RESUMEN
Background: The KIT receptor tyrosine kinase and its ligand, stem cell factor (SCF), control proliferation and survival of mast cells. Thus, targeting KIT signaling may show promise for the treatment of allergic diseases involving mast cells. Recently, we discovered a new compound, MOD000001, as a potential small-molecule KIT kinase inhibitor by using an in silico approach. Objective: We sought to determine whether MOD000001 is highly selective to KIT, inhibits KIT signaling in mast cells, and affects IgE-mediated mast cell activation. Methods: The interaction of MOD000001 with 468 human kinases and its inhibitory activity against KIT were profiled and evaluated by using KINOMEscan (Discover X/Eurofins Corporation, Fremont, Calif) and cell-free kinase assays, respectively. The effects of MOD000001 on SCF-dependent signaling were examined by using primary mouse and human mast cells. The effects of MOD000001 on SCF-induced degranulation and passive cutaneous anaphylaxis reaction were examined in mice. Results: MOD000001 interacted with KIT and inhibited KIT kinase activity with high selectivity. MOD000001 suppressed SCF-induced KIT signaling in mouse and human mast cells and in mice. Passive cutaneous anaphylaxis reaction was suppressed in mice treated with MOD000001 both for a short-term (1 week) and for a long-term (7 weeks). Mice treated with MOD000001 for a long-term, but not for a short-term, showed skin mast cell reduction. Conclusions: MOD000001 is a highly selective KIT inhibitor that can suppress IgE-mediated mast cell activation in vivo. MOD000001 may do so by reducing tissue mast cell numbers or by other unknown mechanisms. The findings suggest potential benefits of MOD000001 for allergic diseases involving IgE-mediated mast cell activation.
RESUMEN
Mutations in MEK1/2 have been described as a resistance mechanism to BRAF/MEK inhibitor treatment. We report the discovery of a novel ATP-competitive MEK1/2 inhibitor with efficacy in wildtype (WT) and mutant MEK12 models. Starting from a HTS hit, we obtained selective, cellularly active compounds that showed equipotent inhibition of WT MEK1/2 and a panel of MEK1/2 mutant cell lines. Using a structure-based approach, the optimization addressed the liabilities by systematic analysis of molecular matched pairs (MMPs) and ligand conformation. Addition of only three heavy atoms to early tool compound 6 removed Cyp3A4 liabilities and increased the cellular potency by 100-fold, while reducing log P by 5 units. Profiling of MAP855, compound 30, in pharmacokinetic-pharmacodynamic and efficacy studies in BRAF-mutant models showed comparable efficacy to clinical MEK1/2 inhibitors. Compound 30 is a novel highly potent and selective MEK1/2 kinase inhibitor with equipotent inhibition of WT and mutant MEK1/2, whose drug-like properties allow further investigation in the mutant MEK setting upon BRAF/MEK therapy.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
Alterations in the gene encoding for the FGFR and upregulation of the VEGFR are found often in cancer, which correlate with disease progression and unfavorable survival. In addition, FGFR and VEGFR signaling synergistically promote tumor angiogenesis, and activation of FGFR signaling has been described as functional compensatory angiogenic signal following development of resistance to VEGFR inhibition. Several selective small-molecule FGFR kinase inhibitors are currently in clinical development. ODM-203 is a novel, selective, and equipotent inhibitor of the FGFR and VEGFR families. In this report we show that ODM-203 inhibits FGFR and VEGFR family kinases selectively and with equal potency in the low nanomolar range (IC50 6-35 nmol/L) in biochemical assays. In cellular assays, ODM-203 inhibits VEGFR-induced tube formation (IC50 33 nmol/L) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50-150 nmol/L). In vivo, ODM-203 shows strong antitumor activity in both FGFR-dependent xenograft models and in an angiogenic xenograft model at similar well-tolerated doses. In addition, ODM-203 inhibits metastatic tumor growth in a highly angiogenesis-dependent kidney capsule syngenic model. Interestingly, potent antitumor activity in the subcutaneous syngenic model correlated well with immune modulation in the tumor microenvironment as indicated by marked decrease in the expression of immune check points PD-1 and PD-L1 on CD8 T cells and NK cells, and increased activation of CD8 T cells. In summary, ODM-203 shows equipotent activity for both FGFR and VEGFR kinase families and antitumor activity in both FGFR and angigogenesis models.
