RESUMO
Measurements of skin surface biomarkers have enormous value for the detailed assessment of skin conditions, both for clinical application and in skin care. The main goals of the current study were to assess whether expression patterns of skin surface hBD-1, hBD-2, IL-1α, CXCL-1, and CXCL-8, examples of proteins known to be involved in psoriasis pathology, are associated with disease severity and whether expression patterns of these proteins on the skin surface can be used to measure pharmacodynamic effects of biological therapy. In this observational study using transdermal analysis patch (TAP), levels of skin surface IL-1α, hBD-1, hBD-2, CXCL-1/2, and CXCL-8 of psoriasis vulgaris (PV) patients over biological therapy were assessed. The Psoriasis Area Severity Index (PASI) and local score for erythema, induration, and desquamation were determined from the exact same skin area as FibroTx TAP measurements. Thirty-seven adult PV patients were included, of which twenty-three were subjected to anti-TNF-α, seven to anti-IL-17A, and seven to anti-IL12/IL-23 therapy. Significantly higher levels of hBD-1, hBD-2, CXCL-1/2, and CXCL-8 were detected on lesional skin compared to the non-lesional skin of the PV patients. In contrast, lower levels of IL-1α were found in lesional skin compared to non-lesional skin. In addition, we observed that the biomarker expression levels correlate with disease severity. Further, we confirmed that changes in the expression levels of skin surface biomarkers during biological therapy correlate with treatment response. Biomarker expression patterns in response to treatment differed somewhat between treatment subtypes. We observed that, in the case of anti-TNF-α therapy, an increase after a steady decrease in the expression levels of CXCL-1/2 and CXCL-8 occurred before the change in clinical scores. Moreover, response kinetics of skin surface proteins differs between the applied therapies-hBD2 expression responds quickly to anti-IL-17A therapy, CXCL-1/2 to anti-IL-12/23, and levels of CXCL-8 are rapidly down-regulated by IL-17A and IL-12/23 therapy. Our findings confirm that the skin surface hBD-2, IL-1α, CXCL-1/2, and CXCL-8 are markers for the psoriasis severity. Further, data obtained during this study give the basis for the conclusion that skin surface proteins CXCL-1/2 and CXCL-8 may have value as therapeutic biomarkers, thus confirming that measuring the 'molecular root' of inflammation appears to have value in scoring disease severity on its own.
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Proteínas de Membrana , Psoríase , Adulto , Humanos , Proteínas de Membrana/metabolismo , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Pele/metabolismo , Psoríase/tratamento farmacológico , Psoríase/metabolismo , Terapia Biológica , Interleucina-12/metabolismo , Biomarcadores/metabolismoRESUMO
BACKGROUND: Skin biomarkers are important tools for characterizing specific disease processes in atopic dermatitis (AD) patients and can be used for monitoring and potentially predicting treatment response. Recent developments of minimally invasive skin sampling methods have made sampling easier and less inconvenient for patients. The objective of this study was to evaluate the non-invasive patch technique developed by FibroTx for skin biomarker analysis. MATERIALS AND METHODS: Ten adult patients with AD were included in the study and treated with topical corticosteroid (diprosone 0.05%) for 2 weeks. Skin surface biomarkers were assessed in three lesional and non-lesional sites before and during treatment using the FibroTx Patch method. Skin tape strips were also collected from the subjects for comparison. RESULTS: The results showed expression of IL-1 cytokine family members, chemokines, and defensins on lesional and non-lesional skin. Several of these markers were strongly reduced by topical treatment. The biomarker expression in skin surface eluates correlated strongly with those seen in skin tape strips from the same subjects. CONCLUSION: These data further support the usefulness of non-invasive sampling methods for assessing inflammatory processes in AD skin and demonstrate that the patch sampling method is a good alternative to skin tape strips.
