RESUMEN
Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform). Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated) DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method's specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector.
RESUMEN
INTRODUCTION: Cardiac implantable electronic devices (CIED) are increasingly being used in end-stage renal disease (ESRD) patients. These patients have a high risk of device infection. OBJECTIVES: To study the optimal management of device infections in patients with ESRD. METHOD: We used the United States Renal Data System (USRDS) to assess the presence of a CIED and associated comorbidities, risk factors for infection, and mortality following device extraction or medical management in ESRD patients with CIED infection. Univariable, multivariable, and survival analyses were performed using USRDS data from 2005 to 2009. RESULTS: Of 546,769 patients, 6.4% had CIED and 8.0% of those developed CIED infection. The major risk factors for device infection were black race, temporary dialysis catheter, and body mass index >25. Patients with artificial valves were excluded from the analysis. Only 28.4% of infected CIED were removed. CIED removal was more common in those with congestive heart failure. The median time to death following diagnosis of a CIED infection was 15.7 months versus 9.2 months for those treated via device extraction versus medical-only therapy (hazard ratio: 0.75; 95% confidence interval: 0.68-0.82). CONCLUSION: Patients with ESRD and infected CIEDs have a poor prognosis. Rates of device extraction are low, but this strategy appears to be associated with modest improvement in survival.
Asunto(s)
Desfibriladores Implantables/efectos adversos , Corazón Auxiliar/efectos adversos , Fallo Renal Crónico/complicaciones , Marcapaso Artificial/efectos adversos , Infecciones Relacionadas con Prótesis/epidemiología , Adolescente , Adulto , Anciano , Bases de Datos Factuales , Remoción de Dispositivos , Femenino , Humanos , Fallo Renal Crónico/mortalidad , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Retrospectivos , Factores de Riesgo , Estados Unidos/epidemiología , Adulto JovenRESUMEN
Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2's normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.
Asunto(s)
Exones , Mutación , Síndromes Mielodisplásicos/genética , Proteínas Nucleares/genética , Ribonucleoproteínas/genética , Animales , Proteína Potenciadora del Homólogo Zeste 2 , Expresión Génica , Ratones , Ratones Mutantes , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteolisis , Empalme del ARN , Factores de Empalme Serina-ArgininaRESUMEN
Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains.
Asunto(s)
Neoplasias Hematológicas/genética , Proteínas Nucleares/genética , Empalme del ARN , Ribonucleoproteínas/genética , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Daño del ADN , Metilación de ADN , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Neoplasias Hematológicas/patología , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Modelos Moleculares , Mutación , Proteínas Nucleares/metabolismo , Sitios de Empalme de ARN , Ribonucleoproteínas/metabolismo , Factor de Empalme U2AF , Dedos de Zinc , ADN Metiltransferasa 3BRESUMEN
BACKGROUND: Chest pain is a common problem in obese patients. Because of the body habitus, the results of noninvasive evaluation for CAD may be limited in this group. METHODS: We reviewed the records of 1446 consecutive patients who had undergone clinically indicated stress echocardiography (SE). We compared major adverse cardiac events (MACE; myocardial infarction, cardiac intervention, cardiac death, subsequent hospitalization for cardiac events, and emergency department visits) at 1 year in normal weight, overweight, and obese subjects with normal SE. RESULTS: Excluding patients with an abnormal and indeterminate SE and those who were lost to follow-up, a retrospective analysis of 704 patients was performed. There were 366 obese patients (BMI ≥ 30), 196 overweight patients (BMI 25-29.9), and 142 patients with normal BMI (18.5-24.9). There was no MACE in the groups at 1-year follow-up after a normal SE. CONCLUSIONS: In obese patients including those with multiple risk factors and symptoms concerning for cardiac ischemia, stress echocardiography is an effective and reliable noninvasive tool for identifying those with a low 1-year risk of cardiac events.