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1.
Nat Commun ; 11(1): 887, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060305

RESUMO

The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell-cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.


Assuntos
Antígenos CD2/metabolismo , Antígeno CD56/metabolismo , Membrana Celular/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Imagem Individual de Molécula/métodos , Antígenos CD2/química , Antígeno CD56/química , Linhagem Celular , Membrana Celular/química , Humanos , Imageamento Tridimensional/métodos , Antígenos Comuns de Leucócito/química
2.
Scand J Immunol ; 91(1): e12839, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31630416

RESUMO

The humanized IgG1κ monoclonal antibody siplizumab and its rat parent monoclonal IgG2b antibody BTI-322 are directed against the CD2 antigen. Siplizumab is species-specific, reacting with human and chimpanzee cells but not with cells from any other species, including other non-human primates. Because siplizumab treatment has recently shown great potential in clinical transplantation, we now present the results of our previous pharmacokinetic, pharmacodynamic and safety studies of both antibodies. Fourteen chimpanzees received 1-3 doses of 0.143 to 5.0 mg/kg iv The effects were followed with flow cytometry on peripheral lymphocytes and staining of lymph nodes. Side effects were recorded. Serum antibody concentrations were followed. Across the doses, a rapid, transient depletion of CD2, CD3, CD4 and CD8 lymphocytes and NK cells was observed for both antibodies. Immune reconstitution was more rapid for BTI-322 compared to siplizumab. Paracortical lymph node T cell depletion was moderate, estimated at 45% with doses of >0.6 mg/kg. Restoration of lymph node architecture was seen after two weeks to two months for all animals. All four subjects receiving BTI-322 experienced AEs on the first dosing day, while the eight subjects dosed with siplizumab experienced few mild, transient AEs. Infusion with siplizumab and BTI-322 resulted in rapid depletion of CD2+ cells in circulation and tissue. Siplizumab had a longer t1/2 and fewer AEs compared to BTI-322.


Assuntos
Anticorpos Monoclonais/farmacocinética , Antígenos CD2/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores , Biópsia , Citocinas/sangue , Feminino , Imunoglobulina G/administração & dosagem , Imunoglobulina G/farmacologia , Imunofenotipagem , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Depleção Linfocítica , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Pan troglodytes , Ratos
3.
Acta Haematol ; 143(1): 33-39, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31216534

RESUMO

Immune-mediated processes are considered important in the pathogenesis of bone marrow failure syndromes (BFS). We previously reported that natural killer group 2D (NKG2D) ligands were expressed on pathological blood cells of patients with BFS and that NKG2D immunity may be involved in bone marrow failure. In addition to membranous NKG2D ligands on the cell surface, soluble NKG2D ligands can exist in plasma. We therefore examined the relationship between soluble NKG2D ligands and blood cell counts in 86 patients with BFS, including aplastic anemia, myelodysplastic syndrome with single lineage dysplasia, and paroxysmal nocturnal hemoglobinuria. Approximately half of the BFS patients were positive for soluble NKG2D ligands in the plasma by enzyme-linked immunosorbent assay, and soluble NKG2D ligand-positive BFS patients exhibited severe cytopenia regardless of membranous NKG2D ligand expression. In vitroanalyses demonstrated that soluble ULBP1, an NKG2D ligand, down-regulated NKG2D receptors on CD2-positive cells in peripheral blood. Moreover, soluble ULBP1 attenuated the cytotoxic effects of peripheral blood mononuclear cells on K562, which express membranous ULBP1. Our results suggest that soluble NKG2D ligands can be easy-to-measure biomarkers for the prediction of activity of immune-meditated bone marrow injury in BFS and that soluble NKG2D ligands suppress redundant immune-mediated bone marrow injury.