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Dermatite Atópica , Eczema , Adulto , Biomarcadores , Citocinas , Dermatite Atópica/tratamento farmacológico , Humanos , PeleRESUMO
It is well established that natural killer (NK) cells confer resistance to many viral diseases, but in only a few instances the molecular mechanisms whereby NK cells recognize virus-infected cells are known. Here we show that CD94, a molecule preferentially expressed by NK cells, is essential for the resistance of C57BL/6 mice to mousepox, a disease caused by the Orthopoxvirus ectromelia virus. Ectromelia virus-infected cells expressing the major histocompatibility complex (MHC) class Ib molecule Qa-1(b) are specifically recognized by the activating receptor formed by CD94 and NKG2E. Because CD94-NKG2 receptors and their ligands are highly conserved in rodents and humans, a similar mechanism may exist during human infections with the smallpox and monkeypox viruses, which are highly homologous to ectromelia virus.
Assuntos
Ectromelia Infecciosa/imunologia , Células Matadoras Naturais/imunologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Linhagem Celular , Movimento Celular , Humanos , Linfonodos/citologia , Linfonodos/imunologia , CamundongosRESUMO
BACKGROUND: The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by effector memory T cells (TEM) and plays an important role in their activation and proliferation. Mycosis fungoides (MF), the most common subtype of cutaneous T-cell lymphoma (CTCL), was recently proposed to be a malignancy of skin-resident TEM. However, the expression of Kv1.3 in CTCL has not been investigated. OBJECTIVES: This study aims to examine the expression of Kv1.3 in situ and in vitro in CTCL. METHODS: The expression of Kv1.3 was examined by immunohistochemistry in skin lesions from 38 patients with MF, 4 patients with Sézary syndrome (SS), and 27 patients with benign dermatosis. In 4 malignant T-cell lines of CTCL (Myla2059, PB2B, SeAx, and Mac2a) and a non-malignant T-cell line (MyLa1850), the expression of Kv1.3 was determined by flow cytometry. The proliferation of those cell lines treated with various concentrations of Kv1.3 inhibitor ShK was measured by 3H-thymdine incorporation. RESULTS: Half of the MF patients (19/38) displayed partial Kv1.3 expression including 1 patient with moderate Kv1.3 positivity, while the other half (19/38) exhibited Kv1.3 negativity. An almost identical distribution was observed in patients with benign conditions, that is, 44.4% (12/27) were partially positive for Kv1.3 including 1 patient with moderate Kv1.3 positivity, while 55.6% (15/27) were Kv1.3 negative. In contrast, 3 in 4 SS patients displayed partial Kv1.3 positivity including 2 patients with weak staining and 1 with moderate staining, while 1 in 4 SS patients was Kv1.3 negative. In addition, all malignant T-cell lines, and a non-malignant T-cell line, displayed low Kv1.3 surface expression with a similar pattern. Whereas 2 cell lines (PB2B and Mac2a) were sensitive to Kv1.3 blockade, the other 2 (Myla2059 and SeAx) were completely resistant. CONCLUSIONS: We provide the first evidence of a heterogeneous Kv1.3 expression in situ in CTCL lesions.
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Dermatite/metabolismo , Canal de Potássio Kv1.3/biossíntese , Linfoma Cutâneo de Células T/metabolismo , Neoplasias Cutâneas/metabolismo , Pele/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Linhagem Celular Tumoral , Criança , Dermatite/patologia , Feminino , Humanos , Imuno-Histoquímica , Canal de Potássio Kv1.3/antagonistas & inibidores , Linfoma Cutâneo de Células T/patologia , Masculino , Pessoa de Meia-Idade , Pele/patologia , Neoplasias Cutâneas/patologia , Adulto JovemRESUMO
Interleukin-1α (IL-1α) and its receptor antagonist IL-1RA play a pivotal role in skin homeostasis and disease. Although the use of biopsies to sample these cytokines from human skin is widely employed in dermatological practice, knowledge about less invasive, in vivo sampling methods is scarce. The aim of this study was to provide an overview of such methods by systematically reviewing studies in Medline, EMBASE, Web of Science and Cochrane Library using combinations of the terms "IL-1α", IL-1RA", "skin", "human", including all possible synonyms. Quality was assessed using the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) checklist. The search, performed on 14 October 2016, revealed 10 different sampling methods, with varying degrees of invasiveness and wide application spectrum, including assessment of both normal and diseased skin, from several body sites. The possibility to sample quantifiable amounts of cytokines from human skin with no or minimal discomfort holds promise for linking clinical outcomes to molecular profiles of skin inflammation.
