RESUMO
Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r/r ALL setting.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Citotoxicidade Imunológica , Imunoterapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/efeitos adversos , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Estudos Clínicos como Assunto , Terapia Combinada , Desenho de Fármacos , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Modelos Biológicos , Neoplasia Residual/diagnóstico , Neoplasia Residual/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Resultado do TratamentoRESUMO
Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Crise Blástica/tratamento farmacológico , Complexo CD3/imunologia , Citotoxicidade Imunológica , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Linfócitos T/imunologia , Animais , Movimento Celular , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos SCID , Células U937RESUMO
BACKGROUND: Targeted therapy options in HER2-negative breast cancer are limited. This open-label, multicenter phase IB dose-escalation trial was conducted to determine safety, tolerability, and antitumor activity of a combination of docetaxel (Taxotere) and increasing doses of adecatumumab, a human IgG1 antibody targeting epithelial cell adhesion molecule (EpCAM), in EpCAM-positive relapsed or primary refractory advanced-stage breast cancer. PATIENTS AND METHODS: Patients pretreated with up to four prior chemotherapy regimens received increasing adecatumumab doses either every 3 weeks (q3w) or weekly (qw) combined with docetaxel (100 mg/m(2) q3w). Primary end points were safety and tolerability. Antitumor activity was evaluated according to RECIST. Clinical benefit was defined as complete or partial response or stable disease for ≥24 weeks. RESULTS: Thirty-one evaluable patients were treated. Most adverse events were mild to moderate in severity. Neutropenia, leukocytopenia, lymphopenia, and diarrhea (dose-limiting) were the most frequent toxic effects. Maximum tolerated doses of adecatumumab given in combination with docetaxel were 550 mg/m(2) q3w and 360 mg/m(2) qw. Clinical benefit was observed in 44% of patients treated with q3w adecatumumab and docetaxel, increasing to 63% in patients with high EpCAM-expressing tumors. CONCLUSION: Combination therapy of adecatumumab and docetaxel is safe, feasible, and potentially active in heavily pretreated advanced-stage breast cancer.
Assuntos
Antígenos de Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/administração & dosagem , Docetaxel , Esquema de Medicação , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Transtornos Leucocíticos/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Taxoides/administração & dosagem , Resultado do TratamentoRESUMO
OBJECTIVE: A pathogenic role for granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)17 in rheumatoid arthritis (RA) has been suggested. In previously published work, the therapeutic potentials of GM-CSF and IL17 blockade in arthritis have been described. In the present study, the simultaneous blockade of both pathways in a mouse model for chronic arthritis was investigated to identify whether this double blockade provides a superior therapeutic efficacy. METHODS: A chronic relapsing arthritis was induced in C57Bl/6 wild type (WT) and C57Bl/6 genetically deficient for IL17 receptor (IL17R knockout (KO)) mice by intra-articular injection of Streptococcal cell wall (SCW) fragments into knees on days 0, 7, 14 and 21. Treatments (intraperitoneal) were given weekly starting on day 14. Animals were analysed for inflammation, joint damage and a range of inflammatory mediators. RESULTS: Joint swelling and cartilage damage were significantly reduced in the IL17R KO mice and in WT mice receiving anti-GM-CSF neutralising mAb 22E9 compared to isotype control antibodies. The therapeutic effect was significantly more pronounced in mice where IL17 and GM-CSF pathways were inhibited (eg, IL17R KO mice treated with 22E9 mAb). Tumour necrosis factor (TNF)alpha blockade had essentially no effect. CONCLUSION: Our data further support the therapeutic potentials of GM-CSF and IL17 blockade in a RA model that is no longer responsive to an established TNFalpha antagonist, moreover, our results suggest that concomitant inhibition of both pathways may provide the basis for a highly effective treatment of chronic RA in patients that are resistant to treatment by TNFalpha inhibitors.
