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1.
Mol Cancer Ther ; 19(6): 1298-1307, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32229606

RESUMO

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/farmacocinética , Leucócitos Mononucleares/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Animais , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacocinética , Inibidores de Checkpoint Imunológico/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/patologia , Proteína 2 Ligante de Morte Celular Programada 1/antagonistas & inibidores , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Distribuição Tecidual , Testes de Toxicidade
2.
Leukemia ; 33(2): 426-438, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30135465

RESUMO

We investigate here how APRIL impacts immune regulatory T cells and directly contributes to the immunosuppressive multiple myeloma (MM) bone marrow (BM) microenvironment. First, APRIL receptor TACI expression is significantly higher in regulatory T cells (Tregs) than conventional T cells (Tcons) from the same patient, confirmed by upregulated Treg markers, i.e., Foxp3, CTLA-4. APRIL significantly stimulates proliferation and survival of Tregs, whereas neutralizing anti-APRIL monoclonal antibodies (mAbs) inhibit these effects. Besides TACI-dependent induction of cell cycle progression and anti-apoptosis genes, APRIL specifically augments Foxp3, IL-10, TGFß1, and PD-L1 in Tregs to further enhance Treg-inhibited Tcon proliferation. APRIL further increases MM cell-driven Treg (iTreg) via TACI-dependent proliferation associated with upregulated IL-10, TGFß1, and CD15s in iTreg, which further inhibits Tcons. Osteoclasts producing APRIL and PD-L1 significantly block Tcon expansion by iTreg generation, which is overcome by combined treatment with anti-APRIL and anti-PD1/PD-L1 mAbs. Finally, APRIL increases IL-10-producing B regulatory cells (Bregs) via TACI on BM Bregs of MM patients. Taken together, these results define novel APRIL actions via TACI on Tregs and Bregs to promote MM cell survival, providing the rationale for targeting APRIL/TACI system to alleviate the immunosuppressive BM milieu and improve patient outcome in MM.


Assuntos
Tolerância Imunológica/imunologia , Terapia de Imunossupressão , Mieloma Múltiplo/imunologia , Osteoclastos/imunologia , Linfócitos T Reguladores/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Humanos , Imunossupressores/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Transdução de Sinais , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética
4.
Front Immunol ; 9: 869, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29760700

RESUMO

Avian influenza A of the subtype H7N9 has been responsible for almost 1,600 confirmed human infections and more than 600 deaths since its first outbreak in 2013. Although sustained human-to-human transmission has not been reported yet, further adaptations to humans in the viral genome could potentially lead to an influenza pandemic, which may have severe consequences due to the absence of pre-existent immunity to this strain at population level. Currently there is no influenza A (H7N9) vaccine available. Therefore, in case of a pandemic outbreak, alternative preventive approaches are needed, ideally even independent of the type of influenza virus outbreak. Bacillus Calmette-Guérin (BCG) is known to induce strong heterologous immunological effects, and it has been shown that BCG protects against non-related infection challenges in several mouse models. BCG immunization of mice as well as human induces trained innate immune responses, resulting in increased cytokine responses upon subsequent ex vivo peripheral blood mononuclear cell restimulation. We investigated whether BCG (Statens Serum Institut-Denmark)-induced trained immunity may protect against a lethal avian influenza A/Anhui/1/2013 (H7N9) challenge. Here, we show that isolated splenocytes as well as peritoneal macrophages of BCG-immunized BALB/c mice displayed a trained immunity phenotype resulting in increased innate cytokine responses upon ex vivo restimulation. However, after H7N9 infection, no significant differences were found between the BCG immunized and the vehicle control group at the level of survival, weight loss, pulmonary influenza A nucleoprotein staining, or histopathology. In conclusion, BCG-induced trained immunity did not result in protection in an oseltamivir-sensitive influenza A/Anhui/1/2013 (H7N9) challenge mouse model.


