RESUMEN
BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer, whose tumour cells often express CD56. While immune checkpoint inhibitors constitute a major advance for treating patients with MCC with advanced disease, new therapeutic options are still urgently required. OBJECTIVES: To produce and evaluate the therapeutic performance of a new antibody-drug conjugate (Adcitmer® ) targeting CD56 in preclinical models of MCC. METHODS: CD56 expression was evaluated in a MCC cohort (immunohistochemistry on a tissue microarray of 90 tumour samples) and MCC cell lines. Interaction of an unconjugated CD56-targeting antibody with CD56+ MCC cell lines was investigated by immunohistochemistry and imaging flow cytometry. Adcitmer® product was generated by the bioconjugation of CD56-targeting antibody to a cytotoxic drug (monomethyl auristatin E) using the McSAF Inside® bioconjugation process. The chemical properties and homogeneity of Adcitmer® were characterized by hydrophobic interaction chromatography. Adcitmer® cytotoxicity was evaluated in vitro and in an MCC xenograft mice model. RESULTS: Similar to previous reports, CD56 was expressed by 66% of MCC tumours in our cohort, confirming its relevance as a therapeutic target. Specific binding and internalization of the unconjugated CD56-targeting antibody was validated in MCC cell lines. The high homogeneity of the newly generated Adcitmer® was confirmed by hydrophobic interaction chromatography. The CD56-mediated cytotoxicity of Adcitmer® was demonstrated in vitro in MCC cell lines. Moreover, Adcitmer® significantly reduced tumour growth in a MCC mouse model. CONCLUSIONS: Our study suggests that Adcitmer® should be further assessed as a therapeutic option in patients with MCC, as an alternative therapy or combined with immune checkpoint inhibitors.
Asunto(s)
Carcinoma de Células de Merkel , Neoplasias Cutáneas , Animales , Carcinoma de Células de Merkel/tratamiento farmacológico , Carcinoma de Células de Merkel/patología , Humanos , Inmunohistoquímica , Ratones , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Neoplasias Cutáneas/patologíaAsunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Interleucina-10/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/etiología , Leucemia Linfocítica Crónica de Células B/metabolismo , Polimorfismo Genético , Receptores de IgG/genética , Rituximab/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos B/patología , Femenino , Humanos , Masculino , Rituximab/administración & dosificación , Resultado del TratamientoRESUMEN
Monoclonal antibodies (mAbs) are usually delivered systemically, but only a small proportion of the drug reaches the lung after intravenous injection. The inhalation route is an attractive alternative for the local delivery of mAbs to treat lung diseases, potentially improving tissue concentration and exposure to the drug while limiting passage into the bloodstream and adverse effects. Several studies have shown that the delivery of mAbs or mAb-derived biopharmaceuticals via the airways is feasible and efficient, but little is known about the fate of inhaled mAbs after the deposition of aerosolized particles in the respiratory system. We used cetuximab, an anti-EGFR antibody, as our study model and showed that, after its delivery via the airways, this mAb accumulated rapidly in normal and cancerous tissues in the lung, at concentrations twice those achieved after intravenous delivery, for early time points. The spatial distribution of cetuximab within the tumor was heterogeneous, as reported after i.v. injection. Pharmacokinetic (PK) analyses were carried out in both mice and macaques and showed aerosolized cetuximab bioavailability to be lower and elimination times shorter in macaques than in mice. Using transgenic mice, we showed that FcRn, a key receptor involved in mAb distribution and PK, was likely to make a greater contribution to cetuximab recycling than to the transcytosis of this mAb in the airways. Our results indicate that the inhalation route is potentially useful for the treatment of both acute and chronic lung diseases, to boost and ensure the sustained accumulation of mAbs within the lungs, while limiting their passage into the bloodstream.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Sistema Respiratorio/metabolismo , Administración por Inhalación , Aerosoles , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Cetuximab , Sistemas de Liberación de Medicamentos , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Neoplasias Pulmonares/tratamiento farmacológico , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Receptores Fc/genéticaRESUMEN
Treatment of common variable immunodeficiency disorders (CVID) is based on replacement therapy using intravenous (i.v.) or subcutaneous (s.c.) immunoglobulin (Ig)G. Interindividual variation of IgG dose is common. A total of 380 CVID patients on stable IgG replacement from two prospective cohorts were analysed. An 'efficiency' index was defined as the ratio of serum IgG trough level minus IgG residual to the average weekly dose of IgG infusion. A reduced efficiency of IgG was associated independently with the i.v. route (P < 0·001) and with the presence of at least one CVID disease-related phenotype (lymphoproliferation, autoimmune cytopenia or enteropathy) (P < 0·001). High IgG efficiency was noted in patients homozygotes for the variable number tandem repeat (VNTR) 3/3 polymorphism of the neonatal Fc receptor gene [IgG Fc fragment receptor transporter alpha chain (FCGRT)] promoter, and this was particularly significant in patients treated with IVIG (P < 0.01). In a multivariate analysis, FCGRT VNTR 3/3 genotype (P = 0·008) and high serum albumin (P < 0·001) were associated independently with increased efficiency of i.v. Ig.
