RESUMEN
Living cells have developed a set of complex signaling responses, which allow them to withstand different environmental challenges. Signaling pathways enable the cell to monitor external and internal states and to articulate the appropriate physiological responses. Cellular signal transmission requires the dynamic formation of spatiotemporal controlled molecular interactions. One of the most important signaling circuits in Saccharomyces cerevisiae is the one controlled by cAMP-Protein Kinase A (PKA). In budding yeast, extracellular glucose and a plethora of signals related with growth and stress conditions regulate the intracellular cAMP levels that modulate PKA activity which in turn regulates a broad range of cellular processes. The cAMP-PKA signaling output requires a controlled specificity of the PKA responses. In this review we discuss the molecular mechanisms that are involved in the establishment of the specificity in the cAMP-PKA signaling pathway in S.cerevisiae.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , AMP Cíclico/genética , Saccharomyces cerevisiae/genética , Glucosa/genética , Fosforilación/genética , Transducción de Señal/genética , Proteínas ras/genéticaRESUMEN
BACKGROUND AND PURPOSE: The percentage of patients with clinical total anterior circulation infarct (TACI) syndrome treated with reperfusion therapies in the absence of intracranial large-vessel occlusion (ILVO) was determined and their characteristics and outcome are described. METHODS: Data from a population-based, prospective, externally audited registry of all stroke patients treated with intravenous thrombolysis (IVT) and endovascular therapies in Catalonia from January 2011 to December 2013 were used. Patients with a baseline TACI and initial stroke severity measured by the National Institute of Health Stroke Scale (NIHSS) ≥ 8, evaluated less than 4.5 h post-onset, for whom a vascular study prior to treatment was available (n = 1070) were selected. Clinical characteristics, outcome and radiological data for patients treated with IVT alone (n = 605) were compared between those with detected ILVO (n = 474) and non-ILVO patients (n = 131). RESULTS: A total of 1070 patients met study criteria; non-ILVO was found in 131 (12.2%). Analysing the 605 patients treated only with IVT, no significant differences were found between non-ILVO and ILVO patients in age, sex, risk factors, time-to-treatment and type of radiological studies performed. Although non-ILVO patients had lower initial stroke severity (P < 0.001) and a better prognosis (P = 0.001), 51.3% had a poor outcome and 16% were deceased at 90 days. In 66.4% of patients without ILVO, a recent anterior territorial infarct was detected. CONCLUSIONS: Intracranial artery patency was observed in 12.2% of TACI patients evaluated within 4.5 h. Although absence of ILVO was associated with slightly better prognosis, more than half had a poor outcome at 3 months.
Asunto(s)
Arteriopatías Oclusivas/epidemiología , Arteriopatías Oclusivas/patología , Infarto de la Arteria Cerebral Anterior/epidemiología , Infarto de la Arteria Cerebral Anterior/patología , Accidente Cerebrovascular/epidemiología , Accidente Cerebrovascular/patología , Anciano , Anciano de 80 o más Años , Arteriopatías Oclusivas/diagnóstico por imagen , Arterias Cerebrales/patología , Procedimientos Endovasculares , Femenino , Humanos , Infarto de la Arteria Cerebral Anterior/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Sistema de Registros , Factores de Riesgo , España/epidemiología , Accidente Cerebrovascular/terapia , Terapia Trombolítica , Resultado del TratamientoRESUMEN
The possible association of three DEFB1 gene polymorphisms with susceptibility to develop ulcerative colitis (UC) and Crohn's disease (CD) was investigated in Brazilian patients and controls. Although a clear and strong association between functional 5'-UTR DEFB1 SNPs and susceptibility/protection to IBDs cannot be drawn, our results suggest a possible involvement of DEFB1 gene in inflammatory bowel diseases, especially with the colonic localization of Crohn's disease.