Assuntos
Vacina BCG/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinação
5.
PLoS One ; 7(6): e38889, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22719976

RESUMO

BACKGROUND: Fungal components have been shown very effective in generating Th17 responses. We investigated whether exposure to a minute amount of C. albicans in the arthritic joint altered the local cytokine environment, leading to enhanced Th17 expansion and resulting in a more destructive arthritis. METHODOLOGY: Chronic SCW arthritis was induced by repeated injection with Streptococcus pyogenes (SCW) cell wall fragments into the knee joint of C57Bl/6 mice, alone or in combination with the yeast of C. albicans or Zymosan A. During the chronic phase of the arthritis, the cytokine levels, mRNA expression and histopathological analysis of the joints were performed. To investigate the phenotype of the IL-17 producing T-cells, synovial cells were isolated and analyzed by flowcytometry. PRINCIPAL FINDINGS: Intra-articular injection of either Zymosan A or C. albicans on top of the SCW injection both resulted in enhanced joint swelling and inflammation compared to the normal SCW group. However, only the addition of C. albicans during SCW arthritis resulted in severe chondrocyte death and enhanced destruction of cartilage and bone. Additionally, exposure to C. albicans led to increased IL-17 in the arthritic joint, which was accompanied by an increased synovial mRNA expression of T-bet and RORγT. Moreover, the C. albicans-injected mice had significantly more Th17 cells in the synovium, of which a large population also produced IFN-γ. CONCLUSION: This study clearly shows that minute amounts of fungal components, like C. albicans, are very potent in interfering with the local cytokine environment in an arthritic joint, thereby polarizing arthritis towards a more destructive phenotype.


Assuntos
Artrite/imunologia , Candida albicans/fisiologia , Células Th17/imunologia , Animais , Candida albicans/imunologia , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase
6.
J Immunother ; 35(2): 169-78, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22306905

RESUMO

Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4 inhibitory receptor (ipilimumab). Enhanced expression of another inhibitory immunoreceptor, programmed death-1 (PD-1), and its ligand, PD-L1, was found to correlate with a poor prognosis in prostate cancer and melanoma. PD-1-blocking antibodies are being developed to modulate antitumor immune responses. To support preclinical and clinical development of anti-PD-1 therapy, we sought to develop biomarker assays that can detect the effect of PD-1-blocking agents in whole blood and peripheral blood mononuclear cells. In this study, we assessed the effect of PD-1 blockade in modulating super antigen (staphylococcus enterotoxin B)-induced and recall antigen (tetanus toxoid)-induced T-cell reactivity in vitro using whole blood and peripheral blood mononuclear cells from patients with advanced melanoma, prostate cancer, and healthy controls. PD-1 blockade was found to shift antigen-induced cellular reactivity toward a proinflammatory Th1/Th17 response, as evidenced by enhanced production of interferon γ, interleukin (IL)-2, tumor necrosis factor α, IL-6, and IL-17 and reduced production of the Th2 cytokines IL-5 and IL-13. It is interesting to note that suppression of Th2 responsivity was seen with whole blood cells only from patients with cancer. Taken together, we identified novel biomarker assays that might be used to determine the functional consequences of PD-1 blockade in peripheral blood cells from patients with cancer. How these assays translate to the local antitumor response remains to be established in a clinical setting.


Assuntos
Biomarcadores Tumorais/imunologia , Citocinas/imunologia , Melanoma/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias da Próstata/imunologia , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Biomarcadores Tumorais/sangue , Separação Celular , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Masculino , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Evasão Tumoral/imunologia
7.
Arthritis Rheum ; 64(6): 1762-70, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22213107

RESUMO

OBJECTIVE: To provide an intermediate step between classic arthritis models and clinical trials, the rheumatoid arthritis (RA) synovium SCID mouse model is a valuable tool for use during preclinical research. We undertook this study to investigate the validity of this humanized mouse model using anti-tumor necrosis factor (anti-TNF) and anti-interleukin-1 (anti-IL-1) treatment and to investigate the direct effect of T cells- and B cell-related therapies on the transplanted RA synovial tissue. METHODS: CB17/SCID mice were engrafted with human RA synovial tissue and systemically treated with anti-TNF, anti-IL-1, anti-IL-17, CTLA-4Ig, anti-CD20, or isotype control antibodies. RESULTS: Validation of the model with anti-TNF treatment significantly reduced serum cytokine levels and decreased histologic inflammation, whereas anti-IL-1 therapy did not show any effect on the RA synovial grafts. In mice engrafted with B cell-rich synovial tissue, anti-CD20 treatment showed clear therapeutic effects. Surprisingly, CTLA-4Ig treatment did not show any effects in this transplantation model, despite prescreening of the synovial tissue for the presence of CD3+ T cells and the costimulatory molecules CD80 and CD86. In contrast, great therapeutic potential was observed for anti-IL-17 treatment, but only when CD3+ T cells were abundantly present in the RA synovial tissue. CONCLUSION: This human RA synovium SCID mouse model enabled us to show that CTLA-4Ig lacks direct effects on T cell activation processes in the synovial tissue. Further evidence was obtained that IL-17 might indeed be an interesting therapeutic target in RA patients with CD3-rich synovial tissue. Further characterization of the RA patients' individual synovial profiles is of great importance for achieving tailored therapy.


