealed a significant decrease of IL-6 levels after immunomodulating treatment. IL-6 changes were accompanied by clear improvement of electroencephalography (EEG) patterns and neuropsychological evaluation. We hypothesize that IL-6 correlates with disease activity and immunomodulating treatment efficacy.
Assuntos
Transtornos Cognitivos/imunologia , Citocinas/imunologia , Transtornos da Linguagem/imunologia , Transtornos do Sono-Vigília/imunologia , Estado Epiléptico/imunologia , Adolescente , Estudos de Casos e Controles , Quimiocina CCL2/imunologia , Quimiocina CCL3/imunologia , Criança , Pré-Escolar , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/fisiopatologia , Transtornos Cognitivos/psicologia , Eletroencefalografia , Feminino , Glucocorticoides/uso terapêutico , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Inflamação , Interleucina-10/imunologia , Interleucina-1alfa/imunologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Oxirredutases Intramoleculares/imunologia , Transtornos da Linguagem/tratamento farmacológico , Transtornos da Linguagem/fisiopatologia , Transtornos da Linguagem/psicologia , Fatores Inibidores da Migração de Macrófagos/imunologia , Masculino , Metilprednisolona/uso terapêutico , Testes Neuropsicológicos , Prednisolona/uso terapêutico , Transtornos do Sono-Vigília/tratamento farmacológico , Transtornos do Sono-Vigília/fisiopatologia , Transtornos do Sono-Vigília/psicologia , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/fisiopatologia , Estado Epiléptico/psicologia , Síndrome , Resultado do TratamentoRESUMO
Current views emphasize TCR diversity as a key feature that differentiates the group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 systems. Whereas TCR sequence motifs define CD1d-reactive NKT cells, the available data do not allow a TCR-based organization of the group 1 CD1 repertoire. The observed TCR diversity might result from donor-to-donor differences in TCR repertoire, as seen for MHC-restricted T cells. Alternatively, diversity might result from differing CD1 isoforms, Ags, and methods used to identify TCRs. Using CD1b tetramers to isolate clones recognizing the same glycolipid, we identified a previously unknown pattern of V gene usage (TRAV17, TRBV4-1) among unrelated human subjects. These TCRs are distinct from those present on NKT cells and germline-encoded mycolyl lipid-reactive T cells. Instead, they resemble the TCR of LDN5, one of the first known CD1b-reactive clones that was previously thought to illustrate the diversity of the TCR repertoire. Interdonor TCR conservation was observed in vitro and ex vivo, identifying LDN5-like T cells as a distinct T cell type. These data support TCR-based organization of the CD1b repertoire, which consists of at least two compartments that differ in TCR sequence motifs, affinity, and coreceptor expression.
Assuntos
Motivos de Aminoácidos/imunologia , Receptores de Antígenos de Linfócitos T/química , Subpopulações de Linfócitos T/química , Antígenos CD1/imunologia , Sequência de Bases , Sequência Conservada/imunologia , Citometria de Fluxo , Glicolipídeos/imunologia , Humanos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologiaRESUMO
BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 µg/mL) compared with those with PAPA syndrome (116 ± 74 µg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (EâK) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Erros Inatos do Metabolismo dos Metais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Alarminas/genética , Alarminas/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Criança , Citocinas/metabolismo , Proteínas do Citoesqueleto/genética , Feminino , Genótipo , Humanos , Complexo Antígeno L1 Leucocitário/genética , Masculino , Erros Inatos do Metabolismo dos Metais/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Fosforilação , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Multimerização Proteica , Pirina , Adulto JovemRESUMO
Cytokines are key components of the innate and adaptive immune system. As pivotal players in the progression or regression of a pathological process, these molecules provide a window through which diseases can be monitored and can thus act as biomarkers. In order to measure cytokine levels, a plethora of protocols can be applied. These methods include bioassays, protein microarrays, high-performance liquid chromatography (HPLC), sandwich enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence and bead based multiplex immunoassays (MIA). Due to the interaction and activity of cytokines, multiplex immunoassays are at the forefront of cytokine analysis by allowing multiple cytokines to be measured in parallel. However, even with optimized protocols, sample standardization needs to occur before these proteins can optimally act as biomarkers. This review describes various factors influencing the levels of cytokines measured in plasma, serum, dried blood spots and tissue biopsies, focusing on sample collection and handling, long term storage and the repetitive use of samples. By analyzing how each of these factors influences protein levels, it is concluded that samples should be stored at low temperatures in order to maintain cytokine stability. In addition, within a study, sample manipulations should be kept the same, with measurement protocols being chosen for their compatibility with the research in question. By having a clear understanding of what factors influence cytokine levels and how to overcome these technical issues, minimally confounded data can be obtained and cytokines can achieve optimal biomarker activity.
