RESUMEN
This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3' junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents. We assessed trueness, precision, efficiency, and linearity of the two assays, and the results demonstrate that both the assays independently assessed and the entire method fulfill European and international requirements for methods for genetically modified organism (GMO) testing, within the dynamic range tested. The homogeneity of the results of the collaborative trial between Europe and Asia is a good indicator of the robustness of the method.
Asunto(s)
Oryza/genética , Plantas Modificadas Genéticamente/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Asia , Europa (Continente) , Oryza/clasificación , Oryza/enzimología , Fosfolipasa D/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/clasificación , Plantas Modificadas Genéticamente/enzimología , Reacción en Cadena en Tiempo Real de la Polimerasa/normasRESUMEN
In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.
Asunto(s)
Oryza/química , Fosfolipasa D/genética , Plantas Modificadas Genéticamente/clasificación , Plantas Modificadas Genéticamente/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ADN de Plantas/análisis , Semillas/química , Semillas/genética , Sensibilidad y Especificidad , Vitamina A/análisisRESUMEN
The purpose of this study is to comparatively evaluate the performance of different wearable systems based on indirect breathing monitoring in terms of susceptibility to motion artifacts. These performances are compared with direct respiratory measurements using a spirometer, which is accurate, reliable, and less sensitive to movement artifacts, but cannot be integrated into truly wearable form. Experiments were carried out on four indirect methods implemented into wearable systems, inductive plethysmography, impedance plethysmography, piezoresistive pneumography, and piezoelectric pneumography, to ascertain the performance of each of them in terms of noise due to movement artifacts, as well as to study the effects of different movements or gestures during each test. A group of volunteers was asked to wear all of the breath monitoring systems simultaneously along with the face mask of the spirometer while carrying out four physical exercises in a gym under controlled conditions. Data are analyzed in the time and frequency domain to estimate the frequency respiration from each wearable system and compare it with those of the spirometer. Results confirmed that all the wearable systems are somehow affected by movement artifacts, but statistical investigation showed that for most of the physical exercises, three out of four, piezoelectric pneumography provided best performance in terms of robustness and reduced susceptibility to movement artifacts.
Asunto(s)
Artefactos , Monitoreo Ambulatorio , Pletismografía , Frecuencia Respiratoria , Procesamiento de Señales Asistido por Computador , Espirometría , Adulto , Vestuario , Femenino , Humanos , Masculino , Monitoreo Ambulatorio/instrumentación , Monitoreo Ambulatorio/métodos , Actividad Motora/fisiología , Pletismografía/instrumentación , Pletismografía/métodos , Espirometría/instrumentación , Espirometría/métodos , Estadísticas no ParamétricasRESUMEN
Preliminary results from a pilot trial on trastuzumab's mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56(+) T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16(+) lymphocytes was influenced by 158 V/F polymorphism of Fc gamma RIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16(+) lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Genes erbB-2 , Receptor ErbB-2/genética , Anticuerpos Monoclonales Humanizados , Antígenos CD/inmunología , Neoplasias de la Mama/cirugía , Femenino , Humanos , Inmunoterapia/métodos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Proyectos Piloto , Receptores de IgG/inmunología , TrastuzumabRESUMEN
Studies investigating the relation of diet to breast cancer have produced conflicting results. We hypothesized that dietary factors associated with breast cancer risk might differentially influence the HER-2 status of the cancers that develop, and investigated this hypothesis by analyzing the data of the ORDET prospective study. We analyzed 8,861 volunteer women residents of the Varese Province, Italy, for whom we had full data. By December 31, 2001, 238 cases had occurred in which HER-2 status was known. Four dietary patterns had been identified previously by factor analysis: salad vegetables (high consumption of raw vegetables and olive oil), prudent (cooked vegetables, poultry, fish), western (potatoes, meat, eggs, butter), and canteen (pasta, tomato sauce, wine). In our study, relative risks (RRs) of developing HER-2-positive and HER-2-negative breast cancers by tertiles of dietary pattern factor scores were assessed by multinomial logistic regression. The salad vegetables dietary pattern had a protective effect against HER-2-positive cancers (RR = 0.25, 95% CI 0.10-0.64, for the highest tertile; p(trend) = 0.001), much stronger than for HER-2-negative cancers (p(heterogeneity) = 0.039). This important finding that a salad vegetables dietary pattern protects mainly against a specific breast cancer subtype indicates that future studies on environmental/dietary risk factors should explicitly take account of the heterogeneity of breast cancer phenotypes.
Asunto(s)
Neoplasias de la Mama/metabolismo , Conducta Alimentaria , Receptor ErbB-2/metabolismo , Verduras , Adulto , Anciano , Dieta , Femenino , Humanos , Italia , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
The immune system of vertebrates detects bacterial DNA as a "danger signal" based on the presence of unmethylated CpG motifs. We examined whether oligodeoxynucleotides (ODNs) with CpG motifs (CpG-ODNs) also induce mobilization of hematopoietic progenitor cells (HPCs). Mice challenged with CpG-ODNs showed an increase in peripheral blood colony-forming units (CFU) with a peak at day 4 after treatment, associated with an increase, starting 30 min after CpG treatment, in serum levels of mouse keratinocyte-derived chemokine (mKC), a functional homolog of human interleukin (IL) 8; production of granulocyte-colony-stimulating factor (CSF) was also detected. Mobilization and mKC induction were sequence-specific and dose-dependent occurring even with low doses of CpG-ODNs. Interestingly, intestinal cells were involved in mKC production. HPC mobilization by CpG-ODNs was dependent on peripheral blood mononuclear cells since mobilization was reduced in neutrophil-depleted mice. Moreover, CpG-ODN treatment significantly increased G-CSF mobilizing capacity. Finally, pretreatment with an anti-mKC neutralizing antibody significantly reduced CpG-induced mobilization, further supporting a role for mKC. Thus, bacterial DNA is a "danger signal" not only for immune cells but also for hematopoietic cells, communicating the need for increased hematopoiesis during infections and for the renewal of the immune system. The HPC mobilization activity of CpG-ODNs will need to be considered in the design of treatment regimens for cancer clinical trials using CpG-ODNs in association with chemotherapy.