Assuntos
Biomarcadores/sangue , Transtornos da Insuficiência da Medula Óssea/diagnóstico , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Aplástica/diagnóstico , Contagem de Células Sanguíneas , Transtornos da Insuficiência da Medula Óssea/complicações , Antígenos CD2/metabolismo , Regulação para Baixo , Proteínas Ligadas por GPI/sangue , Doenças Hematológicas/complicações , Doenças Hematológicas/diagnóstico , Hemoglobinúria Paroxística/diagnóstico , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Adulto Jovem
4.
Thromb Res ; 183: 63-68, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31669825

RESUMO

INTRODUCTION: Immune thrombocytopenia (ITP) is known as an immune-mediated disease and often evolves to chronic type in adult. Corticosteroids only work in around 60% of patients. This study evaluated the roles of subgroup lymphocytes from peripheral blood in ITP adults with different treatment response. METHODS: Between October 2009 and March 2017, 37 adults were newly diagnosed as ITP requiring treatment. The patients were separated into two groups: 23 patients with platelet count <50,000/µL with corticosteroid dependence or second-line treatment (Poor-responder Group), and 14 patients with platelet counts <50,000/µL with standard steroid treatment, which stopped within three months (Good-responder Group). Subgroup lymphocyte percentages of peripheral blood were determined through flow cytometry before treatment. Data analysis with Mann-Whitney test and receiver operating characteristic curves were performed using GraphPad Prism (Version 7). A p-value of <0.05 was considered significant. RESULTS: Lymphocyte percentage was significantly lower in Poor-responder Group than in Good-responder Group (p = 0.008). In subgroup lymphocytes, higher percentages of CD19+ B lymphocytes were found in Good-responder Group (p = 0.03). In Poor-responder Group, a higher CD2+ and CD56+ lymphocytes were observed (p = 0.02 and 0.03). By the cut-off value of percentage of CD56+ lymphocytes with 24.5% or CD2+ lymphocytes with 85.7%, the specificity showed 92.86%. CONCLUSIONS: This study found that newly diagnosed ITP patients with increased percentages of CD56+ or CD2+ lymphocytes in peripheral blood associated with a poorer response to steroid treatment.


Assuntos
Antígenos CD2/metabolismo , Antígeno CD56/metabolismo , Linfócitos/metabolismo , Esteroides/uso terapêutico , Trombocitopenia/tratamento farmacológico , Trombocitopenia/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Esteroides/farmacologia
5.
Pediatr Dermatol ; 36(4): 477-481, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31120154

RESUMO

BACKGROUND: Pediatric mastocytosis differs from adult mastocytosis in its presentation and clinical course. However, the data regarding the immunophenotypic characterization of mast cells in children are limited. Our objective was to evaluate the immunophenotype of mast cells in pediatric mastocytosis and correlate it with the clinical course. METHODS: Biopsy specimens of children with cutaneous mastocytosis were retrieved from the institutions of pathology and were stained for CD25, CD2, and CD30. The percentage of mast cells and the staining intensity were correlated with the clinical data. RESULTS: Twenty-five biopsy specimens were included in the study. Patients' average age was 15.4 at presentation and 37.5 months at biopsy performance. Clinical presentations included maculopapular cutaneous mastocytosis in 79% and mastocytoma in 21% of cases. CD25, CD2, and CD30 were positive in 60%, 44%, and 84% of the biopsy specimens, respectively. The staining score was significantly higher for CD30 as compared to those for CD25 and CD2 (P = 0.02). No correlation was found between the immunophenotype and the clinical form or course of disease. CONCLUSIONS: Our results confirm that CD30 is a sensitive marker for pediatric-onset mastocytosis. Nevertheless, its expression does not correlate with clinical subtype or clinical course. The sensitivity of CD25 is higher than that of CD2 in skin lesions.