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Proteína Antagonista do Receptor de Interleucina 1/isolamento & purificação , Interleucina-1alfa/isolamento & purificação , Pele/metabolismo , Manejo de Espécimes/métodos , Humanos , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1alfa/metabolismoRESUMO
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast-like synoviocytes (FLS) are central components of the aggressive, tumour-like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA-FLS and NK cells. We used cultured RA-FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA-FLS/NK cell cross-talk. We show that RA-FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM-1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA-FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA-E expressed on RA-FLS further enhanced Nishi cell degranulation in co-culture with RA-FLS. Using cultured RA-FLS and the human NK cell line Nishi as an in vitro model system of RA-FLS/NK cell cross-talk, our results suggest that cell-mediated cytotoxicity of RA-FLS may be one mechanism by which NK cells influence local joint inflammation in RA.
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Antígenos de Diferenciação de Linfócitos T/imunologia , Artrite Reumatoide/imunologia , Degranulação Celular/imunologia , Fibroblastos/imunologia , Células Matadoras Naturais/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Membrana Sinovial/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Linhagem Celular , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Receptor 2 Desencadeador da Citotoxicidade Natural/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Regulação para Cima/imunologia , Antígenos HLA-ERESUMO
Although natural killer (NK) cells have been implicated in regulating immune responses, their ability to modulate disease development in autoimmune arthritis has not been analyzed. Here we investigate the contribution of NK cells to regulating collagen-induced arthritis, a well-characterized preclinical model of human rheumatoid arthritis. We find that the disease is induced by the combined action of two CD4(+) T helper (T(H)) subsets: follicular T(H) cells and T(H)17 cells. Both CD4(+) T(H) subsets are highly susceptible to lysis by NK cells after activation. Administration of antibody that activates NK cells through blockade of its inhibitory CD94/NKG2A receptor allows enhanced elimination of pathogenic follicular T(H) and T(H)17 cells and arrest of disease progression. These results suggest that antibody-dependent enhancement of NK activity may yield effective, previously undescribed therapeutic approaches to this autoimmune disorder.
Assuntos
Artrite Experimental/prevenção & controle , Células Matadoras Naturais/fisiologia , Animais , Artrite Experimental/etiologia , Comunicação Celular , Camundongos , Camundongos Endogâmicos C57BL , Subfamília C de Receptores Semelhantes a Lectina de Células NK/fisiologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/fisiologia , Células Th1/fisiologia , Células Th17/fisiologiaRESUMO
Autoimmune encephalomyelitis may be ameliorated experimentally by enhancing NK cell-mediated elimination of activated autoreactive T cells through a mutation that interrupts the interaction between Qa-1(b) and CD94/NKG2A. Here we evaluate the ability of an anti-NKG2A F(ab')(2) Ab to enhance elimination of autoreactive T cells and reduce experimental autoimmune encephalomyelitis (EAE). Anti-NKG2A F(ab')(2) treatment diminishes progression of both myelin oligodendrocyte glycoprotein (MOG)-induced EAE in intact C57BL/6 mice and after adoptive transfer of disease-causing T cells. Analyses of the underlying mechanism revealed that administration of anti-NKG2A F(ab')(2) Ab reduces CD4(+) T recall responses to MOG and skews the proportion of IL-17- and IFNgamma-producing CD4(+) T cells toward the protective IL-4- and IL-10-secreting CD4(+) T cell subpopulations. CD94/NKG2A-dependent inhibition of inflammatory damage to spinal cord is associated with decreased infiltration of T cells and reduced microglia activation in the central nervous system. Because anti-NKG2A F(ab')(2) treatment had no detectable effect on the numbers or activity of T and B lymphocytes and NK cells in peripheral lymphoid tissues, this anti-NKG2A-based approach may represent a safe and effective therapy for this CNS disorder.