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2.
PLoS One ; 14(2): e0210675, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30730971

RESUMO

The role of extracellular vesicles (EVs), specifically exosomes, in intercellular communication likely plays a key role in placental orchestration of pregnancy and maternal immune sensing of the fetus. While murine models are powerful tools to study pregnancy and maternal-fetal immune interactions, in contrast to human placental exosomes, the content of murine placental and pregnancy exosomes remains largely understudied. Using a recently developed in vitro culture technique, murine trophoblast stem cells derived from B6 mice were differentiated into syncytial-like cells. EVs from the conditioned media, as well as from pregnant and non-pregnant sera, were enriched for exosomes. The RNA composition of these murine trophoblast-derived and pregnancy-associated exosome-enriched-EVs (ExoE-EVs) was determined using RNA-sequencing analysis and expression levels confirmed by qRT-PCR. Differentially abundant miRNAs were detected in syncytial differentiated ExoE-EVs, particularly from the X chromosome cluster (mmu-miR-322-3p, mmu-miR-322-5p, mmu-miR-503-5p, mmu-miR-542-3p, and mmu-miR-450a-5p). These were confirmed to be increased in pregnant mouse sera ExoE-EVs by qRT-PCR analysis. Interestingly, fifteen miRNAs were only present within the pregnancy-derived ExoE-EVs compared to non-pregnant controls. Mmu-miR-292-3p and mmu-miR-183-5p were noted to be some of the most abundant miRNAs in syncytial ExoE-EVs and were also present at higher levels in pregnant versus non-pregnant sera ExoE-EVs. The bioinformatics tool, MultiMir, was employed to query publicly available databases of predicted miRNA-target interactions. This analysis reveals that the X-chromosome miRNAs are predicted to target ubiquitin-mediated proteolysis and intracellular signaling pathways. Knowing the cargo of placental and pregnancy-specific ExoE-EVs as well as the predicted biological targets informs studies using murine models to examine not only maternal-fetal immune interactions but also the physiologic consequences of placental-maternal communication.


Assuntos
Exoma , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Gravidez/fisiologia , Trofoblastos/metabolismo , Animais , Vesículas Extracelulares/imunologia , Feminino , Camundongos , MicroRNAs/imunologia , Trofoblastos/imunologia
3.
Nat Commun ; 9(1): 2650, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29985393

RESUMO

During development in the thymus, invariant natural killer T (iNKT) cells commit to one of three major functionally different subsets, iNKT1, iNKT2, and iNKT17. Here, we show that T cell antigen receptor (TCR) signal strength governs the development of iNKT cell subsets, with strong signaling promoting iNKT2 and iNKT17 development. Altering TCR diversity or signaling diminishes iNKT2 and iNKT17 cell subset development in a cell-intrinsic manner. Decreased TCR signaling affects the persistence of Egr2 expression and the upregulation of PLZF. By genome-wide comparison of chromatin accessibility, we identify a subset of iNKT2-specific regulatory elements containing NFAT and Egr binding motifs that is less accessible in iNKT2 cells that develop from reduced TCR signaling. These data suggest that variable TCR signaling modulates regulatory element activity at NFAT and Egr binding sites exerting a determinative influence on the dynamics of gene enhancer accessibility and the developmental fate of iNKT cells.


Assuntos
Diferenciação Celular/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Timócitos/imunologia , Animais , Sítios de Ligação , Diferenciação Celular/genética , Células Cultivadas , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Perfilação da Expressão Gênica/métodos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Subpopulações de Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo
4.
Blood ; 125(1): 185-94, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25301707

RESUMO

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infarto Encefálico/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Trombose/metabolismo , Motivos de Aminoácidos , Animais , Plaquetas/citologia , Artérias Carótidas/patologia , Membrana Celular/metabolismo , Venenos de Crotalídeos/química , Inflamação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/química , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Artéria Cerebral Média/patologia , Fosfoproteínas/metabolismo , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Venenos de Serpentes/química , Quinase Syk
5.
Arthritis Care Res (Hoboken) ; 66(5): 649-57, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24756998