Assuntos
Artrite Experimental/prevenção & controle , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Interleucina-17/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Cartilagem Articular/patologia , Quimiocina CXCL1/biossíntese , Doença Crônica , Interleucina-1beta/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-17/deficiência , Transdução de Sinais/imunologia , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Although epithelial cell adhesion/activating molecule (EpCAM/CD326) is one of the first tumour-associated antigens identified, it has never received the same level of attention as other target proteins for therapy of cancer. It is also striking that ever since its discovery in the late 1970s the actual contribution of EpCAM to carcinogenesis remained unexplored until very recently. With a First International Symposium on EpCAM Biology and Clinical Application this is now changing. Key topics discussed at the meeting were the frequency and level of EpCAM expression on various cancers and its prognostic potential, the role of EpCAM as an oncogenic signalling molecule for cancer cells, recent progress on EpCAM-directed immunotherapeutic approaches in clinical development and the interaction of EpCAM with other proteins, which may provide a basis for a therapeutic window and repression of its growth-promoting signalling in carcinoma. Future research on EpCAM may benefit from a unified nomenclature and more frequent exchange among those who have been working on this cancer target during the past 30 years and will do so in the future.
Assuntos
Antígenos de Neoplasias/fisiologia , Moléculas de Adesão Celular/fisiologia , Neoplasias/etiologia , Antígenos de Neoplasias/química , Moléculas de Adesão Celular/química , Molécula de Adesão da Célula Epitelial , Humanos , Imunoterapia , Neoplasias/mortalidade , Prognóstico , Transdução de SinaisRESUMO
OBJECTIVE: The pathogenic involvement of granulocyte-macrophage colony-stimulating factor (GM-CSF) in arthritis has been put forward. We have investigated the therapeutic effect of GM-CSF neutralisation in the streptococcal cell wall (SCW) arthritis model in mice. In this model, the pathogenic contribution of tumour necrosis factor (TNF)alpha is minor and is expressed only on joint swelling, whereas cartilage proteoglycan depletion is independent of this cytokine. METHODS: Acute monarthritis was induced by injection of SCW bacterial extracts to mouse knees. Treatments (mAb 22E9 at 300, 100, 30 microg; or Enbrel 300 microg) were given twice intraperitoneally 2 h before and 3 days after disease induction. Swelling was assessed by (99m)Tc uptake into knees on days 1 and 2. Local cytokine levels were determined in patellae washouts on day one. Proteoglycan loss from cartilage was scored on histological sections at termination on day four. RESULTS: Treatment with anti-GM-CSF mAb 22E9 showed a dose-related efficacy by decreasing swelling that was significant at the 300 and 100 microg doses in comparison to isotype control, and comparable to dexamethasone (5 mg/ml). Proteoglycan loss from cartilage was also significantly reduced by mAb 22E9 300 microg (p=0.001). This reduced proteoglycan loss observed after GM-CSF neutralisation was not seen after TNFalpha-blockade with Enbrel. Similarly, levels of interleukin 1beta in joints were reduced after treatment with 22E9 mAb (p=0.003) but not in mice receiving Enbrel. CONCLUSIONS: Our findings show a pathogenic role for GM-CSF in this arthritis model, support the therapeutic potential of neutralising this cytokine, and may indicate therapeutic activity of an anti-GM-CSF mAb in TNFalpha-independent disease situations.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/terapia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Doença Aguda , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Cartilagem Articular/metabolismo , Parede Celular/imunologia , Relação Dose-Resposta Imunológica , Interleucina-1beta/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanas/metabolismo , Streptococcus pyogenes/imunologia , Membrana Sinovial/patologiaRESUMO
Epithelial cell adhesion molecule (Ep-CAM; CD326) is used as a target by many immunotherapeutic approaches, but little data are available about Ep-CAM expression in major human malignancies with respect to level, frequency, tumour stage, grade, histologic tumour type and impact on survival. We analysed by immunohistochemical staining tissue microarrays with 4046 primary human carcinoma samples from colon, stomach, prostate and lung cancers for both frequency and intensity of Ep-CAM expression under highly standardised conditions. A total of 3360 samples were analysable. High-level Ep-CAM expression was observed in 97.7% (n=1186) of colon, 90.7% of gastric (n=473), and 87.2% of prostate cancers (n=414), and in 63.9% of lung cancers (n=1287). No detectable Ep-CAM staining was found with only 0.4% of colon, 2.5% of gastric, 1.9% of prostate cancers, and 13.5% of lung cancers. The only significant correlation of Ep-CAM expression with tumour grading was observed in colon cancer where high-level Ep-CAM expression on grade 3 tumours was down to 92.1% (P<0.0001). Adenosquamous and squamous carcinomas of the lung had a lower percentage of high-level Ep-CAM expression compared to adenocarcinomas with 35.4 and 53.6%, respectively, and with 45.5 and 17.3% of tumours being Ep-CAM negative. With the exception of moderately differentiated colon carcinoma, where patients not expressing Ep-CAM on their tumours showed an inferior survival (P=0.0014), correlation of Ep-CAM expression with survival did not reach statistical significance for any of the other cancer indications and subgroups. In conclusion, the data strongly support the notion that Ep-CAM is a prime target for immunotherapies in major human malignancies. This is because the most common human cancers show (i) a low frequency of Ep-CAM-negative tumours, (ii) a high frequency of Ep-CAM expression on cells of a given tumour, and (iii) for most cancers, an insignificant influence of tumour staging, grading and histology on Ep-CAM expression.
