RESUMEN
Purpose: Metastatic melanoma lymph nodes (MMLns) might be challenging to detect on MR-WBI, as both MMLns and normal lymph nodes (NLns) can show restricted water diffusion. Our purpose is to assess the potential contribution of the DIXON sequence in differentiating MMLns from NLns. Material and methods: We followed a cohort of 107 patients with stage IIIb/c and IV skin melanoma for 32 months using MR-WBI with DIXON, STIR, and DWI/ADC sequences. We compared signal intensity (SI) values of MMLns and NLns in the four series of the DIXON sequence (in/out-of-phase, fat_only, and water_only series). The fat fraction (SIfat_only/SIin) and the long:short axis ratio of MMLns were calculated. The fat fraction was also calculated in the fatty hila of NLns. Results: All MMLns (8 from 7 patients) showed SIout>SIin with a mean fat fraction of 10%. In 40 normal fatty hila (25 patients), the proportion of SIout>SIin was 100% and mean fat fraction was 89% (p<0.001 for fat fraction, Mann-Whitney U-test). In the cortex of NLns, a SIout>SIin pattern was identified in 41/113 cases from 19/40 patients. The median long:short axis ratio in MMLns was 1.13 (range 1.03-1.25). Conclusion: The combination of three features of MMLns (SIout>SIin, low-fat fraction and rounded shape) might hold promise in differentiating NLns from MMLns in patients with skin melanoma. Further research is warranted due to the small number of MMLns in our cohort.
RESUMEN
Adjuvant systemic therapy was introduced in the Netherlands as a breast cancer treatment in the early 1980s. In this paper, we describe the trends in the usage of adjuvant systemic treatment in the period 1975-1997 in the Netherlands. The main aim of our study was to assess the effects of adjuvant tamoxifen and polychemotherapy on breast cancer mortality, compared to the effects of the mammography screening programme. The computer simulation model MIcrosimulation SCreening ANalysis, which simulates demography, natural history of breast cancer and screening effects, was used to estimate the effects. Use of adjuvant therapy increased over time, but since 1990 it remained rather stable. Nowadays, adjuvant therapy is given to 88% of node-positive patients aged 50-69 years, while less than 10% of node-negative patients receive any kind of adjuvant treatment. Adjuvant treatment is given independent of the mode of detection (adjusted by nodal status and size). We predict that the reduction in breast cancer mortality due to adjuvant therapy is 7% in women aged 55-74 years, while the reduction due to screening, which was first implemented in women aged 50-69 years in 1990-97, will be 28-30% in 2007. In conclusion, although adjuvant systemic therapy can reduce breast cancer mortality rates, it is anticipated to be less than the mortality reduction caused by mammography screening.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Quimioterapia Adyuvante/tendencias , Tamoxifeno/uso terapéutico , Distribución por Edad , Anciano , Simulación por Computador , Quimioterapia Combinada , Utilización de Medicamentos/tendencias , Femenino , Humanos , Mamografía/tendencias , Tamizaje Masivo , Persona de Mediana Edad , Países Bajos/epidemiología , Tasa de SupervivenciaAsunto(s)
Anticuerpos Antivirales/sangre , Autoanticuerpos/sangre , Proteínas del Tejido Nervioso/inmunología , Enfermedad de Parkinson/etiología , Proteínas de la Matriz Viral/inmunología , Autoanticuerpos/líquido cefalorraquídeo , Corteza Cerebral/virología , ADN Viral/análisis , ADN Viral/líquido cefalorraquídeo , Ensayo de Inmunoadsorción Enzimática , Infecciones por Virus de Epstein-Barr/sangre , Infecciones por Virus de Epstein-Barr/inmunología , Humanos , Mesencéfalo/virología , Enfermedad de Parkinson/sangre , Reacción en Cadena de la Polimerasa , Sinucleínas , Latencia del Virus/inmunologíaRESUMEN
BACKGROUND: The preventive effect of low-dose aspirin in cardiovascular disease is generally attributed to its antiplatelet action caused by differential inhibition of platelet cyclooxygenase-1. However, there is evidence that aspirin also affects release of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha). It is not known whether this is caused by direct action on the cytokine pathway or indirectly through cyclooxygenase inhibition and altered prostanoid synthesis, or both. METHODS: We assessed the capacity of lipopolysaccharide-activated leukocytes in whole blood cultures of eight healthy subjects following a single oral dose of 80 mg aspirin to release TNF-alpha, prostanoid E2 (PGE2) and prostanoid I2 (PGI2), and thromboxane A2 (TXA2). TNF-alpha and prostanoids were determined by enzyme-linked immunoassays. RESULTS: In seven subjects, TNF-alpha release in blood cultures decreased 24h after intake of aspirin. The effect of aspirin on prostanoid release was assessed in three individuals: PGE2 increased in all subjects, PGI2 increased in two and remained unchanged in one, and TXA2 was reduced in two and unchanged in one individual The presence of DFU, a specific inhibitor of cyclooxygenase 2, did not affect the reduction of TNF-alpha release by aspirin, but abolished prostanoid production in all three individuals. CONCLUSION: The capacity of activated leukocytes to release TNF-alpha is reduced by ingestion of low-dose aspirin, independent of changes in prostanoid biosynthesis.
