RESUMEN
Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3+ T-cells and CD163+ tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS30) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1+pSTAT1Y701+) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10+IRF1+STAT1+ M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10+IRF1+STAT1+ macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.
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Carcinoma/metabolismo , Quimiocina CXCL10/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Quísticas, Mucinosas y Serosas/metabolismo , Neoplasias Ováricas/metabolismo , Microambiente Tumoral , Macrófagos Asociados a Tumores/metabolismo , Anciano , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Complejo CD3/metabolismo , Carcinoma/tratamiento farmacológico , Carcinoma/genética , Carcinoma/inmunología , Células Cultivadas , Quimiocina CXCL10/genética , Resistencia a Antineoplásicos , Femenino , Humanos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Neoplasias Quísticas, Mucinosas y Serosas/tratamiento farmacológico , Neoplasias Quísticas, Mucinosas y Serosas/genética , Neoplasias Quísticas, Mucinosas y Serosas/inmunología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Fenotipo , Pronóstico , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
Langerhans cell histiocytosis (LCH) is a rare disorder characterized by tissue accumulation of CD1a+CD207+ LCH cells. In LCH, somatic mutations of the BRAF V600E gene have been detected in tissue LCH cells, bone marrow CD34+ hematopoietic stem cells, circulating CD14+ monocytes, and BDCA1+ myeloid dendritic cells (DC). Targeting BRAF V600E in clonal Langerhans cells (LC) and their precursors is a potential treatment option for patients whose tumors have the mutation. The development of mouse macrophages and LCs is regulated by the CSF1 receptor (CSF1R). In patients with diffuse-type tenosynovial giant cell tumors, CSF1R inhibition depletes tumor-associated macrophages (TAM) with therapeutic efficacy; however, CSF1R signaling in LCs and LCH has not been investigated. We found through IHC and flow cytometry that CSF1R is normally expressed on human CD1a+CD207+ LCs in the epidermis and stratified epithelia. LCs that were differentiated from CD14+ monocytes, BDCA1+ DCs, and CD34+ cord blood progenitors expressed CSF1R that was downregulated upon maturation. Immature LCs migrated toward CSF1, but not IL34. Administration of the c-FMS/CSF1R kinase inhibitors GW2580 and BLZ945 significantly reduced human LC migration. In LCH clinical samples, LCH cells (including BRAF V600E cells) and TAMs retained high expression of CSF1R. We also detected the presence of transcripts for its ligand, CSF1, but not IL34, in all tested LCH cases. CSF1R and CSF1 expression in LCH, and their role in LC migration and differentiation, suggests CSF1R signaling blockade as a candidate rational approach for treatment of LCH, including the BRAF V600E and wild-type forms of the disease.
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Diferenciación Celular , Movimiento Celular , Células Dendríticas/patología , Histiocitosis de Células de Langerhans/patología , Células de Langerhans/patología , Monocitos/patología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Adolescente , Adulto , Anciano , Apoptosis , Linaje de la Célula , Células Cultivadas , Niño , Preescolar , Células Dendríticas/metabolismo , Femenino , Histiocitosis de Células de Langerhans/metabolismo , Humanos , Lactante , Recién Nacido , Células de Langerhans/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Microambiente Tumoral , Adulto JovenRESUMEN
BACKGROUND: Urothelial bladder cancers (UBCs) are distinct in two main molecular subtypes, namely basal and luminal type. Subtypes are also diverse in term of immune contexture, providing a rationale for patient selection to immunotherapy. METHODS: By digital microscopy analysis of a muscle-invasive BC (MIBC) cohort, we explored the density and clinical significance of CD66b+ tumor-associated-neutrophils (TAN) and CD3+ T cells. Bioinformatics analysis of UBC datasets and gene expression analysis of UBC cell lines were additionally performed. RESULTS: Basal type BC contained a significantly higher density of CD66b+ TAN compared to the luminal type. This finding was validated on TCGA, GSE32894 and GSE124305 datasets by computing a neutrophil signature. Of note, basal-type MIBC display a significantly higher level of chemokines (CKs) attracting neutrophils. Moreover, pro-inflammatory stimuli significantly up-regulate CXCL1, CXCL2 and CXCL8 in 5637 and RT4 UBC cell lines and induce neutrophil chemotaxis. In term of survival, a high density of T celsl and TAN was significantly associated to a better outcome, with TAN density showing a more limited statistical power and following a non-linear predicting model. CONCLUSIONS: TAN are recruited in basal type MIBC by pro-inflammatory CKs. This finding establishes a groundwork for a better understanding of the UBC immunity and its relevance.