RESUMO

OBJECTIVE: To create a pediatric rheumatology Top 5 list as part of the American Board of Internal Medicine Foundation's Choosing Wisely campaign. METHODS: Delphi surveys of a core group of representative pediatric rheumatology providers from across North America generated candidate Top 5 items. Items with high content agreement and perceived to be of prevalent use and of high impact were included in a survey of all American College of Rheumatology (ACR) members who identified themselves as providing care to pediatric patients. Items with the highest ratings were subjected to literature review and further evaluation. RESULTS: A total of 121 candidate items were proposed in the initial Delphi survey and were reduced to 28 items in subsequent surveys. These 28 items were sent to 1,198 rheumatology providers who care for pediatric patients, and 397 (33%) responded. Based upon survey data and literature review, the Top 5 items were identified. These items focused on testing for antinuclear antibodies, autoantibody panels, Lyme disease, methotrexate toxicity monitoring, and use of routine radiographs. CONCLUSION: The ACR pediatric rheumatology Top 5 is one of the first pediatric subspecialty-specific Choosing Wisely Top 5 lists and provides an opportunity for patients and providers to discuss appropriate use of health care in pediatric rheumatology.


Assuntos
Comportamento de Escolha , Pesquisas sobre Serviços de Saúde/métodos , Pediatria/normas , Doenças Reumáticas/diagnóstico , Doenças Reumáticas/terapia , Reumatologia/normas , Sociedades Médicas/normas , Antirreumáticos/uso terapêutico , Criança , Humanos , Pediatria/métodos , Reumatologia/métodos , Estados Unidos
6.
Clin Immunol ; 150(2): 201-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24440645

RESUMO

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP(-/-) mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP(-/-) 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection.


Assuntos
Anticorpos Antinucleares/biossíntese , DNA/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Animais , Anticorpos Antinucleares/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Feminino , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias kappa de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/metabolismo , Cadeias lambda de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Receptores de Antígenos de Linfócitos B/metabolismo
8.
Am J Clin Exp Immunol ; 3(3): 107-23, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25628960

RESUMO

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

9.
PLoS One ; 8(10): e75200, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24124476

RESUMO

While neddylation is known to activate cullin (CUL)-RING ubiquitin ligases (CRLs), its role in regulating T cell signaling is poorly understood. Using the investigational NEDD8 activating enzyme (NAE) inhibitor, MLN4924, we found that neddylation negatively regulates T cell receptor (TCR) signaling, as its inhibition increases IL-2 production, T cell proliferation and Treg development in vitro. We also discovered that loss of CUL neddylation occurs upon TCR signaling, and CRLs negatively regulate IL-2 production. Additionally, we found that tyrosine kinase signaling leads to CUL deneddylation in multiple cell types. These studies indicate that CUL neddylation is a global regulatory mechanism for tyrosine kinase signaling.


Assuntos
Proteínas Culina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Morte Celular/fisiologia , Células Cultivadas , Proteínas Culina/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Immunoblotting , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Linfócitos T Reguladores/metabolismo
10.
Immunol Res ; 55(1-3): 116-24, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22956467

RESUMO

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vß5 mice, expressing only the ß-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vß5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vß5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vß5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vß5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antígenos/imunologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas pp60(c-src)/genética
11.
Am J Clin Exp Immunol ; 1(1): 12-19, 2012 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-23097751

RESUMO

OBJECTIVES: Pregnancy leads to rheumatoid arthritis remission in humans. The objective of this study was to determine if the SKG mouse could serve as a model for pregnancy-associated inflammatory arthritis amelioration. In addition, the maternal peripheral blood mononuclear cell (PBMC) transcriptome was assessed to define a biomarker associated with remission. METHODS: Cohorts of zymosan-treated pregnant SKG mice and controls were monitored for arthritis progression. Microarray analysis evaluated alterations in gene expression in maternal PBMCs at embryonic day 14.5 (E14.5) between arthritic and pregnancy-remitted mice. A selected target, serum amyloid A3 (SAA3), was further investigated using quantitative reverse transcriptase PCR (qRT-PCR) and an enzyme-linked immunosorbent assay (ELISA). RESULTS: Pregnancy resulted in complete or partial remission in the majority of the zymosan-treated SKG mice. Twenty-seven transcripts were differentially expressed in the PBMCs between arthritic and pregnancy-remitted mice. Expression and plasma SAA3 levels decreased with pregnancy-induced arthritis amelioration and plasma SAA3 levels correlated with arthritis severity. CONCLUSIONS: These results establish the SKG mouse as a model system to study pregnancy-induced amelioration of arthritis. These studies also establish SAA3 as a biomarker of arthritis amelioration in SKG mice. This model can be used to elucidate the molecular and cellular mechanisms underlying the impact of pregnancy on the maternal immune system that results in arthritis amelioration.