Assuntos
Imunofenotipagem/métodos , Antígeno Ki-1/imunologia , Mastócitos/imunologia , Mastocitose Cutânea/patologia , Mastocitose Cutânea/fisiopatologia , Neoplasias de Tecido Conjuntivo/patologia , Adolescente , Fatores Etários , Biomarcadores/análise , Biópsia por Agulha , Antígenos CD2/imunologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica , Lactente , Subunidade alfa de Receptor de Interleucina-2/imunologia , Israel , Masculino , Mastócitos/patologia , Mastocitoma/imunologia , Mastocitoma/patologia , Mastocitose Cutânea/imunologia , Neoplasias de Tecido Conjuntivo/imunologia , Neoplasias de Tecido Conjuntivo/fisiopatologia , Prognóstico , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Estatísticas não Paramétricas
6.
Inflammation ; 42(3): 1071-1081, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30715690

RESUMO

The exact etiology and pathogenesis of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) are still unknown, as a result, available therapeutic options for patients are far from satisfactory. Therefore, there is a need to develop a valid therapeutic approach that can ameliorate the manifestations of CP/CPPS. Fifty male C57BL/6 mice were randomly divided into five groups of ten mice each. All groups except naïve were subcutaneously injected with 0.2 ml of T2 plus complete Freund adjuvant (CFA) on day 0 and 14 to generate valid CP/CPPS model. After successful CP/CPPS induction, model group was injected with 0.2 ml of normal saline while PLGA, PLGA-OVA, and PLGA-T2 groups were administered intravenously with 0.2 ml mixture of PLGA, PLGA-OVA, and PLGA-T2, respectively. Voiding behavior, pain threshold, and hematoxylin and eosin staining were used to assess micturition habits, pain intensity as well as prostate inflammation. Additionally, TNF-α, CRP, and IL-10 levels in plasma were measured by using ELISA kits. Mice administered with PLGA-T2 showed higher pain threshold, lower urine frequencies, mild edema, and inflammation in prostate tissue in comparison to other groups. Moreover, the expression of TNF-α and CRP levels was markedly decreased while IL-10 expression was increased in the PLGA-T2 treatment group as compared to the other groups. Our results showed that nanoparticles conjugated with autoantigen novel peptide T2 could successfully alleviate or even heal CP/CPPS to some extent in mice. This study provides an easy, useful, and economic tool for ameliorating the manifestations of CP/CPPS that will improve the therapeutic approaches.


Assuntos
Antígenos CD2/uso terapêutico , Nanopartículas/uso terapêutico , Prostatite/tratamento farmacológico , Animais , Autoantígenos/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Proteína C-Reativa/efeitos dos fármacos , Proteína C-Reativa/metabolismo , Modelos Animais de Doenças , Interleucina-10/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
7.
Front Immunol ; 9: 2552, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30455699

RESUMO

Elimination of the latent HIV reservoir remains the biggest hurdle to achieve HIV cure. In order to specifically eliminate HIV infected cells they must be distinguishable from uninfected cells. CD2 was recently identified as a potential marker enriched in the HIV-1 reservoir on CD4+ T cells, the largest, longest-lived and best-characterized constituent of the HIV reservoir. We previously proposed to repurpose FDA-approved alefacept, a humanized α-CD2 fusion protein, to reduce the HIV reservoir in CD2hi CD4+ memory T cells. Here, we show the first evidence that alefacept can specifically target and reduce CD2hi HIV infected cells in vitro. We explore a variety of natural killer (NK) cells as mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) including primary NK cells, expanded NK cells as well as the CD16 transduced NK-92 cell line which is currently under study in clinical trials as a treatment for cancer. We demonstrate that CD16.NK-92 has a natural preference to kill CD2hi CD45RA- memory T cells, specifically CD45RA- CD27+ central memory/transitional memory (TCM/TM) subset in both healthy and HIV+ patient samples as well as to reduce HIV DNA from HIV+ samples from donors well controlled on antiretroviral therapy. Lastly, alefacept can combine with CD16.NK-92 to decrease HIV DNA in some patient samples and thus may yield value as part of a strategy toward sustained HIV remission.