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Encefalomielite Autoimune Experimental/terapia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Linfócitos T/efeitos dos fármacos , Transferência Adotiva , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteínas da Mielina , Glicoproteína Associada a Mielina/química , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
To improve the care of patients with chronic inflammatory skin conditions, such as psoriasis, there is a need for diagnostic methods that can facilitate personalized medicine. This exploratory pilot study aimed to determine whether non-invasive measurements of inflammation-related proteins from psoriatic skin can be sampled using the FibroTx Transdermal Analysis Patch (TAP) to assess disease severity and monitor pharmacodynamic changes. Ten healthy volunteers and 44 psoriasis vulgaris patients were enrolled in the exploratory pilot study. Skin surface protein measurements for healthy and lesional skin were performed using TAP. Patients' scores of psoriasis activity and severity (PASI) were documented, and differences in the thickness of skin layers were determined using sonography. The study assessed the skin surface protein levels of psoriasis patients undergoing whole-body treatment with narrow-band UVB to evaluate whether the levels of the skin surface proteins IL-1α, IL-1RA CXCL-1/2, and hBD-1 were associated with the disease activity and severity measurements. Using TAP technology, it was observed that there were clear differences in levels of IL-1α, IL-1RA, CXCL-1/2, and hBD-1 between psoriasis lesional and non-lesional skin. In addition, a positive correlation between CXCL-1/2 and desquamation, and between CXCL-1/2 and SLEB thickness was observed. During UVB treatment, the TAP measurements revealed a clear reduction of IL-1RA, CXCL 1/2, and hBD-1 on lesional skin. Further, skin surface measurements of IL-1RA and CXCL-1/2 displayed a different profile than those achieved by visual scoring of local inflammation, thus indicating that measuring the 'molecular root' of inflammation appears to have value as an objective, non-invasive biomarker measurement for scoring disease severity.
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CD161 is a C-type lectin-like receptor expressed on human natural killer (NK) cells and subsets of T cells. CD161 has been described as an inhibitory receptor that regulates NK cell-mediated cytotoxicity and IFN-γ production. Its role on T cells has remained unclear. Studies have shown that triggering of CD161 enhances NK T cell proliferation and T cell-IFN-γ production while inhibiting TNF-α production by CD8(+) T cells. Lectin-like transcript 1 (LLT1), the ligand of CD161, was found to be expressed on Toll-like receptor (TLR)-activated plasmacytoid and monocyte-derived dendritic cells (DC) and on activated B cells. Using newly developed anti-LLT1 mAbs, we show that LLT1 is not expressed on the surface of circulating B and T lymphocytes, NK cells, monocytes, and dendritic cells but that LLT1 is up-regulated upon activation. Not only TLR-stimulated dendritic cells and B cells but also T cell receptor-activated T cells and activated NK cells up-regulate LLT1. Interestingly, IFN-γ increases LLT1 expression level on antigen-presenting cells. LLT1 is also induced on B cells upon viral infection such as Epstein-Barr virus or HIV infection and in inflamed tonsils. Finally, expression of LLT1 on B cells inhibits NK cell function but costimulates T cell proliferation or IFN-γ production, and coengagement of CD161 with CD3 increases IL-17 secretion. Altogether, our results point toward a role for LLT1/CD161 in modulating immune responses to pathogens.