Asunto(s)
Neoplasias de los Músculos/metabolismo , Neutrófilos/inmunología , Linfocitos T/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Anciano , Anciano de 80 o más Años , Complejo CD3/metabolismo , Antígeno Carcinoembrionario/metabolismo , Línea Celular Tumoral , Movimiento Celular/inmunología , Supervivencia Celular/inmunología , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Bases de Datos Genéticas , Femenino , Silenciador del Gen , Humanos , Inmunohistoquímica , Interleucina-8/genética , Interleucina-8/metabolismo , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Neoplasias de los Músculos/genética , Neoplasias de los Músculos/secundario , Neutrófilos/citología , Neutrófilos/metabolismo , Estudios Retrospectivos , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Linfocitos T/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Urothelial bladder cancer (UBC) are classified into luminal and basal subtypes showing distinct molecular features and clinical behaviour. Recent in silico data have proposed the activation on the Signal Transducer and Activator of Transcription 3 (STAT3) as relevant transcription factor in UBC. To answer this question, we have combined the retrospective analysis of clinical samples, functional assays on cell lines, interrogation of public UBC datasets and a murine model of basal-type UBC. Immunohistochemistry on a retrospective UBC cohort uncovered that STAT3 Y705 phosphorylation (pSTAT3) is significantly increased in infiltrating basal-type UBC compared to luminal UBC. In vitro, STAT3 silencing in UBC cell lines significantly reduced tumor cell viability and invasion. Gene expression profile of UBC cell lines combined with the analysis of the Cancer Genome Atlas (TCGA) and GSE32894 UBC datasets showed that increased expression of a set of STAT3 targets predicts basal-type, propensity to local progression and worse prognosis. MYC and FOSL1 represent relevant STAT3 downstream targets, as validated by their co-localization in pSTAT3+ UBC cancer cells. These findings were largely reproduced in the BBN-induced murine model of basal-type UBC. Of note, FOSL1 protein resulted strongly expressed in the non-papillary UBC pathway and FOSL1-regulated transcripts were significantly enriched in the transition from NMIBC to MIBC, as indicated by the interrogation of the GSE32894 dataset. The blockade of the STAT3 pathway might represent a novel treatment option for these neoplasms. Monitoring pSTAT3 and the downstream targets, particularly FOSL1, could provide meaningful levels of UBC stratification.
RESUMEN
Neutrophils are known to perform a series of effector functions that are crucial for the innate and adaptive responses, including the synthesis and secretion of a variety of cytokines. In light of the controversial data in the literature, the main objective of this study was to more in-depth reevaluate the capacity of human neutrophils to express and produce cytokines of the IL-17 family in vitro. By reverse transcription quantitative real-time PCR, protein measurement via commercial ELISA, immunohistochemistry (IHC) and immunofluorescence (IF), flow cytometry, immunoblotting, chromatin immunoprecipitation (ChIP), and ChIP-seq experiments, we found that highly pure (>99.7%) populations of human neutrophils do not express/produce IL-17A, IL-17F, IL-17AF, or IL-17B mRNA/protein upon incubation with a variety of agonists. Similar findings were observed by analyzing neutrophils isolated from active psoriatic patients. In contrast with published studies, IL-17A and IL-17F mRNA expression/production was not even found when neutrophils were incubated with extremely high concentrations of IL-6 plus IL-23, regardless of their combination with inactivated hyphae or conidia from Aspergillus fumigatus. Consistently, no deposition of histone marks for active (H3K27Ac) and poised (H3K4me1) genomic regulatory elements was detected at the IL-17A and IL-17F locus of resting and IL-6 plus IL-23-stimulated neutrophils, indicating a closed chromatin conformation. Concurrent experiments revealed that some commercial anti-IL-17A and anti-IL-17B antibodies (Abs), although staining neutrophils either spotted on cytospin slides or present in inflamed tissue samples by IHC/IF, do not recognize intracellular protein having the molecular weight corresponding to IL-17A or IL-17B, respectively, in immunoblotting experiments of whole neutrophil lysates. By contrast, the same Abs were found to more specifically recognize other intracellular proteins of neutrophils, suggesting that their ability to positively stain neutrophils in cytospin preparations and, eventually, tissue samples derives from IL-17A- or IL-17B-independent detections. In sum, our data confirm and extend, also at epigenetic level, previous findings on the inability of highly purified populations of human neutrophils to express/produce IL-17A, IL-17B, and IL-17F mRNAs/proteins in vitro, at least under the experimental conditions herein tested. Data also provide a number of justifications explaining, in part, why it is possible to false positively detect IL-17A+-neutrophils.