Assuntos
Citocinas/sangue , Imunoensaio/normas , Manejo de Espécimes/normas , Biomarcadores/sangue , Teste em Amostras de Sangue Seco , Humanos , Análise Serial de Proteínas , Padrões de Referência , Temperatura , Fatores de TempoRESUMO
Adenoviruses (HAdV) can cause life threatening infections, especially in paediatric patients after allogeneic stem cell transplantation (SCT). Yet, no effective antiviral medication is available. One treatment option is adoptive transfer of HAdV-specific T-cells from the graft donor into the patient. Especially CD4+ T-cells are critical to control HAdV infection. To allow for applicability of CD4+ T-cells in adoptive therapy, sufficient numbers of HAdV-specific T-cells with low levels of residual alloreactive T-cells are required. In this study, we explored the possibility to selectively expand and isolate functional HAdV-specific T-cells from PBMCs in response to 15-mer peptides using artificial antigen-presenting cells (aAPCs), composed of liposomes harbouring HAdV-peptide/HLA-Class-II complexes. HAdV-specific T-cells generated using this method produce mainly pro-inflammatory cytokines, express perforin and granzyme B, kill HAdV-infected cells effectively and are not alloreactive. Thus, the generation and isolation of HAdV-specific CD4+ T-cells seem a critical step towards specific adoptive therapy for HAdV infections after allogeneic SCT.
Assuntos
Infecções por Adenovirus Humanos/imunologia , Linfócitos T CD4-Positivos/imunologia , Perforina/imunologia , Adulto , Células Apresentadoras de Antígenos/imunologia , Antígenos/farmacologia , Proliferação de Células/efeitos dos fármacos , Granzimas/imunologia , Humanos , Leucócitos Mononucleares/citologia , Teste de Cultura Mista de Linfócitos , Peptídeos/farmacologiaRESUMO
During the last decade research has focused on the application of FOXP3(+) regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Inflamação/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T Reguladores/fisiologia , Adolescente , Artrite Juvenil/imunologia , Artrite Juvenil/metabolismo , Artrite Juvenil/patologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/terapia , Subpopulações de Linfócitos B/imunologia , Células Cultivadas , Criança , Pré-Escolar , Ativação Enzimática , Humanos , Terapia de Imunossupressão , Imunoterapia Adotiva , Inflamação/patologia , Inflamação/terapia , Masculino , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Falha de Tratamento , Regulação para CimaRESUMO
Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disorder characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE may involve defects in the post-transcriptional regulation of gene expression. MicroRNAs (miRNAs) are non-coding RNAs that control the expression of genes at the post-transcriptional level. Here, we performed a genome-wide miRNA profiling study to examine whether miRNA-mediated mechanisms are affected in human mTLE. miRNA profiles of the hippocampus of autopsy control patients and two mTLE patient groups were compared. This revealed segregated miRNA signatures for the three different patient groups and 165 miRNAs with up- or down-regulated expression in mTLE. miRNA in situ hybridization detected cell type-specific changes in miRNA expression and an abnormal nuclear localization of select miRNAs in neurons and glial cells of mTLE patients. Of several cellular processes implicated in mTLE, the immune response was most prominently targeted by deregulated miRNAs. Enhanced expression of inflammatory mediators was paralleled by a reduction in miRNAs that were found to target the 3'-untranslated regions of these genes in reporter assays. miR-221 and miR-222 were shown to regulate endogenous ICAM1 expression and were selectively co-expressed with ICAM1 in astrocytes in mTLE patients. Our findings suggest that miRNA changes in mTLE affect the expression of immunomodulatory proteins thereby further facilitating the immune response. This mechanism may have broad implications given the central role of astrocytes and the immune system in human neurological disease. Overall, this work extends the current concepts of human mTLE pathogenesis to the level of miRNA-mediated gene regulation.
Assuntos
Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/imunologia , Genes MHC da Classe II , MicroRNAs , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrócitos/patologia , Sequência de Bases , Estudos de Casos e Controles , Epilepsia do Lobo Temporal/patologia , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Hipocampo/patologia , Humanos , Mediadores da Inflamação/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neuroglia/patologia , Neurônios/fisiologiaRESUMO
In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17-secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells "convert" to Th17/1 under conditions that mimic the disease site, namely low TGFbeta and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.
