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1.
Cancer Cell Int ; 23(1): 89, 2023 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-37165394

RESUMO

BACKGROUND: Cancer stem-like cells (CSCs) are a subpopulation of tumor cells responsible for tumor initiation, metastasis, chemoresistance, and relapse. Recently, CSCs have been identified in Uveal Melanoma (UM), which represents the most common primary tumor of the eye. UM is highly resistant to systemic chemotherapy and effective therapies aimed at improving overall survival of patients are eagerly required. METHODS: Herein, taking advantage from a pan Fibroblast Growth Factor (FGF)-trap molecule, we singled out and analyzed a UM-CSC subset with marked stem-like properties. A hierarchical clustering of gene expression data publicly available on The Cancer Genome Atlas (TCGA) was performed to identify patients' clusters. RESULTS: By disrupting the FGF/FGF receptor (FGFR)-mediated signaling, we unmasked an FGF-sensitive UM population characterized by increased expression of numerous stemness-related transcription factors, enhanced aldehyde dehydrogenase (ALDH) activity, and tumor-sphere formation capacity. Moreover, FGF inhibition deeply affected UM-CSC survival in vivo in a chorioallantoic membrane (CAM) tumor graft assay, resulting in the reduction of tumor growth. At clinical level, hierarchical clustering of TCGA gene expression data revealed a strong correlation between FGFs/FGFRs and stemness-related genes, allowing the identification of three distinct clusters characterized by different clinical outcomes. CONCLUSIONS: Our findings support the evidence that the FGF/FGFR axis represents a master regulator of cancer stemness in primary UM tumors and point to anti-FGF treatments as a novel therapeutic strategy to hit the CSC component in UM.

2.
Clin Immunol ; 237: 108974, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35278713

RESUMO

Dedicator of Cytokinesis 8 (DOCK8) deficiency is a rare form of autosomal recessive combined immunodeficiency. The effect of DOCK8 deficiency on Natural Killer cell biology has not been fully elucidated yet. Thus, we undertook a detailed phenotypic and functional evaluation of NK cells from seven patients with DOCK8 deficiency. Patients' immature CD56bright NK cells were defective in IFN-γ secretion, while their mature CD56dim NK cells showed impaired cytotoxicity, partially rescued upon rIL-2 addition. Cross-linking of NK cell receptors revealed a specific defect in the CD3 zeta chain-dependent activation pathway in DOCK8 deficiency. Lack of DOCK8, but not of WASP, impaired CCR7 expression on human CD56bright NK cells, a critical receptor for their migration to secondary lymph nodes. Evaluation of a patient's lymph node showed a severe reduction in NK cells that showed increased intracellular expression of CCR7. Our data suggest that DOCK8 deficiency variably affects NK cell homeostasis in humans.


Assuntos
Citocinese , Fatores de Troca do Nucleotídeo Guanina , Células Matadoras Naturais , Receptores CCR7 , Antígeno CD56/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Proteína da Síndrome de Wiskott-Aldrich
3.
Int J Mol Sci ; 20(4)2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791364

RESUMO

The crosstalk between cancer cells and host cells is a crucial prerequisite for tumor growth and progression. The cells from both the innate and adaptive immune systems enter into a perverse relationship with tumor cells to create a tumor-promoting and immunosuppressive tumor microenvironment (TME). Epithelial ovarian cancer (EOC), the most lethal of all gynecological malignancies, is characterized by a unique TME that paves the way to the formation of metastasis and mediates therapy resistance through the deregulation of immune surveillance. A characteristic feature of the ovarian cancer TME is the ascites/peritoneal fluid, a malignancy-associated effusion occurring at more advanced stages, which enables the peritoneal dissemination of tumor cells and the formation of metastasis. The standard therapy for EOC involves a combination of debulking surgery and platinum-based chemotherapy. However, most patients experience disease recurrence. New therapeutic strategies are needed to improve the prognosis of patients with advanced EOC. Harnessing the body's natural immune defenses against cancer in the form of immunotherapy is emerging as an innovative treatment strategy. NK cells have attracted attention as a promising cancer immunotherapeutic target due to their ability to kill malignant cells and avoid healthy cells. Here, we will discuss the recent advances in the clinical application of NK cell immunotherapy in EOC.


Assuntos
Imunomodulação , Imunoterapia , Células Matadoras Naturais/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Biomarcadores Tumorais , Terapia Combinada , Feminino , Humanos , Imunomodulação/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Terapia de Alvo Molecular , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Evasão Tumoral/imunologia
4.
N Engl J Med ; 372(25): 2409-22, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26083206

RESUMO

Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).