Assuntos
Alefacept/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Latência Viral/efeitos dos fármacos , Transferência Adotiva , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Biomarcadores/metabolismo , Antígenos CD2/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , DNA Viral/genética , Quimioterapia Combinada , Proteínas Ligadas por GPI/metabolismo , HIV-1/imunologia , Humanos , Memória Imunológica/imunologia , Células Jurkat , Células Matadoras Naturais/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Receptores de IgG/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
8.
J Comput Aided Mol Des ; 32(11): 1295-1313, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30368623

RESUMO

The CD2-CD58 protein-protein interaction is known to favor the recognition of antigen presenting cells by T cells. The structural, energetics, and dynamical properties of three known cyclic CD58 ligands, named P6, P7, and RTD-c, are studied through molecular dynamics (MD) simulations and molecular docking calculations. The ligands are built so as to mimic the C and F ß-strands of protein CD2, connected via turn inducers. The MD analyses focus on the location of the ligands with respect to the experimental binding site and on the direct and water-mediated hydrogen bonds (H bonds) they form with CD58. Ligand P6, with a sequence close to the experimental ß-strands of CD2, presents characteristics that explain its higher experimental affinity, e.g., the lower mobility and flexibility at the CD58 surface, and the larger number and occurrence frequency of ligand-CD58 H bonds. For the two other ligands, the structural modifications lead to changes in the binding pattern with CD58 and its dynamics. In parallel, a large set of molecular docking calculations, carried out with various search spaces and docking algorithms, are compared to provide a consensus view of the preferred ligand binding modes. The analysis of the ligand side chain locations yields results that are consistent with the CD2-CD58 crystal structure and suggests various binding modes of the experimentally identified hot spot of the ligands, i.e., Tyr86. P6 is shown to form a number of contacts that are also present in the experimental CD2-CD58 structure.


Assuntos
Antígenos CD2/química , Antígenos CD58/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação de Hidrogênio , Ligantes , Ligação Proteica , Conformação Proteica , Termodinâmica
9.
Biotechniques ; 65(3): 149-157, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30227746

RESUMO

Protein-protein interactions (PPI) by homo-, hetero- or oligo-merization in the cellular environment regulate cellular processes. PPI can be inhibited by antibodies, small molecules or peptides, and this inhibition has therapeutic value. A recently developed method, the proximity ligation assay (PLA), provides detection of PPI in the cellular environment. However, most applications using this assay are for proteins expressed in the same cell. We employ PLA for the first time to study PPI of cell surface proteins on two different cells. Inhibition of PPI using a peptide inhibitor is also quantified using this assay; PLA is used to detect PPI of CD2 and CD58 between Jurkat cells (T cells) and human fibroblast-like synoviocyte-rheumatoid arthritis cells that are important in the immune response in the autoimmune disease rheumatoid arthritis. This assay provides direct evidence of inhibition of PPI of two proteins on different cell surfaces.


Assuntos
Biotecnologia/métodos , Antígenos de Histocompatibilidade Classe II/análise , Proteínas de Membrana/química , Proteínas/química , Antígenos CD2/análise , Antígenos CD2/metabolismo , Antígenos CD58/análise , Antígenos CD58/metabolismo , Células Cultivadas , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Células Jurkat , Proteínas de Membrana/análise , Modelos Moleculares , Ligação Proteica , Sinoviócitos
10.
Sci Rep ; 8(1): 12479, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30127338

RESUMO

How membrane proteins distribute and behave on the surface of cells depends on the molecules' chemical potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. Here, we present a method we call hydrodynamic trapping that can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding above a lipid membrane. The chemical potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this method to study four representative proteins, with different height-to-width ratios and molecular properties; from globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The data we obtain illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins, as well as underlining the general applicability of the method.