Assuntos
Antígenos de Neoplasias/biossíntese , Moléculas de Adesão Celular/biossíntese , Neoplasias do Colo/genética , Neoplasias Pulmonares/genética , Estadiamento de Neoplasias , Neoplasias da Próstata/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais , Moléculas de Adesão Celular/fisiologia , Neoplasias do Colo/patologia , Molécula de Adesão da Célula Epitelial , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoterapia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/patologia , Análise Serial de Proteínas , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Análise de SobrevidaRESUMO
MT201 is a fully human monoclonal IgG1 antibody with moderate affinity for epithelial cell adhesion molecule (Ep-CAM) being clinically developed for the treatment of carcinomas. Like many other clinically validated IgG1 monoclonal antibodies, MT201 primarily acts by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Here, we analysed ADCC and CDC induced by MT201 and, as reference, trastuzumab against a panel of nine human breast cancer cell lines expressing distinct surface levels of Ep-CAM and human epithelial growth factor receptor type 2 antigen. Maximal cell lysis by ADCC by MT201 and trastuzumab in the presence of peripheral mononuclear cells did not significantly differ when averaged over the nine cell lines, but showed marked differences with respect to individual cell lines. The extent of cell lysis at intermediate surface target density was highly variable, suggesting a dominant influence of other susceptibility factors. Only one breast cancer cell line was eliminated via CDC, but only by MT201. Resistance to CDC appeared to correlate with high expression levels of complement resistance factors. Our present data as well as recent data on the prevalence and prognostic relevance of Ep-CAM expression in metastatic breast cancer suggest that Ep-CAM-specific monoclonal IgG1 antibodies may have a significant therapeutic potential in the treatment of breast cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular/farmacologia , Ativação do Complemento/efeitos dos fármacos , Animais , Anticorpos Monoclonais Humanizados , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Humanos , Receptor ErbB-2/biossíntese , TrastuzumabRESUMO
Recently, we have shown that a novel recombinant bispecific single-chain antibody construct (bscCD19 x CD3), induces highly efficacious lymphoma-directed cytotoxicity mediated by unstimulated peripheral T lymphocytes. Functional analysis of bscCD19 x CD3 has so far been exclusively performed with human B lymphoma cell lines and T cells from healthy donors. Here we analysed the properties of bscCD19 x CD3 using primary B cells and autologous T cells from healthy volunteers or patients with B-cell chronic lymphocytic leukaemia (B-CLL). We show that bscCD19 x CD3 induces T-cell-mediated depletion of nonmalignant B cells in all four cases and depletion of primary lymphoma cells in 22 out of 25 cases. This effect could be observed at low effector-to-target (E:T) ratios and in the majority of cases without additional activation of autologous T cells by IL-2. Even in samples derived from patients heavily pretreated with different chemotherapy regimens, strong cytotoxic effects of bscCD19 x CD3 could be observed. The addition of bscCD19 x CD3 to patients' cells resulted in an upregulation of activation-specific cell surface antigens on autologous T cells and elevated levels of CD95 on lymphoma B cells. Although anti-CD95 antibody CH-11 failed to induce apoptosis in lymphoma cells, we provide evidence that B-CLL cell depletion by bscCD3 x CD3 is mediated at least in part by apoptosis via the caspase pathway.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antígenos CD19/imunologia , Complexo CD3/imunologia , Citotoxicidade Imunológica , Leucemia Linfocítica Crônica de Células B/terapia , Depleção Linfocítica , Linfócitos T/imunologia , Idoso , Idoso de 80 Anos ou mais , Anexina A5/metabolismo , Especificidade de Anticorpos , Linfócitos B/imunologia , Inibidores de Caspase , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Imunoterapia/métodos , Interleucina-2/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