Asunto(s)
Antígeno B7-H1/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Linfocitos T/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Neoplasias Renales/metabolismo , Células Asesinas Naturales/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) significantly accelerates progression to allograft cirrhosis. Current biochemical parameters to monitor progression of chronic HCV after OLT have yielded low specificity and sensitivity. Here we investigated the HCV-specific immunity and serum levels of soluble CD30 (sCD30), a novel marker of Th2 immunity, in patients with and without allograft cirrhosis. Patients with hepatic inflammation but no cirrhosis (HIN, n=20) revealed elevated serum interferon (IFN)-gamma and high frequency of IFN-gamma producing CD4 T(h1) cells compared to those with hepatic cirrhosis (HFC, n=20) that had high interleukin (IL)-5 and IL-5 producing CD4 T(h2) cells. Patients with HFC, but not HIN, were found to have significantly higher levels of sCD30. Therefore, we conclude that lack of optimal Th1-type CD4 T cells is associated with HCV-induced allograft cirrhosis. Further, sCD30 may represent a novel marker for surveillance of hepatic cirrhosis in transplant recipients with chronic HCV infection.
Asunto(s)
Hepatitis C/sangre , Hepatitis C/cirugía , Antígeno Ki-1/sangre , Cirrosis Hepática/virología , Trasplante de Hígado/efectos adversos , Adulto , Anciano , Antígenos CD/sangre , Biomarcadores/sangre , Femenino , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C/patología , Humanos , Interferón gamma/sangre , Cirrosis Hepática/diagnóstico , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/virología , Trasplante HomólogoRESUMEN
BACKGROUND: Chronic human lung allograft rejection, represented by bronchiolitis obliterans syndrome (BOS), is the single most important factor that limits the long-term survival following lung transplantation (LT). However, the pathogenesis of BOS remains unclear. We hypothesized that the early posttransplant inflammation would promote the development of donor anti-human leukocyte antigen (HLA) alloimmunity and predispose to BOS. METHODS: Serum levels of interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, IP-10, MIG, MCP-1, MIP-1alpha, MIP-1beta, RANTES, tumor necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IFN-gamma, granulocyte-macrophage colony-stimulating factor, IL-1Ralpha, and IL-2R were serially analyzed in 31 BOS+ and matched 31 BOS- patients using quantitative multiplex bead immunoassays. Donor-specific HLA class II cellular immunity was analyzed using enzyme-linked immunospot (ELISPOT) by testing recipient peripheral blood mononuclear cells against mismatched donor HLA-DR peptides. Anti-HLA class II antibodies were monitored using flow panel reactive antibodies. RESULTS: There was early posttransplant elevation in basal serum levels of proinflammatory chemokines IP-10 and MCP-1 and Th1-cytokines IL-1beta, IL-2, IL-12, and IL-15 in BOS+ patients, compared to BOS- and normal subjects. In addition, a threefold decline in IL-10 levels was found during BOS development. BOS+ patients revealed increased development of HLA class II alloantibodies and Th1-predominant donor-specific cellular immunity with high frequency of IFN-gamma and low IL-5 producing T-cells. CONCLUSION: Early posttransplant elevation of proinflammatory mediators is associated with alloimmunity and chronic human lung allograft rejection.
Asunto(s)
Rechazo de Injerto/sangre , Rechazo de Injerto/inmunología , Trasplante de Pulmón/efectos adversos , Trasplante de Pulmón/inmunología , Bronquiolitis Obliterante/sangre , Bronquiolitis Obliterante/inmunología , Enfermedad Crónica , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inflamación/sangre , Inflamación/inmunología , Mediadores de Inflamación/sangre , Mediadores de Inflamación/inmunología , Pulmón , Masculino , Persona de Mediana Edad , Células TH1/metabolismo , Factores de Tiempo , Trasplante Homólogo/efectos adversos , Trasplante Homólogo/inmunologíaRESUMEN
BACKGROUND: In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipient's immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation. METHODS: We retrospectively analyzed seven patients that were transplanted with islets under the auspices of the Juvenile Diabetes Research Foundation and Islet Cell Resource Center/National Institutes of Health. Humoral sensitization towards donor antigens both prior to and following islet transplantation was detected by FLOW panel reactive antibodies (PRA) and donor-specific cellular sensitization was detected by performing enzyme-linked immunospot assay analysis for cytokines interferon-gamma and interleukin-2. RESULTS: Our analysis demonstrates that humoral and cellular sensitization to histocompatibility antigens prior to and after islet transplantation are associated with the failure of transplanted islets CONCLUSION: Patient selection based on sensitization to donor HLA may be one of the factors crucial for the success of islet transplant. Further, in some patients, rejection of islets can be associated with sensitization to mismatched donor histocompatibility antigens.