Assuntos
Proteínas de Membrana/metabolismo , Membranas/metabolismo , Antígenos CD2/metabolismo , Antígenos CD4/metabolismo , Humanos , Hidrodinâmica , Antígenos Comuns de Leucócito/metabolismo , Bicamadas Lipídicas/metabolismo , Estreptavidina/metabolismo
11.
Front Immunol ; 9: 1204, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29904386

RESUMO

CD58 and CD2 have long been known as a pair of reciprocal adhesion molecules involved in the immune modulations of CD8+ T and NK-mediated cellular immunity in humans and several other mammals. However, the functional roles of CD58 and CD2 in CD4+ T-mediated adaptive humoral immunity remain poorly defined. Moreover, the current functional observations of CD58 and CD2 were mainly acquired from in vitro assays, and in vivo investigation is greatly limited due to the absence of a Cd58 homology in murine models. In this study, we identified cd58 and cd2 homologs from the model species zebrafish (Danio rerio). These two molecules share conserved structural features to their mammalian counterparts. Functionally, cd58 and cd2 were significantly upregulated on antigen-presenting cells and Cd4+ T cells upon antigen stimulation. Blockade or knockdown of Cd58 and Cd2 dramatically impaired the activation of antigen-specific Cd4+ T and mIgM+ B cells, followed by the inhibition of antibody production and host defense against bacterial infections. These results indicate that CD58/CD2 interaction was required for the full activation of CD4+ T-mediated adaptive humoral immunity. The interaction of Cd58 with Cd2 was confirmed by co-immunoprecipitation and functional competitive assays by introducing a soluble Cd2 protein. This study highlights a new costimulatory mechanism underlying the regulatory network of adaptive immunity and makes zebrafish an attractive model organism for the investigation of CD58/CD2-mediated immunology and disorders. It also provides a cross-species understanding of the evolutionary history of costimulatory signals from fish to mammals as a whole.


Assuntos
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Imunidade Humoral , Peixe-Zebra/imunologia , Peixe-Zebra/metabolismo , Imunidade Adaptativa , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Antígenos CD2/química , Antígenos CD2/genética , Antígenos CD58/química , Antígenos CD58/genética , Clonagem Molecular , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Modelos Moleculares , Ligação Proteica , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Análise de Sequência de DNA , Relação Estrutura-Atividade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Peixe-Zebra/genética
12.
Immun Inflamm Dis ; 6(2): 221-233, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29314730

RESUMO

INTRODUCTION: Impaired proliferation and production of IL2 are the hallmarks of experimental T cell tolerance. However, in most autoimmune diseases, auto-reactive T cells do not display hyper proliferation, but inflammatory phenotypes. METHODS: We have now demonstrated that the transcription factors Egr2 and 3 are important for the control of inflammatory cytokine production by tolerant T cells, but not for tolerance induction. RESULTS: In the absence of Egr2 and 3, T cell tolerance, as measured by impaired proliferation and production of IL2, can still be induced, but tolerant T cells produced high levels of inflammatory cytokines. Egr2 and 3 regulate expression of differentiation repressors and directly inhibit T-bet function in T cells. Indeed, decreased expression of differentiation repressors, such as Id3 and Tcf1, and increased expression of inflammatory transcription factors, such as RORγt and Bhlhe40 were found in Egr2/3 deficient T cells under tolerogenic conditions. In addition, T-bet was co-expressed with Egr2 in tolerant T cells and Egr2/3 defects leads to production of high levels of IFNγ in tolerant T cells. CONCLUSIONS: Our findings demonstrated that despite impaired proliferation and IL2 production, tolerant T cells can display inflammatory responses in response to antigen stimulation and this is controlled at least partly by Egr2 and 3.


