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1.
J Clin Immunol ; 39(5): 476-485, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31144250

RESUMO

OBJECTIVES: Mutations affecting the TMEM173 gene cause STING-associated vasculopathy with onset in infancy (SAVI). No standard immunosuppressive treatment approach is able to control disease progression in patients with SAVI. We studied the efficacy and safety of targeting type I IFN signaling with the Janus kinase inhibitor, ruxolitinib. METHODS: We used DNA sequencing to identify mutations in TMEM173 in patients with peripheral blood type I IFN signature. The JAK1/2 inhibitor ruxolitinib was administered on an off-label basis. RESULTS: We identified three patients with SAVI presenting with skin involvement and progressive severe interstitial lung disease. Indirect echocardiographic signs of pulmonary hypertension were present in one case. Following treatment with ruxolitinib, we observed improvements of respiratory function including increased forced vital capacity in two patients, with discontinuation of oxygen therapy and resolution of echocardiographic abnormalities in one case. Efficacy was persistent in one patient and only transitory in the other two patients. Clinical control of skin complications was obtained, and one patient discontinued steroid treatment. One patient, who presented with kidney involvement, showed resolution of hematuria. One patient experienced increased recurrence of severe viral respiratory infections. Monitoring of peripheral blood type I interferon signature during ruxolitinib treatment did not show a stable decrease. CONCLUSIONS: We conclude that targeting type I IFN receptor signaling may represent a promising therapeutic option for a subset of patients with SAVI syndrome and severe lung involvement. However, the occurrence of viral respiratory infection might represent an important cautionary note for the application of such form of treatment.

2.
Oncoimmunology ; 7(1): e1378843, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29296542

RESUMO

GD2-redirected chimeric antigen receptor (CAR) T lymphocytes represent a promising therapeutic option for immunotherapy of neuroblastoma (NB). However, despite the encouraging therapeutic effects observed in some hematological malignancies, clinical results of CAR T cell immunotherapy in solid tumors are still modest. Tumor driven neo-angiogenesis supports an immunosuppressive microenvironment that influences treatment responses and is amenable to targeting with antiangiogenic drugs. The latter agents promote lymphocyte tumor infiltration by transiently reprogramming tumor vasculature, and may represent a valid combinatorial approach with CAR T cell immunotherapy. In light of these considerations, we investigated the anti-NB activity of GD2-CAR T cells combined with bevacizumab (BEV) in an orthotopic xenograft model of human NB. Two weeks after tumor implantation, mice received BEV or GD2-CAR T cells or both by single intravenous administration. GD2-CAR T cells exerted a significant anti-NB activity only in combination with BEV, even at the lowest concentration tested, which per se did not inhibit tumor growth. When combined with BEV, GD2-CAR T cells massively infiltrated tumor mass where they produced interferon-γ (IFN-γ), which, in turn, induced expression of CXCL10 by NB cells. IFN-γ, and possibly other cytokines, upregulated NB cell expression of PD-L1, while tumor infiltrating GD2-CAR T cells expressed PD-1. Thus, the PD-1/PD-L1 axis can limit the anti-tumor efficacy of the GD2-CAR T cell/BEV association. This study provides a strong rationale for testing the combination of GD2-CAR T cells with BEV in a clinical trial enrolling NB patients. PD-L1 silencing or blocking strategies may further enhance the efficacy of such combination.

3.
Pharmacol Rep ; 68(3): 654-61, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27026293

RESUMO

BACKGROUND: We describe the potentiation of antiproliferative and apoptotic activities triggered by cis-diamminedichloroplatinum(II) (DDP), and obtained in vitro by the co-administration of procainamide hydrochloride (PdHCl) in murine P388, and human A2780 and A549 cells. METHODS: We determined the antiproliferative and apoptotic activities of DDP and PdHCl combinations by different techniques. Moreover, cell cycle analysis, restriction enzyme inhibition followed by agarose gel electrophoresis, and TUNEL analysis of tumour cells in vivo were also used to strengthen our hypothesis. RESULTS: Our results show that PdHCl may significantly increase the inhibition of cell proliferation and apoptosis. Experiments in vivo showed that the co-administration of DDP and PdHCl increased the percentage of apoptotic cells compared to DDP alone treatment, both in subcutaneous (sc) and intraperitoneal (ip) P388 tumours. We finally demonstrated that the co-administration of PdHCl prevents DNA digestion accounting for a restriction enzyme inhibition that in some cases was greater than that obtained by DDP alone. Moreover, when PdHCl was mixed with the reaction products (RP) of DDP (RP-PdHCl) we obtained a restriction enzyme inhibition greater for some enzymes (Bsp1407I, Hin1II, and Psp1406I) than that obtained by the DDP-PdHCl solution. CONCLUSIONS: On the whole our data demonstrate that the class I antiarrhythmic drug PdHCl may increase the antiproliferative activity of DDP by improving its triggering of apoptosis, and that this phenomenon may be likely linked to the formation of a new Pt compound.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Procainamida/farmacologia , Animais , Antiarrítmicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Camundongos , Mapeamento por Restrição
4.
Immunobiology ; 221(2): 291-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26547104

RESUMO

In a previous study, lack of IL-12 signaling in il12rb2 knock-out mice was found to predispose to lung adenocarcinoma (LAC). We asked whether specific polymorphisms of the human IL12RB2 gene may confer susceptibility to LAC. We studied IL12RB2 single nucleotide polymorphisms (SNPs) spanning from the promoter to the first untranslated exon of the gene. Genotypes of 49 individuals with LAC were compared with those of 93 healthy subjects. Two allele variants were found to be associated with increased susceptibility to LAC. One haplotype (hap), hap18, was more frequent in patients (18%) versus controls (6%) and significantly associated with increased probability of disease occurrence. Furthermore, IL-12 driven STAT4 phosphorylation in T cell blasts from healthy individuals was found to correlate with both single allele variants and haplotypes. In conclusion, genetically determined low signaling activity of IL-12R predisposes to the development of LAC.


Assuntos
Adenocarcinoma/genética , Predisposição Genética para Doença , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-12/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Alelos , Animais , Estudos de Casos e Controles , Éxons , Feminino , Regulação da Expressão Gênica , Frequência do Gene , Haplótipos , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Regiões Promotoras Genéticas , Receptores de Interleucina-12/imunologia , Risco , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Regiões não Traduzidas
5.
Mol Ther ; 21(5): 1034-43, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23481325

RESUMO

Low expression of surface major histocompatibility complex (MHC) class I molecules and defects in antigen processing machinery make human neuroblastoma (NB) cells appropriate targets for MHC unrestricted immunotherapeutic approaches. Human T-cell receptor (TCR) Vγ9Vδ2 lymphocytes exert MHC-unrestricted antitumor activity and are activated by phosphoantigens, whose expression in cancer cells is increased by aminobisphosphonates. With this background, we have investigated the in vivo anti-NB activity of human Vγ9Vδ2 lymphocytes and zoledronic acid (ZOL). SH-SY-5Y human NB cells were injected in the adrenal gland of immunodeficient mice. After 3 days, mice received ZOL or human Vγ9Vδ2 T cells or both agents by intravenous administration once a week for 4 weeks. A significantly improved overall survival was observed in mice receiving Vγ9Vδ2 T cells in combination with ZOL. Inhibition of tumor cell proliferation, angiogenesis and lymphangiogenesis, and increased tumor cell apoptosis were detected. Vγ9Vδ2 T lymphocytes were attracted to NB-tumor masses of mice receiving ZOL where they actively modified tumor microenvironment by producing interferon-γ (IFN-γ), that in turn induced CXCL10 expression in NB cells. This study shows that human Vγ9Vδ2 T cells and ZOL in combination inhibit NB growth in vivo and may provide the rationale for a phase I clinical trial in patients with high-risk NB.


Assuntos
Transferência Adotiva , Difosfonatos/farmacologia , Imidazóis/farmacologia , Neuroblastoma/imunologia , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular Tumoral , Quimiocina CXCL10/metabolismo , Terapia Combinada , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Difosfonatos/administração & dosagem , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Imidazóis/administração & dosagem , Imunofenotipagem , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Neovascularização Patológica , Neuroblastoma/mortalidade , Neuroblastoma/terapia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido Zoledrônico
6.
J Mol Med (Berl) ; 90(9): 1025-35, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22307522

RESUMO

Expansions of a polyalanine (polyA) stretch in the coding region of the PHOX2B gene cause congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by the absence of adequate control of autonomic breathing. Expansion of polyA in PHOX2B leads to protein misfolding and accumulation into inclusions. The mechanisms that regulate mutant protein degradation and turnover have been poorly elucidated. Here, we investigate the regulation of degradation of wild-type and polyA-expanded PHOX2B. We show that expanded PHOX2B is targeted for degradation through the ubiquitin-proteasome system, resulting in lowered levels of the mutant protein relative to its wild-type counterpart. Moreover, we show that mutant PHOX2B forms ubiquitin-positive inclusions, which sequester wild-type PHOX2B. This sequestration correlates with reduced transcriptional activity of endogenous wild-type protein in neuroblastoma cells. Finally, we show that the E3 ubiquitin ligase TRIM11 plays a critical role in the clearance of mutant PHOX2B through the proteasome. Importantly, clearance of mutant PHOX2B by TRIM11 correlates with a rescue of PHOX2B transcriptional activity. We propose that CCHS is partially caused by a dominant-negative effect of expanded PHOX2B due to the retention of the wild-type protein in pathogenic aggregates. Our results demonstrate that TRIM11 is a novel modifier of mutant PHOX2B toxicity and represents a potential therapeutic target for CCHS.


Assuntos
Proteínas de Homeodomínio/metabolismo , Hipoventilação/congênito , Peptídeos/metabolismo , Apneia do Sono Tipo Central/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Células HeLa , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/genética , Humanos , Hipoventilação/genética , Hipoventilação/metabolismo , Proteínas Mutantes/análise , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Apneia do Sono Tipo Central/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas com Motivo Tripartido , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/análise
7.
Neurobiol Dis ; 45(1): 508-18, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21964250

RESUMO

Heterozygous in frame duplications of the PHOX2B gene, leading to polyalanine (polyAla) expansions ranging from +5 to +13 residues of a 20-alanine stretch, have been identified in the vast majority of patients affected with Congenital Central Hypoventilation Syndrome (CCHS), a rare neurocristopathy characterized by absence of adequate autonomic control of respiration with decreased sensitivity to hypoxia and hypercapnia. Ventilatory supports such as tracheostomy, nasal mask or diaphragm pacing represent the only options available for affected. We have already shown that the severity of the CCHS phenotype correlates with the length of polyAla expansions, ultimately leading to formation of toxic intracytoplasmic aggregates and impaired PHOX2B mediated transactivation of target gene promoters, such as DBH. At present, there is no specific treatment to reduce cell aggregates and to ameliorate patients' respiration. In this work, we have undertaken in vitro analyses aimed at assessing the effects of molecules on the cellular response to polyAla PHOX2B aggregates. In particular, we tested 17-AAG, ibuprofen, 4-PBA, curcumin, trehalose, congo red and chrysamine G for their ability to i) recover the nuclear localisation of polyAla expanded PHOX2B, ii) rescue of PHOX2B mediated transactivation of the DBH promoter, and iii) clearance of PHOX2B (+13 Ala) aggregates. Our data have suggested that 17-AAG and curcumin are effective in vitro in both rescuing the nuclear localization and transactivation activity of PHOX2B carrying the largest expansion of polyAla and promoting the clearance of aggregates of these mutant proteins inducing molecular mechanisms such as ubiquitin-proteasome (UPS), autophagy and heat shock protein (HSP) systems.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Hipoventilação/congênito , Peptídeos/genética , Apneia do Sono Tipo Central/genética , Fatores de Transcrição/genética , Animais , Benzoatos/farmacologia , Benzoquinonas/farmacologia , Compostos de Bifenilo/farmacologia , Células COS , Células Cultivadas , Vermelho Congo/farmacologia , Curcumina/farmacologia , Células HeLa , Proteínas de Homeodomínio/metabolismo , Humanos , Hipoventilação/genética , Hipoventilação/metabolismo , Ibuprofeno/farmacologia , Lactamas Macrocíclicas/farmacologia , Peptídeos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Apneia do Sono Tipo Central/metabolismo , Fatores de Transcrição/metabolismo , Trealose/farmacologia
8.
Clin Dev Immunol ; 2011: 730828, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21776288

RESUMO

Glucocorticoid administration before cardiopulmonary bypass (CPB) can reduce the systemic inflammatory response and improve clinical outcome. Long pentraxin PTX3 is a novel inflammatory parameter that could play a protective cardiovascular role by regulating inflammation. Twenty-nine children undergoing open heart surgery were enrolled in the study. Fourteen received dexamethasone (1st dose 1.5 mg/Kg i.v. or i.m. the evening before surgery; 2nd dose 1.5 mg/kg i.v. before starting bypass) and fifteen children served as control. Blood PTX3, short pentraxin C-reactive protein (CRP), interleukin-1 receptor II (IL-1RII), fibrinogen and partial thromboplastin time (PTT) were assayed at different times. PTX3 levels significantly increased during CPB in dexamethasone-treated (+D) and dexamethasone-untreated (-D) subjects, but were significantly higher in +D than -D patients. CRP levels significantly increased both in +D and -D patients in the postoperative days, with values significantly higher in -D than +D patients. Fibrinogen and PTT values were significantly higher in -D than +D patients in the 1st postoperative day. IL-1RII plasma levels increased in the postoperative period in both groups. Dexamethasone prophylaxis in pediatric patients undergoing CPB for cardiac surgery is associated with a significant increase of blood PTX3 that could contribute to decreasing inflammatory parameters and improving patient clinical outcome.


Assuntos
Anti-Inflamatórios/uso terapêutico , Proteína C-Reativa/metabolismo , Ponte Cardiopulmonar , Dexametasona/uso terapêutico , Inflamação/prevenção & controle , Componente Amiloide P Sérico/metabolismo , Feminino , Humanos , Lactente , Inflamação/metabolismo , Mediadores da Inflamação/sangue , Masculino
9.
Cancer Immunol Immunother ; 60(10): 1485-95, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21660451

RESUMO

The high molecular weight melanoma-associated antigen (HMW-MAA) and the cytoplasmic melanoma-associated antigen (cyt-MAA/LGALS3BP) are expressed in melanoma. Their serum levels are increased in melanoma patients and correlate with clinical outcome. We investigated whether these molecules can serve as prognostic markers for neuroblastoma (NB) patients. Expression of cyt-MAA and HMW-MAA was evaluated by flow cytometry in NB cell lines, patients' neuroblasts ((FI)-NB), and short-term cultures of these latter cells (cNB). LGALS3BP gene expression was evaluated by RT-qPCR on (FI)-NB, cNB, and primary tumor specimens. Soluble HMW-MAA and cyt-MAA were tested by ELISA. Cyt-MAA and HMW-MAA were expressed in NB cell lines, cNB, and (FI)-NB samples. LGALS3BP gene expression was higher in primary tumors and cNB than in (FI)-NB samples. Soluble cyt-MAA, but not HMW-MAA, was detected in NB cell lines and cNBs supernatants. NB patients' serum levels of both antigens were higher than those of the healthy children. High cyt-MAA serum levels at diagnosis associated with higher incidence of relapse, independently from other known risk factors. In conclusion, both HMW-MAA and cyt-MAA antigens, and LGALS3BP gene, were expressed by NB cell lines and patients' neuroblasts, and both antigens' serum levels were increased in NB patients. Elevated serum levels of cyt-MAA at diagnosis correlated with relapse, supporting that cyt-MAA may serve as early serological biomarker to individuate patients at higher risk of relapse that may require a more careful follow-up, after being validated in a larger cohort of patients at different time-points during follow-up. Given its immunogenicity, cyt-MAA may also be a potential target for NB immunotherapy.


Assuntos
Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Proteínas de Transporte/sangue , Glicoproteínas/sangue , Neuroblastoma/sangue , Separação Celular , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Neuroblastoma/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
PLoS One ; 5(7): e11763, 2010 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-20668702

RESUMO

BACKGROUND: In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations. METHODOLOGY/PRINCIPAL FINDINGS: T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4(+) T cells, ii) CXCR3 in CD8(+) T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vdelta2gamma9 T cells, and upregulated CXCR4 expression in TCR Vdelta2gamma9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4(+) T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8(+) T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vdelta2gamma9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T(FH)) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T(FH) and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T(FH) cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, beta-arrestin and SHP2 was modulated by sHLA-G treatment. CONCLUSIONS/SIGNIFICANCE: Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Quimiotaxia/efeitos dos fármacos , Antígenos HLA/farmacologia , Antígenos de Histocompatibilidade Classe I/farmacologia , Receptores de Quimiocinas/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Antígenos HLA-G , Humanos , Imuno-Histoquímica , Imunomodulação/efeitos dos fármacos , Imunomodulação/imunologia , Técnicas In Vitro , Tonsila Palatina/citologia , Fosforilação/efeitos dos fármacos , Receptores CCR2/metabolismo , Receptores CXCR3/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR5/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Exp Cell Res ; 316(13): 2152-65, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20471977

RESUMO

Alexander disease is a rare, untreatable and usually fatal neurodegenerative disorder caused by heterozygous mutations of the glial fibrillary acidic protein (GFAP) gene which ultimately lead to formation of aggregates, containing also alphaB-Crystallin, HSP27, ubiquitin and proteasome components. Recent findings indicate that up-regulation of alphaB-Crystallin in mice carrying GFAP mutations may temper the pathogenesis of the disease. Neuroprotective effects of ceftriaxone have been reported in various animal models and, noteworthy, we have recently shown that the chronic use of ceftriaxone in a patient affected by an adult form of Alexander disease could halt its progression and ameliorate some of the symptoms. Here we show that ceftriaxone is able to reduce the intracytoplasmic aggregates of mutant GFAP in a cellular model of Alexander disease. Underlying mechanisms include mutant GFAP elimination, concurrent with up-regulation of HSP27 and alphaB-Crystallin, polyubiquitination and autophagy. Ceftriaxone has also been shown to modulate the proteasome system, thus decreasing NF-kappaB activation and GFAP promoter transcriptional regulation, which further accounts for the down-modulation of GFAP protein levels. These mechanisms provide previously unknown neuroprotective targets of ceftriaxone and confirm its potential therapeutic role in patients with Alexander disease and other neurodegenerative disorders with astrocyte involvement.


Assuntos
Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Proteínas de Choque Térmico HSP27/genética , Cadeia B de alfa-Cristalina/genética , Astrocitoma/tratamento farmacológico , Astrocitoma/metabolismo , Autofagia , Western Blotting , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Técnicas In Vitro , Luciferases/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Multimerização Proteica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Ubiquitina/metabolismo , Cadeia B de alfa-Cristalina/metabolismo
12.
Stem Cells ; 27(3): 693-702, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19096038

RESUMO

The immunomodulatory activities of human mesenchymal stem cells (MSCs) provide a rational basis for their application in the treatment of immune-mediated diseases, such as graft versus host disease and multiple sclerosis. The effects of MSCs on invariant natural killer T (iNKT) and gammadelta T cells, both involved in the pathogenesis of autoimmune diseases, are unknown. Here, we investigated the effects of MSCs on in vitro expansion of these unconventional T-cell populations. MSCs inhibited iNKT (Valpha24(+)Vbeta11(+)) and gammadelta T (Vdelta2(+)) cell expansion from peripheral blood mononuclear cells in both cell-to-cell contact and transwell systems. Such inhibition was partially counteracted by indomethacin, a prostaglandin E(2) inhibitor. Block of indoleamine 2,3-deoxygenase and transforming growth factor beta1 did not affect Valpha24(+)Vbeta11(+) and Vdelta2(+) cell expansion. MSCs inhibited interferon-gamma production by activated Valpha24(+)Vbeta11(+) and impaired CD3-mediated proliferation of activated Valpha24(+)Vbeta11(+) and Vdelta2(+) T cells, without affecting their cytotoxic potential. MSCs did not inhibit antigen processing/presentation by activated Vdelta2(+) T cells to CD4(+) T cells. In contrast, MSCs were lysed by activated Vdelta2(+) T cells through a T-cell receptor-dependent mechanism. These results are translationally relevant in view of the increasing interest in MSC-based therapy of autoimmune diseases.


Assuntos
Citotoxicidade Imunológica/imunologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Células T Matadoras Naturais/imunologia , Linfócitos T/imunologia , Anti-Inflamatórios não Esteroides/farmacologia , Proliferação de Células , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Citometria de Fluxo , Humanos , Indometacina/farmacologia , Interferon gama/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Prostaglandinas E/antagonistas & inibidores , Prostaglandinas E/metabolismo
13.
Eur J Hum Genet ; 16(4): 462-70, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18197187

RESUMO

Alexander disease is a neurological genetic disorder characterized by progressive white-matter degeneration, with astrocytes containing cytoplasmic aggregates, called Rosenthal fibers, including the intermediate filament glial fibrillary acidic protein (GFAP). The age of onset of the disease defines three different forms, infantile, juvenile and adult, all due to heterozygous GFAP mutations and characterized by a progressive less severe phenotype from infantile to adult forms. In an Italian family with a recurrent mild adult onset of Alexander disease, we have identified two GFAP mutations, coupled on a same allele, leading to p.[R330G; E332K]. Functional studies on this complex allele revealed less severe aggregation patterns compared to those observed with p.R239C GFAP mutant, associated with a severe Alexander disease phenotype. Moreover, in addition to confirming the involvement of the ubiquitin-proteasome system in cleaning cells from aggregates and a dominant effect of the novel mutant protein, in cells expressing the mild p.[R330G; E332K] mutant we have observed that indirect alphaB-crystallin overexpression, induced by high extracellular potassium concentration, could completely rescue the correct filament organization while, under the same experimental conditions, in cells expressing the severe p.R239C mutant only a partial rescue effect could be achieved.


Assuntos
Doença de Alexander/genética , Alelos , Proteína Glial Fibrilar Ácida/genética , Mutação , Adulto , Idade de Início , Doença de Alexander/fisiopatologia , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Ubiquitina/metabolismo , Vimentina/metabolismo , Cadeia B de alfa-Cristalina/metabolismo
14.
Cancer Res ; 67(13): 6433-41, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17616704

RESUMO

HLA-G is overexpressed in different tumors and plays a role in immune escape. Because no information is available on HLA-G in relation to human neuroblastoma, we have investigated the expression of membrane-bound and secretion of soluble isoforms of HLA-G in neuroblastoma and functionally characterized their immunosuppressive activities. At diagnosis, serum soluble HLA-G (sHLA-G) levels were significantly higher in patients than in age-matched healthy subjects. In addition, patients who subsequently relapsed exhibited higher sHLA-G levels than those who remained in remission. Neuroblastoma patient sera selected according to high sHLA-G concentrations inhibited natural killer (NK) cell and CTL-mediated neuroblastoma cell lysis. Such lysis was partially restored by serum depletion of sHLA-G. In 6 of 12 human neuroblastoma cell lines, low HLA-G surface expression was not up-regulated by IFN-gamma. Only the ACN cell line secreted constitutively sHLA-G. IFN-gamma induced de novo sHLA-G secretion by LAN-5 and SHSY5Y cells and enhanced that by ACN cells. Primary tumor lesions from neuroblastoma patients tested negative for HLA-G. Neuroblastoma patients displayed a higher number of sHLA-G-secreting monocytes than healthy controls. Incubation of monocytes from normal donors with IFN-gamma or pooled neuroblastoma cell line supernatants significantly increased the proportion of sHLA-G-secreting cells. In addition, tumor cell supernatants up-regulated monocyte expression of CD68, HLA-DR, CD69, and CD71 and down-regulated IL-12 production. Our conclusions are the following: (a) sHLA-G serum levels are increased in neuroblastoma patients and correlate with relapse, (b) sHLA-G is secreted by monocytes activated by tumor cells rather than by tumor cells themselves, and (c) sHLA-G dampens anti-neuroblastoma immune responses.


Assuntos
Regulação Neoplásica da Expressão Gênica , Antígenos HLA/sangue , Antígenos de Histocompatibilidade Classe I/sangue , Neuroblastoma/sangue , Neuroblastoma/imunologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Antígenos HLA-G , Humanos , Sistema Imunitário , Imuno-Histoquímica/métodos , Imunossupressores/farmacologia , Leucócitos Mononucleares/metabolismo , Modelos Biológicos , Monócitos/metabolismo , Neurônios/metabolismo , Recidiva
15.
Int J Biochem Cell Biol ; 39(2): 327-39, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17045833

RESUMO

Polyalanine expansions in the PHOX2B gene have been detected in the vast majority of patients affected with congenital central hypoventilation syndrome, a neurocristopathy characterized by absence of adequate control of breathing, especially during sleep, with decreased sensitivity to hypoxia and hypercapnia. The correlation between length of the alanine expanded tracts and severity of congenital central hypoventilation syndrome respiratory phenotype has been confirmed by length-dependent cytoplasmic PHOX2B retention with formation of aggregates. To deepen into the molecular mechanisms mediating the effects of PHOX2B polyalanine expansions, we have set up experiments aimed at assessing the fate of cells characterized by PHOX2B polyalanine aggregates. In particular, we have observed that activation of the heat shock response by the drug geldanamycin is efficient both in preventing formation and in inducing clearance of PHOX2B pre-formed polyalanine aggregates in COS-7 cells expressing PHOX2B-GFP fused proteins, and ultimately also in rescuing the PHOX2B ability to transactivate the Dopamine-beta-Hydroxilase promoter. In addition, we have demonstrated elimination of PHOX2B mutant proteins by the proteasome and autophagy, two cellular mechanisms already been involved in the clearance of proteins containing expanded polyglutamine and polyalanine tracts. Moreover, our data suggest that geldanamycin effects on PHOX2B aggregates may be also mediated by the proteasome pathway. Finally, analysis of cellular toxicity due to polyalanine aggregates has confirmed the occurrence of cell apoptosis consequent to expression of PHOX2B carrying the longest expanded alanine tract and shown that geldanamycin can delay cell progression toward the most advanced apoptotic stages.


Assuntos
Benzoquinonas/farmacologia , Núcleo Celular/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Lactamas Macrocíclicas/farmacologia , Peptídeos/química , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Autofagia , Benzoquinonas/toxicidade , Células COS , Dopamina beta-Hidroxilase/genética , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Lactamas Macrocíclicas/toxicidade , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Transfecção , Expansão das Repetições de Trinucleotídeos , Ubiquitina/metabolismo
16.
Neoplasia ; 8(10): 833-42, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17032500

RESUMO

Several observations suggest a potential role of T-cell-mediated immunity in the control of neuroblastoma (NB). However, the generation of NB-specific cytotoxic T lymphocytes (CTL) on T-cell priming with tumor mRNA-transfected dendritic cells (DC) has never been investigated before. In the present study, the feasibility of this strategy has been analyzed, both in healthy donors and in NB patients. Monocyte-derived DC were raised from three human leukocyte antigen (HLA) A2+ NB patients and seven HLA-A1+ or HLA-A2+ healthy donors transfected with mRNA from four NB cell lines and cocultured with autologous CD8+ lymphocytes. Expanded CTL expressed an effector/memory phenotype and a T cytotoxic 1-like profile of cytokine secretion. CTL specificity was demonstrated by interferon-gamma release on incubation with HLA-matched NB cell lines. The latter cell lines, but not autologous T-cell blasts, were lysed by CTL in an HLA-restricted manner. Cytotoxicity was found to involve the release of granzyme B. When tested for reactivity against NB-associated antigens, CTL from normal individuals recognized anaplastic lymphoma-associated kinase (ALK) and preferentially expressed antigen of melanoma (PRAME) peptides only, whereas patients' CTL reacted also to survivin, telomerase, and tyrosine hydroxylase peptides. This study demonstrates that DC transfected with NB mRNA induce the generation of patients' CTL specific for different NB-associated antigens, supporting the feasibility of NB T-cell immunotherapy.


Assuntos
Células Dendríticas/fisiologia , Imunoterapia/métodos , Neuroblastoma/imunologia , Neuroblastoma/terapia , Linfócitos T Citotóxicos/imunologia , Quinase do Linfoma Anaplásico , Antígenos de Neoplasias/imunologia , Antígeno HLA-A2 , Humanos , Proteínas Tirosina Quinases/imunologia , RNA Mensageiro , Receptores Proteína Tirosina Quinases , Transfecção , Células Tumorais Cultivadas
17.
Microbes Infect ; 8(2): 552-60, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16324868

RESUMO

The aim of this study was to investigate T cell immunity to Toxoplasma gondii (Tg) in pregnant women with primary toxoplasmosis. This issue has never been addressed before in humans and available information derives from murine models. Peripheral blood mononuclear cells (PBMC) from pregnant women with primary Tg infection were stimulated with Tg tachyzoites, excretory-secretory antigens (ESA) or recombinant surface antigen-1 (rSAG-1), and tested for proliferation, immunophenotype, cytokine production and antigen specific cytotoxic activity. Pregnant women with primary toxoplasmosis displayed a significant decrease of the CD4/CD8 T cell ratio and a significant increase of circulating T cell receptor (TCR) gammadelta+ cells as compared to their uninfected counterparts. T cells from Tg infected pregnant women proliferated to Tg tachyzoites, ESA or rSAG-1. Most tachyzoite and ESA specific T cell blasts were CD4+, whereas SAG-1 specific blasts were CD4+ and CD8+. ESA and tachyzoite specific T cell blasts displayed a Th1 or Th0 cytokine profile with overexpression of IFN-gamma. This pattern was unchanged upon in vitro exposure of T cells to progesterone, tested at a concentration close to that reached in vivo at the maternal-fetal interface. Finally, tachyzoite or ESA specific T cell blasts lysed, through a granule exocytosis dependent mechanism, autologous lymphoblastoid cell lines presenting Tg antigens. In conclusion, pregnant women with primary toxoplasmosis mounted in vitro Tg-specific Th1/Th0 responses whose impact on neonatal infection warrants further investigation.


Assuntos
Ativação Linfocitária , Complicações Parasitárias na Gravidez/imunologia , Linfócitos T/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Relação CD4-CD8 , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Imunofenotipagem , Gravidez , Complicações Parasitárias na Gravidez/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Toxoplasmose/parasitologia
18.
Oncogene ; 24(29): 4634-44, 2005 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-15897905

RESUMO

Low expression of human leukocyte antigen (HLA) class I in human tumors may be related to defects of the antigen-processing machinery (APM) components. Neuroblastoma cells are virtually HLA class I negative, but (i) the underlying mechanisms are unknown, and (ii) expression of the APM components has never been investigated. Here we have used a panel of novel monoclonal antibodies to proteasomal and immunoproteasomal components, chaperons and transporter associated with antigen processing (TAP) to characterize 24 stroma-poor neuroblastoma tumors and six neuroblastoma cell lines. Primary tumors showed defects in the expression of zeta, tapasin, TAP1 or TAP2, HLA class I heavy chain and beta2 microglobulin, LMP2 and LMP7, as compared to normal adrenal medulla. Neuroblastoma cell lines displayed roughly similar patterns of APM expression in comparison to primary tumors. Incubation of neuroblastoma cell lines with interferon-gamma caused upregulation of HLA class I molecules and reduced lysis by killer inhibitory receptor HLA ligand-matched NK cells. Defects in APM components explain reduced peptide loading on HLA class I molecules, their instability and failure to be expressed on the cell surface. HLA class I upregulation by interferon-gamma, although enhancing neuroblastoma cell recognition by cytotoxic T cells, dampens their susceptibility to NK cells.