Asunto(s)
Diabetes Mellitus Tipo 1/cirugía , Histocompatibilidad/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Diabetes Mellitus Tipo 1/sangre , Antígenos HLA/inmunología , Prueba de Histocompatibilidad , Humanos , Insulina/sangre , Trasplante Homólogo , Resultado del TratamientoRESUMEN
Allografts transplanted across ABO incompatibility or human leucocyte antigen (HLA)-sensitization undergoes antibody (Ab) mediated hyperacute rejection. Depleting anti-graft Ab from the recipient by plasmapheresis prior to transplantation can prevent this Ab-mediated rejection. Under these conditions, allografts have been shown to function even when the Ab rebound in the recipients. We have developed an in vitro model using human aortic endothelial cells (EC) and elucidated the ability of W6/32 HLA class I monoclonal Ab to provide signals following binding to MHC class I molecules. Using this model, we show that ECs undergo caspase 3-dependent cell death by apoptosis upon exposure to saturating concentrations of W6/32 and complement. In contrast, exposure of ECs to sub-saturating concentrations of W6/32 conferred resistance towards Ab/complement-mediated lysis that has been termed accommodation. Accommodated ECs exhibited a significant increase in the expression of anti-apoptotic genes Bcl-xL, Bcl-2 and Heme Oxygenase-1 and the induction of Phosphatidylinositol 3 kinase (PI3K) and cyclic adenosine monophosphate (cAMP) dependent protein kinase A activities that facilitate the phosphorylation of Bad at positions Ser(136) and Ser(112). In conclusion, exposure of sub-saturating concentrations of HLA class I Ab results in the induction of signals downstream that confers resistance to endothelial cells against Ab-complement mediated cell death. Together, the observations made in this study will provide the basis for delineating the molecular mechanisms involved in mediating accommodation and developing strategies to induce accommodation in grafts prior to transplantation in highly sensitized patients.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Células Endoteliales/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos HLA/inmunología , Isoanticuerpos/farmacología , Transducción de Señal/efectos de los fármacos , Sistema del Grupo Sanguíneo ABO/inmunología , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Células Cultivadas , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/citología , Células Endoteliales/enzimología , Regulación de la Expresión Génica/inmunología , Rechazo de Injerto/enzimología , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Humanos , Isoanticuerpos/inmunología , Trasplante de Órganos , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/inmunología , Trasplante HomólogoRESUMEN
BACKGROUND: Chronic lung allograft rejection in the form of bronchiolitis obliterans syndrome and its histopathologic correlate, obliterative bronchiolitis (OB), are a major source of morbidity and mortality after lung transplantation. Murine heterotopic tracheal transplants into fully allogeneic mismatched recipients develop obliterative airway disease (OAD), which is a suitable model of OB. Using this murine heterotopic tracheal allograft model, we evaluated the effect of pirfenidone, a novel antifibrotic agent, on the development of OAD. METHODS: Mice transplanted with complete MHC-mismatched tracheal allografts received pirfenidone (0.5%) in pulverized food according to different schedules: daily for the first 14 days after transplantation or daily for the duration of the study beginning on posttransplantation days 0, 5, or 10. RESULTS: Mice on a continuous daily regimen of pirfenidone failed to develop evidence of chronic allograft rejection at the termination of the study (60 days). Mice receiving pirfenidone limited to the early posttransplantation period had delayed onset of OAD to 60 days. Forty percent (2/5) of mice receiving a continuous regimen of pirfenidone beginning on day 5 after transplantation had no evidence of OAD at 28 days. However, when the drug was started on day 10, all mice developed OAD by 28 days. CONCLUSIONS: Our results demonstrate a delay of onset or abrogation of OAD when pirfenidone is administered in the early posttransplantation period. These findings suggest that pirfenidone is a candidate drug to be evaluated for prevention of the fibrotic changes seen in OB in human recipients of lung transplants.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/prevención & control , Piridonas/farmacología , Tráquea/trasplante , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Rechazo de Injerto , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Complicaciones Posoperatorias/tratamiento farmacológico , Complicaciones Posoperatorias/prevención & control , Trasplante HomólogoRESUMEN