Asunto(s)
Aspirina/administración & dosificación , Inhibidores de la Ciclooxigenasa/administración & dosificación , Leucocitos/metabolismo , Prostaglandinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Células Sanguíneas/citología , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona , Epoprostenol , Femenino , Furanos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Leucocitos/citología , Leucocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas , Tromboxano A2RESUMEN
The basic Helix-Loop-Helix (bHLH) proteins are transcription factors that play important roles during the development of various metazoans including fly, nematode, and vertebrates. They are also involved in human diseases, particularly in cancerogenesis. We made an extensive search for bHLH sequences in the completely sequenced genomes of Caenorhabditis elegans and of Drosophila melanogaster. We found 35 and 56 different genes, respectively, which may represent the complete set of bHLH of these organisms. A phylogenetic analysis of these genes, together with a large number (>350) of bHLH from other sources, led us to define 44 orthologous families among which 36 include bHLH from animals only, and two have representatives in both yeasts and animals. In addition, we identified two bHLH motifs present only in yeast, and four that are present only in plants; however, the latter number is certainly an underestimate. Most animal families (35/38) comprise fly, nematode, and vertebrate genes, suggesting that their common ancestor, which lived in pre-Cambrian times (600 million years ago) already owned as many as 35 different bHLH genes.
Asunto(s)
Genómica , Secuencias Hélice-Asa-Hélice/genética , Familia de Multigenes , Filogenia , Factores de Transcripción/genética , Secuencia de Aminoácidos/genética , Animales , Caenorhabditis elegans/genética , Bases de Datos Factuales , Drosophila melanogaster/genética , Duplicación de Gen , Genes/genética , Genes de Helminto/genética , Genes de Insecto/genética , Proteínas del Helminto/genética , Humanos , Proteínas de Insectos/genética , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Epstein-Barr virus (EBV) gene expression in tumor cells of posttransplant lymphoproliferative disorder (PTLD) patients resembles that of EBV transformed B-cell lines (LCL). EBV-specific cytotoxic T-lymphocytes can be generated by stimulating peripheral blood lymphocytes with autologous LCL. We describe a standardized method for the growth inactivation and cryopreservation of LCL for optimal T-cell stimulation and analyzed the function and phenotype of responding T-cells. LCL growth was completely blocked by mitomycin C treatment (McLCL) and McLCL could be cryopreserved while retaining excellent APC function. McLCL stimulated both CD4(+) and CD8(+) T-cells as measured by HLA-DR and CD25 expression using FACS analysis. EBV-specific CTL activity and T-cell proliferation were induced and immunocytochemical staining showed CD4(+) and (granzyme B positive) CD8(+) T-cells rosetting with McLCL. Granzymes A and B, IFN-gamma, and IL-6 were detected at significant levels in the supernatant. Thus, ex vivo T-cell activation with cryopreserved McLCL results in activation of both CD4(+) and CD8(+) T-cells producing a Th1-like cytokine profile, making this a suitable protocol for adoptive therapy of PTLD.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Herpesvirus Humano 4/inmunología , Mitomicina/farmacología , Traslado Adoptivo/métodos , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , División Celular/efectos de los fármacos , Línea Celular Transformada , Criopreservación/métodos , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Granzimas , Herpesvirus Humano 4/fisiología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Activación de Linfocitos/efectos de los fármacos , Fenotipo , Formación de Roseta , Serina Endopeptidasas/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células Tumorales CultivadasRESUMEN