The most effective immediate cure for coronary stenosis is stent-supported angioplasty. Restenosis due to neointima proliferation represents a major limitation. We investigated the expression of 2435 genes in atherectomy specimens and blood cells of patients with restenosis, normal coronary artery specimens, and cultured human smooth muscle cells (SMCs). Of the 223 differentially expressed genes, 37 genes indicated activation of interferon-gamma (IFN-gamma) signaling in neointimal SMCs. In cultured SMCs, IFN-gamma inhibited apoptosis. Genetic disruption of IFN-gamma signaling in a mouse model of restenosis significantly reduced the vascular proliferative response. Our data suggest an important role of IFN-gamma in the control of neointima proliferation.
Assuntos
Perfilação da Expressão Gênica , Interferon gama/fisiologia , Animais , Apoptose/genética , Ciclo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/patologia , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/patologia , Humanos , Interferon gama/genética , Interferon gama/farmacologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Músculo Liso Vascular/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Dendritic cells (DC) generated from peripheral blood monocytes have been used with promising results as a new approach for the immunotherapy of cancer. However, at least four different subpopulations of peripheral blood monocytes have been recognized and their contribution to the generation of functional DC is not known. Recently, the monoclonal antibody M-DC8 has been shown to react with 0.2 - 1 % of blood leukocytes. We have identified M-DC8(+) cells as monocytes which represent about 40 % of CD14(low)CD16(+) monocytes. Similar to M-DC8(-) monocytes, they develop in the presence of GM-CSF and IL-4 into a very homogenous population of cells with DC phenotype and function. M-DC8(+) DC show on average a twofold higher expression of HLA class I and class II molecules than M-DC8(-) DC. These DC produce IL-12p75 both in response to LPS and to CD40 ligation. M-DC8(+) DC induced a strong Th1 immune response and were two to four old more potent than M-DC8(-) DC for the priming of cord blood T cells. M-DC8(+) monocytes can be used as a source of very potent dendritic cells with the potential to significantly improve the efficacy of DC-based immunotherapies.
Assuntos
Anticorpos Monoclonais/imunologia , Células Dendríticas/imunologia , Monócitos/imunologia , Antígenos CD/biossíntese , Antígeno B7-2 , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Reagentes de Ligações Cruzadas , Células Dendríticas/classificação , Sangue Fetal/citologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Humanos , Imunofenotipagem , Interleucina-12/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Monócitos/classificação , Monócitos/citologia , Receptores de Quimiocinas/biossíntese , Receptores de IgG/imunologia , Coloração e Rotulagem , Subpopulações de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Regulação para CimaRESUMO
BACKGROUND: Restenosis due to neointima formation is the major limitation of stent-supported balloon angioplasty. Despite abundant animal data, molecular mechanisms of neointima formation have been investigated on only a limited basis in patients. This study sought to establish a method for profiling gene expression in human in-stent neointima and to identify differentially expressed genes that may serve as novel therapeutic targets. METHODS AND RESULTS: We retrieved tissue specimens from patients with symptomatic in-stent restenosis using a novel helix cutter atherectomy device. cDNA samples prepared from neointima (n=10) and, as a control, from the media of normal arteries (n=14) were amplified using a novel polymerase chain reaction protocol and hybridized to cDNA arrays. Immunohistochemistry characterized the atherectomy material as neointima. cDNA arrays readily identified differentially expressed genes. Some of the differentially expressed genes complied with expected gene expression patterns of neointima, including downregulation of desmin and upregulation of thrombospondin-1, cyclooxygenase-1, and the 70-kDa heat shock protein B. Additionally, we discovered previously unknown gene expression patterns, such as downregulation of mammary-derived growth inhibitor and upregulation of FK506-binding protein 12 (FKBP12). Upregulation of FKBP12 was confirmed at the protein level in neointimal smooth muscle cells. CONCLUSIONS: Gene expression patterns of human neointima retrieved by helix-cutter atherectomy can be reliably analyzed by cDNA array technology. This technique can identify therapeutic targets in patients, as exemplified by the findings regarding FKBP12. FKBP12 is the receptor for Rapamycin (sirolimus), which in animal models reduced neointima formation. Our study thus yields a rationale for the use of Rapamycin to prevent restenosis in patients.