Asunto(s)
Biomarcadores Farmacológicos , Inmunodeficiencia Variable Común/tratamiento farmacológico , Antígenos de Histocompatibilidad Clase I/genética , Inmunoglobulinas Intravenosas/administración & dosificación , Receptores Fc/genética , Adulto , Estudios de Cohortes , Inmunodeficiencia Variable Común/inmunología , Femenino , Humanos , Inmunoglobulinas Intravenosas/efectos adversos , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Repeticiones de Minisatélite/genética , Fenotipo , Polimorfismo Genético , Regiones Promotoras Genéticas/genética , Estudios Prospectivos , Activación Transcripcional/genética , Resultado del TratamientoRESUMEN
STAT5 transcription factors are involved in normal B lymphocyte development and in leukemogenesis. We show that the inhibition of STAT5A expression or activity in the NALM6, 697 and Reh leukemic pre-B cell lines, results in a higher spontaneous apoptosis and an increased FAS-induced cell death. However, the molecular mechanisms underlying the altered pre-B cell survival are unclear. We used a proteomic approach to identify proteins that are differentially regulated in cells expressing (NALM6Δ5A) or not a dominant negative form of STAT5A. Among the 14 proteins identified, six were involved in the control of the oxidative stress like glutathione (GSH) synthetase and DJ-1. Accordingly, we showed increased levels of reactive oxygen species (ROS) in NALM6Δ5A cells and suppression of the increased sensitivity to Fas-mediated apoptosis by the GSH tripeptide. Similar results were observed when NALM6 cells were treated with TAT-STAT5Δ5A fusion proteins or STAT5A shRNA. In addition, the 697 and Reh pre-B cells were found to share number of molecular changes observed in NALM6Δ5A cells including ROS generation, following inhibition of STAT5 expression or function. Our results point out to a hitherto undescribed link between STAT5 and oxidative stress and provide new insights into STAT5 functions and their roles in leukemogenesis.
Asunto(s)
Leucemia de Células B/metabolismo , Estrés Oxidativo , Células Precursoras de Linfocitos B/metabolismo , Factor de Transcripción STAT5/fisiología , Apoptosis , Línea Celular , Humanos , Leucemia de Células B/patología , Interferencia de ARN , Factor de Transcripción STAT5/genéticaRESUMEN
Nasal-type natural killer (NK) cell lymphoma is an infrequent aggressive malignant disease with very poor prognosis. We aimed to explore the possible role of the transcription factor STAT3 in the pathophysiology of this malignancy, as it was involved in oncogenesis and chemoresistance. For this, we established and characterized a continuous interleukin 2-dependent NK cell line (MEC04) from a patient with a fatal nasal-type NK-cell lymphoma. Cells harbored poor cytotoxic activity against K562 cells, and spontaneously secreted interferon-gamma, interleukin-10 and vascular-endothelium growth factor in vitro. STAT3 was phosphorylated in Y705 dimerization residue in MEC04 cells and restricted to the nucleus. Y705 STAT3 phosphorylation involved JAK2, as exposure of cells to AG490 inhibitor inhibited Y705 STAT3 phosphorylation. By using recombinant transducible TAT-STAT3-beta (beta isoform), TAT-STAT3Y705F (a STAT3 protein mutated on Y705 residue, which prevents STAT3 dimerization) and peptides inhibiting specifically STAT3 dimerization, we inhibited STAT3 phosphorylation and cell growth, with cell death induction. Finally, STAT3 was phosphorylated in Y705 residue in the nuclei of lymphoma cells in eight/nine patients with nasal-type NK/T-cell lymphoma and in YT, another NK cell line. Our results suggest that STAT3 protein has a major role in the oncogenic process of nasal-type NK-cell lymphomas, and may represent a promising therapeutical target.