Asunto(s)
Interleucina-17/biosíntesis , Neutrófilos/metabolismo , Células Cultivadas , Humanos , Técnicas In Vitro , Neutrófilos/inmunologíaRESUMEN
AIM: To investigate the presence of human papillomavirus (HPV) DNA in squamous cell carcinoma (SCC) of the lung, and to examine the protein expression and genomic status of p16 and their correlation. MATERIALS AND METHODS: Fifty cases of surgically removed primary lung SCC were analyzed. HPV detection was performed by Polymerase Chain Reaction (PCR) of L1 region and E6/E7 region of high-risk viral genotype. p16 protein and gene analysis were carried out by immunohistochemistry and Fluorescence In Situ Hybridization (FISH), respectively. RESULTS: HPV DNA was found in two out of 50 cases (4%, p>0.05). In five cases, p16 protein expression was positive. The data showed that in 45/50 cases (90%, p<0.05) HPV DNA and p16 were both negative, in 2/50 cases (4%) both were positive, and in 3/50 (6%) cases, HPV DNA was negative and p16 positive. FISH analysis for p16 gene showed aneusomia of chromosome 9 with or without loss of p16 gene in all cases (100%, p<0.05). CONCLUSION: Our study shows that in pulmonary SCC, there is no association between the presence of HPV DNA and the expression of p16 protein. Furthermore, the loss of the p16 gene and the instability of chromosome 9 were frequently found in HPV DNA-negative cases.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virología , Genes p16 , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/virología , Papillomaviridae , Anciano , ADN Viral/análisis , ADN Viral/genética , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
The study of proto-oncogene Her-2/neu using the fluorescence in situ hybridization (FISH) technique in routinely paraffin-embedded formalin-fixed tissue has become commonplace over the past decade and mandatory among invasive breast cancer expressing a score 2+ by immunohistochemical analysis of c-erbB2 protein. The patient's eligibility for treatment with the biological drug trastuzumab/herceptin is based on the evidence of a Her-2/neu proto-oncogene amplification (ratio Her-2/neu/CEP-17>2.2). However, although the exclusion is declared in the absence of Her-2/neu gene amplification (ratio Her-2/neu/CEP-17 <1.8) according to the American Society of Clinical Oncology/College of American Pathologists recommendations, there are borderline cases (1.8
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Neoplasias de la Mama/diagnóstico , Carcinoma/diagnóstico , Receptor ErbB-2/genética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Femenino , Amplificación de Genes/genética , Genotipo , Humanos , Inmunohistoquímica/métodos , Inmunofenotipificación , Hibridación Fluorescente in Situ/métodos , Queratinas/metabolismo , Terapia Molecular Dirigida , Selección de Paciente , Proto-Oncogenes Mas , TrastuzumabRESUMEN
The expressions of p16, Ki-67, and L1 proteins and human papillomavirus DNA were investigated using polymerase chain reaction (HPV/PCR) and catalyzed signal-amplified colorimetric DNA in situ hybridization (CSAC/ISH) as potential molecular markers for the diagnosis and transforming potential of low cervical intraepithelial neoplasia (CIN1). Ki-67 and p16 protein expression increased linearly from control cases to more dysplastic cases (CIN1, CIN2, and CIN3), peaking in squamous cell carcinoma cases (P<0.05). In contrast, L1 expression was inversely correlated with malignant transformation. Patients with CIN1 were divided into 4 groups: L1p16, L1p16, L1p16, and L1p16, and the immunohistochemical results were combined with HPV/PCR, L1/PCR, and high-risk E6/E7 genome and CSAC/ISH data. Malignant transformation correlated with L1p16 patients (100% of CIN2, CIN3, and squamous cell carcinoma cases) and was evident in approximately 23% of CIN1 cases. In addition, the presence of HPV/DNA was evident in 52% of CIN1 cases, and within the L1p16 group. In 4 of 7 cases, the high-risk E6/E7 HPV genome was present and in 1 case it was integrated into the host DNA, as confirmed using CSAC/ISH. In patients with CIN1, investigating the presence of HPV/DNA using PCR and the presence of the high-risk E6/E7 genome is necessary to distinguish high-risk oncogenic patient groups from low-risk groups. This study highlights the importance of combining immunohistochemical analysis with HPV/PCR and CSAC/ISH to identify patients with CIN1 with a risk of neoplastic progression.