Asunto(s)
Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-8/biosíntesis , Oligodesoxirribonucleótidos/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL2 , Femenino , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/metabolismo , Interleucina-8/antagonistas & inhibidores , Interleucina-8/sangre , Queratinocitos/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Ratones , Ratones Endogámicos C57BL , Monocinas/antagonistas & inhibidores , Monocinas/metabolismoRESUMEN
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a profound dysregulation of the host's immune system at both the humoral and cellular level. We investigated to see if this dysregulation could be due partly to thymus dysfunction by quantifying the number of signal joint T-cell receptor excision circles (sjTRECs) in peripheral blood mononuclear cells of 30 untreated B-CLL patients at diagnosis and in age-matched healthy controls. sjTRECs were found decreased, normal and elevated in 19, 9 and 2 patients, respectively, in comparison to age-matched controls. We next speculated that sjTREC levels might be related to an accredited B-CLL prognostic marker represented by the somatic hypermutation (SHM) status of the variable heavy chain (V(H)) genes. Eight of 17 patients with SHMs had sjTREC levels in the range of or higher than normal donors, whereas only 3 of the 13 patients lacking SHMs had normal sjTREC levels. After a 5-year observation period in 16 patients for whom a clinical follow-up was available, only 2 of 10 patients with SHMs progressed vs. 5 of 6 patients without SHMs and 3 of 7 patients with normal or higher sjTREC levels progressed vs. 4 of 9 with low sjTREC levels. Our study demonstrates that sjTREC levels are decreased in >60% of B-CLL patients and suggests a potential role of thymus in the immune dysfunction of these patients.
Asunto(s)
Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Mutación , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Secuencia de Bases , Cartilla de ADN , Reparación del ADN , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Valores de Referencia , Timo/inmunologíaRESUMEN
Isotype switch recombination together with somatic mutation of immunoglobulin variable genes is indicative of B-cell maturation stage. Because aberrant isotype switch events occur in a proportion of gastric mucosa-associated lymphoid tissue (MALT) lymphomas, we tested whether gastric MALT lymphomas with or without aberrant rearrangements in the switch regions differ in B-cell maturation stage. Southern blot analysis of DNA from six gastric MALT lymphoma cases revealed the presence of aberrant isotype switch events in three cases. Somatic common mutations were present in all immunoglobulin variable heavy chain genes of the six cases, and homology with the closest germline ranged from 89.5% to 98.8%. Replacement versus silent mutation ratio analysis of complementarity-determining regions and frameworks indicated the positive selective pressure of an antigen in four cases. In the remaining two cases, protein translated from the third complementarity-determining region suggested the selective pressure of an autoantigen. The three cases with aberrant isotype switch events showed no uncommon mutations, whereas two of three cases without evidence of aberrant isotype switch showed high levels of such mutations. Moreover the three cases with aberrant isotype switch, compared with the three cases without, showed an increased number of common mutations and of N segment additions. These data raise the possibility of two distinct subsets of gastric low-grade MALT lymphomas, one with aberrant isotype switch and no intraclonal diversification, and one with no aberrant isotype switch but with intraclonal diversification. The first subset may originate from a post-germinal center environment and the second from a germinal center. Alternatively, the first subset may derive from the second after maturation or after a transformation event that blocks the mutational process.
Asunto(s)
Mucosa Gástrica/patología , Cambio de Clase de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Linfoma de Células B de la Zona Marginal/diagnóstico , Neoplasias Gástricas/diagnóstico , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión de Anticuerpos , Southern Blotting , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Secuencia de Consenso , ADN de Neoplasias/análisis , Humanos , Isotipos de Inmunoglobulinas , Región de Cambio de la Inmunoglobulina/genética , Inmunohistoquímica , Linfoma de Células B de la Zona Marginal/genética , Datos de Secuencia Molecular , Fenotipo , Neoplasias Gástricas/genéticaRESUMEN
We investigated 38 cases of B-cell chronic lymphocytic leukemia (B-CLL) for the presence of non-productive rearrangements in the S(mu) regions and defined for the first time the molecular nature of these rearrangements. Southern blot analysis revealed S(mu) region rearrangements in 13 cases (34%) and polymerase chain reactions (PCRs) indicated that these rearrangements consisted of internal deletions of the S(mu) region. Long-distance PCRs localized the S(mu) deletions in the V(H)DJ(H) rearranged allele in most cases. We investigated if S(mu) deletions were related to V(H) somatic mutations that, together with isotype switch recombination, are indicative of the B-cell maturation stage. No significant correlation between the presence of S(mu) deletions and V(H) somatic mutations was found, indicating that the two processes are independent in B-CLL. Moreover no significant correlation between S(mu) deletions and prognosis was observed. Having shown that S(mu) internal deletions are not chromosome translocations rules out their involvement in the onset of malignancy, while their localization in the V(H)DJ(H) rearranged alleles suggests a possible role in the stabilization of the isotype of the expressed immunoglobulin.