Asunto(s)
Células Asesinas Naturales/patología , Linfoma de Células T/etiología , Neoplasias Nasales/etiología , Factor de Transcripción STAT3/fisiología , Animales , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Femenino , Humanos , Interferón gamma/biosíntesis , Janus Quinasa 2/fisiología , Linfoma de Células T/genética , Linfoma de Células T/inmunología , Linfoma de Células T/patología , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Neoplasias Nasales/genética , Neoplasias Nasales/inmunología , Neoplasias Nasales/patología , Fosforilación , Factor de Transcripción STAT3/antagonistas & inhibidores , Proteína bcl-X/fisiologíaRESUMEN
The aim of this study was to determine the prevalence of celiac disease (CD) markers in a French cohort of 84 children type 1 diabetics. Detection of antitransglutaminase (AtTG), antiendomysium (AEA) and antigliadin (AGA) antibodies was performed. Group 1 included 81 (96.4%) diabetic patients with negative antibodies. Group 2 included 3 patients (3.6%) with positive serological markers: 1 AGA-AEA-AtTG and 1 AEA-AtTG with proved histological diagnosis and 1 AGA positive with negative histology. No statistically significant difference was observed between the groups with regard to age, duration of diabetes, familial target stature, and ratios Height/Age and Weight/Height. Presence of CD serological markers was related to a lower level of HbA1c. Prevalence of CD serological markers is important in this French cohort but lower than other countries.
Asunto(s)
Autoanticuerpos/sangre , Enfermedad Celíaca/sangre , Diabetes Mellitus Tipo 1/sangre , Adolescente , Biomarcadores/sangre , Enfermedad Celíaca/inmunología , Niño , Preescolar , Estudios de Cohortes , Diabetes Mellitus Tipo 1/inmunología , Francia , Gliadina/inmunología , Humanos , Transglutaminasas/inmunologíaRESUMEN
OBJECTIVE: To report on an uncommon association of agranulocytosis in primary Sjögren's syndrome (SS). METHODS: The clinical, haematological, and immunological features of seven patients with primary SS associated with a chronic (>6 months) agranulocytosis, and the outcome of the patients, were analysed. RESULTS: Patients were white women with an unexplained agranulocytosis. They all had non-erosive arthritis and three had a thrombocytopenia or Evan's syndrome. In three patients, transient or durable expansion of T lymphocytes was present in the peripheral blood or in the bone marrow, but evolved independently from neutrophil counts. There was no paroxysmal nocturnal haemoglobinuria clone or antibodies to neutrophil surface antigens. In vitro bone marrow culture was normal (four patients) or showed a decrease in colony forming unit-granulocyte monocyte (CFU-GM) and colony forming unit-erythroblast (CFU-E) (one patient). Serum levels of soluble Fas ligand (sFasL) were normal, and granulocyte-colony stimulating factor (G-CSF) concentrations were either normal or raised. One patient was treated with steroids associated with intravenous immunoglobulins and achieved a lasting response. Two other patients were treated with steroids and methotrexate, with poor efficacy. Short courses of subcutaneous G-CSF produced a transient and mild response in all three patients. Complete recovery of the neutrophils occurred temporarily during pregnancy in two patients. After a mean follow up of 34.8 months (range 6-139) all patients were alive and none developed serious infections. CONCLUSION: A subset of patients with primary SS and non-destructive arthritis may develop a chronic but well tolerated agranulocytosis that is usually poorly responsive to steroids and oral methotrexate.