Basic Helix-Loop-Helix (bHLH) transcription factors control various aspects of the formation of the nervous system in the metazoans. In Drosophila some bHLH (such as the achaete-scuteatonal, and amos genes) act as proneural genes, directing ectodermal cells toward a neural fate. Their vertebrate orthologs, however, probably do not assume such a neural determination function, but rather control the decision made by neural precursors to generate neurons and not glial cells, as well as the progression of neuronal precursors toward differentiation into mature neurons. The proneural function of Drosophila bHLH genes may be an innovation that occurs in the evolutive lineage that leads to arthropods. In addition, although neural bHLH appear to be involved in the specification of neuronal identities, they probably do not confer by themselves neuronal type-specific properties to the cells. Rather, neural bHLH allow neural cells to correctly interpret specification and positional cues provided by other factors. Although bHLH genes are often expressed in complementary subsets of neural cells and/or expressed sequentially in those cells, the coding regions of the various neural bHLH appear largely interchangeable. We propose that the specific expression patterns have been acquired, following gene duplications, by subfunctionalization, i.e., the partitioning of ancestral expression patterns among the duplicates and, by extension, we propose that subfunctionalization is a key process to understand the evolution of neural bHLH genes.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Evolución Molecular , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/crecimiento & desarrollo , Factores de Transcripción/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila , Secuencias Hélice-Asa-Hélice , Proteínas del Tejido Nervioso/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is expressed in malignancies with latency type II and III and is an important transforming protein. To further study this protein LMP1 was expressed by and purified from recombinant baculovirus infected Sf9 cells. Expression levels of LMP1 in EBV transformed B cell lines and Sf9 cells were analyzed using a newly developed quantitative LMP1-capture ELISA. Highest expression was found in the cell line X50/7 (6.2 ng/10(7) cells), whereas expression levels of recombinant LMP1 (bLMP1) in Sf9 cells reached 506 ng/10(7) cells. Surprisingly bLMP1 could also be detected in the culture medium as a stable full-length protein. Highest expression in Sf9 cells (506 ng/10(7) cells) was observed at 48 h post infection and in the culture medium (1590 ng/ml) at 96 h post infection. Before purification bLMP1 was solubilised using 0.22 m octyl-beta-glucoside at pH 6.0. Purification of bLMP1 using Q-Sepharose FF yielded 10-80 times enriched bLMP1 fractions, indicating that Q-Sepharose can be used for pre-purification. A one-step monoclonal antibody based immunoaffinity chromatography yielded highly purified bLMP1. Although the overall yields (20 microg purified LMP1 from 100 ml culture supernatant) and protein concentrations were low, higher concentrations of >95% purified BLMP1 could be reached after freeze drying.