Assuntos
Antirreumáticos/farmacologia , Artrite Reumatoide/tratamento farmacológico , Complexo CD3/imunologia , Imunoconjugados/farmacologia , Interleucina-17/antagonistas & inibidores , Membrana Sinovial/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Abatacepte , Animais , Anticorpos Neutralizantes , Artrite Reumatoide/imunologia , Feminino , Camundongos , Membrana Sinovial/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
8.
Bioorg Med Chem Lett ; 21(12): 3823-7, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21565498

RESUMO

The identification of a potent, selective, and orally available MK2 inhibitor series is described. The initial absence of oral bioavailability was successfully tackled by moving the basic nitrogen of the spiro-4-piperidyl moiety towards the electron-deficient pyrrolepyridinedione core, thereby reducing the pK(a) and improving Caco-2 permeability. The resulting racemic spiro-3-piperidyl analogues were separated by chiral preparative HPLC, and the activity towards MK2 inhibition was shown to reside mostly in the first eluting stereoisomer. This led to the identification of new MK2 inhibitors, such as (S)-23, with low nanomolar biochemical inhibition (EC(50) 7.4 nM) and submicromolar cellular target engagement activity (EC(50) 0.5 µM).


Assuntos
Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Piperidinas/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Compostos de Espiro/síntese química , Administração Oral , Animais , Ligação Competitiva , Disponibilidade Biológica , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular/química , Estrutura Molecular , Piperidinas/química , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Ratos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por Substrato
9.
Arthritis Res Ther ; 12(3): R101, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20497523

RESUMO

INTRODUCTION: The immune modulatory role of estrogens in inflammation is complex. Both pro- and anti-inflammatory effects of estrogens have been described. Estrogens bind both estrogen receptor (ER)alpha and beta. The contribution of ERalpha and ERbeta to ER-mediated immune modulation was studied in delayed type hypersensitivity (DTH) and in experimental arthritis METHODS: ER-mediated suppression of rat adjuvant arthritis (AA) was studied using ethinyl-estradiol (EE) and a selective ERbeta agonist (ERB-79). Arthritis was followed for 2 weeks. Next, effects of ER agonists (ethinyl-estradiol, an ERalpha selective agonist (ERA-63) and a selective ERbeta agonist (ERB-79) on the development of a tetanus toxoid (TT)-specific delayed type hypersensitivity response in wild type (WT) and in ERalpha- or ERbeta-deficient mice were investigated. Finally, EE and ERA-63 were tested for their immune modulating potential in established collagen induced arthritis in DBA/1J mice. Arthritis was followed for three weeks. Joint pathology was examined by histology and radiology. Local synovial cytokine production was analyzed using Luminex technology. Sera were assessed for COMP as a biomarker of cartilage destruction. RESULTS: EE was found to suppress clinical signs and symptoms in rat AA. The selective ERbeta agonist ERB-79 had no effect on arthritis symptoms in this model. In the TT-specific DTH model, EE and the selective ERalpha agonist ERA-63 suppressed the TT-specific swelling response in WT and ERbetaKO mice but not in ERalphaKO mice. As seen in the AA model, the selective ERbeta agonist ERB-79 did not suppress inflammation. Treatment with EE or ERA-63 suppressed clinical signs in collagen induced arthritis (CIA) in WT mice. This was associated with reduced inflammatory infiltrates and decreased levels of proinflammatory cytokines in CIA joints. CONCLUSIONS: ERalpha, but not ERbeta, is key in ER-mediated suppression of experimental arthritis. It remains to be investigated how these findings translate to human autoimmune disease.