Assuntos
Proteína 2 de Resposta de Crescimento Precoce/imunologia , Proteína 3 de Resposta de Crescimento Precoce/imunologia , Tolerância Imunológica/genética , Inflamação/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Autoantígenos/imunologia , Transplante de Medula Óssea , Antígenos CD2/imunologia , Antígenos CD2/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteína 3 de Resposta de Crescimento Precoce/genética , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Enterotoxinas/administração & dosagem , Enterotoxinas/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Humanos , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/metabolismo , Quimeras de Transplante/imunologia
13.
Theranostics ; 8(21): 6070-6087, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30613283

RESUMO

Cancer immunotherapy has proven high efficacy in treating diverse cancer entities by immune checkpoint modulation and adoptive T-cell transfer. However, patterns of treatment response differ substantially from conventional therapies, and reliable surrogate markers are missing for early detection of responders versus non-responders. Current imaging techniques using 18F-fluorodeoxyglucose-positron-emmission-tomograpy (18F-FDG-PET) cannot discriminate, at early treatment times, between tumor progression and inflammation. Therefore, direct imaging of T cells at the tumor site represents a highly attractive tool to evaluate effective tumor rejection or evasion. Moreover, such markers may be suitable for theranostic imaging. Methods: We mainly investigated the potential of two novel pan T-cell markers, CD2 and CD7, for T-cell tracking by immuno-PET imaging. Respective antibody- and F(ab´)2 fragment-based tracers were produced and characterized, focusing on functional in vitro and in vivo T-cell analyses to exclude any impact of T-cell targeting on cell survival and antitumor efficacy. Results: T cells incubated with anti-CD2 and anti-CD7 F(ab´)2 showed no major modulation of functionality in vitro, and PET imaging provided a distinct and strong signal at the tumor site using the respective zirconium-89-labeled radiotracers. However, while T-cell tracking by anti-CD7 F(ab´)2 had no long-term impact on T-cell functionality in vivo, anti-CD2 F(ab´)2 caused severe T-cell depletion and failure of tumor rejection. Conclusion: This study stresses the importance of extended functional T-cell assays for T-cell tracer development in cancer immunotherapy imaging and proposes CD7 as a highly suitable target for T-cell immuno-PET imaging.


Assuntos
Transferência Adotiva/métodos , Antígenos CD7/análise , Imunoterapia/métodos , Imagem Molecular/métodos , Neoplasias/terapia , Linfócitos T/química , Linfócitos T/imunologia , Animais , Antígenos CD2/análise , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Camundongos , Transplante de Neoplasias , Tomografia por Emissão de Pósitrons/métodos , Traçadores Radioativos , Compostos Radiofarmacêuticos/administração & dosagem
14.
Int Immunol ; 29(10): 479-485, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29106539

RESUMO

Lineage-specific Cre Tg mice are widely used to delineate the functions of genes in a tissue-specific manner. Several T-cell-specific promoter cassettes have been developed; however, the activities of those promoters in non-T cells have not been investigated extensively. Here, we report that CD2-Cre-mediated deletion of Erk proteins by generating CD2-Cre × Erk1-/-Erk2flox/flox (Erk∆CD2-Cre) mice results in abnormal cartilage hyperplasia. Histological analysis revealed that this abnormality is caused by aberrant hyperplasia of chondrocytes. The presence of Erk-deficient T cells is not required for this chondroma formation, as it was similarly observed in the absence of T cells in a CD3ε-deficient background. In addition, adoptive transfer of bone marrow cells from Erk∆CD2-Cre mice to wild-type recipients did not cause chondroma formation, suggesting that Erk-deficient non-immune cells are responsible for this abnormality. By tracing Cre-expressed tissues using a ROSA26-STOP-RFP allele, we found that the chondroma emitted RFP fluorescence, indicating that functional Cre is expressed in hyperplastic chondrocytes in Erk∆CD2-Cre mice. Furthermore, RFP+ chondrocytes were also found in an Erk-sufficient background, albeit without aberrant growth. These results suggest that unexpected expression of CD2-driven Cre in chondrocytes generates Erk-deficient chondrocytes, resulting in hyperplastic cartilage formation. Recently, two independent reports showed that CD4-Cre-mediated Ras-Erk signaling ablation led to similar abnormal cartilage formation (Guittard, G., Gallardo, D. L., Li, W. et al. 2017. Unexpected cartilage phenotype in CD4-Cre-conditional SOS-deficient mice. Front. Immunol. 8:343; Wehenkel, M., Corr, M., Guy, C. S. et al. 2017. Extracellular signal-regulated kinase signaling in CD4-expressing cells inhibits osteochondromas. Front. Immunol. 8:482). Together with these reports, our study suggests that an unexpected link exists between T-like cell and chondrocyte lineages during ontogeny.