Asunto(s)
Proteínas de la Cápside , Antígeno Ki-67/biosíntesis , Proteínas de Neoplasias , Proteínas Oncogénicas Virales , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Biomarcadores de Tumor/análisis , Proteínas de la Cápside/biosíntesis , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Transformación Celular Neoplásica/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina , ADN Viral , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Proteínas de Neoplasias/biosíntesis , Proteínas Oncogénicas Virales/biosíntesis , Papillomaviridae , Infecciones por Papillomavirus/complicaciones , Reacción en Cadena de la Polimerasa , Pronóstico , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/virologíaRESUMEN
BACKGROUND: Human papillomavirus DNA (HPV DNA) and p16 and p53 protein expressions were investigated for their role in transforming dysplasia into squamous cell carcinoma of the oral cavity in a non-smoker and non-drinker patient group. MATERIALS AND METHODS: A total of 56 oral biopsies from non-smoker and non-drinker patients were analyzed. The specimens were grouped into three categories: group 1 included 31 cases of hyperplastic mucosa and mild dysplasia, group 2 included 14 cases of moderate and severe dysplasia, while group 3 comprised 11 cases of invasive squamous cell carcinomas. In all cases, immunohistochemical methods were performed to detect p16 and p53 protein expressions. The nested polymerase chain reaction for HPV (nested HPV-PCR) and the catalyzed signal-amplified colorimetric DNA in situ hybridization (CSAC-ISH) methods were applied for HPV DNA detection and typing of high-risk genotype. RESULTS: P16 protein, absent from all specimens of group 1, was especially noted in group 2 (92.86%) and in group3 (54.55%). Five out of 14 of group 2 cases (35.71%) and 3/11 (27.27%) of group 3 were HPV DNA positive. The HPVs detected were of both high-risk and low-risk genotype. The analysis of the relationship between HPV and p16 protein expression revealed that all the group 2 and 3 samples with HPV DNA, overexpressed p16 protein. CONCLUSION: The results suggest that HPV could be a molecular marker in group 2 and 3 specimens in non-smoker and non-drinker patients. The virus may play an etiological role in carcinogenesis in the oral cavity. The association between HPV and p16 overexpression suggests a molecular mechanism similar to that found in cervical cancer.
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Carcinoma de Células Escamosas/virología , Neoplasias de la Boca/virología , Infecciones por Papillomavirus/epidemiología , Lesiones Precancerosas/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/patología , Niño , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , ADN Viral/análisis , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/patología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/complicaciones , Lesiones Precancerosas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/biosíntesis , Adulto JovenRESUMEN
Decay of mitochondria, energy failure and increased oxidative stress are features commonly detected in brains from Alzheimer's disease (AD) patients. Recent findings indicate that neuronal heme deficiency may contribute to the appearance of those cytopathologies and potentially alter the course of AD. We repressed heme synthesis in cells by inhibiting ferrochelatase enzyme with small interfering RNA and N-methylprotoporphyrin IX. The treatments induced a severe perturbation of mitochondria and energy production, with decrease of the subunit II of Cytochrome c Oxidase, alteration of the membrane potential and a 50% reduction of intracellular ATP. The state and processing of the Amyloid Precursor Protein (APP) was also affected, with the appearance of APP aggregates and a significant decrease (30-40%) of sAPPalpha secretion, associated with perturbation of ADAM10 and TACE, enzymes involved in the alpha-secretase cleavage. The production of sAPPbeta was increased, without augment of Amyloid beta generation. Our findings strengthen the hypothesis that a reduced availability of heme may play a role in AD pathogenesis.