BACKGROUND: Presence of donor-specific antibodies (Abs) is detrimental to posttransplant allograft function. Some sensitized recipients have successfully undergone transplantation after pretransplant conditioning regimen using plasmapheresis and/or intravenous immunoglobulin therapy, but underlying mechanisms that confer such allograft protection are undefined. METHODS: We developed a single human leukocyte antigen (HLA)-mismatched heterotopic murine heart transplant model (HLA-A2 into HLA-A2-sensitized-C57BL/6) to determine whether pretreatment of donors with low concentration of HLA class I (W6/32) or control Ab (C1.18.4) will confer protection. Expression levels of survival genes, Bcl-2 and heme oxygenase-1, were analyzed by gene array analysis and quantitative real-time polymerase chain reaction. Expression levels of cytokine panel were analyzed by Luminex. Role of Bcl-2 in the induction of allograft protection was analyzed by silencing the Bcl-2 expression in the donor hearts using a small hairpin (shRNA) specific for Bcl-2. RESULTS: Control Ab-pretreated hearts were rejected in less than 5 days demonstrating hemorrhage, Ab, and C4 deposition. In contrast, W6/32-pretreated hearts were rejected at 15 days (P<0.05) that was prolonged to 25 days with antilymphocyte serum treatment. W6/32-pretreated hearts on day 5 exhibited increased expression of Bcl-2 (5.5-folds), Bcl-xl (5.5-folds), and heme oxygenase-1 (4.4-folds); decreased expression of ICAM-1, VCAM-1 (3.2-fold), along with reduced levels of cytokines interleukin (IL)-1ß (4.4-folds), tumor necrosis factor α (3.7-folds), IL-6 (7.5-folds), IL-12 (2.3-folds) and chemokines monocyte chemotactic protein 1 (4.5-folds), MIG (4.4-folds), MIP-1α (3.4-folds), and IL-8 (3.1-folds). Silencing of Bcl-2 in accommodated hearts before transplant resulted in loss of protection with rejection (9±3 vs. 15±2days, P<0.05). CONCLUSION: Pretreatment of hearts with low levels of anti-HLA Abs increases expression of antiapoptotic genes that inhibits caspases, leading to decreased inflammatory cytokines and chemokines, which promote allograft survival.
Asunto(s)
Anticuerpos Antiidiotipos/uso terapéutico , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Trasplante de Corazón/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Rechazo de Injerto/inmunología , Antígenos HLA/genética , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Factores de TiempoRESUMEN
Anti-HLA class I Abs are associated with the development of bronchiolitis obliterans syndrome (BOS) after lung transplantation. BOS is characterized histologically by fibrosis and airway epithelial cell apoptosis. We have previously shown that anti-HLA class I Abs induce proliferation, growth factor production and apoptosis in airway epithelial cells in vitro. Thus, this study was designed to determine whether anti-HLA class I Abs alone could induce obliterative airway disease (OAD) in heterotopic murine tracheal allografts. Toward this, HLA-A*0201-transgenic tracheal allografts were transplanted into Rag1-deficient mice treated with the W6/32 anti-HLA class I mAb. Allografts were harvested at days +30, +45, +60 and +90. Allografts displayed epithelial metaplasia by day +45, epithelial destruction and mild cellular infiltration by day +60 and complete lumen obliteration and moderate cellular infiltration by day +90. Anti-HLA class I Abs induced the production of several growth factors and growth factor receptors and apoptosis of parenchymal cells in the allograft. In addition, anti-HLA class I Abs induced macrophages and granulocytes infiltration. The results from this study demonstrate that anti-HLA class I Abs play an important role in the pathogenesis of OAD by inducing growth factor production, apoptosis and chemotaxis of inflammatory cells.
Asunto(s)
Anticuerpos/farmacología , Antígenos de Histocompatibilidad Clase I/inmunología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Trasplante/métodos , Animales , Apoptosis , Bronquiolitis Obliterante/etiología , Proliferación Celular , Quimiotaxis , Células Epiteliales/citología , Epitelio/patología , Fibrosis , Granulocitos/metabolismo , Sustancias de Crecimiento/metabolismo , Antígenos HLA-A/genética , Antígeno HLA-A2 , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunohistoquímica , Inflamación , Trasplante de Pulmón/efectos adversos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Tráquea/patología , Trasplante HomólogoRESUMEN
Allografts transplanted across HLA-sensitization results in an antibody-mediated rejection known as hyperacute rejection. Depleting anti-graft antibodies from the recipient by plasmapheresis prior to transplantation can prevent this rejection. We developed an in vitro model using polyclonal HLA class I antibodies obtained from highly sensitized patients awaiting transplantation,and analyzed their ability to provide signals following binding to human aortic endothelial cells (EC). Using this model, we show that EC undergo caspase 3-dependent cell death by apoptosis upon exposure to saturating concentrations of HLA class I antibodies and complement accompanied by loss of Akt activation and phosphorylation of Bad. In contrast, exposure of EC to sub-saturating concentrations of HLA class I antibodies conferred resistance towards antibody/complement-mediated lysis termed accommodation. Accommodated EC exhibited reduction in the expression of the adhesion molecules ICAM-1 and VCAM-1 and a significant increase in the expression of anti-apoptotic genes Bcl-xL, Bcl-2 and heme oxygenase-1. Further, induction of phosphatidylinositol 3-kinase (PI3K) and Akt activities that facilitate the phosphorylation of Bad were also noted. In conclusion, exposure of sub-saturating concentrations of HLA class I antibodies results in the induction of PI3K/Akt pathway that confers resistance to endothelial cells against antibody/complement-mediated cell death.
Asunto(s)
Anticuerpos/inmunología , Proteínas Portadoras/metabolismo , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Adhesión Celular/fisiología , Células Endoteliales/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos , Proteínas Proto-Oncogénicas c-akt , Molécula 1 de Adhesión Celular Vascular/metabolismo , Proteína Letal Asociada a bclRESUMEN
Tropical pulmonary eosinophilia (TPE) is an occult manifestation of filariasis, brought about by helminth parasites Wuchereria bancrofti and Brugia malayi. Treatment of patients suffering from TPE involves the administration of diethyl carbamazine and Ivermectin. Although the drugs are able to block acute inflammation, they are not able to alleviate chronic basal inflammation. We have attempted to examine the disease by targeting two important components; namely filarial parasitic sheath proteins (FPP) induced apoptosis and pro-inflammatory cytokine response in human laryngeal carcinoma cells of epithelial origin (HEp-2) cells an epithelial cell line. Earlier studies by us have shown that FPP exposure induced apoptosis in these cells. In this study with hydrocortisone, calpain inhibitor (ALLN) and phorbol myristate acetate (PMA) treatments we demonstrate that apoptosis is inhibited as shown by [3H] thymidine incorporation studies, propidium iodide staining and Annexin V staining. Hydrocortisone at a dose, which inhibits cell death also down regulated, the expression of pro-inflammatory cytokines IL-6 and IL-8. These findings give us insights into the multifaceted approach one may adopt to target critical signalling molecules using appropriate inhibitors, which could eventually be used to reduce lung damage in TPE.
Asunto(s)
Citocinas/biosíntesis , Sistemas de Liberación de Medicamentos/métodos , Inflamación/tratamiento farmacológico , Eosinofilia Pulmonar/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Línea Celular , Citocinas/antagonistas & inhibidores , Citocinas/genética , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glicoproteínas/administración & dosificación , Humanos , Hidrocortisona/administración & dosificación , Inflamación/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Eosinofilia Pulmonar/metabolismo , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/administración & dosificaciónRESUMEN
BACKGROUND: A novel breast cancer-associated antigen, mammaglobin-A, is expressed in 80% of primary breast tumors. The characterization of immune responses against this highly expressed breast cancer-specific antigen would be of value in the development of new therapeutic strategies for breast cancer. METHODS: We developed an in vivo model using human leukocyte antigen-A*0201/human CD8+ (HLA-A2+/hCD8+) double-transgenic mice to define the epitopes and to study the level of protection acquired by mammaglobin-A cDNA vaccination toward mammaglobin-A+/HLA-A2+ breast cancer cell lines. Mammaglobin-A epitopes were identified using an HLA class I peptide binding prediction computer program, and their activity was verified using gamma interferon ELISPOT and cytotoxicity assays. RESULTS: We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A*0201 molecule (Mam-A2.1-7). CD8+ cytotoxic T lymphocytes (CTLs) from HLA-A2+/hCD8+ mice reacted to the Mam-A2.1 (amino acids [aa] 83-92, LIYDSSLCDL), Mam-A2.2 (aa 2-10, KLLMVLMLA), Mam-A2.4 (aa 66-74, FLNQTDETL), and Mam-A2.6 (aa 32-40, MQLIYDSSL) epitopes. CD8+ CTLs from breast cancer patients also recognized a similar epitope pattern as did those in the HLA-A2+/hCD8 mice and reacted to the Mam-A2.1, Mam-A2.2, Mam-A2.3, Mam-A2.4, and Mam-A2.7 epitopes. Passive transfer of mammaglobin-A-reactive CTLs into SCID (severe combined immunodeficient) beige mice with actively growing mammaglobin-A+ tumors resulted in statistically significant regression (P<.001) in the growth of the tumors. CONCLUSIONS: The HLA-A2+/hCD8+ mouse represents a valuable animal model to characterize the HLA-A*0201-restricted CD8+ CTL immune response to mammaglobin-A in vivo, and the data reported here demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and/or prevention of breast cancer.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/farmacología , Inmunoterapia/métodos , Proteínas de Neoplasias/farmacología , Uteroglobina/farmacología , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , ADN Complementario/uso terapéutico , Modelos Animales de Enfermedad , Epítopos , Femenino , Humanos , Mamoglobina A , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/uso terapéutico , Linfocitos T Citotóxicos/inmunología , Uteroglobina/genética , Uteroglobina/uso terapéuticoRESUMEN
Tropical pulmonary eosinophilia is in part caused by the hyperimmune responsiveness of the lung tissue against the antigens of degenerating microfilariae. We have previously shown that the activation of the transcription factor NF-kappaB is essential for the synthesis and release of multiple pro-inflammatory cytokines in HEp-2 human airway epithelial cells following exposure to filarial parasitic sheath proteins (FPS). Neither the antigenic component nor the receptor involved in this activation is known. Herein we provide evidence that FPS activation of NF-kappaB can be augmented by the cell surface expression of CD14. CD14 expression, however, is not sufficient to transduce FPS signals for NF-kappaB activation, since its expression in different cell types does not always furnish the capacity to respond to FPS. We also show that NF-kappaB activation by FPS treatment can be distinguished from that induced by bacterial lipolysaccharide, an agent that can also activate NF-kappaB in a CD14-dependent fashion. These observations suggest that the capacity of certain lung epithelial cells to interact with microfilarial antigens, activate NF-kappaB in a CD14-dependent manner and produce pro-inflammatory cytokines may be a contributory factor to immune responses manifested by tropical pulmonary eosinophilia.
Asunto(s)
Proteínas I-kappa B , Receptores de Lipopolisacáridos/biosíntesis , FN-kappa B/metabolismo , Animales , Antibacterianos/farmacología , Western Blotting , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Endotoxinas/farmacología , Eosinófilos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Pulmón/microbiología , Ratones , Inhibidor NF-kappaB alfa , Fosforilación , Polimixina B/farmacología , Unión Proteica , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
A breast cancer-associated antigen, mammaglobin-A, is specifically expressed in 80% of primary breast tumors. The definition of immune responses against this highly expressed breast cancer-specific antigen should be of great value in the development of new therapeutic strategies for breast cancer. Thus, the purpose of this study was to identify HLA-A2-restricted mammaglobin-A-derived epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL). We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A2 molecule (Mam-A2.1-7) by means of a HLA class I-peptide binding computer algorithm from the Bioinformatics & Molecular Analysis Section of the National Institutes of Health. Subsequently, we determined that CD8+ CTLs from breast cancer patients reacted to the Mam-A2.1 (83-92, LIYDSSLCDL), Mam-A2.2 (2-10, KLLMVLMLA), Mam-A2.3 (4-12, LMVLMLAAL), Mam-A2.4 (66-74, FLNQTDETL), and Mam-A2.7 (32-40, TINPQVSKT) epitopes using an IFN-gamma ELISPOT assay. Interestingly, healthy individuals also showed high reactivity to the Mam-A2.2 epitope. Two CD8+ CTL lines generated in vitro against TAP-deficient T2 cells loaded with the candidate epitopes showed significant cytotoxic activity against the Mam-A2.1-4 epitopes. These CD8+CTL lines recognized a HLA-A2+breast cancer cell line expressing the Mam-A2.1 epitope. In addition, DNA vaccination of HLA-A2+/human CD8+ double-transgenic mice with a DNA construct encoding the Mam-A2.1 epitope and the HLA-A2 molecule induced a significant expansion of epitope-specific CD8+ CTLs that recognize the same HLA- A2+/Mam-A2.1+ breast cancer cell line. In conclusion, these results demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and prevention of breast cancer.