RESUMO
OBJECTIVES: Invariant NKT (iNKT) cells play a role in regulating the function of autoreactive B cells before their entry into germinal centres. Absence and/or reduction of iNKT cells have been demonstrated in patients with systemic lupus erythematosus (SLE) together with an increase of autoreactive B cell activity. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease in which lymphocyte infiltration and organisation in lymphoid structures of inflamed salivary glands occurs. The aim of the study was to investigate the percentage and function of iNKT in the salivary glands and peripheral blood of patients with pSS. METHODS: Minor salivary gland biopsies were obtained from patients with pSS and with non-specific chronic sialoadenitis (nSS). Flow cytometry analysis of CD1d/α-GalactosylCeramide (α-GalCer) tetramer positive cells, producing IFN-γ and IL-17, and quantitative gene expression analysis by TaqMan real-time PCR for Vα24 were performed on salivary glands biopsies and peripheral blood samples obtained from patients and controls. Flow cytometry and immunofluorescence analysis for autoreactive B lymphocytes and ELISA for anti-SSA antibodies (Ab) detection were also performed. RESULTS: An increase of iNKT was detected ex vivo in peripheral blood of pSS patients; after α-GalCer stimulation this subset produce IL-17 and IFN-iNKT were undetectable in the salivary glands of pSS patients and anti-SSA specific B cells were found in target tissue. Invariant NKT cells were able to inhibit autoantibody production by B cells obtained from salivary glands of pSS. CONCLUSIONS: Impaired iNKT migration to inflamed sites might induce the activation of autoreactive B cells specific for SSA-antigen in salivary glands of pSS patients.
Assuntos
Proliferação de Células , Ativação Linfocitária , Células T Matadoras Naturais/imunologia , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologia , Anticorpos Antinucleares/biossíntese , Anticorpos Antinucleares/imunologia , Formação de Anticorpos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biópsia , Estudos de Casos e Controles , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Contagem de Linfócitos , Células T Matadoras Naturais/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Síndrome de Sjogren/sangue , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/genéticaRESUMO
OBJECTIVE: IL-9 has been shown to be upregulated before the clinical onset of articular disease in RA. The exact role of IL-9 and Th9 cells in RA, however, has not yet been adequately studied. The aim of this study was to evaluate the expression of IL-9 and IL-9-expressing cells in RA patients. METHODS: IL-9, IL-9R, PU.1, IL-9, thymic stromal lymphopoietin (TSLP), IL-4 and TGF-ß expression was assessed by real-time-PCR in the synovial tissues of RA and OA patients. IL-9, IL-9R, IL-4, TSLP and TGF-ß were also investigated by immunohistochemistry. Peripheral CD4(+) T cell subsets were studied by flow cytometry analysis before and after incubation with citrullinated peptides. RESULTS: IL-9 was overexpressed in RA synovial tissues and correlated with the degree of histological organization of B and T cells in ectopic lymphoid structures. The majority of IL-9-producing cells were identified as CD3(+) cells. Increased mRNA and protein expression of IL-9R, IL-4, TSLP and TGF-ß was also observed in RA synovial tissue. Blood peripheral Th9 cells were expanded by citrullinated peptides. CONCLUSION: These results indicate that Th9 cells and IL-9 were frequently detected in peripheral blood mononuclear cells and synovia of RA patients. A possible pathogenic role for Th9 in RA is discussed.