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Precursor de Proteína beta-Amiloide/metabolismo , Hemo/antagonistas & inhibidores , Hemo/metabolismo , Fenómenos Fisiológicos/efectos de los fármacos , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Adenosina Trifosfato/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Recuento de Células/métodos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Complejo IV de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/farmacología , Ferroquelatasa/antagonistas & inhibidores , Ferroquelatasa/genética , Humanos , Proteínas de la Membrana/metabolismo , Neuroblastoma , Fragmentos de Péptidos/metabolismo , Carbonilación Proteica/efectos de los fármacos , Protoporfirinas/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
BACKGROUND: Age-related macular degeneration (ARMD) and Alzheimer's disease (AD) are neurodegenerative disorders that share a high prevalence among elderly people, the extracellular deposition of beta-amyloid and the involvement of genetic factors in their aetiology. Genetic linkage with the chromosome regions 10q26 and 10q24-25 have been shown for ARMD and AD, respectively. The rs10490924 polymorphism, the major determinant of the 10q26 association with ARMD, determines the A69S substitution in the LOC387715/ARMS2 gene. Little information is available about the expression of the gene in humans. METHODS: We analysed the expression of the gene by RT-PCR in the brain and we looked for nucleotide variations in the gene sequence by DHPLC. RESULTS: We found specific gene transcripts in the hippocampus, cortex and cerebellum. The genetic analysis identified two other common variations, which determine the R3H change (rs10490923) and a premature stop codon (rs2736911), respectively. The analysis of their distribution in 213 AD patients and 149 controls revealed a trend for a reduced frequency of the variant allele of rs2736911 in AD patients (p = 0.038), with an odds ratio of 0.631. CONCLUSION: The LOC387715/ARMS2 gene is expressed in the human brain, and it may concur to the individual risk for AD.
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Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/genética , Polimorfismo Genético , Proteínas/genética , Anciano , Anciano de 80 o más Años , Apolipoproteína E4/genética , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/epidemiología , Humanos , Degeneración Macular/epidemiología , Degeneración Macular/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
The gamma-secretase complex is a multimeric aspartyl protease which plays a pivotal role in the production of amyloid beta-peptide, the main component of senile plaques in Alzheimer's disease (AD). APH-1a and APH-1b have been recently identified as important subunits of the gamma-secretase complex. We previously studied sequence variations in both genes and their association with AD in a small Italian population. The rare polymorphism c + 651T > G in APH-1b showed a possible interaction with the Apolipoprotein E (APOE) epsilon4 allele in the AD population sample. We extended our genetic analysis to 449 AD patients and 435 controls and, in AD cases, we found a significant interaction (P=0.001) between the allelic variants in the two genes, resulting in a marked increase of the relative risk for AD (OR=28.6). Despite the amino acid substitution does not seem to modify either the intracellular localization or the half-life of APH-1b protein, these data suggest that a cooperative mechanism involving APOE and APH-1b plays a role in the susceptibility to develop AD.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Apolipoproteína E4/genética , Predisposición Genética a la Enfermedad/genética , Proteínas de la Membrana/genética , Péptido Hidrolasas/genética , Polimorfismo de Nucleótido Simple/genética , Anciano , Enfermedad de Alzheimer/fisiopatología , Sustitución de Aminoácidos/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Apolipoproteína E4/metabolismo , Células COS , Chlorocebus aethiops , Análisis Mutacional de ADN , Endopeptidasas , Femenino , Frecuencia de los Genes/genética , Pruebas Genéticas , Variación Genética/genética , Genotipo , Células HeLa , Humanos , Italia , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Mutación/genética , Péptido Hidrolasas/metabolismo , TransfecciónAsunto(s)
Predisposición Genética a la Enfermedad , Infecciones por VIH/complicaciones , VIH-1 , Hemocromatosis/complicaciones , Hemocromatosis/genética , Mutación , Enfermedades del Sistema Nervioso Periférico/etiología , Estudios de Casos y Controles , Femenino , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Análisis de RegresiónRESUMEN
Senile plaques and neurofibrillary tangles (NFT) are the prominent lesions in the brain of Alzheimer's disease (AD) patients. NFT are mainly composed of an abnormally phosphorylated form of tau protein, which has lost its function to bind microtubules and promote their assembly. Tau hyperphosphorylation critically decreases tau function and precedes neurodegeneration. The majority of tau phosphorylation sites are Ser/Thr-Pro motifs, which are known to exist in two distinct cis and trans conformations. The prolyl isomerase Pin1 catalyses the conversion of those conformations. Pin1 binds to tau specifically at the Thr231-Pro site and restores tau function, either by inducing conformational changes or facilitating dephosphorylation. It has been shown that Pin1 expression levels inversely correlate with the predicted vulnerability of different brain areas to neurodegeneration and soluble Pin1 is depleted in neurons from AD brains; furthermore, Pin1 knock-out mice develop signs and symptoms of tau-related pathologies late in life. It seems that Pin1 plays an important role in maintaining tau function, thereby preserving neuronal homeostasis and preventing age-dependent neurodegeneration. DNA sequence variations in Pin1 gene may affect its expression level or function and influence the individual risk for developing AD. We screened by denaturing high performance liquid chromatography the genomic DNA of 120 AD subjects and 134 age-matched controls and we found very few and rare sequence variations in the promoter region and in exons 2 and 3. We conclude that Pin1 is a very well conserved gene, whose rare nucleotide variations have no effect on the individual genetic risk for AD.