Assuntos
Constrição Patológica/genética , Proteína 1A de Ligação a Tacrolimo/genética , Túnica Íntima/patologia , Idoso , Aterectomia Coronária , Constrição Patológica/etiologia , Constrição Patológica/metabolismo , Constrição Patológica/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Recidiva , Reprodutibilidade dos Testes , Stents/efeitos adversos , Túnica Média/patologia , Regulação para CimaRESUMO
The transcription factor, nuclear factor-kappaB (NF-kappaB) has been studied extensively due to its prominent role in the regulation of immune and inflammatory genes, apoptosis, and cell proliferation. It has been known for more that a decade that NF-kappaB is a redox-sensitive transcription factor. The contribution of redox regulation and the location of potential redox-sensitive sites within the NF-kappaB activation pathway are subject to intense debate due to many conflicting reports. Redox regulation of NF-kappaB has been extensively addressed in this journal and the reader is referred to two comprehensive reviews on the subject [1,2]. With the identification of signaling intermediates proximal to the degradation of the inhibitor, IkappaB, the number of potential redox-sensitive sites is rapidly increasing. The purpose of this review is to address recent insights into the NF-kappaB signaling cascades that are triggered by proinflammatory cytokines such as TNF-alpha and IL-1beta. In addition, the role of nitrogen monoxide (.NO) in the regulation of NF-kappaB will be reviewed. Opportunities for redox regulation that occur upstream of IkappaB-alpha degradation, as well as the potential for redox control of phosphorylation of NF-kappaB subunits, will be discussed. Redox-sensitive steps are likely to depend on the nature of the NF-kappaB activator, the type of reactive oxygen or nitrogen species involved, the selectivity of signaling pathways activated, as well as the cell type under investigation. Lastly, it is discussed how redox regulation of NF-kappaB activation is likely to involve multiple subcellular compartments.
Assuntos
Regulação da Expressão Gênica/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/fisiologia , Transcrição Gênica , Animais , Dimerização , Perfilação da Expressão Gênica , Humanos , Proteínas I-kappa B/fisiologia , Inflamação , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Biológicos , NF-kappa B/química , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/fisiologia , Oxirredução , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/fisiologiaRESUMO
A critical step in the activation of NF-kappa B is the phosphorylation of I kappa Bs by the I kappa B kinase (IKK) complex. IKK alpha and IKK beta are the two catalytic subunits of the IKK complex and two additional molecules, IKK gamma/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-kappa B signaling. IKAP and IKK gamma belong to distinct cellular complexes. Quantitative association of IKK gamma was observed with IKK alpha and IKK beta. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKK gamma or IKAP blocked tumor necrosis factor alpha induction of an NF-kappa B-dependent reporter construct, but IKAP in addition affected several NF-kappa B-independent promoters. Whereas specific down-regulation of IKK gamma protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-kappa B activation, a similar reduction of IKAP protein levels had no effect on NF-kappa B signaling. Using solely IKK alpha, IKK beta, and IKK gamma, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKK gamma is a stoichiometric component of the IKK complex, obligatory for NF-kappa B signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-kappa B activation.