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Regiones no Traducidas 5' , Enfermedades Inflamatorias del Intestino/genética , beta-Defensinas/genética , Adulto , Brasil , Estudios de Casos y Controles , Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
Prostate cancer is the second most common cancer in men, with a significant increase in incidence and mortality in men over 50 years of age. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study is to evaluate the association between the KIR genes and HLA alleles in patients with prostate cancer and healthy controls. Two hundred patients with prostate cancer and 185 healthy controls were typed for HLA class I and KIR genes by PCR-SSP. When both groups were compared, no significant differences were found for HLA-C group 1 and group 2, HLA-Bw4, HLA-A3 and A11. No difference was seen either in KIR frequency between patients with prostate cancer and controls. In conclusion, our data suggest no potential role for the KIR gene system in prostate cancer.
Asunto(s)
Frecuencia de los Genes , Genes MHC Clase I , Genotipo , Neoplasias de la Próstata/genética , Receptores KIR/genética , Brasil/epidemiología , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Estudios de Asociación Genética/métodos , Prueba de Histocompatibilidad/métodos , Humanos , Células Asesinas Naturales , Ligandos , Masculino , Reacción en Cadena de la Polimerasa/métodosRESUMEN
OBJECTIVES: To evaluate the adequacy of vitamin D treatment based on clinic evidence in a Primary Care Center as well as to analyze some characteristics of the prescriptions made. MATERIALS AND METHODS: Descriptive cross-sectional study. Primary Care. Patients above 14 years old with vitamin D prescription. Main variable was the therapeutic adequacy with vitamin D compounds (adequacy was considered when there was a clinical indication for treatment and blood vitamin D levels below 20ng/ml). Other clinical variables were collected. Frequency and association measures were used for statistical analysis. Level of statistical significance was considered <0.05. RESULTS: 430 patients, 346 women (80.5%, 95% CI=77-84). Record of vitamin D values in 216 (50.2%, 95% CI=45-55). Screening/treatment indications in 219 patients (50.9%, 95% CI=46-56), of those in 150 patients vitamin D values were recorded (68.5%, 95% CI=62-75), average (±SD) was 21.22±12ng/ml, deficiency criteria in 86 (57.3%, 95% CI=51-64), insufficiency in 37 (24.7%, 95% CI=19-30) and sufficiency in 27 (18%, 95% CI=13-23). 86 patients (20%, 95% CI=16-24) had treatment indications plus vitamin D deficiency with no differences between genders. CONCLUSIONS: Only 20% of the patients had treatment indications plus vitamin D deficiency. Female predominance. Just over half had indications for screening of serological vitamin D values and/or indications for treatment with vitamin D compounds.
Asunto(s)
Deficiencia de Vitamina D , Vitamina D , Adolescente , Estudios Transversales , Femenino , Humanos , Masculino , Prescripciones , Atención Primaria de Salud , Deficiencia de Vitamina D/diagnóstico , Deficiencia de Vitamina D/tratamiento farmacológico , Deficiencia de Vitamina D/epidemiologíaRESUMEN
Killer immunoglobulin-like receptors (KIR) regulate the activity of natural killer and T cells through an interaction with specific human leucocyte antigen (HLA) class I molecules on target cells. Diversity in KIR gene content, KIR allelic and haplotype polymorphism has been observed between different ethnic groups. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania and Africa remain poorly studied. The aim of this study was to analyse the variability of KIR genes in 200 healthy nonrelated individuals from the Southern Brazilian population. KIR genes and HLA-A, -B and -Cw were genotyped using polymerase chain reaction-sequence-specific primers. Southern Brazilian population demonstrated several similarities to states that are closer geographically and distinct differences with Northern Brazil in the frequency of genes KIR2DS1, 2DS2, 2DS3, 2DS5, 3DL1, 3DS1, 2DL1 and 2DL2. The activating gene KIR2DS5 was the least frequent locus found in our group. Interaction of KIR/HLA was more common in the 2DS1-/2DL1+/C2+ association. This study demonstrated the diversity of KIR genes and of KIR/HLA association in a Caucasian group of Southern Brazil, establishing differences and similarities to other different populations.