Assuntos
Doenças Genéticas Inatas/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/genética , Mutação , Linfócitos T/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Pré-Escolar , Evolução Fatal , Feminino , Proteínas Ativadoras de GTPase , Genes Recessivos , Doenças Genéticas Inatas/terapia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Transplante de Células-Tronco Hematopoéticas , Humanos , Síndromes de Imunodeficiência/terapia , Lactente , Células Matadoras Naturais/imunologia , Masculino , Linhagem , Linfócitos T/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
5.
Cell Biol Int ; 42(3): 353-364, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29105212

RESUMO

The aim of this study was to investigate the in vitro effect of Silicon, in the soluble form of sodium orthosilicate, combined and not with the concentrated growth factors (CGF), a platelet-rich preparation, on three different human cell lines of fibroblasts (NHDF), endothelial cells (HUVEC), and osteoblasts (HOBs). Each cell type was treated with sodium orthosilicate at the final concentration of 0.5 mM and 1 mM, CGF, and sodium orthosilicate combined with CGF, for 72 h. At the end of the experimental period, the in vitro effect on cell growth, proliferation, and metabolic activity was evaluated by performing a simple cell count, using an automated cell counter and by evaluating the expression of the intracellular proliferation marker Ki-67, using Fluorescence-activated cell sorting (FACS) analysis. Moreover, the expression of other cell markers and active molecules, such as Collagen type I, Osteopontin, Vascular Endothelial Growth Factor, and endothelial Nitric Oxide Synthase, was evaluated, through immunohistochemistry. Results obtained showed that the combined use of CGF and sodium orthosilicate stimulates cell growth, proliferation, and metabolic activity, suggesting that this treatment could be effective in tissue regeneration.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Compostos de Silício/farmacologia , Plaquetas , Contagem de Células , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Osteoblastos/efeitos dos fármacos , Compostos de Silício/metabolismo , Sódio
6.
J Allergy Clin Immunol ; 139(1): 335-346.e3, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27372564

RESUMO

BACKGROUND: Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to T cells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. OBJECTIVE: We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. METHODS: We performed multiparametric cytofluorimetric analysis of PD-1+ NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. RESULTS: We provide unequivocal evidence that PD-1 is highly expressed (PD-1bright) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56dim but not CD56bright NK cells and is confined to fully mature NK cells characterized by the NKG2A-KIR+CD57+ phenotype. Proportions of PD-1bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. CONCLUSIONS: We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody-mediated disruption of PD-1/programmed death ligand interaction.


Assuntos
Células Matadoras Naturais/imunologia , Receptor de Morte Celular Programada 1/imunologia , Degranulação Celular , Proliferação de Células , Citocinas/imunologia , Feminino , Humanos , Células Matadoras Naturais/fisiologia , Neoplasias Ovarianas/imunologia , Fenótipo
7.
J Allergy Clin Immunol ; 140(2): 553-564.e4, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28069426

RESUMO

BACKGROUND: Gain-of-function (GOF) mutations affecting the coiled-coil domain or the DNA-binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. OBJECTIVE: We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. METHODS: Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. RESULTS: Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN-γ in response to IL-15 and reduced proliferation after stimulation with IL-2 or IL-15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN-α is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL-15, whereas STAT5 phosphorylation and DNA binding to IL-2 receptor α (IL2RA) are reduced or not affected in response to the same cytokine. CONCLUSION: These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities.


Assuntos
Candidíase Mucocutânea Crônica/genética , Células Matadoras Naturais/imunologia , Fator de Transcrição STAT1/genética , Adolescente , Adulto , Candidíase Mucocutânea Crônica/imunologia , Criança , Citocinas/farmacologia , Feminino , Expressão Gênica , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Fosforilação , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT5/metabolismo
8.
Clin Immunol ; 175: 99-108, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27923702

RESUMO

NFKB1, a component of the canonical NF-κB pathway, was recently reported to be mutated in a limited number of CVID patients. CVID-associated mutations in NFKB2 (non-canonical pathway) have previously been shown to impair NK cell cytotoxic activity. Although a biological function of NFKB1 in non-human NK cells has been reported, the role of NFKB1 mutations for human NK cell biology and disease has not been investigated yet. We decided therefore to evaluate the role of monoallelic NFKB1 mutations in human NK cell maturation and functions. We show that NFKB1 mutated NK cells present impaired maturation, defective cytotoxicity and reduced IFN-γ production upon in vitro stimulation. Furthermore, human IL-2 activated NFKB1 mutated NK cells fail to up-regulate the expression of the activating marker NKp44 and show reduced proliferative capacity. These data suggest that NFKB1 plays an essential novel role for human NK cell maturation and effector functions.