Assuntos
Artrite Juvenil/imunologia , Interleucina-17/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Células Th1/imunologia , Sequência de Aminoácidos , Artrite Juvenil/genética , Artrite Juvenil/metabolismo , Sequência de Bases , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Criança , Citometria de Fluxo , Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Dados de Sequência Molecular , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores CCR6/genética , Receptores CCR6/imunologia , Receptores CCR6/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Proteínas com Domínio T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th1/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mesial temporal lobe epilepsy (mTLE) is a chronic and often treatment-refractory brain disorder characterized by recurrent seizures originating from the hippocampus. The pathogenic mechanisms underlying mTLE remain largely unknown. Recent clinical and experimental evidence supports a role of various inflammatory mediators in mTLE. Here, we performed protein expression profiling of 40 inflammatory mediators in surgical resection material from mTLE patients with and without hippocampal sclerosis, and autopsy controls using a multiplex bead-based immunoassay. In mTLE patients we identified 21 upregulated inflammatory mediators, including 10 cytokines and 7 chemokines. Many of these upregulated mediators have not previously been implicated in mTLE (for example, CCL22, IL-7 and IL-25). Comparing the three patient groups, two main hippocampal expression patterns could be distinguished, pattern I (for example, IL-10 and IL-25) showing increased expression in mTLE + HS patients compared to mTLE-HS and controls, and pattern II (for example, CCL4 and IL-7) showing increased expression in both mTLE groups compared to controls. Upregulation of a subset of inflammatory mediators (for example, IL-25 and IL-7) could not only be detected in the hippocampus of mTLE patients, but also in the neocortex. Principle component analysis was used to cluster the inflammatory mediators into several components. Follow-up analyses of the identified components revealed that the three patient groups could be discriminated based on their unique expression profiles. Immunocytochemistry showed that IL-25 IR (pattern I) and CCL4 IR (pattern II) were localized in astrocytes and microglia, whereas IL-25 IR was also detected in neurons. Our data shows co-activation of multiple inflammatory mediators in hippocampus and neocortex of mTLE patients, indicating activation of multiple pro- and anti-epileptogenic immune pathways in this disease.
Assuntos
Citocinas/metabolismo , Epilepsia do Lobo Temporal/patologia , Hipocampo/metabolismo , Sistema Imunitário/metabolismo , Neocórtex/metabolismo , Regulação para Cima/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Citocinas/genética , Epilepsia do Lobo Temporal/imunologia , Feminino , Hipocampo/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neocórtex/patologia , Neuroglia/metabolismo , Neurônios/metabolismo , Análise de Componente PrincipalRESUMO
OBJECTIVES: Peptide-based immune tolerance induction is considered an attractive treatment option for autoimmune diseases. The authors have developed a novel method that can enhance the induction of protective peptide-specific T-cell responses, using a rat arthritis model. The authors focused on the Toll-like receptor 9 ligand CpG, which was shown to stimulate regulatory T-cell proliferation when added to plasmacytoid dendritic cells (pDC) using in-vitro cultures. METHODS: The peptide used is a heat shock protein 60 epitope (p1) that elicits tolerogenic peptide-specific immune responses in human arthritis patients and was recently shown to have protective capacity as a bystander antigen in the rat adjuvant arthritis model. Rats were treated with three nasal doses of p1, CpG or a combination of p1 and CpG. Antigen-presenting cells were studied in nose-draining lymph nodes (mandibular lymph nodes; MLN) after nasal treatment, and T-cell responses were analysed in joint-draining lymph nodes after arthritis induction. RESULTS: Nasal co-administration of p1/CpG significantly augmented the arthritis-protective effect of p1, while CpG treatment alone did not. Co-treatment of p1/CpG increased both the number and activation status of pDC in draining MLN, which was accompanied by amplified p1-specific T-cell proliferation and interleukin (IL)-10 production. During early arthritis, p1-specific IL-10 production was identified at the site of inflammation. P1 and p1/CpG-treated rats showed a greater amount of CD4+FoxP3+ regulatory T cells in the joint-draining lymph nodes, which correlated with lower arthritis scores. CONCLUSIONS: These clinical and immunological data suggest the use of CpG as a potent adjuvant for mucosal peptide-specific immune therapy in arthritis.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Artrite Reumatoide/imunologia , Chaperonina 60/imunologia , Oligodesoxirribonucleotídeos/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Administração Intranasal , Animais , Artrite Experimental/imunologia , Chaperonina 60/administração & dosagem , Células Dendríticas/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Ativação Linfocitária/imunologia , Masculino , Oligodesoxirribonucleotídeos/administração & dosagem , Ratos , Ratos Endogâmicos Lew , Linfócitos T Reguladores/imunologia , Receptor Toll-Like 9/agonistas , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Adipokines have recently emerged as a novel group of mediators with important roles in inflammatory and immune responses and in the process of wound healing. This study investigated the involvement of several adipokines in the future development of proliferative vitreoretinopathy (PVR) following reattachment