Asunto(s)
Agranulocitosis/complicaciones , Síndrome de Sjögren/complicaciones , Adulto , Anciano , Agranulocitosis/tratamiento farmacológico , Agranulocitosis/inmunología , Anticuerpos Antinucleares/análisis , Antirreumáticos/uso terapéutico , Examen de la Médula Ósea/métodos , Femenino , Factor Estimulante de Colonias de Granulocitos/sangre , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Humanos , Metotrexato/uso terapéutico , Persona de Mediana Edad , Neutropenia/etiología , Embarazo , Síndrome de Sjögren/tratamiento farmacológico , Síndrome de Sjögren/inmunología , Esteroides/administración & dosificación , Resultado del TratamientoRESUMEN
Glucocorticoids reduce in vivo the number of eosinophils and are effective in treatment of blood and tissue hypereosinophilia. Dexamethasone is a synthetic glucocorticoid known to induce in vitro apoptosis of eosinophils of healthy donors, and apoptosis may be a mechanism induced by glucocorticoids to reduce eosinophilia. Here we confirm that dexamethasone exerts a pro-apoptotic effect on eosinophils isolated from healthy subjects but that dexamethasone is not always a pro-apoptotic agent towards eosinophils from hypereosinophilic patients. Implications of these results in the resistance of some patients to a treatment by glucocorticoids are discussed.
Asunto(s)
Antiinflamatorios/farmacología , Apoptosis , Dexametasona/farmacología , Eosinófilos/efectos de los fármacos , Glucocorticoides/farmacología , Síndrome Hipereosinofílico/sangre , Separación Celular , Eosinófilos/citología , Humanos , Síndrome Hipereosinofílico/inmunologíaRESUMEN
Activation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-3/farmacología , Proteínas de la Leche , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transactivadores/fisiología , Animales , Apoptosis , Línea Celular , Proteínas de Unión al ADN/genética , Genes Dominantes , Genes Reporteros , Genes bcl-2 , Células Madre Hematopoyéticas/metabolismo , Ratones , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Factor de Transcripción STAT5 , Eliminación de Secuencia , Transactivadores/genética , Transcripción Genética , Proteína bcl-XRESUMEN
Cell proliferation and differentiation are under the control of cytokines and growth factors. Different signaling pathways are involved in the transmission of a specific signal through successive phosphorylation and dephosphorylation of proteins leading to gene transcription necessary for growth and differentiation. The cytokines and growth factors activate the Stat family of transcription factors. The Jak-Stat pathway is essential for cytokine signal transduction. Dysregulation of this cascade might lead to uncontrolled hematopoiesis. Studies have been carried out to examine the functionality of this pathway in cells from patients with acute leukemia. Members of the Stat protein family (Stat1, Stat3 and Stat5) are constitutively activated in cells collected from some acute leukemias suggesting dysregulation of the Jak-Stat pathway. Evidence of the existence of constitutively activated spliced variants of Stat3 and Stat5 proteins are described. The mechanisms of such activation remain to be clarified.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Leucemia/metabolismo , Proteínas de la Leche , Transducción de Señal , Transactivadores/metabolismo , Enfermedad Aguda , Proteínas de Unión al ADN/genética , Humanos , Leucemia/genética , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Transactivadores/genéticaRESUMEN
The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de la Leche , Proteínas de Saccharomyces cerevisiae , Eliminación de Secuencia , Transactivadores/metabolismo , Factores de Transcripción , Transcripción Genética , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Caseínas/genética , Bovinos , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/metabolismo , Genes Reporteros , Humanos , Cinética , Luciferasas/biosíntesis , Datos de Secuencia Molecular , Mutagénesis , Oligodesoxirribonucleótidos , Fenotipo , Fosfotirosina/análisis , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Factor de Transcripción STAT5 , Homología de Secuencia de Aminoácido , Ovinos , Transactivadores/biosíntesis , Transactivadores/química , Transfección , Proteínas Supresoras de TumorRESUMEN
Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta-casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1-6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus-positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.
Asunto(s)
Linfoma de Burkitt/genética , Proteínas de Unión al ADN/metabolismo , Regulación Leucémica de la Expresión Génica , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/genética , Herpesvirus Humano 4/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mieloide/genética , Proteínas de la Leche , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Transducción de Señal/fisiología , Transactivadores/metabolismo , Transcripción Genética , Infecciones Tumorales por Virus/genética , Enfermedad Aguda , Secuencia de Bases , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Núcleo Celular/metabolismo , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Infecciones por Herpesviridae/virología , Humanos , Interleucina-10/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mieloide/patología , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Infecciones Tumorales por Virus/virologíaRESUMEN
A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.
Asunto(s)
Células Sanguíneas/metabolismo , Proteínas de Unión al ADN/sangre , Regulación Leucémica de la Expresión Génica , Leucemia/sangre , Proteínas de la Leche , Proteínas de Neoplasias/sangre , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Transactivadores/sangre , Enfermedad Aguda , Adulto , Anciano , Secuencia de Bases , ADN de Neoplasias/sangre , Humanos , Leucemia/genética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Transcripción GenéticaRESUMEN
IL-5, which is produced mainly by activated T cells and allergically triggered mast cells, is a major survival and differentiation factor for eosinophils, and therefore, is of relevance to diseases associated with this type of cell infiltration, most importantly asthma. In this study, we have examined the transcriptional regulation of human IL-5 in a mouse mast cell line, CPII, stimulated with IgE and Ag. We report that an inducible activity in the region between -177 and -80, and a constitutive activity between -80 and -70, in the promoter of the human gene, are both necessary for the allergically triggered activation. A computer-assisted search for transcription factor binding motifs revealed a nuclear factor of activated T cell (NF-AT) and a GATA consensus site in the two regions. Corresponding binding activities were detected to be present in nuclear extracts from the mouse mast cell line by defined NF-AT and GATA binding sites as probes for a gel shift analysis. Competition analysis, in combination with probes from the human IL-5 promoter, confirmed that these factors indeed bind to the consensus sequences identified by computer analysis. An oligonucleotide spanning the IL-5 NF-AT consensus site is shown to confer allergic stimulation to a basal IL-5 promoter only in conjunction with the GATA site downstream, indicating that an inducible NF-AT-like factor cooperates with a constitutive member of the GATA transcription factor family in mediating the allergic stimulation of the human IL-5 gene.
Asunto(s)
Antígenos/fisiología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/inmunología , Inmunoglobulina E/fisiología , Interleucina-5/genética , Proteínas Nucleares , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Línea Celular , Electroforesis/métodos , Vectores Genéticos/biosíntesis , Interleucina-5/biosíntesis , Mastocitos , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Reacción en Cadena de la PolimerasaRESUMEN
The existence of a functional receptor for secretory component (SC) on the eosinophil membrane might explain the preferential degranulation induced by secretory IgA (sIgA) when compared to serum IgA. Indeed, flow cytometry analysis revealed that purified human SC could bind to a subpopulation (4-59%) of blood eosinophils purified from 19 patients with eosinophilia. Binding of radiolabeled human SC could be competitively inhibited using unlabeled SC or secretory IgA but not with serum IgA or IgG. Immunoprecipitation and immunosorbent chromatography using human SC revealed the presence of a major component at 15 kDa in eosinophil extracts as well as in culture supernatants but not in neutrophils. The 15-kDa protein eluted from the human SC immunosorbent was able to bind to SC or to sIgA but not to serum IgA. Eosinophils preincubated with human SC or sIgA released eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) after addition of anti-SC or anti-IgA monoclonal antibody as respective cross-linking reagents. These results indicated that binding of free or complexed SC to human eosinophils could induce eosinophil degranulation. Furthermore, the dose-dependent inhibition by SC of mediator release induced by sIgA but not by serum IgA, suggested that the receptor for SC could be involved in the preferential degranulation mediated by sIgA. These results indicate a novel pathway of eosinophil activation and its potential involvement in mucosal immunity, particularly in inflammatory diseases associated with infiltration of eosinophils and the enhanced production of sIgA.