Assuntos
Artrite Experimental/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Inflamação/metabolismo , Animais , Artrite Experimental/tratamento farmacológico , Citocinas/metabolismo , Modelos Animais de Doenças , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Etinilestradiol/análogos & derivados , Etinilestradiol/uso terapêutico , Feminino , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Ratos , Membrana Sinovial/metabolismo , Toxoide Tetânico/efeitos adversos
10.
J Immunol ; 183(6): 4127-34, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19717518

RESUMO

CD97 is a member of the EGF-TM7 family of adhesion class receptors, with a proposed role in inflammatory cell recruitment. Neutralization of murine CD97 with the anti-mCD97 mAb 1B2 was efficacious in prevention of murine collagen-induced arthritis, a model with features resembling rheumatoid arthritis. Here, the therapeutic potential of neutralizing CD97 in arthritis was studied with emphasis on the 1B2 pharmacokinetics. Mice with established arthritis were treated with anti-mCD97 or anti-TNF-alpha serum. Ab pharmacokinetics and biodistribution were studied in diseased and nondiseased mice using labeled 1B2. The impact of CD97 expression on Ab pharmacokinetics was studied using CD97 knockout mice. Treatment with 1B2 showed an efficacy comparable to anti-TNF-alpha treatment. Pharmacokinetic analysis of 1B2 in wild-type and CD97 knockout mice indicated a dose-dependent Ab clearance, due to specific interaction with CD97. Biodistribution studies showed accumulation of 1B2 in spleen and lung. In vitro studies using murine splenocytes revealed that CD97 when bound to Ab was internalized. Moreover, soluble CD97 was detected in the supernatant, suggesting Ag shedding. Finally, in arthritic mice, higher levels of soluble CD97 were found and 1B2 treatment led to specific targeting of inflamed paws, resulting in a higher clearance rate of 1B2 in arthritic mice than in wild-type mice. In conclusion, our data support a therapeutic value of CD97 neutralization in experimental arthritis. The pharmacokinetic profile of the 1B2 Ab illustrates the complexity of Ab elimination from an organism and stresses the importance of understanding Ag-Ab interactions when developing therapeutic mAbs.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/tratamento farmacológico , Glicoproteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/farmacocinética , Antígenos , Relação Dose-Resposta a Droga , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Farmacocinética , Receptores Acoplados a Proteínas G , Baço/metabolismo , Distribuição Tecidual
11.
Ann N Y Acad Sci ; 1069: 401-13, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16855167

RESUMO

For dehydroepiandrosterone (DHEA) both immunosuppressive and immuno-stimulating properties have been described. The immunosuppressive effects may be explained by the conversion of DHEA into androgens and/or estrogens. The described immuno-stimulating effects of DHEA may be due to the conversion of DHEA into 7alpha-hydroxy-DHEA (7alpha-OH-DHEA) by the activity of the p450 enzyme, Cyp7b. 7alpha-OH-DHEA is thought to have anti-glucocoticoid activity preventing the anti-inflammatory action of endogenous glucocorticoids. To investigate a putative role of Cyp7b in the arthritic process, tissues from both the murine collagen-induce arthritis (CIA) model and from patients with rheumatoid arthritis (RA) were studied. We determined the Cyp7b expression levels in synovial tissue and the level of 7alpha-OH-DHEA in both serum and arthritic joints of mice with CIA. Our studies showed that the severity of arthritis correlates with increased Cyp7b activity. Next, we investigated Cyp7b expression and activity in RA patients where the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are known to control the disease process. Fibroblast-like synoviocytes (FLS), isolated from RA synovial biopsies were found to express Cyp7b mRNA. In addition, Cyp7b enzymatic activity was detected in these cells. We also investigated whether Cyp7b activity is regulated by cytokines. Proinflammatory (e.g., TNF-alpha and IL-1beta) cytokines were found to stimulate Cyp7b activity and the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) was found to suppress Cyp7b activity in FLS. Next, we studied which signal transduction pathway is involved in the TNF-alpha-mediated induction of Cyp7b activity in human FLS. The results show a role for nuclear factor kappa B (NFkappaB) and activator protein-1 (AP-1) in the regulation of Cyp7b expression. Finally, we established that the effects of DHEA or 7alpha-OH-DHEA on the immune system can not be explained by glucocorticoid receptor (GR) engagement. The role of the p450 enzyme Cyp7b in DHEA metabolism and its relevance in the arthritic process will be discussed.