13.
J Immunol ; 186(4): 1923-33, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21220694

RESUMO

GM-CSF is an important cytokine involved in myeloid differentiation and inflammatory processes. Signaling through the GM-CSFR also plays a critical role in the generation of monocyte-derived dendritic cells (DC). In this article, we report that the Src-like adaptor protein (SLAP) functions as a negative regulator of the GM-CSFR. In bone marrow-derived DC (BM-DC) lacking SLAP and the closely related SLAP2, downregulation of GM-CSFRß is impaired, leading to enhanced phosphorylation of Jak2 and prolonged activation of Akt and Erk1/2 in response to GM-CSF stimulation. Compared with wild-type bone marrow, SLAP/SLAP2(-/-) bone marrow gave rise to similar numbers of CD11c(+) and CD11b(+) DC, but SLAP/SLAP2(-/-) BM-DC failed to acquire high levels of MHC class II, CD80, and CD86, indicating an impairment in maturation. Furthermore, MHC class II expression in SLAP/SLAP2(-/-) BM-DC was rescued by decreasing GM-CSF concentration, suggesting that enhanced GM-CSF signaling mediates the block in maturation. In addition, SLAP/SLAP2(-/-) BM-DC produced less IL-12 and TNF-α in response to LPS compared with controls and failed to stimulate T cells in an MLR. Ag-specific T cell activation assays showed that SLAP/SLAP2(-/-) BM-DC were less robust at inducing IFN-γ secretion by DO11.10 T cells. These results indicated that SLAP-mediated GM-CSFR regulation is important for the generation of functionally mature monocytic DC.


Assuntos
Diferenciação Celular/imunologia , Subunidade beta Comum dos Receptores de Citocinas/fisiologia , Células Dendríticas/imunologia , Inibidores do Crescimento/fisiologia , Monócitos/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Diferenciação Celular/genética , Subunidade beta Comum dos Receptores de Citocinas/antagonistas & inibidores , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Monócitos/metabolismo , Monócitos/patologia , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Transdução de Sinais/genética
14.
J Immunol ; 186(4): 2273-81, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21248251

RESUMO

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease.


Assuntos
Artrite Experimental/prevenção & controle , Doenças Autoimunes/prevenção & controle , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doença Crônica , Modelos Animais de Doenças , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteína-Tirosina Quinase ZAP-70/genética
15.
EMBO J ; 29(7): 1285-98, 2010 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-20150895

RESUMO

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.


Assuntos
Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Animais , Dinaminas/metabolismo , Regulação da Expressão Gênica , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Lisina/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Linfócitos T/citologia
16.
Pediatr Rheumatol Online J ; 7: 21, 2009 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-20028520

RESUMO

PURPOSE: We investigated the etiology of acute hepatitis in three children with systemic Juvenile Idiopathic Arthritis (sJIA) taking Interleukin-1 receptor antagonist (IL1RA). METHODS: Laboratory and clinical data for three children with sJIA diagnosed at ages 13 months to 8 years who developed acute hepatitis during treatment with IL1RA were reviewed for evidence of sJIA flare, infection, macrophage activation syndrome (MAS), malignancy, and drug reaction. RESULTS: In all patients, hepatitis persisted despite cessation of known hepatotoxic drugs and in absence of known infectious triggers, until discontinuation of IL1RA. Liver biopsies had mixed inflammatory infiltrates with associated hepatocellular injury suggestive of an exogenous trigger. At the time of hepatitis, laboratory data and liver biopsies were not characteristic of MAS. In two patients, transaminitis resolved within one week of discontinuing IL1RA, the third improved dramatically in one month. CONCLUSIONS: Although sJIA symptoms improved significantly on IL1RA, it appeared that IL1RA contributed to the development of acute hepatitis. Hepatitis possibly occurred as a result of an altered immune response to a typical childhood infection while on IL1RA. Alternatively, hepatitis could have represented an atypical presentation of MAS in patients with sJIA taking IL1RA. Further investigation is warranted to determine how anti-IL1 therapies alter immune responsiveness to exogenous triggers in patients with immune dysfunction such as sJIA. Our patients suggest that close monitoring for hepatic and other toxicities is indicated when treating with IL1RA.