Assuntos
Proteínas de Transporte/análise , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Quinase I-kappa B , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Especificidade por Substrato , Fatores de Elongação da TranscriçãoRESUMO
The pleiotropic transcription factor NF-kappaB is localized in the cytoplasm bound to its inhibitory subunit IkappaB. The predominant form of NF-kappaB is a p50/p65 heterodimer which can be released from IkappaB-alpha and migrate to the nucleus. Previous studies have shown that IkappaB-alpha-/- mice die 8 to 10 days postnatally, showing runting and a severe dermatitis. However, the organ distribution of mouse IkappaB-alpha, the exon-intron structure, and the chromosomal localization of ikba have not been determined so far. A mouse Sv129 genomic DNA library was screened with a human IkappaB-alpha/MAD-3 cDNA probe. One clone (P1) was isolated, spanning the complete ikba gene and the promoter/enhancer region. We show that the exon-intron structure between mouse and pig ikba is completely conserved. In contrast to human ikba, the ankyrin repeat 5 is not interrupted by an intron. Furthermore, the mouse ikba promoter contains 6 putative NF-kappaB binding sequences, which are conserved in mouse, pig, and human, underlining the importance of NF-kappaB as a key regulator of ikba transcription. The deduced amino acid sequence shows >90% similarity between mouse, pig, and human ikba. Chromosome mapping localized the mouse ikba gene to chromosome 12. Northern blot analysis demonstrated predominant expression in lymphoid tissue (lymph node and thymus). However, IkappaB-alpha mRNA was detected as well in liver tissue, the gastrointestinal tract, and the reproductive tract. The cloning and determination of the structure are a prerequisite for the construction of vectors for conditional gene targeting experiments.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas I-kappa B , NF-kappa B/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Coloração Cromossômica , Sequência Conservada , Éxons , Dosagem de Genes , Biblioteca Genômica , Humanos , Íntrons , Camundongos , Inibidor de NF-kappaB alfa , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Suínos/genéticaRESUMO
Hypoxia, reoxygenation, and the tyrosine phosphatase inhibitor pervanadate activate the transcription factor NF-kappaB, involving phosphorylation of its inhibitor IkappaB-alpha on tyrosine 42. This modification does not lead to degradation of IkappaB by the proteasome/ubiquitin pathway, as is seen on stimulation of cells with proinflammatory cytokines. It is currently unknown how tyrosine-phosphorylated IkappaB is removed from NF-kappaB. Here we show that p85alpha, the regulatory subunit of PI3-kinase, specifically associates through its Src homology 2 domains with tyrosine-phosphorylated IkappaB-alpha in vitro and in vivo after stimulation of T cells with pervanadate. This association could provide a mechanism by which newly tyrosine-phosphorylated IkappaB is sequestered from NF-kappaB. Another mechanism by which PI3-kinase contributed to NF-kappaB activation in response to pervanadate appeared to involve its catalytic p110 subunit. This was evident from the inhibition of pervanadate-induced NF-kappaB activation and reporter gene induction by treatment of cells with nanomolar amounts of the PI3-kinase inhibitor wortmannin. The compound had virtually no effect on tumor necrosis factor- and interleukin-1-induced NF-kappaB activities. Wortmannin did not inhibit tyrosine phosphorylation of IkappaB-alpha or alter the stability of the PI3-kinase complex but inhibited Akt kinase activation in response to pervanadate. Our data suggest that both the regulatory and the catalytic subunit of PI3-kinase play a role in NF-kappaB activation by the tyrosine phosphorylation-dependent pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas I-kappa B , Células Jurkat , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Ativação Transcricional/genética , Fator de Necrose Tumoral alfa/farmacologia , Vanadatos/farmacologia , Wortmanina , Domínios de Homologia de src/genéticaAssuntos
Proteínas de Ligação a DNA/química , Inflamação , NF-kappa B/química , Anquirinas/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas I-kappa B , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B , Conformação Proteica , Fator de Transcrição RelARESUMO
We have shown that an estradiol-dependent activation of human T cell leukemia virus-I Tax leads to the inhibition of cell proliferation and to the induction of apoptosis. The present study demonstrates that a hormone-dependent activation of Tax promotes an enhanced prooxidant state in stably transfected Jurkat cells as measured by changes in the intracellular levels of glutathione and H2O2; these changes are followed by apoptotic cell death. Additional stimulation of the CD3/TCR pathway enhances the oxidative and apoptotic effects. Both Tax-mediated apoptosis and oxidative stress can be potently suppressed by antioxidants, as is seen with the administration of recombinant thioredoxin (adult T cell leukemia-derived factor) or pyrrolidine dithiocarbamate. Hormone-induced Tax activation induces a long-lasting activation of NF-kappaB, which is a major target of reactive oxygen intermediates. The long-term exposure of Jurkat cells to hormone eventually results in a selection of cell clones that have lost Tax activity. A subsequent transfection of these apparently "nonresponsive" clones allows the recovery of Tax responses in these cells. Our observations indicate that changes in the intracellular redox status may be a determining factor in Tax-mediated DNA damage, apoptosis, and selection against the long-term expression of Tax function.