Asunto(s)
Variación Genética , Receptores KIR/genética , Población Blanca/genética , Adolescente , Adulto , Brasil , Femenino , Frecuencia de los Genes , Genotipo , Antígenos HLA-A/genética , Humanos , Masculino , Persona de Mediana Edad , Familia de Multigenes , Adulto JovenRESUMEN
Marine food-reliant subsistence systems such as those in the African Middle Stone Age (MSA) were not thought to exist in Europe until the much later Mesolithic. Whether this apparent lag reflects taphonomic biases or behavioral distinctions between archaic and modern humans remains much debated. Figueira Brava cave, in the Arrábida range (Portugal), provides an exceptionally well preserved record of Neandertal coastal resource exploitation on a comparable scale to the MSA and dated to ~86 to 106 thousand years ago. The breadth of the subsistence base-pine nuts, marine invertebrates, fish, marine birds and mammals, tortoises, waterfowl, and hoofed game-exceeds that of regional early Holocene sites. Fisher-hunter-gatherer economies are not the preserve of anatomically modern people; by the Last Interglacial, they were in place across the Old World in the appropriate settings.
Asunto(s)
Dieta , Hombre de Neandertal , Exoesqueleto , Animales , Arqueología , Océano Atlántico , Aves , Cuevas , Peces , Mamíferos , Nueces , Pinus , Portugal , Alimentos Marinos , TortugasAsunto(s)
Sustitución de Aminoácidos/genética , Predisposición Genética a la Enfermedad , Enfermedades Inflamatorias del Intestino/genética , Polimorfismo de Nucleótido Simple/genética , Receptor Toll-Like 9/genética , Brasil , Estudios de Casos y Controles , Frecuencia de los Genes/genética , HumanosRESUMEN
Small vessel vascular disease is a spectrum of different conditions that includes lacunar infarction, alteration of deep white matter, or microbleeds. Hypertension is the main risk factor, although the atherothrombotic lesion may be present, particularly in large-sized lacunar infarctions along with other vascular risk factors. MRI findings are characteristic and the lesions authentic biomarkers that allow differentiating the value of risk factors and defining their prognostic value.
Asunto(s)
Enfermedades de los Pequeños Vasos Cerebrales , Biomarcadores , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/diagnóstico por imagen , Enfermedades de los Pequeños Vasos Cerebrales/diagnóstico por imagen , Enfermedades de los Pequeños Vasos Cerebrales/etiología , Enfermedades de los Pequeños Vasos Cerebrales/fisiopatología , Enfermedades de los Pequeños Vasos Cerebrales/terapia , Demencia Vascular/etiología , Complicaciones de la Diabetes , Humanos , Hiperhomocisteinemia/complicaciones , Hipertensión/complicaciones , Inflamación , Imagen por Resonancia Magnética , Neuroimagen , Pronóstico , Insuficiencia Renal Crónica/complicaciones , Factores de Riesgo , Accidente Vascular Cerebral Lacunar/diagnóstico por imagen , Accidente Vascular Cerebral Lacunar/etiología , Tomografía Computarizada por Rayos X , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/patologíaRESUMEN
The presence and characteristics of androgen receptors (ARs) have been described by our group in one human melanoma cell line. We have now investigated their presence in two other human melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, as well as in biopsies from human metastatic melanoma. Scatchard analysis revealed a single binding component for both cell lines, the apparent dissociation constant obtained being 15 nM, with a binding capacity of 280 fmol/mg total cell protein, for IIB-MEL-LES cells and 14 nM, with a binding capacity of 206 fmol/mg total cell protein for IIB-MEL-IAN cells. When specificity was assessed, not only androgen and anti-androgen but also non-androgenic compounds were able to compete for [3H]R1881 binding, as seen before. When immunocytochemistry of IIB-MEL-LES and IIB-MEL-IAN cells was performed for ARs, both cell lines were deeply stained in the nucleus, whereas no staining was found for oestrogen or progesterone receptors. Every specimen of melanoma metastases tested for the presence of ARs was deeply stained, and in the majority the intensity of the staining was high. Several hormones and anti-hormones were tested for their ability to affect cell proliferation. In both cell lines, testosterone, dihydrotesterone, oestradiol and progesterone significantly stimulated cell proliferation, and this was reversed by hydroxyflutamide, bicalutamide or tamoxifen.