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Interferon gama/metabolismo , Lectinas Tipo C/biossíntese , Subfamília B de Receptores Semelhantes a Lectina de Células NK/biossíntese , Receptores de Superfície Celular/biossíntese , Animais , Linhagem Celular , Células-Tronco Hematopoéticas/citologia , Humanos , Sistema Imunitário , Interferons/metabolismo , Leucócitos Mononucleares/citologia , Camundongos , Modelos Biológicos , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Lectin-like transcript 1 (LLT1) encoded by CLEC2D gene is a C-type lectin-like molecule interacting with human CD161 (NKR-P1A) receptor expressed by natural killer cells and subsets of T cells. Using RT-PCR and sequencing, we identified several CLEC2D alternatively spliced transcript variants generated by exon skipping. In addition to the reported transcript variants 1 (LLT1) and 2, we identified a novel splice variant 4 and transcripts coding for putative soluble proteins. CLEC2D transcripts were detected primarily in hematopoietic cell lines and were found to be co-induced by the same activation signals. Although very low amounts of putative soluble CLEC2D protein isoforms could be produced by transfectants, CLEC2D isoforms 2 and 4 were efficiently expressed. By contrast to LLT1, which was detected on the cell surface, isoform 2 and 4 remained in the endoplasmic reticulum where they formed homodimers or heterodimers with LLT1. They failed to interact with CD161, leaving LLT1 as the sole ligand for this receptor. CLEC2D therefore uses gene splicing to generate protein isoforms that are structurally distinct and that have different biological activities.
Assuntos
Processamento Alternativo , Lectinas Tipo C/genética , Receptores de Superfície Celular/genética , Transcrição Gênica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células Jurkat , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Subfamília B de Receptores Semelhantes a Lectina de Células NK/química , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias/terapia , Receptores KIR/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/patologia , Receptores KIR/antagonistas & inibidores , Receptores KIR2DL1/química , Receptores KIR2DL1/genética , Receptores KIR2DL1/imunologia , Receptores KIR2DL2/química , Receptores KIR2DL2/genética , Receptores KIR2DL2/imunologia , Receptores KIR2DL3/genética , Receptores KIR2DL3/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by a subset of chronically activated memory T cells and plays an important role in their activation and proliferation. Here, we show that primary malignant T cells isolated from patients with Sézary syndrome (SS) express Kv1.3 and are sensitive to potent Kv1.3 inhibitors ShK and Vm24, but not sensitive to a less potent inhibitor [N17A/F32T]-AnTx. Kv1.3 blockade inhibits CD3/CD28-induced proliferation and IL-9 expression by SS cells in a concentration-dependent manner. In parallel, CD3/CD28-mediated CD25 induction is inhibited, whereas Kv1.3 blockade has no effect on apoptosis or cell death as judged by Annexin V and PI staining. In conclusion, we provide the first evidence that malignant T cells in SS express functional Kv1.3 channels and that Kv1.3 blockade inhibits activation-induced proliferation as well as cytokine and cytokine receptor expression in malignant T cells, suggesting that Kv1.3 is a potential target for therapy in SS.