Assuntos
Artrite Reumatoide/imunologia , Interleucina-9/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Citocinas/biossíntese , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-4/biossíntese , Interleucina-9/biossíntese , Interleucina-9/genética , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Membrana Sinovial/imunologia , Fator de Crescimento Transformador beta/biossíntese , Adulto Jovem , Linfopoietina do Estroma do TimoRESUMO
OBJECTIVES: To investigate the in vitro and ex-vivo effect of IL-6 inhibition on the balance between Th1, Th2, Th17 and Treg cells. METHODS: Ten consecutive adult patients with active early rheumatoid arthritis (ERA) and ten healthy volunteers were included in the study. The percentages of Th1, Th2, Th17 and Treg cells were analysed by flow cytometry in the peripheral blood mononuclear cells obtained from controls and from RA patients at the time of first evaluation and just before the third TCZ infusion. The in vitro effect of TCZ on the different subsets of CD4+ T cells and the expression levels of Th1, Th2, Th17 and Treg-related cytokines was also assessed. RESULTS: Treatment with TCZ, both ex vivo and in vitro, resulted in a significant reduction of the percentage of Th1, Th17 and Treg cells with a concomitant significant increase of Th2 cell subsets. The reduction of the different subsets of T lymphocytes was associated with an intense staining with Annexin V, suggesting an apoptotic-related cell reduction. A significant decrease of Th1, Th17 and Treg cytokines and a concomitant increase of IL-4 was also observed after TCZ treatment in PBMC isolated from RA patients. CONCLUSIONS: TCZ could modify the immune imbalance in RA inducing apoptosis of Th1, Th17 and Treg cells and promoting the appearance of a Th2 response.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Interleucina-6/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Adulto , Apoptose/efeitos dos fármacos , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression.
Assuntos
Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/metabolismo , Senescência Celular/fisiologia , Melanócitos/metabolismo , Proteínas Nucleares/metabolismo , Adulto , Western Blotting , Dioxigenases , Humanos , Imuno-Histoquímica , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Análise Serial de TecidosRESUMO
Deubiquitinating enzymes are key regulators of the ubiquitin-proteasome system and cell cycle, and their dysfunction leads to tumorigenesis. Our in vivo drop-out screens in patient-derived xenograft models identify USP7 as a regulator of melanoma. We show that USP7 downregulation induces cellular senescence, arresting melanoma growth in vivo and proliferation in vitro in BRAF- and NRAS-mutant melanoma. We provide a comprehensive understanding of targets and networks affected by USP7 depletion by performing a global transcriptomic and proteomics analysis. We show that RRM2 is a USP7 target and is regulated by USP7 during S phase of the cell cycle. Ectopic expression of RRM2 in USP7-depleted cells rescues the senescent phenotype. Pharmacological inhibition of USP7 by P5091 phenocopies the shUSP7-induced senescent phenotype. We show that the bifunctional histone deacetylase (HDAC)/LSD1 inhibitor domatinostat has an additive antitumor effect, eliminating P5091-induced senescent cells, paving the way to a therapeutic combination for individuals with melanoma.
Assuntos
Inibidores de Histona Desacetilases , Melanoma , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases , Histona Desmetilases/genética , Humanos , Melanoma/patologia , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas B-raf/genética , Tiofenos , Peptidase 7 Específica de Ubiquitina/metabolismo , UbiquitinasRESUMO
Metastases are the primary cause of cancer-related deaths. The underlying molecular and biological mechanisms remain, however, elusive, thus preventing the design of specific therapies. In melanomas, the metastatic process is influenced by the acquisition of metastasis-associated mutational and epigenetic traits and the activation of metastatic-specific signaling pathways in the primary melanoma. In the current study, we investigated the role of an adaptor protein of the Shc family (ShcD) in the acquisition of metastatic properties by melanoma cells, exploiting our cohort of patient-derived xenografts (PDXs). We provide evidence that the depletion of ShcD expression increases a spread cell shape and the capability of melanoma cells to attach to the extracellular matrix while its overexpression switches their morphology from elongated to rounded on 3D matrices, enhances cells' invasive phenotype, as observed on collagen gel, and favors metastasis formation in vivo. ShcD overexpression sustains amoeboid movement in melanoma cells, by suppressing the Rac1 signaling pathway through the confinement of DOCK4 in the cytoplasm. Inactivation of the ShcD signaling pathway makes melanoma cells more sensitive to therapeutic treatments. Consistently, ShcD expression predicts poor outcome in a cohort of 183 primary melanoma patients.
RESUMO
The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.