Asunto(s)
Proteínas Oncogénicas Virales/metabolismo , Proteínas de la Matriz Viral/metabolismo , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Baculoviridae/genética , Callithrix , Línea Celular , Línea Celular Transformada , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática/métodos , Insectos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sefarosa , Transfección , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/aislamiento & purificaciónRESUMEN
Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated malignancies and is essential for EBV-genome maintenance. Antibodies to EBNA1 are abundantly detected in serum of most EBV carriers but EBNA1 escapes recognition by effector T-lymphocytes. To further study the functional and immunological characteristics of EBNA1 it is important to have sufficient quantities of purified EBNA1 available. This paper describes a simple, reproducible method for the production and purification of EBV-encoded EBNA1 expressed in insect cells (bEBNA1). For quantification of EBNA1 expression levels in cell lines and for monitoring bEBNA1 purification and overall yields we developed a quantitative and EBNA1-specific capture ELISA. We observed that EBV-positive cell lines express EBNA1 at different levels, with the B cell lymphoblastoid cell line X50/7 having the highest production. However, much larger quantities (380-fold) were obtained by expressing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells. Scaling-up experiments revealed that bEBNA1 expression kinetics and protein stability are identical in 1-liter stirred bioreactors when compared to expression in stationary culture flasks. Optimal expression was reached after 72 h following inoculation at 1 pfu/cell, when insect cell viability was about 50%. For purification the nuclear fraction containing most of the bEBNA1 (>95%) was isolated. Solubilized bEBNA1 was purified by a one-step oriP DNA-Sepharose affinity purification procedure, using biotinylated PCR-amplified family of repeats (FR)-domain products immobilized onto streptavidin agarose. A >200-fold specific enrichment was reached and yields of bEBNA1 with an estimated purity of >95%.
Asunto(s)
Baculoviridae/genética , Antígenos Nucleares del Virus de Epstein-Barr/biosíntesis , Antígenos Nucleares del Virus de Epstein-Barr/aislamiento & purificación , Animales , Reactores Biológicos , Línea Celular , Supervivencia Celular , Cromatografía de Afinidad/métodos , Técnicas de Cultivo/métodos , ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Immunoblotting , Unión Proteica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y Especificidad , Spodoptera/citología , Spodoptera/virologíaRESUMEN
In neoplastic cells of EBV-positive lymphoid malignancies latent membrane protein (LMP1) is expressed. Because no adequate cellular immune response can be detected against LMP1, we investigated whether LMP1 had a direct effect on T lymphocyte activation. In this study we show that nanogram amounts of purified recombinant LMP1 (rLMP1) strongly suppresses activation of T cells. By sequence alignment two sequences (LALLFWL and LLLLAL) in the first transmembrane domain of LMP1 were identified showing strong homology to the immunosuppressive domain (LDLLFL) of the retrovirus-encoded transmembrane protein p15E. The effects of rLMP1 and LMP1-derived peptides were tested in T cell proliferation and NK cytotoxicity assays and an Ag-induced IFN-gamma release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides showed strong inhibition of T cell proliferation and NK cytotoxicity, while acetylated LALLFWL peptides had an even stronger effect. In addition, Ag-specific IFN-gamma release was severely inhibited. To exert immunosuppressive effects in vivo, LMP1 has to be excreted from the cells. Indeed, LMP1 was detected in supernatant of EBV-positive B cell lines (LCL), and differential centrifugation in combination with Western blot analysis of the pellets indicated that LMP1 is probably secreted by LCL in the form of exosomes. The amount of secreted LMP1 in B cell cultures is well below the immunosuppressive level observed with rLMP1. Our results demonstrate direct immunosuppressive properties of LMP1 (fragments) and suggest that EBV-positive tumor cells may actively secrete LMP1 and thus mediate immunosuppressive effects on tumor-infiltrating lymphocytes. Moreover, we demonstrate, for the first time, that transmembrane protein-mediated immunosuppression is not solely restricted to RNA tumor viruses, but can also be found in DNA tumor viruses.
Asunto(s)
Herpesvirus Humano 4/inmunología , Inmunosupresores/farmacología , Proteínas de la Matriz Viral/fisiología , Secuencia de Aminoácidos , Fraccionamiento Celular , Línea Celular Transformada , Sistema Libre de Células/química , Sistema Libre de Células/inmunología , Sistema Libre de Células/virología , Citotoxicidad Inmunológica/inmunología , Epítopos de Linfocito T/inmunología , Herpesvirus Humano 4/genética , Humanos , Inmunosupresores/química , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Linfocitos T/inmunología , Linfocitos T/virología , Toxoide Tetánico/inmunología , Células Tumorales Cultivadas , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
In Drosophila, Hedgehog (Hh) is a key regulator of limb development and activates decapentaplegic (dpp), a gene encoding a TGFbeta-related factor that controls growth and patterning of the limbs. During wing development, Hh also has morphogen-like Dpp-independent functions, controlling the morphogenesis of the central part of the wing through the activation of the evolutionarily conserved transcription factors encoded by the iroquois and collier genes. The ways in which Hh forms an activity gradient to lay the basis of patterning of the adult wing are described here. As the signal transduction pathway of Hh is strongly conserved during evolution and human Hh may be implicated in congenital diseases and cancers, these observations provide important advances which may help in understanding the function of Hh proteins in normal and pathological development and tumourigenesis in humans.