17.
Immunol Rev ; 232(1): 218-28, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19909366

RESUMO

Src-like adapter proteins (SLAP and SLAP-2) constitute a family of proteins that are expressed in a variety of cell types but are studied most extensively in lymphocytes. They have been shown to associate with proximal components of the T-cell receptor (TCR) and B-cell receptor (BCR) signaling complexes. An interaction of SLAP with c-Cbl leads to the ubiquitination and degradation of phosphorylated components of the TCR- and BCR-signaling complexes. The absence of this process in immature SLAP-deficient T and B cells leads to increased immunoreceptor levels due to decreased intracellular retention and degradation. We propose a model in which SLAP-dependent regulation of immunoreceptor levels allows for finer control of immunoreceptor signaling. Thus, SLAP functions to dampen immunoreceptor signaling, thereby influencing lymphocyte development and repertoire selection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Antígenos/metabolismo , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Humanos , Proteínas Proto-Oncogênicas c-cbl/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/imunologia , Receptores de Antígenos/imunologia , Transdução de Sinais/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Ubiquitinação/imunologia
18.
J Immunol Methods ; 349(1-2): 1-8, 2009 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-19720064

RESUMO

Antinuclear antibodies (ANA) have been detected in patients with systemic rheumatic diseases and are used in the screening and/or diagnosis of autoimmunity in patients as well as mouse models of systemic autoimmunity. Indirect immunofluorescence (IIF) on HEp-2 cells is the gold standard for ANA screening. However, its usefulness is limited in diagnosis, prognosis and monitoring of disease activity due to the lack of standardization in performing the technique, subjectivity in interpreting the results and the fact that it is only semi-quantitative. Various immunological techniques have been developed in an attempt to improve upon the method to quantify ANA, including enzyme-linked immunosorbent assays (ELISAs), line immunoassays (LIAs), multiplexed bead immunoassays and IIF on substrates other than HEp-2 cells. Yet IIF on HEp-2 cells remains the most common screening method for ANA. In this study, we describe a simple quantitative method to detect ANA which combines IIF on HEp-2 coated slides with analysis using a near-infrared imaging (NII) system. Using NII to determine ANA titer, 86.5% (32 of 37) of the titers for human patient samples were within 2 dilutions of those determined by IIF, which is the acceptable range for proficiency testing. Combining an initial screening for nuclear staining using microscopy with titration by NII resulted in 97.3% (36 of 37) of the titers detected to be within two dilutions of those determined by IIF. The NII method for quantitative ANA measurements using serum from both patients and mice with autoimmunity provides a fast, relatively simple, objective, sensitive and reproducible assay, which could easily be standardized for comparison between laboratories.


Assuntos
Anticorpos Antinucleares/sangue , Doenças Autoimunes/sangue , Técnica Indireta de Fluorescência para Anticorpo/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
Proc Natl Acad Sci U S A ; 103(48): 18202-7, 2006 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17110436

RESUMO

Src-like adaptor protein (SLAP) and c-Cbl recently have been shown to cooperate in regulating T cell receptor (TCR) levels in developing T cells. SLAP also is expressed in developing B cells, and its deficiency leads to alterations in B cell receptor (BCR) levels and B cell development. Hence, we hypothesized that SLAP and c-Cbl may cooperate during B cell development to regulate BCR levels. In mice deficient in both SLAP and c-Cbl, we found that B cell development is altered, suggesting that they function through intersecting pathways. To study the mechanism by which SLAP and c-Cbl alter BCR levels, we coexpressed them in a mature mouse B cell line (Bal-17). First we determined that SLAP associates with proximal components of the BCR complex after stimulation and internalization. Coexpression of SLAP and c-Cbl in Bal-17 led to decreased surface and total BCR levels. This decrease in BCR levels depended on intact Src homology 2 (SH2) and C-terminal domains of SLAP. In addition, a mutation in the SH2 domain of SLAP blocked its colocalization with c-Cbl and the BCR complex, whereas deletion of the C terminus did not affect its localization. Last, coexpression of SLAP and c-Cbl altered BCR complex recycling. This alteration in BCR complex recycling depended on enzymatically active c-Cbl and Src family kinases, as well as the intact SH2 and C-terminal domains of SLAP. These data suggest that SLAP has a conserved function in B and T cells by adapting c-Cbl to the antigen-receptor complex and targeting it for degradation.


Assuntos
Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl/deficiência , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Receptores de Antígenos de Linfócitos B/imunologia
20.
Nat Immunol ; 7(1): 57-66, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16327786

RESUMO

The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.


Assuntos
Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Animais , Complexo CD3/imunologia , Complexo CD3/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Células Jurkat , Camundongos , Camundongos Mutantes , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transfecção
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