Assuntos
Artrite/imunologia , Artrite/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Desidroepiandrosterona/metabolismo , Sistema Endócrino/imunologia , Sistema Endócrino/metabolismo , Animais , Artrite/genética , Linhagem Celular , Colesterol 7-alfa-Hidroxilase/genética , Desidroepiandrosterona/farmacologia , Regulação da Expressão Gênica , Glucocorticoides/antagonistas & inibidores , Glucocorticoides/metabolismo , Humanos , Interleucina-1/genética , Articulações/imunologia , Articulações/metabolismo , Camundongos , NF-kappa B/metabolismo , RNA Mensageiro/genética , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
12.
Arthritis Res Ther ; 7(6): R1271-80, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16277680

RESUMO

Glucocorticoids have successfully been used in the treatment of rheumatoid arthritis. Data suggest that 7alpha-hydroxy-dehydroepiandrosterone (7alpha-OH-DHEA), an immunostimulating metabolite of dehydroepiandrosterone, can block glucocorticoid-induced immune suppression. Formation of 7alpha-OH-DHEA is catalyzed by activity of cytochrome p450 enzyme 7b (Cyp7b). Recently, we reported that tumour necrosis factor (TNF)-alpha, IL-1alpha, IL-1beta and IL-17 enhance Cyp7b mRNA expression and induce a concomitant increase in the formation of 7alpha-OH-DHEA by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis patients. The aim of this study was to elucidate which signal transduction pathway is involved in the TNF-alpha-mediated induction of Cyp7b activity in FLS. We studied the effects of inhibitors of different signal transduction pathways on Cyp7b activity in FLS by measuring Cyp7b mRNA expression using reverse transcription PCR and by measuring the formation of 7alpha-OH-DHEA. We applied SN50, an inhibitor of nuclear translocation of transcription factors (i.e. activator protein-1 [AP-1] and nuclear factor-kappaB [NF-kappaB]); PSI, a proteasome inhibitor that prevents IkappaB degradation and thereby NF-kappaB release; SP600125, a c-Jun N-terminal kinase (JNK) inhibitor; and the mitogen-activated protein kinase inhibitors PD98059 (extracellular signal-regulated kinase) and SB203580 (p38). Cyp7b is constitutively expressed in RA FLS and can be activated in response to TNF-alpha. SN50 and PSI prevented the TNF-alpha-induced increase in Cyp7b activity, whereas the mitogen-activated protein kinase inhibitors PD98059 and SB203580 had no effect. In addition, inhibition of Cyp7b mRNA expression and activity was observed with SN50, PSI and SP600125, suggesting that NF-kappaB and AP-1 induce Cyp7b transcription. These findings suggest that NF-kappaB and AP-1 are involved in the TNF-alpha-enhanced formation of the dehydroepiandrosterone metabolite 7alpha-OH-DHEA. Our results are in accordance with presence of AP-1 and NF-kappaB binding sites in the Cyp7b promoter.


Assuntos
Artrite Reumatoide/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Desidroepiandrosterona/metabolismo , NF-kappa B/metabolismo , Esteroide Hidroxilases/metabolismo , Membrana Sinovial/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Bases , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Família 7 do Citocromo P450 , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esteroide Hidroxilases/genética , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Transcrição Gênica/efeitos dos fármacos
13.
Arthritis Rheum ; 52(3): 770-8, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15751070

RESUMO

OBJECTIVE: The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collagen-induced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7alpha-OH-DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: The presence of 7alpha-OH-DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7alpha-OH-DHEA with the use of high-performance liquid chromatography. RESULTS: The CYP7B enzyme product 7alpha-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-17 up-regulated CYP7B activity in an FLS cell line 2-10-fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor beta inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFalpha increased CYP7B activity in all cell lines tested. CONCLUSION: Exposure to the proinflammatory cytokines TNFalpha, IL-1alpha, IL-1beta, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7alpha-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.