Assuntos
Células Matadoras Naturais/imunologia , Subunidade p50 de NF-kappa B/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Interferon gama/imunologia , Interleucina-2/imunologia , Células K562 , Masculino , NF-kappa B/imunologia
9.
J Immunol ; 192(3): 1241-8, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24395917

RESUMO

The interaction of NK cells with dendritic cells (DCs) results in reciprocal cell activation through the interaction of membrane proteins and the release of soluble factors. In this article, we report that in NK-DC cocultures, among a set of 84 cytokines investigated, activin A was the second highest induced gene, with CXCL8 being the most upregulated one. Activin A is a member of the TGF-ß superfamily and was previously shown to possess both proinflammatory and anti-inflammatory activities. In NK-DC cocultures, the induction of activin A required cell contact and was dependent on the presence of proinflammatory cytokines (i.e., IFN-γ, TNF-α, and GM-CSF), as well as on NK cell-mediated DC killing. CD1(+) DCs were the main activin A producer cells among myeloid blood DC subsets. In NK-DC cocultures, inhibition of activin A by follistatin, a natural inhibitory protein, or by a specific blocking Ab, resulted in the upregulation of proinflammatory cytokine release (i.e., IL-6, IL-8, TNF-α) by DCs and in the increase of DC maturation. In conclusion, our study reports that activin A, produced during NK-DC interactions, represents a relevant negative feedback mechanism that might function to prevent excessive immune activation by DCs.


Assuntos
Ativinas/fisiologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Ativinas/biossíntese , Ativinas/genética , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígeno B7-2/biossíntese , Antígeno B7-2/genética , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/genética , Citotoxicidade Imunológica , Cães , Retroalimentação Fisiológica , Folistatina/farmacologia , Síndrome de Hermanski-Pudlak/patologia , Humanos , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Interleucina-15/farmacologia , Interleucina-8/biossíntese , Interleucina-8/genética , Células Matadoras Naturais/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Monócitos/citologia , Monócitos/efeitos dos fármacos , Receptores de Células Matadoras Naturais/biossíntese , Receptores de Células Matadoras Naturais/genética , Regulação para Cima , Antígeno CD83
10.
Eur J Immunol ; 44(5): 1526-34, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24496997

RESUMO

X-linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM-associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B-cell lymphomas. Here, we show that in the absence of SLAM-associated protein, co-engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM-dependent signaling pathways including activating killer Ig-like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN-γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM-1 and NKG2D triggering receptors. Thus, while CD48(+) B-EBV and lymphoma B cells devoid of NKG2D and DNAM-1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross-talk of inhibitory 2B4 with triggering NK (and T) receptors.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Células Matadoras Naturais/imunologia , Transtornos Linfoproliferativos/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígeno CD48 , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/patologia , Feminino , Herpesvirus Humano 4/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Células Matadoras Naturais/patologia , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/patologia , Masculino , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Receptores Imunológicos/genética , Transdução de Sinais/genética , Família de Moléculas de Sinalização da Ativação Linfocitária
12.
J Allergy Clin Immunol ; 134(6): 1381-1387.e7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24985396

RESUMO

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, heterogeneous, hyperinflammmatory disorder. Prompt identification of inherited forms resulting from mutation in genes involved in cellular cytotoxicity can be crucial. X-linked lymphoproliferative disease 1 (XLP1), due to mutations in SH2D1A (Xq25) encoding signaling lymphocyte activation molecule-associated protein (SAP), may present with HLH. Defective SAP induces paradoxical inhibitory function of the 2B4 coreceptor and impaired natural killer (NK) (and T) cell response against EBV-infected cells. OBJECTIVE: To characterize a cohort of patients with HLH and XLP1 for SAP expression and 2B4 function in lymphocytes, proposing a rapid diagnostic screening to direct mutation analysis. METHODS: We set up rapid assays for 2B4 function (degranulation or (51)Cr-release) to be combined with intracellular SAP expression in peripheral blood NK cells. We studied 12 patients with confirmed mutation in SH2D1A and some family members. RESULTS: The combined phenotypic/functional assays allowed efficient and complete diagnostic evaluation of all patients with XLP1, thus directing mutation analysis and treatment. Nine cases were SAP(-), 2 expressed SAP with mean relative fluorescence intensity values below the range of healthy controls (SAP(dull)), and 1, carrying the R55L mutation, was SAP(+). NK cells from all patients showed inhibitory 2B4 function and defective killing of B-EBV cells. Carriers with SH2D1A mutations abolishing SAP expression and low percentage of SAP(+) cells showed neutral 2B4 function at the polyclonal NK cell level. Three novel SH2D1A mutations have been identified. CONCLUSIONS: Study of SAP expression is specific but may have insufficient sensitivity for screening XLP1 as a single tool. Combination with 2B4 functional assay allows identification of all cases.