surgery for rhegmatogenous retinal detachment (RRD). A multiplex immunoassay was used to measure 6 different adipokines in 75 subretinal fluid samples collected during reattachment surgery for primary RRD. Twenty-one patients who developed a redetachment due to postoperative PVR after scleral buckling surgery (PVR group) were compared with age-, sex-, and storage-time-matched RRD samples from 54 patients with an uncomplicated postoperative course (RRD group). Levels of adiponectin (P = 0.006), cathepsin S (P = 0.001), and leptin (P = 0.041) were significantly elevated in the PVR group as compared to the RRD group. Levels of tissue inhibitor of metalloproteinase (TIMP)-1 were significantly lower in the PVR group than in the RRD group (P = 0.044). After correction for diabetes, body mass index (BMI), macular involvement, and preoperative PVR, the association between postoperative PVR development and adiponectin, cathepsin S, and TIMP-1 remained statistically significant (P < 0.05), whereas the significant correlation between PVR and elevated leptin levels was lost (P = 0.068). There were no significant differences in levels of chemerin (P = 0.351) and adipsin (P = 0.915). Of all adipokines investigated, multivariate logistic regression analysis showed that adiponectin was the exclusive predictor of the development of postoperative PVR after scleral buckling surgery (P = 0.003). Our findings indicate that, at the time of surgery for primary RRD, an altered expression of certain adipokines is associated with the future development of postoperative PVR.
Assuntos
Adipocinas/metabolismo , Descolamento Retiniano/metabolismo , Líquido Sub-Retiniano/metabolismo , Adulto , Idoso , Catepsinas/metabolismo , Tamponamento Interno , Feminino , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Recidiva , Descolamento Retiniano/diagnóstico , Descolamento Retiniano/cirurgia , Recurvamento da Esclera , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Vitrectomia , Vitreorretinopatia Proliferativa/etiologia , Vitreorretinopatia Proliferativa/metabolismoRESUMO
Glycogen storage disease type 1b (GSD 1b) is caused by mutations in the Glucose-6-phosphate transporter and is characterized by impaired glucose homeostasis. In addition, GSD-1b is associated with chronic neutropenia resulting in recurrent infections and inflammatory bowel disease. It is unclear whether the neutropenia is solely due to enhanced apoptosis of mature neutrophils or whether aberrant neutrophil development may also contribute. Here we demonstrate that hematopoietic progenitors from GSD-1b patients are not impaired in their capacity to develop into mature neutrophils. However, optimal survival of neutrophil progenitors from GSD-1b patients requires high glucose levels (> 200 mg dl(-1)), suggesting that even under normoglycemic conditions these cells are more prone to apoptosis. Furthermore, analysis of cytokine levels in peripheral blood suggests an inflammatory state with an inverse correlation between the level of inflammation and the number of neutrophils. Finally, in some patients, with low numbers of peripheral blood neutrophils, high numbers of neutrophils were observed in the intestine. Together, these results suggest that the neutropenia observed in GSD-1b patients is not caused by impaired maturation, but may be caused by both increased levels of apoptosis and egress of neutrophils from the blood to the inflamed tissues.
Assuntos
Doença de Depósito de Glicogênio Tipo I/patologia , Células-Tronco Hematopoéticas/metabolismo , Neutropenia/patologia , Neutrófilos/patologia , Adolescente , Animais , Antiporters/deficiência , Antiporters/genética , Apoptose/genética , Apoptose/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Criança , Pré-Escolar , Citocinas/sangue , Feminino , Glucose/metabolismo , Doença de Depósito de Glicogênio Tipo I/sangue , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/metabolismo , Homeostase/genética , Homeostase/fisiologia , Humanos , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Transporte de Monossacarídeos/deficiência , Proteínas de Transporte de Monossacarídeos/genética , Neutropenia/sangue , Neutropenia/genética , Neutropenia/metabolismo , Neutrófilos/metabolismo , Taxa de SobrevidaRESUMO
Macrophages contribute significantly to the pathology of many chronic inflammatory diseases, including rheumatoid arthritis (RA), asthma, and chronic obstructive pulmonary disease. Macrophage activation and survival are tightly regulated by reversible acetylation and deacetylation of histones, transcription factors, and structural proteins. Although histone deacetylase (HDAC) inhibitors (HDACis) demonstrate therapeutic effects in animal models of chronic inflammatory disease, depressed macrophage HDAC activity in patients with asthma, chronic obstructive pulmonary disease, or RA may contribute to inflammation in these diseases, potentially contraindicating the therapeutic administration of HDACis. In this study, we directly examined whether HDACis could influence the activation of macrophages derived from the inflamed joints of patients with RA. We found that inhibition of class I/II HDACs or class III sirtuin HDACs potently blocked the production of IL-6 and TNF-alpha by macrophages from healthy donors and patients with RA. Two HDACis, trichostatin A and nicotinamide, selectively induced macrophage apoptosis associated with specific downregulation of the antiapoptotic protein Bfl-1/A1, and inflammatory stimuli enhanced the sensitivity of macrophages to HDACi-induced apoptosis. Importantly, inflammatory and angiogenic cytokine production in intact RA synovial biopsy explants was also suppressed by HDACis. Our study identifies redundant, but essential, roles for class I/II and sirtuin HDACs in promoting inflammation, angiogenesis, and cell survival in RA.