Assuntos
Artrite Reumatoide/imunologia , Sistema Enzimático do Citocromo P-450/biossíntese , Desidroepiandrosterona/análogos & derivados , Desidroepiandrosterona/imunologia , Esteroide Hidroxilases/biossíntese , Membrana Sinovial/imunologia , Linhagem Celular , Sistema Enzimático do Citocromo P-450/imunologia , Família 7 do Citocromo P450 , Citocinas/imunologia , Citocinas/fisiologia , Desidroepiandrosterona/metabolismo , Regulação para Baixo , Humanos , Interleucina-1/fisiologia , Interleucina-10/fisiologia , Esteroide Hidroxilases/imunologia , Líquido Sinovial/metabolismo , Membrana Sinovial/citologia , Fator de Crescimento Transformador beta/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima
14.
Arthritis Rheum ; 50(10): 3346-53, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15476247

RESUMO

OBJECTIVE: The endogenous steroid dehydroepiandrosterone (DHEA) has been reported to play a role in rheumatoid arthritis (RA). DHEA is metabolized by the P450 enzyme CYP7B into 7alpha-OH-DHEA, which has immunostimulating properties. This study was undertaken to investigate the putative role of CYP7B in arthritis using murine collagen-induced arthritis (CIA), an interleukin-1beta (IL-1beta)-dependent model. METHODS: DBA/1J mice were immunized and administered a booster with type II collagen. The presence of 7alpha-OH-DHEA was determined in both arthritic and nonarthritic joints and the serum of CIA mice by radioimmunoassay. CYP7B messenger RNA (mRNA) expression was analyzed in synovial biopsy samples, and in fibroblast-like synoviocytes (FLS) isolated from these synovial biopsy samples, by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, the regulatory role of IL-1beta on CYP7B activity in FLS was determined using RT-PCR, Western blotting, and high-performance liquid chromatography. RESULTS: In knee joint synovial biopsy samples from arthritic mice, 7alpha-OH-DHEA levels were 5-fold higher than in nonarthritic mice. Elevated levels of 7alpha-OH-DHEA were accompanied by an increase in CYP7B mRNA expression and were positively correlated with disease severity. In serum, no differences in 7alpha-OH-DHEA levels were observed between arthritic and nonarthritic mice. Incubation of FLS with IL-1beta resulted in a dose-dependent increase in 7alpha-OH-DHEA formation. In addition, IL-1beta enhanced CYP7B mRNA and CYP7B protein levels in FLS. CONCLUSION: Disease progression in CIA is correlated with enhanced CYP7B activity, which leads to locally enhanced 7alpha-OH-DHEA levels. Elevated IL-1beta levels within the arthritic joint may regulate this increase in CYP7B activity.


Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Desidroepiandrosterona/metabolismo , Interleucina-1/fisiologia , Esteroide Hidroxilases/metabolismo , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/enzimologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/enzimologia , Western Blotting , Cromatografia Líquida de Alta Pressão , Colágeno , Sistema Enzimático do Citocromo P-450/genética , Masculino , Camundongos , Camundongos Endogâmicos DBA , RNA Mensageiro/análise , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Esteroide Hidroxilases/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia
15.
Rouxs Arch Dev Biol ; 201(5): 275-283, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28305831

RESUMO

Lucifer Yellow-Dextran labelling of lower layer cells (LLC), sometimes together with upper layer cells (ULC), of the 64-cellBarbus conchonius embryo resulted in labelled primordial germ cells (PGCs) at 12 h after fertilization (a.f.) in about 25% of cases. The presence of labelled PGCs was independent of the location of the injected blastomere with respect to the later orientation of the embryonic axis. After injection of an ULC alone, however, labelled PGCs were never found. Also, the distribution of labelled somatic cells differed between the ULC- and LLC-injected embryos. When we found fluorescent PGCs, only a few of them were labelled, suggesting that either a single predecessor exists earlier than the 64-cell stage or that the formation of germ cells is a polyclonal process. Tracing the fluorescent cells at successive stages of development shows an extensive mixing with unlabelled cells during the epiboly stage, which might well be the cause of partly unpredictable cell lineages. The chance of being committed to a specific fate is different for the ULC and LLC descendants. This might be due to relatively limited cell mixing between these two cell populations.

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