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INTRODUCTION: The need for biomarkers which can predict disease course and treatment response in rheumatoid arthritis (RA) is evident. We explored whether clinical and imaging responses to biologic disease modifying anti-rheumatic drug treatment (bDMARD) were associated with the individual's mediator production in explants obtained at baseline. METHODS: RA Patients were evaluated by disease activity score 28 joint C-reactive protein (DAS 28-)), colour Doppler ultrasound (CDUS) and 3 Tesla RA magnetic resonance imaging scores (RAMRIS). Explants were established from synovectomies from a needle arthroscopic procedure prior to initiation of bDMARD. Explants were incubated with the bDMARD in question, and the productions of interleukin-6 (IL-6), monocyte chemo-attractive protein-1 (MCP-1) and macrophage inflammatory protein-1-beta (MIP-1b) were measured by multiplex immunoassays. The changes in clinical and imaging variables following a minimum of 3 months bDMARD treatment were compared to the baseline explant results. Mixed models and Spearman's rank correlations were performed. P-values below 0.05 were considered statistically significant. RESULTS: 16 patients were included. IL-6 production in bDMARD-treated explants was significantly higher among clinical non-responders compared to responders (P = 0.04), and a lack of suppression of IL-6 by the bDMARDS correlated to a high DAS-28 (ρ = 0.57, P = 0.03), CDUS (ρ = 0.53, P = 0.04) and bone marrow oedema (ρ = 0.56, P = 0.03) at follow-up. No clinical association was found with explant MCP-1 production. MIP-1b could not be assessed due to a large number of samples below the detection limit. CONCLUSIONS: Synovial explants appear to deliver a disease-relevant output testing which when carried out in advance of bDMARD treatment can potentially pave the road for a more patient tailored treatment approach with better treatment effects.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Processamento de Imagem Assistida por Computador/métodos , Interleucina-6/análise , Membrana Sinovial/patologia , Adulto , Idoso , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/metabolismo , Estudos de Casos e Controles , Quimiocina CCL2/metabolismo , Feminino , Humanos , Técnicas In Vitro , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Prognóstico , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Técnicas de Cultura de Tecidos , Ultrassonografia Doppler em Cores/métodosRESUMO
OBJECTIVE: Recently we serendipitously identified a patient with systemic lupus erythematosus (SLE) who was positive for autoantibodies to CD94/natural killer receptor group 2A (NKG2A). The present study was undertaken to investigate the occurrence and function of autoantibodies targeting lectin-like NK cell receptors in SLE. METHODS: Sera from 203 SLE patients and 90 healthy individuals were analyzed, by flow cytometry, for Ig binding to Ba/F3 cells transfected with CD94/NKG2A, CD94/NKG2C, or NKG2D. Autoantibodies identified were characterized with regard to interference with HLA-E binding, effect on NK cell activation in response to HLA-E-transfected K562 cells, and capacity to facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). Levels of autoantibodies were determined in longitudinally sampled sera, and correlations with disease activity (SLE Disease Activity Index 2000) and severity (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index) were investigated. RESULTS: Anti-CD94/NKG2A autoantibodies were identified in 7 SLE patients. The autoantibodies from 6 patients inhibited binding of HLA-E to CD94/NKG2A, whereas those from the seventh patient augmented this binding. Autoantibodies from 2 patients also reacted with the activating receptor CD94/NKG2C, with inhibition of the binding of HLA-E to CD94/NKG2C observed in 1 case and enhancement of this binding in the other. None of the sera contained anti-NKG2D autoantibodies. The levels of anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies correlated with disease activity and with a more severe SLE phenotype. Mechanistically, anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies both interfered with HLA-E-mediated regulation of NK cell activation and facilitated the elimination of target cells expressing CD94/NKG2A or CD94/NKG2C through ADCC. CONCLUSION: Anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies occur in a subset of patients with clinically active SLE. Given their capacity to deplete certain NK cell subsets and interfere with particular NK cell function, such autoantibodies may promote the pathogenesis of SLE.
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Autoanticorpos/sangue , Lúpus Eritematoso Sistêmico/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/imunologia , Humanos , Lúpus Eritematoso Sistêmico/sangue , Ativação Linfocitária/imunologiaRESUMO
Non-Hodgkin's lymphomas (NHLs) are malignant neoplasms which are clinically and biologically diverse. Their incidence is constantly increasing and despite treatment advances, there is a need for novel targeted therapies. Here, we identified Lectin-like transcript 1 (LLT1) as a biomarker of germinal center (GC)-derived B-cell NHLs. LLT1 identifies GC B cells in reactive tonsils and lymph nodes and its expression is maintained in B-cell NHLs which derive from GC, including Burkitt lymphoma (BL), follicular lymphoma (FL), and GC-derived diffuse large B-cell lymphoma (DLBCL). We further show that LLT1 expression by tumors dampens natural killer (NK) cell functions following interaction with its receptor CD161, uncovering a potential immune escape mechanism. Our results pinpoint LLT1 as a novel biomarker of GC-derived B-cell NHLs and as a candidate target for innovative immunotherapies.