Assuntos
Artrite Reumatoide/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Adulto , Idoso , Apoptose/efeitos dos fármacos , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Western Blotting , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Ácidos Hidroxâmicos/farmacologia , Inflamação/genética , Inflamação/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor , Niacinamida/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To identify serum protein biomarkers that might distinguish patients with early inflammatory arthritis (IA) with psoriatic arthritis (PsA) from those with rheumatoid arthritis (RA) and may be used to support appropriate early intervention. METHODS: The serum proteome of patients with PsA and patients with RA was interrogated using nano-liquid chromatography mass spectrometry (nano-LC-MS/MS) (n = 64 patients), an aptamer-based assay (SomaScan) targeting 1,129 proteins (n = 36 patients), and a multiplexed antibody assay (Luminex) for 48 proteins (n = 64 patients). Multiple reaction monitoring (MRM) assays were developed to evaluate the performance of putative markers using the discovery cohort (n = 60 patients) and subsequently an independent cohort of PsA and RA patients (n = 167). RESULTS: Multivariate machine learning analysis of the protein discovery data from the 3 platforms revealed that it was possible to differentiate PsA patients from RA patients with an area under the curve (AUC) of 0.94 for nano-LC-MS/MS, 0.69 for bead-based immunoassay measurements, and 0.73 for aptamer-based analysis. Subsequently, in the separate verification and evaluation studies, random forest models revealed that a subset of proteins measured by MRM could differentiate PsA and RA patients with AUCs of 0.79 and 0.85, respectively. CONCLUSION: We present a serum protein biomarker panel that can separate patients with early-onset IA with PsA from those with RA. With continued evaluation and refinement using additional and larger patient cohorts, including those with other arthropathies, we suggest that the panel identified here could contribute to improved clinical decision making.
Assuntos
Artrite Psoriásica/sangue , Artrite Psoriásica/diagnóstico , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Proteínas Sanguíneas/análise , Adulto , Biomarcadores/sangue , Estudos Transversais , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To investigate how meningococcal C vaccination in patients with remitting (oligoarticular) or progressive (polyarticular) juvenile idiopathic arthritis (JIA) influences the specific T-cell response to both the vaccine and heat shock protein 60, a regulatory auto-antigen in JIA. METHODS: Twenty six oligoarticular, 28 polyarticular JIA patients and 20 healthy adults were studied before and after MenC vaccination in a prospective follow-up study. T-cell proliferation assay, flow cytometry, carboxyfluorescein diacetate succinimidyl ester staining and multiplex immunoassay were performed to quantify and qualify the antigen-specific immune responses. RESULTS: Peripheral blood mononuclear cells (PBMC) from polyarticular JIA exemplified higher antigen-specific CD4 T-cell proliferation, interleukin 2 (IL-2) and tumour necrosis factor alpha (TNFα) production when compared with oligoarticular JIA or healthy individuals after vaccination. Furthermore, in polyarticular JIA antigen-induced CD4+CD25(bright) or CD4+FOXP3+ T cells did not increase upon vaccination. CONCLUSION: Polyarticular JIA CD4+FOXP3+ T cells did not respond to vaccination and demonstrated a higher percentage of cells irrespective of vaccination when compared with oligoarticular JIA. These cells are either activated T cells and/or regulatory cells unable to regulate the antigen-specific immune response after vaccination. When compared with oligoarticular JIA, the increased IL-2 and TNFα production underline the immune hyperresponsiveness of polyarticular JIA PBMC to an antigenic trigger. As this may hold a risk for derailment, these findings could provide a cellular basis for the presumed relationship between environmental triggers and disease in human autoimmune diseases.