Assuntos
Antígenos CD/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Linfo-Histiocitose Hemofagocítica/diagnóstico , Transtornos Linfoproliferativos/diagnóstico , Receptores Imunológicos/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/imunologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Masculino , Mutação , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Adulto Jovem
14.
Biochim Biophys Acta Rev Cancer ; 1879(3): 189104, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38701937

RESUMO

Uveal melanoma (UM) is the most common primary ocular tumor in the adult population. Even though these primary tumors are successfully treated in 90% of cases, almost 50% of patients ultimately develop metastasis, mainly in the liver, via hematological dissemination, with a median survival spanning from 6 to 12 months after diagnosis. In this context, chemotherapy regimens and molecular targeted therapies have demonstrated poor response rates and failed to improve survival. Among the multiple reasons for therapy failure, the presence of cancer stem-like cells (CSCs) represents the main cause of resistance to anticancer therapies. In the last few years, the existence of CSCs in UM has been demonstrated both in preclinical and clinical studies, and new molecular pathways and mechanisms have been described for this subpopulation of UM cells. Here, we will discuss the state of the art of CSC biology and their potential exploitation as therapeutic target in UM.


Assuntos
Melanoma , Células-Tronco Neoplásicas , Neoplasias Uveais , Neoplasias Uveais/patologia , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/metabolismo , Humanos , Melanoma/patologia , Melanoma/tratamento farmacológico , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Animais , Resistencia a Medicamentos Antineoplásicos , Terapia de Alvo Molecular , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia
16.
Blood ; 118(7): 1806-17, 2011 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-21772053

RESUMO

Subjects affected by Signal Transducer and Activator of Transcription 1 (STAT1) deficiency have lethal bacterial and viral infections. Complete STAT1 deficiency is inherited as an autosomal recessive disease; partial STAT1 deficiency is inherited as an autosomal recessive or autosomal dominant trait. Here, we report a patient who developed disseminated mycobacteriosis early in life and had several viral infections, including herpetic skin infection and interstitial pneumonia by cytomegalovirus with severe respiratory distress. Molecular analysis of STAT1 showed a novel homozygous mutation affecting a splice site, leading to exon 3 skipping and to synthesis of a lower molecular weight STAT1 protein. This mutation leads to marked reduction of STAT1 phosphorylation; the electromobility shift assay showed a complete defect of DNA-binding activity, which accounts for the complete impairment of peripheral blood mononuclear cell functional response to both IFN-γ and IFN-α. Moreover, analysis of natural killer cells showed a defective STAT1 phosphorylation in response to IFN-α and impaired basal cytolytic activity, suggesting that the STAT1-dependent pathway might be important for natural killer cell function. These results suggested that exon 3 skipping of STAT1 leads to abnormal signaling in response to IFN-γ and IFN-α, which is associated with susceptibility to intracellular pathogens and viruses.


Assuntos
Infecções por Citomegalovirus/genética , Interferon-alfa/imunologia , Interferon gama/imunologia , Mutação , Infecções por Mycobacterium/genética , Fator de Transcrição STAT1/genética , Pré-Escolar , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/imunologia , Humanos , Células Matadoras Naturais/imunologia , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/genética , Doenças Pulmonares Intersticiais/imunologia , Infecções por Mycobacterium/complicações , Infecções por Mycobacterium/imunologia , Splicing de RNA
17.
Immunobiology ; 228(3): 152381, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37086690

RESUMO

Dominant negative mutations in CARD11 have been reported in patients with immune dysregulation, severe atopic features, and variable T cell alterations. Data on Natural killer (NK) cells from affected patients are lacking. We report on a 12-year-old boy with severe atopic dermatitis, food induced anaphylaxis and hypogammaglobulinemia harbouring a novel de novo heterozygous variant c.169G > A; p.Glu57Lys in CARD11. The dominant negative effect of this mutation was confirmed on both CD4+ and CD8+. CTLA4+Foxp3+CD4+ Tregs were severely reduced. Patient's NK cells showed reduced expression of NKp46, NKG2D and CD69. Patient's CD56bright NK cells showed in vitro impaired production of IFN-γ. Steady state pS6 levels on patient's NK cells were increased and remained elevated upon IL2 + IL12 + IL18 overnight stimulation. Overall, the effect of CARD11 mutation on mTORC1 differs between T and NK cells. These findings may explain the increased susceptibility to viral infections and the reduced immune surveillance in affected patients.


Assuntos
Células Matadoras Naturais , Linfócitos T , Masculino , Humanos , Criança , Mutação , Homeostase , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética
20.
Cells ; 11(18)2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36139434

RESUMO

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling.


Assuntos
Proteínas Proto-Oncogênicas c-akt , Rabdomiossarcoma Embrionário , Animais , Criança , Reparo do DNA , Proteína Quinase Ativada por DNA/genética , Desoxiglucose , Doxorrubicina/farmacologia , Glucose , Glicólise , Hexoquinase/metabolismo , Histonas/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Lovastatina , Inibidores de MTOR , Ácido Mevalônico , Oxirredutases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rabdomiossarcoma Embrionário/tratamento farmacológico , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/genética
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