Assuntos
Antígenos CD2/imunologia , Condroma/metabolismo , Integrases/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Cartilagem/imunologia , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/imunologia , Condrócitos/metabolismo , Condrócitos/patologia , Condroma/imunologia , Integrases/imunologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/deficiência , Proteína Quinase 3 Ativada por Mitógeno/imunologia
15.
Sci Rep ; 7(1): 8383, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28814758

RESUMO

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.


Assuntos
Anticorpos Monoclonais/genética , Antígenos CD2/imunologia , Proteína 9 Associada à CRISPR/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Galactosiltransferases/genética , Suínos/genética , Animais , Animais Geneticamente Modificados , Feminino , Fibroblastos , Técnicas de Introdução de Genes , Marcação de Genes , Humanos , Masculino , Técnicas de Transferência Nuclear , Gravidez , Reprodutibilidade dos Testes , Transgenes
16.
J Am Chem Soc ; 139(37): 12947-12955, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28820257

RESUMO

N-Glycosylation is an important co- and/or post-translational modification that occurs on the vast majority of the one-third of the mammalian proteome that traverses the cellular secretory pathway, regulating glycoprotein folding and functions. Previous studies on the sequence requirements for N-glycosylation have yielded the Asn-X-Ser/Thr (NXS/T) sequon and the enhanced aromatic sequons (Phe-X-Asn-X-Thr and Phe-X-X-Asn-X-Thr), which can be efficiently N-glycosylated. To further investigate the influence of sequence variation on N-glycosylation efficiency in the context of a five-residue enhanced aromatic sequon, we used the human CD2 adhesion domain (hCD2ad) to screen the i-2, i-1, i+1, and i+2 residues flanking Asn at the i position. We found that aromatic residues, especially Trp, and sulfur-containing residues at the i-2 position improved N-glycosylation efficiency, while positively charged residues such as Arg suppressed N-glycosylation. Thiol, hydroxyl, and aliphatic-based side chains at the i-1 position had higher N-glycosylation efficiency, and Cys, in particular, compensated for the negative effect of Arg at the i-2 position. Small residues and Ser at the i+1 position increased the likelihood of N-glycosylation, and Thr is better than Ser at the i+2 position. We devised an algorithm for prediction of N-glycosylation efficiency using the SAS software, employing the 120 sequences studied as a training set. We then introduced the optimized-enhanced aromatic sequons into other glycoproteins and observed an enhancement in N-glycan occupancy that was further supported by modeling the high-affinity interaction between the optimized sequence on hCD2ad and a human oligosaccharyltransferase (OST) subunit. The findings in this study provide useful information for enhancing or suppressing N-glycosylation at a site of interest and valuable data for a better understanding of OST-catalyzed N-glycosylation.


Assuntos
Antígenos CD2/metabolismo , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Antígenos CD2/química , Glicosilação , Hexosiltransferases/química , Humanos , Proteínas de Membrana/química , Modelos Moleculares
18.
Biomed Khim ; 63(3): 255-265, 2017 May.
Artigo em Russo | MEDLINE | ID: mdl-28781259