Asunto(s)
Hormonas/fisiología , Melanoma/metabolismo , Melanoma/secundario , Receptores Androgénicos/metabolismo , Adulto , Unión Competitiva , Biopsia , División Celular/efectos de los fármacos , División Celular/fisiología , Interpretación Estadística de Datos , Dihidrotestosterona/farmacología , Femenino , Hormonas/farmacología , Humanos , Inmunohistoquímica , Masculino , Melanoma/patología , Melanoma Amelanótico/metabolismo , Melanoma Amelanótico/patología , Melanoma Amelanótico/secundario , Receptores de Esteroides/metabolismo , Células Tumorales CultivadasRESUMEN
Monoclonal antibody (MAb) FC-5.01, raised against the undifferentiated breast cancer cell line IIB-BR-G, has been recently shown to react with CD63. The antigen (Ag) recognized by MAb FC-5.01 is expressed in plasma membranes of IIB-BR-G and other neoplastic cells, as well as in activated platelets and endothelial cells, as detected by indirect immunofluorescence performed at 4 degrees C on live cells. In permeabilized cells, MAb FC-5.01 colocalizes with acridine orange in acidic vesicles (lysosomal/endosomal compartment). Scatchard plot analysis performed on IB-BR-G cells demonstrated a 1.4+/-0.4 x 10(7) M(-1) affinity constant and 2.1 x 10(6) antigenic sites per cell. MAb FC-5.01 is not able to mediate C fixation or ADCC toward CD63+ cells, but the FC-5.01-CD63 complex is efficiently internalized into cytoplasmic vesicles, as shown by an acid wash immunofluorescence assay. Cellular catabolism of the antibody bound by IIB-BR-G cells was studied using [125I]-FC-5.01. At 18 h, >70% of the radioactivity was present in the supernatant as degraded fragments (TCA-soluble). After internalization, rapid Ag re-expression could be demonstrated in IIB-BR-G cells. MAb FC-5.01 diminished migration of CD63+ cells in a Boyden chamber assay. Some of the above-mentioned properties would enable the use of MAb FC-5.01 as a vehicle to target different compounds inside CD63+ cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/ultraestructura , Antígenos CD/inmunología , Neoplasias de la Mama/inmunología , Gránulos Citoplasmáticos/inmunología , Glicoproteínas de Membrana Plaquetaria/inmunología , Reacciones Antígeno-Anticuerpo , Transporte Biológico/inmunología , Neoplasias de la Mama/patología , Femenino , Humanos , Tetraspanina 30 , Células Tumorales CultivadasRESUMEN
A monoclonal antibody (MAb) designated FC-5.01 (IgG2a) was generated that binds to several human carcinomas and malignant melanoma. It has revealed no or very low reactivity with most human normal tissues, except for the fact that FC-5.01 binds to some cells from the neuroendocrine system, macrophages, and some renal proximal convolute tubules with an intracellular pattern. Biochemical studies indicate that FC-5.01 recognizes a heterogeneous glycoprotein (broadband between 30-60 kDa) in melanoma tumors. The epitopes reside in the protein core and are presumably conformational, with disulphide bonds implicated in MAb recognition. The current study presents evidence that MAb FC-5.01 reacts with CD63 antigen (Ag), which has been initially described as a melanoma associated Ag, and is a member of the tetraspan family. Reactivity of MAb FC-5.01 with CD63 was demonstrated by Western blot, immunodepletion assay, and FACS analysis of the CD63-negative melanoma cells (KM3) after transfection with the genomic copy of CD63. The epitope recognized by MAb FC-5.01 was shown to be different from the epitope recognized by another anti-CD63 MAb, ME491, by an inhibition radioimmunoassay. Opposite to what has been stated for MAb ME491, no significant differences were found in CD63 expression between primary and metastatic melanoma using MAb FC-5.01.
Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Antígenos CD/aislamiento & purificación , Antígenos de Neoplasias/aislamiento & purificación , Melanoma/inmunología , Glicoproteínas de Membrana Plaquetaria/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Carcinoma/inmunología , Citometría de Flujo , Humanos , Melanoma/secundario , Radioinmunoensayo , Tetraspanina 30 , Distribución TisularRESUMEN
INTRODUCTION: Cavernous angiomas account for 5-13% of all vascular malformations. In 75% of cases they are situated in the posterior fossa and up to 30% are associated with abnormal venous drainage. The main complication is haemorrhage; the presence of a neurological focus without radiological evidence of bleeding is very rare. CASE REPORT: We report the case of a 54-year-old male with cardiovascular risk factors who presented symptoms that progressively deteriorated over a 72-hour period involving the left lower cranial nerves, sensory impairment and coordination disorder, compatible with Wallenberg's syndrome. Two computerised axial tomography scans of the brain were normal and so a tentative diagnosis of ischemic stroke in progression was proposed. Five days later, magnetic resonance imaging (MR) revealed the presence of a venous angioma and associated abnormal venous drainage. CONCLUSIONS: Cavernous angiomas present a dynamic balance between intracavernous bleeding and thrombosis, with very slow venous blood flow. Upsetting this balance leads to an increase in the intracavernous pressure and involvement of the surrounding tissue, with no radiological expression of bleeding. In these cases MR scanning helps to distinguish between a vascular malformation with reduced blood flow and a clinical picture of ischemic stroke of an arterial origin.
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Hemangioma Cavernoso del Sistema Nervioso Central , Síndrome Medular Lateral , Hemangioma Cavernoso del Sistema Nervioso Central/complicaciones , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Malformaciones Arteriovenosas Intracraneales/complicaciones , Malformaciones Arteriovenosas Intracraneales/patología , Síndrome Medular Lateral/diagnóstico , Síndrome Medular Lateral/etiología , Síndrome Medular Lateral/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Accidente Cerebrovascular/diagnósticoRESUMEN
INTRODUCTION: Cerebral venous thrombosis (CVT) is an infrequent process in systemic lupus erythematosus. We report the case of a female patient whose initial manifestation of lupus was a CVT. CASE REPORT: A 30-year-old female who presented headaches and diminished visual acuity; on exploring the patient bilateral papilloedema was found. Magnetic resonance imaging revealed the presence of a venous thrombosis in the superior and transversal longitudinal sinus. Complementary explorations showed high levels of antinuclear antibodies with leukopenia and proteinuria. Antiphospholipid antibodies were negative. Following treatment with anticoagulants, the patient's condition improved both clinically and radiologically. Months later a biopsy was performed and revealed a grade IV diffuse glomerulonephritis. CONCLUSIONS: In systemic lupus erythematosus, phenomena such as CVT can be the initial form of presentation of the disease. The presence of antiphospholipid antibodies plays a partial role in CVT; other phenomena, such as inflammatory processes, should also be taken into account.
