RESUMEN
Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.
Asunto(s)
Neoplasias Cardíacas/patología , Captura por Microdisección con Láser/métodos , Microscopía Confocal/métodos , Miocardio/patología , Mixoma/patología , Actinas/biosíntesis , Actinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Calbindina 2/biosíntesis , Calbindina 2/genética , Diferenciación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias Cardíacas/genética , Neoplasias Cardíacas/cirugía , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Miocardio/metabolismo , Mixoma/genética , Mixoma/cirugía , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , ARN Neoplásico/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVE: To determine whether the allelic frequency variation of the HS1.2 enhancer of the immunoglobulin heavy chain (IgH) 3' regulatory region (3'RR-1) locus represents a risk factor for systemic lupus erythematosus (SLE) and to identify a possible functional difference in the two most frequent alleles (*1 and *2) in binding nuclear factor- κB (NF-κB) and Sp1. METHODS: The frequency of the enhancer HS1.2 alleles was determined in two cohorts of patients with SLE (n=293) and in 1185 controls. Electrophoretic mobility shift assays (EMSA) were carried out with B cell nuclear extracts with different probes of HS1.2 alleles *1 and *2 to map the consensus binding sites of the nuclear factors. A confirmatory cohort of 121 patients with SLE was also included. RESULTS: The frequency of allele *2 of the HS1.2 enhancer was significantly increased in patients with SLE compared with controls (OR 1.60, 95% CI 1.33 to 1.92, p<0.001). EMSA experiments showed the presence of the Sp1 binding site in both alleles whereas only allele *2 carried the consensus for the NF-κB factor. The presence versus absence of allele *2 in patients with SLE correlated with a higher concentration of IgM levels and with the expression of B cell activating factor receptor (BAFF-R). CONCLUSIONS: The increased frequency of allele *2 in patients with SLE identifies a new genetic risk factor for SLE. A possible biological effect of the polymorphism could be the difference observed in the localisation of an NF-κB binding site which is specific for allele *2 and absent in allele *1. These observations suggest a functional effect of the HS1.2 enhancer in this disease.
Asunto(s)
Predisposición Genética a la Enfermedad , Cadenas Pesadas de Inmunoglobulina/genética , Lupus Eritematoso Sistémico/genética , Polimorfismo Genético , Adulto , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Femenino , Frecuencia de los Genes , Humanos , Inmunoglobulinas/genética , Masculino , Datos de Secuencia Molecular , FN-kappa B/genética , Factores de RiesgoRESUMEN
The complex of the yeast Lsm1p-7p proteins with Pat1p is an important mRNA decay factor that is involved in translational shutdown of deadenylated mRNAs and thus prepares these mRNAs for degradation. While the Lsm proteins are highly conserved, there is no unique mammalian homolog of Pat1p. To identify proteins that interact with human LSm1, we developed a novel immunoprecipitation technique that yields virtually pure immunocomplexes. Mass-spec analysis therefore identifies mostly true positives, avoiding tedious functional screening. The method unambiguously identified the Pat1p homolog in HeLa cells, Pat1b. When targeted to a reporter mRNA, Pat1b represses gene expression by inducing deadenylation of the mRNAs. This demonstrates that Pat1b, unlike yPat1p, acts as an mRNA-specific deadenylation factor, highlighting the emerging importance of deadenylation in the mRNA regulation of higher eukaryotes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inmunoprecipitación/métodos , ARN Mensajero/metabolismo , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Poli A/análisis , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Estabilidad del ARN , ARN Mensajero/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Selective IgA deficiency (IGAD) is the most common primary immunodeficiency, yet its pathogenesis is elusive. The IG (heavy) H chain human 3' Regulatory Region harbors three enhancers and has an important role in Ig synthesis. HS1.2 is the only polymorphic enhancer of the 3' RRs. We therefore evaluated HS1.2 allelic frequencies in 88 IGAD patients and 101 controls. Our data show that IGAD patients have a highly significant increase of homozygousity of the allele *1 (39% in the IGAD patients and 15% in controls), with an increase of 2.6-fold. Allele *4 has a similar trend of allele *2, both showing a significant decrease of frequency in IGAD. No relationship was observed between allele *1 frequencies and serum levels of IgG. However, allele *1 was associated in IGAD patients with relatively low IgM levels (within the 30th lowest percentile of patients). The HS1.2 polymorphism influences Ig seric production, but not IgG switch, in fact 30th lowest or highest percentile of IgG in patients did not associate to different frequencies of HS1.2 alleles. The control on normal healthy subjects did not correlate high or low levels of IgM or IgG with HS1.2 allelic frequence variation. Overall our candidate gene approach confirms that the study of polymorphisms in human diseases is a valid tool to investigate the function of these Regulatory Regions that confers multiple immune features.
Asunto(s)
Alelos , Elementos de Facilitación Genéticos/inmunología , Deficiencia de IgA/genética , Deficiencia de IgA/inmunología , Inmunoglobulina M/sangre , Región de Flanqueo 3'/inmunología , Adolescente , Secuencia de Bases , Niño , Preescolar , Femenino , Frecuencia de los Genes/inmunología , Humanos , Deficiencia de IgA/sangre , Inmunoglobulina G/sangre , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Región de Cambio de la Inmunoglobulina/genética , Masculino , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos/inmunología , Adulto JovenRESUMEN
The enhancer DNase-hypersensitive region 1,2 (HS1,2), a member of the Ig heavy-chain 3' regulatory region (3'RR) cluster, is active in human B cells transfected with reporter genes and in mouse is activated in late maturation. HS1,2-A contains binding sites for several transcription factors. There are four known alleles, that is, (*)1, (*)2, (*)3, and (*)4, which differ in their lengths in transcription factor binding. We showed that in celiac disease the frequency of the (*)2 allele is increased. Both dermatitis herpetiformis (DH) and psoriasis can be associated with different frequencies with celiac disease. Thus, we further investigate the frequency of allele (*)2 in DH, plaque psoriatic, and psoriatic arthritis patients. HS1,2-A allele frequencies were investigated in 37 DH, 61 plaque psoriatic, 28 psoriatic arthritis patients, and 265 healthy donors, age- and sex-matched, from the same geographical area. The frequency of the (*)2 allele changes from 0.39 in controls to 0.63 in DH, 0.59 in plaque psoriasis and 0.75 in psoriatic arthritis (P between 10(-4)-10(-5)). Our data evidence an increased frequency of the (*)2 allele of HS1,2-A in these cutaneous immune-related disorders. We suggest a related genetic predisposition in these pathogeneses.