Asunto(s)
Proteínas de Drosophila , Drosophila/crecimiento & desarrollo , Proteínas de Insectos/metabolismo , Alas de Animales/crecimiento & desarrollo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Humanos , Proteínas de Insectos/genética , Modelos Biológicos , Morfogénesis , Isoformas de Proteínas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
AIMS: To examine the expression of Epstein-Barr virus (EBV) transcripts encoding proteins homologous to important human proteins in diverse EBV associated diseases. The proteins were: BHRF1 (homologous to Bcl-2), BDLF2 (homologous to cyclin B1), BARF1 (homologous to intercellular cell adhesion molecule 1 (ICAM-1)), and BCRF1 (viral IL-10 (vIL-10), homologous to human IL-10 (hIL-10)). METHODS: Six cases of oral hairy leukoplakia, seven of Hodgkin's disease, eight of T cell non-Hodgkin's lymphoma, and nine of nasopharyngeal carcinoma were examined at the mRNA level using either the reverse transcriptase polymerase chain reaction (RT-PCR) or nucleic acid sequence based amplification (NASBA). Different primer sets allowed the differentiation by RT-PCR of the latent (Cp/Wp driven) and lytic (Hp driven) transcripts of BHRF1. A specific NASBA reaction was developed for the detection of vIL-10 and BDLF2 transcripts and this was tested initially on cell lines and later on clinical samples. RESULTS: vIL-10 and BDLF2 were expressed almost exclusively in oral hairy leukoplakia, whereas BARF1 transcripts were present in all cases of nasopharyngeal carcinoma, with weak expression in one oral hairy leukoplakia and isolated cases of lymphoid malignancy. Both BHRF1 transcripts were detected across the range of tissues tested, but strong expression of lytic BHRF1 transcripts was seen only in oral hairy leukoplakia. CONCLUSIONS: vIL-10 and BDLF2 transcripts are expressed during productive EBV infection and are unlikely to be important in the pathogenesis of EBV associated malignancies. BARF1 appears to be expressed preferentially during viral latency and is more closely associated with malignant rather than benign epithelial proliferations. The alternative transcripts derived from the BHRF1 open reading frame may have very different roles during latent or productive infection.
Asunto(s)
Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Neoplasias/virología , Homología de Secuencia de Aminoácido , Transcripción Genética , Proteínas Virales/genética , Expresión Génica , Humanos , Interleucina-10/genética , Leucoplasia Vellosa/virología , Linfoma/virología , Glicoproteínas de Membrana/genética , Neoplasias Nasofaríngeas/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
A competitive quantitative PCR (Q-PCR) assay combined with simple silica-based DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competitive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentration. The IS has the same amplicon length and base composition as the wild-type (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Competitive coamplification yields two PCR products that are quantified by enzyme immunoassay or by electrochemiluminescence detection, with probes specific for the 23 differing internal nucleotides. The Q-PCR has a sensitivity of 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quantification of known amounts of plasmid containing the WT EBNA-1 target. Furthermore, we determined EBV genome copy numbers in different cell lines. For EBV quantification in clinical samples, DNA was isolated from lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor peripheral blood samples were positive by Q-PCR. In most of these samples, viral load was less than 2,000 EBV copies/ml of blood. In peripheral blood samples from two AIDS-related non-Hodgkin's lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy. In Burkitt's lymphoma patients, up to 4,592,000 EBV genome copies/ml of blood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship between EBV load and clinical parameters.