Assuntos
Apoptose/imunologia , Produtos do Gene tax/fisiologia , Líquido Intracelular/metabolismo , Ativação Linfocitária , Oxidantes/metabolismo , Linfócitos T/metabolismo , Anticorpos Monoclonais/imunologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Complexo CD3/imunologia , Sinergismo Farmacológico , Estradiol/farmacologia , Produtos do Gene tax/biossíntese , Produtos do Gene tax/efeitos dos fármacos , Produtos do Gene tax/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/biossíntese , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , TransfecçãoRESUMO
The transcription factor NF-kappaB coordinates the activation of numerous genes in response to pathogens and pro-inflammatory cytokines, and is, therefore, vital in the development of acute and chronic inflammatory diseases. NF-kappaB is activated by phsophorylation of its inhibitory subunit, IkappaB-alpha, on serine residues 32 and 36 by cytokine-activated IKB kinases (IKKs); this phosphorylation precedes rapid degradation of IkappaB. IKK-alpha and IKK-beta isozymes are found in large complexes of relative molecular mass 700,000-900,000 (M(r) 70K-90K), but little is known about other components that organize and regulate these complexes. IKK-alpha was independently discovered as a NF-kappaB-inducing kinase (NIK)-associated protein in a yeast two-hybrid screen, and IKK-beta was also identified by homology screening. It is, however, unknown whether NIK is part of the IKK complex. Here we isolate large, interleukin-1-inducible IKK complexes that contain NIK, IKK-alpha, IKK-beta, IkappaB-alpha, NF-kappaB/RelA and a protein of M(r) 150K. This latter component is a new protein, termed IKK-complex-associated protein (IKAP), which can bind NIK and IKKs and assemble them into an active kinase complex. We show that IKAP is a scaffold protein and a regulator for three different kinases involved in pro-inflammatory cytokine signalling.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Linhagem Celular Transformada , Clonagem Molecular , Indução Enzimática , Quinase I-kappa B , Interleucina-1/metabolismo , Dados de Sequência Molecular , Matriz Nuclear/enzimologia , Fosforilação , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Elongação da Transcrição , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND & AIMS: Transcription factors of the nuclear factor kappaB (NF-kappaB) family play an important role in the regulation of genes involved in inflammation. In inflammatory bowel diseases, proinflammatory cytokines known to be regulated by NF-kappaB are involved. The aim of this study was to investigate the role of NF-kappaB activation during mucosal inflammation in situ. METHODS: A monoclonal antibody, alpha-p65mAb, was applied for immunofluorescence and immunohistochemical analysis that recognizes activated NF-kappaB. Electrophoretic mobility shift assay was used to directly demonstrate the presence of active DNA-binding NF-kappaB. RESULTS: Using the alpha-p65mAb antibody, activated NF-kappaB could be found in biopsy specimens from inflamed mucosa but was almost absent in uninflamed mucosa. The number of cells showing NF-kappaB activation correlated with the degree of mucosal inflammation but was not significantly different between inflamed mucosa from patients with Crohn's disease, ulcerative colitis, and nonspecific colitis or diverticulitis. NF-kappaB activation was localized in macrophages and in epithelial cells as identified by double-labeling techniques. Electrophoretic mobility shift assay with isolated lamina propria mononuclear cells and epithelial cells confirmed these results. CONCLUSIONS: This study shows for the first time the activation of NF-kappaB during human mucosal inflammation in situ. In addition to macrophages, epithelial cells contained activated NF-kappaB, indicating an involvement in the inflammatory process.