RESUMO

The effect of different concentrations of the glucocorticoid (GC) methylprednisolone (MP) on CD4+CD95+HLA-DR+ T-cells and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes in the in vitro system was investigated. T cells were obtained from healthy donors and patients with rheumatoid arthritis (RA).Under conditions of TCR-activation, MP increased the number of CD4+HLA-DR+CD95+ cells in CD3+CD45RO+ cultures obtained from RA patients and did not change their content in the control group. In general, MP decreased production of proinflammatory factors (IFN-, IL-2, IL-17, IL-21 and TNF-) by TCR-activated CD3+CD45RO+ cells from healthy donors and RA, consistent with the overall immunosuppressive mechanism of GC action. The correlation between CD4+CD45RO+HLA-DR+CD95+ T-cell contents and parameters reflecting production of proinflammatory mediators (IL-17, IL-21 and TNF-) in RA patients indicates maintenance of the pro-inflammatory potential of this T-cell population exposed to GC action. We suggest that relative resistance of CD4+CD45RO+CD95+HLA-DR+ T-cells of RA patients to the suppressor effect of GC leads to maintenance and even enhancement in the functional capacities of autoreactive cells in the pathogenesis of RA.


Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Metilprednisolona/farmacologia , Adulto , Anticorpos/farmacologia , Artrite Reumatoide/patologia , Antígenos CD2/genética , Antígenos CD2/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Antígenos CD4/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica/imunologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Interleucinas/biossíntese , Interleucinas/imunologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária , Masculino , Cultura Primária de Células , Transdução de Sinais , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Receptor fas/genética , Receptor fas/imunologia
19.
Pol J Vet Sci ; 20(1): 5-12, 2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-28525335

RESUMO

It is very difficult to cure pregnant females suffering from infections, because of the risk which might occur during treatment by several, even herbal, medications. Many of these substances, among them extracts from plants, have antimicrobial, anti-inflammatory and immunostimulatory properties owing to their polyphenols content, but also may reveal unwanted effects on the fetal development because of their anti-angiogenic properties. The aim of the present study was to elucidate whether daily feeding pregnant and nursing mice 0.2 mg/kg epigallocatechin (EGC), previously recognized as angiogenesis inhibitor, may lead to abnormalities in morphology of spleen and in some parameters of immune function of their adult, 6-week old progeny. Morphometry of EGC offspring spleens revealed lower number of lymphatic nodules and their larger diameter than those found in the control offspring. Cellularity of spleens was lower in EGC offspring than in the controls. Cytometric analysis showed that this decline concerns lymphocytes with CD335 (p<0.001), CD19 (p<0.01) and CD4 (p<0.05) markers. No differences were observed in the humoral response to the immunization with SRBC, and in the proliferative response of splenocytes to mitogens PHA, ConA and LPS.


Assuntos
Catequina/análogos & derivados , Baço/efeitos dos fármacos , Baço/patologia , Anormalidades Induzidas por Medicamentos , Animais , Animais Recém-Nascidos , Animais Lactentes , Antígenos CD2/imunologia , Catequina/toxicidade , Eritrócitos/metabolismo , Feminino , Lactação , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Complicações na Gravidez , Ovinos/sangue , Baço/citologia
20.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 25(2): 592-595, 2017 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-28446317

RESUMO

Lymphocyte function-associated antigen-3 (LFA-3/CD58) is a cell-surface glycoprotein, it can bind to CD2 and activate the costimulation pathways of T lymphocytes and natural killer (NK) cells, maximizing the cytolysis of target cells by cytotoxic T lymphocytes (CTL) and NK cells. Some studies have demonstrated that in acute lymphoblastic leukemia(ALL) and lymphomas, lack of CD58 on the tumor cells may fail to activate the T lymphocytes and NK cells, resulting in feeble cytotoxic effect and subsequently escape from immune surveillance, making the disease become more complicated and liable to relapse. Therefore, this article aims to review the structure, biological characteristics of CD58 on the tumor cells and its relationship with ALL and lymphomas.


Assuntos
Antígenos CD58/fisiologia , Linfoma/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Anticorpos Monoclonais , Antígenos CD , Antígenos CD2 , Humanos , Células Matadoras Naturais , Linfócitos T Citotóxicos
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