Asunto(s)
Eosinófilos/inmunología , Inmunoglobulina A Secretora/fisiología , Receptores de Superficie Celular/fisiología , Componente Secretorio/fisiología , Unión Competitiva , Degranulación de la Célula/inmunología , Peroxidasa del Eosinófilo , Citometría de Flujo , Humanos , Peroxidasas/metabolismo , Pruebas de PrecipitinaRESUMEN
Macrophage cell-surface protein 2 (Mac-2), a galactose specific S-type lectin identified in inflammatory macrophages, presents a high degree of homology with the rat IgE-binding protein (epsilon BP). In the present study, we show by different experimental approaches that human eosinophils can express Mac-2/epsilon BP. Flow cytometry analysis revealed that a large proportion of eosinophilic patients expressing binding sites for IgE on their eosinophil membrane, were able to bind anti-Mac-2 monoclonal antibody (mAb). Northern blot performed with eosinophil RNA hybridized with the human Mac-2 or epsilon BP cDNA probes revealed that eosinophils presented a unique transcript at 1.2 kb. Immunoprecipitation of eosinophil extracts with anti-Mac-2 mAb revealed the presence of a molecule of 29 kDa corresponding to Mac-2 protein, as well as one additional molecule of 15 kDa, absent from control alveolar macrophages. The function of these molecules was investigated in a radiolabeled IgE binding assay. Anti-Mac-2 mAb as well as galactose and lactose saccharides significantly inhibited the binding of radiolabeled human myeloma IgE protein to eosinophils. Moreover, the dose-dependent inhibition by anti-Mac-2 mAb of IgE-dependent eosinophil-mediated cytotoxicity towards parasite targets indicated the role of these IgE-binding molecules in the function of human eosinophils. These results suggest that in addition to transmembrane receptors, lectin-type molecules can participate in the IgE-dependent effector function of eosinophils.
Asunto(s)
Antígenos de Diferenciación/análisis , Citotoxicidad Inmunológica , Eosinófilos/química , Inmunoglobulina E/fisiología , Lectinas/análisis , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/fisiología , Sitios de Unión , Eosinófilos/inmunología , Citometría de Flujo , Galectina 3 , Humanos , ARN Mensajero/análisis , Ratas , Receptores de IgE/fisiologíaRESUMEN
It has been suggested that neutrophils may be involved in the late-phase reaction of immunoglobulin E (IgE)-dependent hypersensitivity states. However, the identity of neutrophil-associated molecules inducing the release of mediators remains unclear. In this report, we demonstrate that human neutrophils from normal donors or from patients with inflammatory disorders could bind myeloma IgE proteins, especially after desialylation. Northern blot, immunoprecipitation, and flow cytometry analyses revealed that neutrophils did not express Fc epsilon RII/CD23, but rather Mac-2/epsilon binding protein (BP), belonging to the S-type lectin family. Similarly to IgA used as positive control, myeloma IgE proteins, as well as polyclonal IgE antibodies with or without antibody specificity, were both capable of inducing a neutrophil respiratory burst. Anti-Mac-2 but not anti-CD23 mAb strongly decreased the IgE-dependent activation of neutrophils, induced either by the specific antigen or by anti-IgE antibodies. These findings open new perspectives on the functional role of neutrophils in IgE-associated diseases including allergic states or parasitic infections.
Asunto(s)
Antígenos de Diferenciación/análisis , Inmunoglobulina E/fisiología , Neutrófilos/inmunología , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/fisiología , Citometría de Flujo , Galectina 3 , Humanos , Mieloma Múltiple/inmunología , Neutrófilos/fisiología , Pruebas de Precipitina , ARN Mensajero/análisisRESUMEN
The hypereosinophilic syndrome (HES) has been previously described as a clinicobiological entity characterized by a blood eosinophil count of over 1.5 x 10(9)/L of unknown cause associated with several clinical complications. In reality, HES is a heterogeneous group of diseases with variable and unpredictable progress in visceral lesions, thought to be related to the deleterious effects of tissue eosinophil infiltration. Various criteria for discrimination between benign and severe forms of HES have been described. These previous retrospective clinical investigations, using biological and clinical markers, have defined different stages of HES. It appears more relevant, however, to consider elements of disease activity by studying mechanisms of induction of persistent hypereosinophilia. The T-cell dependence of blood eosinophilia has led us to evaluate various markers of T-cell activation in particular. In the present review, we report previous results and perspectives suggested by the study of the interleukin 2 receptor in HES.