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Trombosis Intracraneal/patología , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/patología , Trombosis de la Vena/patología , Adulto , Anticuerpos Antinucleares/sangre , Anticoagulantes/uso terapéutico , Femenino , Humanos , Trombosis Intracraneal/tratamiento farmacológico , Trombosis Intracraneal/etiología , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/tratamiento farmacológico , Angiografía por Resonancia Magnética , Resultado del Tratamiento , Trombosis de la Vena/tratamiento farmacológico , Trombosis de la Vena/etiologíaRESUMEN
Our aim in this work was to further characterize the complexity and specificity of the three different isoforms (Tpk1, Tpk2 and Tpk3) of the catalytic and regulatory (Bcy1) subunits of PKA in Saccharomyces cerevisiae. We thus analyzed the subcellular localization of the PKA subunits in living cells by using strains carrying GFP (green fluorescent protein) fused to each PKA subunit. During exponential growth on glucose, both Bcy1 and Tpk2 localized in the nucleus, whereas Tpk1 and Tpk3 showed a mixed pattern of nucleo-cytoplasmic localization. During exponential growth on glycerol and during stationary phase, the PKA subunits showed mostly cytoplasmic localization, with the peculiarity that Tpk2 and Tpk3 but not Bcy1, were found associated to P-bodies and EGP bodies. Tpk3 was accumulated into P-bodies during glucose deprivation and hyper osmotic stress. Deletion of Tpk3 altered the kinetics of P-body formation. Analysis of protein expression showed that the relative expression pattern of each Tpk changes from low levels under fermentative metabolism to higher levels during stationary phase. The increase in Tpk levels produced an imbalance with Bcy1 levels. Our data suggest that the signaling specificity through PKA in yeast could be mediated by a particular subcellular localization of each isoform of Tpk.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/enzimología , Medios de Cultivo/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Citoplasma/enzimología , Gránulos Citoplasmáticos/enzimología , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimologíaRESUMEN
INTRODUCTION: Bilateral facio-pharyngo-laryngo-glosso-masticatory palsy with automatic-voluntary dissociation is known as Foix-Chavany-Marie (FCM) syndrome. It is usually due to bilateral cortical lesions involving both anterior opercula (biopercular syndrome). We describe three patients with FCM syndrome associated with ischemic lesions in two atypical localizations: a) bilateral subcortical infarcts, and b) unilateral opercular infarct. CASES REPORT: Patient 1, a 66 year old male, was admitted for a sudden onset of anarthria and facial, lingual and masticatory paralysis. Neurological examination revealed automatic-voluntary dissociation of facial motility. MRI showed an acute left subcortical infarct and multiple bilateral subcortical ischemic lesions. Patient 2, a 61 year old male, also suffered a sudden onset of anarthria, with bilateral facial and lingual paralysis and automatic-voluntary dissociation together with sudden onset swallowing alteration. MRI showed a single ischemic lesion involving the left operculum. Patient 3, a 36 year old male, presented sudden onset of dysarthria, dysphagia and bilateral facial palsy with automatic-voluntary dissociation and loss of force in left upper limb. MRI showed an acute right opercular infarct and no contralateral lesions. CONCLUSIONS: FCM syndrome is not only due to bilateral opercular lesions but can also be seen in bilateral subcortical and even unilateral opercular lesions.
Asunto(s)
Parálisis Facial/fisiopatología , Parálisis/fisiopatología , Adulto , Anciano , Parálisis Facial/diagnóstico , Parálisis Facial/patología , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Parálisis/diagnóstico , Parálisis/patología , SíndromeRESUMEN
FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15+) cells were investigated. A 51Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15+ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1-50 micrograms/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8 +/- 2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15-. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with 125I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9 x 10(7) M-1 and 2.8 x 10(6) antigenic sites/cell. 125I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15+ cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Neoplasias de la Mama/inmunología , Proteínas del Sistema Complemento/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos de Neoplasias/metabolismo , Activación de Complemento , Citotoxicidad Inmunológica , Endocitosis , Humanos , Técnicas In Vitro , Células Tumorales CultivadasRESUMEN
The Lewis(x) (Le(x)) trisaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutrophils (PMN) and several human neoplasias, such as breast and gastrointestinal carcinomas and chronic myeloid leukemias. FC-2.15 is an IgM murine mAb that specifically recognizes Le(x) and has been previously shown to mediate the in vitro lysis of Le(x)(+) cells by human complement. In a phase I clinical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. In order to characterize FC-2.15 further and determine the physiological relevance of Le(x) binding, the reactivity of FC-2.15 on PMN was investigated under several conditions. Flow cytometry revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scatchard analysis demonstrated an affinity constant of 5.14 x 10(9) M(-1) and 1.11 x 10(6) antigen sites/cell. In vitro, the binding of Le(x) epitopes by FC-2.15 induced PMN homotypic aggregation, only 28.4 +/- 4.1% remaining as single cells. When PMN and the Le(x)(+) MCF-7 breast cancer cells were co-incubated, FC-2.15 induced heterotypic aggregation. In 51Cr-release assays employing human complement, FC-2.15 lysed 93.4 +/- 7.9% of PMN and 87.8 +/- 10.7% of MCF-7 cells. However, when the effect of FC-2.15 was tested in ex vivo circulating blood, no lytic activity against PMN was detected, whereas MCF-7 cells were still lysed. Blood smears demonstrated that FC-2.15 induced PMN agglutination and heterotypic aggregates when MCF-7 cells were present. A pretreatment of PMN with colchicine impaired PMN agglutination both in vitro (single PMN = 81.15 +/- 4.35%) and in ex vivo circulating blood. In the latter condition, FC-2.15-lytic activity was restored, suggesting that PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on microtubule integrity and that PMN agglutination hinders their lysis. Moreover, when 51Cr-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslinking of Le(x) epitopes by FC-2.15 induces PMN to form homotypic aggregates. It is suggested that the neutropenia observed in FC-2.15-treated patients would be due to PMN agglutination and margination, rather than lysis. In addition, FC-2.15 appears to be able to lyse Le(x)(+) tumor cells in circulation.