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BACKGROUND: The skin proteome contains valuable information on skin condition, but also on how skin may evolve in time and may respond to treatments. Despite the potential of measuring regulatory-, effector- and structural proteins in the skin for biomarker applications in clinical dermatology and skin care, convenient diagnostic tools are lacking. The aim of the present study was to develop a highly versatile and non-invasive diagnostic tool for multiplex measurements of protein biomarkers from the surface of skin. RESULTS: The Transdermal Analyses Patch (TAP) is a novel molecular diagnostic tool that has been developed to capture biomarkers directly from skin, which are quantitatively analyzed in spot-ELISA assays. Optimisation of protocols for TAP production and biomarker analyses makes TAP measurements highly specific and reproducible. In measurements of interleukin-1α (IL-1α), IL-1 receptor antagonist (IL-1RA) and human ß-defensin (hBD-1) from healthy skin, TAP appears far more sensitive than skin lavage-based methods using ELISA. No side-effects were observed using TAP on human skin. CONCLUSION: TAP is a practical and valuable new skin diagnostic tool for measuring protein-based biomarkers from skin, which is convenient to use for operators, with minimal burden for patients.
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Intestinal M play an important role in maintaining gut homeostasis. However, little is known about these cells, their precursors, and their role in intestinal inflammation. Here, we characterize the CD14(+) mononuclear cell populations in intestinal mucosa and blood in patients with CD. Among the LP CD14(+) M, we identified three distinct HLA-DR(+)-expressing subsets. Compared with uninflamed, inflamed mucosa contained a marked increase in the proportion of the CD14(hi)HLA-DR(dim) cellular subset. This subset resembled the classical blood monocytes with low CD16, HLA-DR, and CX3CR1 expression. Classical monocytes migrated efficiently toward CCL2 and released the highest levels of MMP-1 and proinflammatory cytokines when stimulated with immune complexes or LPS. Our findings strongly suggest that it is the classical and not the intermediate or nonclassical monocytes that are the precursors to the dominating intestinal CD14(hi)HLA-DR(dim) subset. This enhances our understanding of CD pathology and may provide new options in treatment.
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Doença de Crohn/imunologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Adolescente , Adulto , Idoso , Antígenos de Diferenciação Mielomonocítica/imunologia , Doença de Crohn/sangue , Doença de Crohn/patologia , Feminino , Citometria de Fluxo , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/imunologia , Humanos , Mucosa Intestinal/patologia , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Adulto JovemRESUMO
INTRODUCTION: Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans. METHODS: RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1ß (MIP-1ß) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model. RESULTS: Tissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman's ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman's ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman's ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman's ρ = 0.50), BME score (P = 0.04, approximate Spearman's ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman's ρ = 0.35). MIP-1ß was associated with CDUS activity (P = 0.02, approximate Spearman's ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman's ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance. CONCLUSIONS: The association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1ß, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.
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Artrite Reumatoide/metabolismo , Biomarcadores/metabolismo , Mediadores da Inflamação/metabolismo , Membrana Sinovial/metabolismo , Adulto , Idoso , Artrite Reumatoide/diagnóstico por imagem , Quimiocina CCL2/metabolismo , Quimiocina CCL4/metabolismo , Estudos Transversais , Feminino , Articulação da Mão/diagnóstico por imagem , Articulação da Mão/patologia , Humanos , Imunoensaio/métodos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Radiografia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Membrana Sinovial/diagnóstico por imagem , Sinovite/diagnóstico , Sinovite/diagnóstico por imagem , Técnicas de Cultura de Tecidos , Ultrassonografia Doppler em Cores/métodosRESUMO
In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-α. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation.