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1.
Haematologica ; 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296574

RESUMO

CD38 is expressed in several types of non-Hodgkin lymphoma and constitutes a promising target for antibody-based therapy. Daratumumab (Darzalex) is a first-in-class anti-CD38 antibody approved for the treatment of relapsed/refractory multiple myeloma. It has also demonstrated clinical activity in Waldenstrom macroglobulinaemia and amyloidosis. Here, we have evaluated the activity and mechanism of action of daratumumab in preclinical in vitro and in vivo models of mantle cell lymphoma, follicular lymphoma and diffuse large B cell lymphoma, as monotherapy or in combination with standard chemo-immunotherapy. In vitro, daratumumab engages Fc-mediated cytotoxicity by antibody-dependent cell cytotoxicity and antibody-dependent cell phagocytosis in all lymphoma subtypes. In the presence of human serum, complement-dependent cell cytotoxicity was marginally engaged. We demonstrated by Selective Plane Illumination Microscopy that daratumumab fully penetrated a 3D lymphoma organoid and decreased organoid volume. In vivo, daratumumab completely prevents tumor outgrowth in models of mantle cell and follicular lymphoma, and shows comparable activity to rituximab in a disseminated in vivo model of blastic mantle cell lymphoma. Moreover, daratumumab improves overall survival in a mouse model of transformed CD20dim follicular lymphoma, where rituximab showed limited activity. Daratumumab potentiates the antitumor activity of CHOP and R-CHOP in mantle cell and follicular lymphoma xenografts. Furthermore, in a patient-derived diffuse large B cell lymphoma xenograft model, daratumumab anti-tumor activity was comparable to R-CHOP and the addition of daratumumab to either CHOP or R-CHOP led to full tumor regression. In summary, daratumumab constitutes a novel therapeutic opportunity in certain scenarios and these results warrant further clinical development.

2.
Hum Pathol ; 70: 6-13, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28601659

RESUMO

In endometrioid endometrial carcinomas (EECs), microcystic, elongated, and fragmented (MELF) myoinvasion is associated with easily overlooked lymph node metastases; however, the role of immunohistochemistry in their detection and their clinical significance have not been addressed. We identified MELF in 43 of 101 (43%) myoinvasive EECs. Nodes were removed in 49 (49%), 25 with MELF and 24 without MELF. Metastases were initially reported in 3 of the former (12%) and 2 of the latter (8%). All negative nodes were reviewed, and cytokeratin immunohistochemistry was performed. Three metastases were identified in the MELF group but none in the EECs without MELF. By immunohistochemistry, metastatic nodal isolated tumor cells (ITCs) were found in 6 of the remaining 19 MELF-positive cases. In contrast, lymph node metastases were detected in only 2 of the 22 EECs without MELF. MELF-positive cases had more lymph node metastases (P=.03) than myoinvasive EECs without MELF. At follow-up, all 6 patients with grade 1-2 EECs and nodal ITCs/micrometastases were alive (5 no evidence of disease and 1 with perineal disease). In contrast, 3 of 4 patients with grade 3 EECs and nodal ITCs/micrometastases died of disease, and the other patient was alive with tumor. In MELF, the frequency of ITCs/micrometastases in apparently negative lymph nodes is high. In patients with grade 1-2 EEC who had not received chemotherapy, the presence of nodal ITCs/micrometastases did not affect survival. In contrast, in high-grade tumors, ITCs/micrometastases were associated with unfavorable prognosis. Immunohistochemistry should be done in MELF-positive cases to detect occult lymph node metastases, especially in high-grade tumors.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Endometrioide/química , Carcinoma Endometrioide/secundário , Neoplasias do Endométrio/química , Neoplasias do Endométrio/patologia , Imuno-Histoquímica , Linfonodos/química , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/mortalidade , Carcinoma Endometrioide/terapia , Neoplasias do Endométrio/mortalidade , Neoplasias do Endométrio/terapia , Feminino , Humanos , Linfonodos/patologia , Metástase Linfática , Pessoa de Meia-Idade , Miométrio/química , Miométrio/patologia , Gradação de Tumores , Invasividade Neoplásica , Micrometástase de Neoplasia , Valor Preditivo dos Testes , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento
3.
Hum Pathol ; 56: 180-8, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27346574

RESUMO

Carcinogenesis is a multistep process in which cancer cells and tumor stroma cells play important roles. T lymphocytes are immune constituents of tumor stroma and play a crucial function in anti-tumor response. By immunohistochemistry and flow cytometry, we studied T cytotoxic (CTLs) and T helper lymphocyte distribution and percentage in the tumor microenvironment and peripheral blood from 35 patients with endometrioid endometrial carcinomas (EEC). We also studied 23 healthy donors' blood samples as a control group. Tumor and non-tumoral endometrium samples were obtained. Immunohistochemistry revealed a high number of CTLs and T helper lymphocytes in the tumor stroma of myoinvasive EECs. T lymphocytes were mostly located in the invasive front. By flow cytometry, the percentages of CTLs and T helper lymphocytes were significantly higher in the tumor compared with the non-neoplastic endometrium (P = .0492 and P = .002). The mean fluorescence intensity of CD8 staining was lower in the tumor compared to the non-neoplastic endometrium (P = .001). There was also reduction of the mean fluorescence intensity of CD8 staining on peripheral blood from patients with grade 3 EECs compare to the peripheral blood from healthy donors (P = .0093). No alterations in the expression of granzymes A and B were found in the CTLs from the EEC cases. Finally, in a proteome profiler cytokine array we found that the growth differentiation factor 15 (GDF15) increased in blood in parallel to the tumor grade. EECs are capable of down-regulating CD8 expression of CTLs. Most likely, this effect is mediated by a soluble molecule present in plasma and is not a result of anergy or exhaustion state.


Assuntos
Biomarcadores Tumorais/análise , Antígenos CD8/análise , Carcinoma Endometrioide/imunologia , Neoplasias do Endométrio/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Carcinoma Endometrioide/sangue , Carcinoma Endometrioide/patologia , Citocinas/sangue , Regulação para Baixo , Neoplasias do Endométrio/sangue , Neoplasias do Endométrio/patologia , Feminino , Fator 15 de Diferenciação de Crescimento/sangue , Humanos , Linfócitos do Interstício Tumoral/patologia , Pessoa de Meia-Idade , Gradação de Tumores , Linfócitos T Citotóxicos/patologia , Microambiente Tumoral
4.
J Immunol ; 192(1): 418-26, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24307736

RESUMO

LPS induces the expression of NO synthase 2 (nos2) in macrophages. The expression of this molecule is one of the hallmarks of classical activation. In this paper, we describe that trichostatin A (TSA), which inhibits deacetylase activity, blocks LPS-dependent nos2 expression. TSA specifically inhibits LPS-dependent genes of secondary response, which require new protein synthesis for their induction but not those belonging to the primary response, which do not depend on this process. Deacetylase activity acts at the transcriptional level because RNA polymerase II was not bound after LPS stimulus when we added TSA. A link between the global acetylation caused by HDAC inhibitor and gene promoter recruitment of CDK8 was found. This Mediator complex subunit associates with Med 12, Med13, and cyclin C to form a submodule that is a transcriptional negative regulator. We also found that TSA reduces C/EBPß phosphorylation without affecting its binding to DNA. Taken together, these results shed light on the molecular mechanisms involved in the transcriptional regulation of LPS-treated macrophages and on how TSA targets critical LPS-induced genes, such as nos2 and tnf-α, in inflammatory macrophage response.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Quinase 8 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Inativação Gênica , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a TATA-Box/metabolismo , Iniciação da Transcrição Genética , Transcrição Genética/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Eur J Immunol ; 42(11): 3028-37, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22865229

RESUMO

The amount of arginine available at inflammatory loci is a limiting factor for the growth of several cells of the immune system. IL-4-induced activation of macrophages produced arginase-1, which converts arginine into ornithine, a precursor of polyamines and proline. Trichostatin A (TSA), a pan-inhibitor of histone deacetylases (HDACs), inhibited IL-4-induced arginase-1 expression. TSA showed promoter-specific effects on the IL-4-responsive genes. While TSA inhibited the expression of arginase-1, fizz1, and mrc1, other genes, such as ym,1 mgl1, and mgl2, were not affected. The inhibition of arginase-1 occurred at the transcriptional level with the inhibition of polymerase II binding to the promoter. IL-4 induced STAT6 phosphorylation and binding to DNA. These activities were not affected by TSA treatment. However, TSA inhibited C/EBPß DNA binding. This inhibitor induced acetylation on lysine residues 215-216, which are critical for DNA binding. Finally, using macrophages from STAT6 KO mice we showed that STAT6 is required for the DNA binding of C/EBPß. These results demonstrate that the acetylation/deacetylation balance strongly influences the expression of arginase-1, a gene of alternative activation of macrophages. These findings also provide a molecular mechanism to explain the control of gene expression through deacetylase activity.


Assuntos
Arginase/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Interleucina-4/farmacologia , Macrófagos/imunologia , Acetilação , Animais , Arginase/genética , Arginase/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Ácidos Hidroxâmicos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosforilação , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT6/imunologia , Estatísticas não Paramétricas
7.
Mol Immunol ; 47(4): 825-32, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19897249

RESUMO

The closest region of the promoter of MHC II genes and particularly three conserved boxes (X, Y and S) are fundamental for the transcriptional regulation. A second set of conserved sequences is present approximately 1200-1500 bp upstream in opposite orientation. In transient transfection experiments in IFN-gamma-treated macrophages and in B lymphocytes, we determined the expression of a fragment of 2035 bp of the I-Abeta gene, which contains the upstream boxes. Mutation of the distal boxes increased induction, thereby suggesting a repressive effect on transcription. In vitro, the proximal and distal ends of I-Abeta promoter were ligated in the presence of nuclear extracts from untreated macrophages but not when the extracts were obtained from IFN-gamma-stimulated cells. The mutation of distal or proximal boxes resulted in a decrease in the ligation assay. The addition of recombinant CIITA to untreated nuclear extracts decreased the capacity of the promoter to be ligated. Finally, we observed increased capacity to ligate the promoter in extracts from B cells lacking CIITA, but not from B cells lacking RFXANK. These results allow us to postulate a model where the proteins in the proximal and distal conserved sequences interact. When CIITA is induced, these proteins make an enhanceosome, allowing chromatin to open and initiate transcription.


Assuntos
Antígenos de Histocompatibilidade Classe II/genética , Região de Controle de Locus Gênico/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Animais , Linhagem Celular , Sequência Conservada , Humanos , Camundongos , Modelos Genéticos , Conformação de Ácido Nucleico
8.
J Immunol ; 180(9): 5898-906, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18424709

RESUMO

After interaction with its receptor, GM-CSF induces phosphorylation of the beta-chain in two distinct domains in macrophages. One induces activation of mitogen-activated protein kinases and the PI3K/Akt pathway, and the other induces JAK2-STAT5. In this study we describe how trichostatin A (TSA), which inhibits deacetylase activity, blocks JAK2-STAT5-dependent gene expression but not the expression of genes that depend on the signal transduction induced by the other domain of the receptor. TSA treatment inhibited the GM-CSF-dependent proliferation of macrophages by interfering with c-myc and cyclin D1 expression. However, M-CSF-dependent proliferation, which requires ERK1/2, was unaffected. Protection from apoptosis, which involves Akt phosphorylation and p21(waf-1) expression, was not modified by TSA. GM-CSF-dependent expression of MHC class II molecules was inhibited because CIITA was not induced. The generation of dendritic cells was also impaired by TSA treatment because of the inhibition of IRF4, IRF2, and RelB expression. TSA mediates its effects by preventing the recruitment of RNA polymerase II to the promoter of STAT5 target genes and by inhibiting their expression. However, this drug did not affect STAT5A or STAT5B phosphorylation or DNA binding. These results in GM-CSF-treated macrophages reveal a relationship between histone deacetylase complexes and STAT5 in the regulation of gene expression.


Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Histona Desacetilases/imunologia , Macrófagos/imunologia , Fator de Transcrição STAT5/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D , Inibidor de Quinase Dependente de Ciclina p21/imunologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/imunologia , Ciclinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Fator Regulador 2 de Interferon/imunologia , Fator Regulador 2 de Interferon/metabolismo , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase II/imunologia , RNA Polimerase II/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
J Immunol ; 176(10): 5918-24, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16670299

RESUMO

Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases L-arginine transport only through the y(+) system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in L-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages.


Assuntos
Arginina/metabolismo , Transportador 2 de Aminoácidos Catiônicos/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Transdução de Sinais/imunologia , Animais , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Transportador 2 de Aminoácidos Catiônicos/fisiologia , Células Cultivadas , Ativação de Macrófagos/genética , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout
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