Asunto(s)
Aglutinación , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Neoplasias de la Mama/inmunología , Antígeno Lewis X/inmunología , Neutrófilos/fisiología , Animales , Agregación Celular , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Neutropenia/etiología , Células Tumorales CultivadasRESUMEN
Monoclonal antibody (MAb) FC-2.15 recognizes Lewis x antigen (Le(x)-Ag) expressed on the cell surface of most human breast cancer cells. FC-2.15 displays important human complement (C')-mediated cytotoxicity (CMC) against its target cells. In this study the reactivity of FC-2.15 against drug resistant-breast cancer cells was investigated, as well as the possibility to combine the antitumor activities of this MAb with adriamycin (Adr) or taxol. Since resistant clones with altered expression of tumor-associated antigens usually emerge after chemotherapy, the expression of Le(x)-Ag was analyzed in Adr(R) MCF-7 breast cancer cells (Adr resistant subline) and in tumor samples from nine patients with locally advanced breast carcinoma who were treated with FEC chemotherapy. A flow cytometry assay showed that most of Adr(R) MCF-7 cells, as well as wild type (WT) cells, expressed Le(x)-Ag; however, the Le(x) epitope is probably bound to different backbones in these cells. When the cytotoxic ability of FC-2.15 against WT and Adr(R) MCF-7 cells was compared, it was found that a 90 min treatment with FC-2.15 plus C' induced similar CMC against both cell lines. An important cytolysis was obtained at 5 microg/ml FC-2.15, reaching a plateau at 25 microg/ml, at which cell population was diminished to 21.1% for WT and 27.9 for Adr(R) MCF-7 cells. Regarding human tumors, Le(x)-Ag expression was evaluated in samples obtained before and in most cases after chemotherapy, and it could be observed that: 1) before treatment, tumor samples from all patients analyzed (responders and non-responders to chemotherapy) were FC-2.15-positive; 2) the presence of Le(x)-Ag was not modified after treatment. The combined action of Adr or taxol with FC-2.15 was then evaluated. WT and Adr(R) MCF-7 cells were cultured with Adr or taxol followed by an incubation with different FC-2.15 concentrations plus C'. When the effect of Adr alone was determined, ID50 were 1 x 10(-7) M for WT and 4.2 x 10(-5) M for Adr(R) MCF-7 cells. The cytotoxic ability of taxol alone was also tested, and ID50 were 6.4 x 10(-9) M for WT and 3.1 x 10(-6) M for Adr(R) MCF-7 cells. When FC-2.15 was added to Adr or taxol, the cytotoxicity of the drug-FC-2.15 combined treatment was always higher than the isolated effects, showing additive cytotoxicity at the different concentrations tested and with both cell lines. Our results suggest that FC-2.15 may be a useful agent against breast tumor cells which survive chemotherapy with Adr or taxol.