Asunto(s)
ADN Viral/sangre , Herpesvirus Humano 4/genética , Reacción en Cadena de la Polimerasa/métodos , Linfoma de Burkitt/virología , Humanos , Técnicas para Inmunoenzimas , Mediciones Luminiscentes , Células Tumorales CultivadasRESUMEN
BACKGROUND: The secreted Hedgehog (Hh) proteins have been implicated as mediators of positional information in vertebrates and invertebrates. A gradient of Hh activity contributes to antero-posterior (A/P) patterning of the fly wing. In addition to inducing localised expression of Decapentaplegic (Dpp), which in turn relays patterning cues at long range, Hh directly patterns the central region of the wing. RESULTS: We show that short-range, dose-dependent Hh activity is mediated by activation of the transcription factor Collier (Col). In the absence of col activity, longitudinal veins 3 and 4 (L3 and L4) are apposed and the central intervein is missing. Hh expression induces col expression in a narrow stripe of cells along the A/P boundary through a dual-input mechanism: inhibition of proteolysis of Cubitus-interruptus (Ci) and activation of the Fused (Fu) kinase. Col, in cooperation with Ci, controls the formation of the central intervein by activating the expression of blistered (bs), which encodes the Drosophila serum response factor (D-SRF), the activity of which is required for the adoption and maintenance of the intervein cell fate. Furthermore, col is allelic to knot, a gene involved in the formation of the central part of the wing. This finding completes our understanding of the sectorial organisation of the Drosophila wing. CONCLUSIONS: Col, the Drosophila member of the COE family (Col/Olf-1/EBF) of non-basic, helix-loop-helix (HLH)-containing transcription factors, is a mediator of the short-range organising activity of Hh in the Drosophila wing. Our results support the idea that Hh controls target gene expression in a concentration-dependent manner and highlight the importance of the Fu kinase in this differential regulation. The high degree of evolutionary conservation of the COE proteins and the diversity of developmental processes controlled by Hh signalling raises the possibility that the specific genetic interactions depicted here may also operate in vertebrates.
Asunto(s)
Proteínas de Drosophila , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Insectos/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas Hedgehog , Mutación , Fenotipo , Transducción de Señal , Alas de Animales/crecimiento & desarrolloRESUMEN
Human antibody responses to latent membrane protein 1 (LMP1) in patients with Epstein-Barr virus (EBV)-related disease syndromes were analyzed in detail. Only by immunoblot analysis with purified recombinant LMP1 and by IFA on recombinant LMP1-expressing insect cells could human antibodies directed against LMP1 be detected. Low serum levels of LMP1-directed antibodies could be detected in 3 of 8 EBV-positive Hodgkin's disease patients, 3 of 40 nasopharyngeal carcinoma patients, 2 of 23 Burkitt's lymphoma patients, and 1 of 27 non-Burkitt's lymphoma patients. No LMP1-directed antibodies could be detected in healthy EBV carriers, infectious mononucleosis patients, or patients with chronic EBV disease. All sera contained significant levels of EBV antibodies directed against the immunodominant EBV proteins and peptides. From this study, it can be concluded that LMP1 is a protein with a very low immunogenicity for the humoral immune response in humans.
Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Proteínas de la Matriz Viral/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/inmunología , Linfoma de Burkitt/inmunología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Enfermedad de Hodgkin/inmunología , Humanos , Immunoblotting , Mononucleosis Infecciosa/inmunología , Linfoma/inmunología , Ratones , Datos de Secuencia Molecular , Neoplasias Nasofaríngeas/inmunología , Péptidos/síntesis química , Proteínas Recombinantes , Proteínas de la Matriz Viral/genéticaRESUMEN
Nucleic acid sequence-based amplification (NASBA) assays were developed for direct detection of Epstein-Barr virus (EBV) transcripts encoding EBV nuclear antigen 1 (EBNA1), latent membrane proteins (LMP) 1 and 2, and BamHIA rightward frame 1 (BARF1) and for the noncoding EBV early RNA 1 (EBER1). The sensitivities of all NASBAs were at least 100 copies of specific in vitro-generated RNA. Furthermore, 1 EBV-positive JY cell in a background of 50,000 EBV-negative Ramos cells (the relative sensitivity) was detected by using the EBNA1, LMP1, and LMP2 NASBA assays. The relative sensitivity of the EBER1 NASBA was 100 EBV-positive cells, which was probably related to the loss of small RNA molecules during the isolation. The BARF1 and LMP2 NASBAs were evaluated on clinical material. BARF1 expression was found in 6 of 7 nasopharyngeal carcinomas (NPC) but in 0 of 22 Hodgkin's disease (HD) cases, whereas LMP2 expression was found in 7 of 7 NPCs and in 17 of 22 HD cases. For detection of EBNA1 transcripts in HLs (n = 12) and T- and B-cell non-Hodgkin's lymphomas (n = 3 and n = 2, respectively), NASBA was compared with reverse transcriptase (RT) PCR. Two samples were positive only with NASBA, and two other samples were positive only with RT-PCR; for all other samples, the RT-PCR and NASBA results were in agreement. We conclude that NASBA is suitable for sensitive and specific detection of the above-mentioned EBV transcripts, regardless of their splicing patterns and the presence of EBV DNA. The EBNA1, LMP2, and BARF1 NASBAs developed in this study proved to be reliable assays for detection of the corresponding transcripts in EBV-positive clinical material.