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Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Ovillos Neurofibrilares/enzimología , Isomerasa de Peptidilprolil/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/fisiopatología , Secuencia de Bases/genética , Encéfalo/enzimología , Encéfalo/fisiopatología , Química Encefálica/genética , Secuencia Conservada/genética , Análisis Mutacional de ADN , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Pruebas Genéticas , Genotipo , Humanos , Isoenzimas/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Peptidilprolil Isomerasa de Interacción con NIMA , Fosforilación , Proteínas tau/metabolismoRESUMEN
The apolipoprotein E ( APOE ) gene in humans contains two single-base polymorphisms in exon 4, which result in three common alleles, conventionally named epsilon2 , epsilon3 and epsilon4 . Numerous studies have shown an important association between the epsilon4 variant and an increased risk of Alzheimer's disease; other data suggest a possible linkage of APOE genetic heterogeneity with lipid profile and an increased risk of atherothrombotic stroke and coronary heart disease. APOE genotyping is therefore an increasingly common assay in laboratory medicine. The most widely used technique for APOE genotyping is based upon restriction isotyping, i.e., amplification of the fragment of exon 4 containing the most common sequence variations, followed by enzymatic digestion of the amplicon and fragments analysis by gel electrophoresis. We developed a novel, reliable and fast method that exploits the sensitivity and specificity of denaturing high-performance liquid chromatography in detecting single-nucleotide polymorphisms. We show that, in most cases, with a single chromatographic separation it is possible to correctly identify the six different APOE allelic patterns, without any manipulation after the polymerase chain reaction amplification. When compared to restriction isotyping, our method is much faster, less labor intensive and similarly inexpensive.
Asunto(s)
Apolipoproteínas E/genética , Haplotipos , Alelos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/estadística & datos numéricos , ADN/genética , Variación Genética , Heterocigoto , Humanos , Mutagénesis Sitio-Dirigida , Desnaturalización de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Sensibilidad y EspecificidadRESUMEN
Recent biological studies indicate the importance of anterior-pharynx defective-1 (APH-1) proteins in Alzheimer's disease (AD) pathogenesis. We scanned APH-1 genes for the presence of sequence variations by denaturing high performance liquid chromatography and analyzed their distribution in an Italian sample of 113 AD patients and 132 controls. We found six different polymorphisms: three of them, all in APH-1b, predict an aminoacid substitution (T27I, V199L and F217L); the others are either silent or in non-coding regions. None of them is significantly associated with the disease; data stratification by the apolipoprotein E epsilon4 carrier status show a trend for coexistence of the transversion c+651T>G (F217L) with the epsilon4 allele. Our data suggest that polymorphisms in APH-1a/b coding regions are not linked with higher risk for sporadic AD in our Italian population sample.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas de la Membrana/genética , Polimorfismo Genético/genética , Anciano , Anciano de 80 o más Años , Alelos , Sustitución de Aminoácidos/genética , Intervalos de Confianza , Endopeptidasas , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Péptido HidrolasasRESUMEN
The amyloid cascade hypothesis suggests that amyloid precursor protein (APP) proteolytic processing is a key event in the pathogenesis of Alzheimer's disease (AD). The enzymes beta-site APP cleaving enzyme (BACE) and A disintegrin and metalloproteinase 10 (ADAM10) play an important role in APP proteolysis. We measured by real time quantitative polymerase chain reaction their mRNA levels in hippocampal and cerebellar sections from 13 AD and 12 control brains. BACE mRNA amounts were similar in patients and controls. In contrast, ADAM10 mRNA levels were twofold higher in AD samples, but without relationship to the severity of anatomical damage. The data do not support the hypothesis that alteration of BACE and ADAM10 expression are associated with development of late stages of AD.