Assuntos
Antígenos HLA/biossíntese , Interferon gama/biossíntese , Neuroblastoma/imunologia , Anticorpos Monoclonais/imunologia , Criança , Pré-Escolar , Regulação para Baixo , Feminino , Genes MHC Classe I , Humanos , Lactente , Masculino , Neuroblastoma/patologia , Células Estromais , Células Tumorais Cultivadas , Regulação para Cima
19.
J Rheumatol ; 31(10): 2048-54, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15468374

RESUMO

OBJECTIVE: It has been suggested that CD45RO+CD27+ T cells represent recently activated memory cells, whereas CD45RO+CD27- T cells are activated memory T cells in the process of differentiating into effector cells. We investigated (1) CCR7 and CCR5 expression and (2) modulation of cytokine expression in "early" (CD27+) and "differentiated" (CD27-) memory CD4+ T cells from peripheral blood and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA). METHODS: SF CD4+CD45RO+CD27+ and CD27- memory T cells from 6 patients with JIA were tested by flow cytometry for intracellular interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) after in vitro priming with CD3 and CD28 mAb in the presence of IL-4, and subsequent culture with IL-2. RESULTS: SF CD4+CD45RO+CD27+ cells contained higher proportions of CCR7+ (median 46% vs 23%) and lower proportions of CCR5+ (73% vs 90%) cells than paired CD27- T cells. Both CD27+ and CD27- memory T helper cells from SF displayed a higher IFN-gamma/IL-4 ratio than their peripheral blood counterparts. No significant difference was observed in the percentage of IFN-gamma-expressing cells between CD27+ (32%, range 4-47%) and CD27- (29.4%, range 5-52%) memory T helper cells from SF. CONCLUSION: Irrespective of their differentiation stage, both CD27+ and CD27- SF memory T helper cells were found to switch from a proinflammatory to an antiinflammatory pattern of cytokine production.


Assuntos
Artrite Juvenil/imunologia , Citocinas/imunologia , Memória Imunológica , Receptores CCR5/imunologia , Receptores de Quimiocinas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Diferenciação Celular , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária , Masculino , Receptores CCR7 , Líquido Sinovial/citologia , Líquido Sinovial/imunologia , Subpopulações de Linfócitos T , Linfócitos T Auxiliares-Indutores/citologia
20.
Ann N Y Acad Sci ; 1028: 69-80, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15650233

RESUMO

Neuroblastoma (NB) is a neuroectodermal tumor that affects children in the first years of life. Half of NB cases present with metastatic disease at diagnosis and have a poor prognosis, in spite of the most advanced chemotherapeutic protocols combined with autologous hematopoietic stem cell transplantation. Among the new avenues for NB treatment that are being explored, immunotherapy has attracted much interest. Emphasis has been placed on monoclonal antibodies directed to tumor-associated antigens--in particular the disialoganglioside GD2--that have been tested in the clinical setting with promising results. In addition, stimulation of cell-mediated antitumor effector mechanisms have been attempted-for example, by recombinant interleukin (IL)-2 administration. Nonetheless, the issue of the immunogenicity of human NB cells has never been thoroughly addressed. Here we shall review the work carried out in our lab in recent years and show that NB cells express tumor-associated antigens, such as MAGE-3, but lack constitutive expression of costimulatory molecules and surface HLA class I and II molecules. As such, NB cells are likely to be ignored by the host T cell compartment, since expression of HLA and costimulatory molecules on antigen presenting cells are sine qua non conditions for efficient peptide presentation to T cells and for the subsequent activation and clonal expansion of the latter cells. Notably, in vitro experiments with NB cell lines demonstrated that surface HLA class I molecules and the CD40 costimulatory molecule were upregulated following cell incubation with recombinant interferon-gamma. Interaction of CD40 with recombinant CD40 ligand induced apoptosis of NB cells through a caspase 8-dependent mechanism. Collectively, these results indicate that the immunogenicity of human NB cells is very low but suggest that manipulation by cytokine administration or gene transfer can increase their immunogenic potential. On the other hand, NB cells represent an excellent target for natural killer cells, the potential role of which in immunotherapy of NB is now being investigated.


Assuntos
Antígenos de Neoplasias/química , Imunoterapia/métodos , Neuroblastoma/imunologia , Anticorpos Monoclonais/química , Apresentação do Antígeno , Apoptose , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/biossíntese , Antígenos CD40/química , Ligante de CD40/química , Vacinas Anticâncer , Linhagem Celular Tumoral , Citocinas/metabolismo , Gangliosídeos/química , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Interferon gama/química , Interleucina-2/administração & dosagem , Metástase Neoplásica , Proteínas de Neoplasias/química , Neuroblastoma/metabolismo , Neuroblastoma/terapia , Peptídeos/química , Prognóstico
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