Asunto(s)
Herpesvirus Humano 4/genética , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Bases , Línea Celular Transformada , Cartilla de ADN/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Expresión Génica , Herpesvirus Humano 4/aislamiento & purificación , Herpesvirus Humano 4/patogenicidad , Enfermedad de Hodgkin/virología , Humanos , Linfoma no Hodgkin/virología , Neoplasias Nasofaríngeas/virología , Empalme del ARN , ARN Viral/análisis , ARN Viral/genética , Sensibilidad y Especificidad , Transcripción Genética , Proteínas de la Matriz Viral/genética , Proteínas Virales/genéticaRESUMEN
Groups of genes sharing similar motifs may be used at different steps of a same developmental process. In this review, we discuss the significance of this phenomenon in the case of the basic Helix-Loop-Helix (bHLH) proteins that are involved at different steps of the development of the peripheral nervous system (PNS) of Drosophila.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Drosophila/genética , Sistema Nervioso/embriología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Vertebrados/embriología , Vertebrados/genéticaRESUMEN
The embryonic peripheral nervous system of Drosophila contains two main types of sensory neurons: type I neurons, which innervate external sense organs and chordotonal organs, and type II multidendritic neurons. Here, we analyse the origin of the difference between type I and type II in the case of the neurons that depend on the proneural genes of the achaete-scute complex (ASC). We show that, in Notch- embryos, the type I neurons are missing while type II neurons are produced in excess, indicating that the type I/type II choice relies on Notch-mediated cell communication. In contrast, both type I and type II neurons are absent in numb- embryos and after ubiquitous expression of tramtrack, indicating that the activity of numb and the absence of tramtrack are required to produce both external sense organ and multidendritic neural fates. The analysis of string- embryos reveals that when the precursors are unable to divide they differentiate mostly into type II neurons, indicating that the type II is the default neuronal fate. We also report a new mutant phenotype where the ASC-dependent neurons are converted into type II neurons, providing evidence for the existence of one or more genes required for maintaining the alternative (type I) fate. Our results suggest that the same mechanism of type I/type II specification may operate at a late step of the ASC-dependent lineages, when multidendritic neurons arise as siblings of the external sense organ neurons and, at an early step, when other multidendritic neurons precursors arise as siblings of external sense organ precursors.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Neuronas/citología , Proteínas Represoras , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Unión al ADN/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Hormonas Juveniles/genética , Proteínas de la Membrana/genética , Morfogénesis , Mutación , Receptores Notch , Órganos de los Sentidos/citología , Órganos de los Sentidos/embriología , Factores de Transcripción/genética , Cromosoma XRESUMEN
A major issue in development is to understand how local heterogeneities are interpreted to determine specific cell fates. The sense organs of Drosophila provide an accessible system for addressing this issue. Most sense organs comprise four types of cells, and their differentiation is the outcome of a complex developmental programme comprising several steps. Recent results illuminate, for several of these steps, the nature of the local heterogeneities and the mechanism used to interpret them in terms of cell fate decisions.