RESUMEN
Frozen section studies are a useful method to rapidly define tumor malignancy and identify the extent of surgical resection. However, diagnosis with a frozen section is qualitative and sometimes difficult. Therefore a quantitative method for grading tumors is desired. We have already reported a technique of intraoperative flow cytometry (iFC) that supports intraoperative histopathological examination of frozen sections. In this study, we report an advanced system named "Fully Automatic Rapid DNA Ploidy Analyzer" with a tissue pretreatment function and a freeze-dried reagent kit for cell staining. To evaluate our system, we analyzed samples from glioma patients who underwent open surgery for brain tumors. We observed obvious difference of the Malignancy Index (MI) between neoplastic and perilesional brain tissue (26.0 ±22.1% and 4.1 ±2.5%, respectively, P<0.001). Cut-off level for identification of the tumor in the biopsy specimen was 6.8% which provided 86% sensitivity and 81% specificity. We also obtained a good correlation between the MI and histological grade (WHO grading). Our new system also enabled finishing the process from sample preparation to the end of analysis in ten minutes or less. These results demonstrate that our fully automatic rapid DNA ploidy analyzer is feasible for rapid determination of glioma presence in a surgical biopsy sample.
Asunto(s)
ADN de Neoplasias/análisis , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Diagnóstico Molecular/métodos , Ploidias , Adulto , Automatización , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/cirugía , Femenino , Citometría de Flujo , Glioma/diagnóstico , Glioma/patología , Glioma/cirugía , Humanos , Periodo Intraoperatorio , Masculino , Juego de Reactivos para DiagnósticoRESUMEN
The downstream DNA region of the fimbrilin gene (fimA), which encodes the major subunit protein of Porphyromonas gingivalis fimbriae, was fully sequenced. Gene products, expressed from this region in Escherichia coli, were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their partial amino acid sequences were determined to verify open reading frames (ORFs) found in the region by DNA sequencing. Four ORFs, designated ORF1, ORF2, ORF3 and ORF4, were found in the 5.8-kb PstI fragment downstream from fimA, which was previously cloned and partially characterized by Yoshimura, Takahashi, Hibi, Takasawa, Kato, and Dickinson (Infect. Immun. 61: 5181-5189, 1993). The direction of transcription of all the ORFs was the same as that of fimA. The 50 and 80 kDa encoded proteins, ORF2 and ORF3, respectively, have been reported to be minor components associated with fimbriae. The 15 and 19 kDa proteins, ORF1 and ORF4, respectively, have been expressed in E. coli but not identified in P. gingivalis. However, all the gene products of the ORFs, expressed in E. coli, appeared to contain intact signal peptides based on their N-terminal amino acid sequences.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Fimbrias , Fimbrias Bacterianas/química , Genes Bacterianos , Porphyromonas gingivalis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Codón , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Porphyromonas gingivalis/química , Señales de Clasificación de Proteína/análisis , Proteínas Recombinantes/química , Análisis de Secuencia de ADNRESUMEN
A humanized monoclonal IgG1 antibody, designated hC4G1, recognizes the fibrinogen receptor glycoprotein (GP)IIb/IIIa on platelets and inhibits platelet aggregation. When the F(ab')2 fragment of hC4G1 (F(ab')2 hC4G1) was administered to cynomolgus monkeys, all the monkeys showed inhibition of platelet aggregation ex vivo. Unexpectedly, a significant decrease in platelet count was observed in 5 of 18 monkeys. Antibodies against F(ab')2 hC4G1 were detected in the plasma of these monkeys by ELISA. Antibody activity in the plasma of these monkeys was significantly correlated with the intensity of platelet decrease (r = 0.84). The natural monkey antibodies to F(ab')2 hC4G1 were directed against the C-terminal region of F(ab')2 fragment common to all human and humanized IgG antibodies. Natural homo-reactive antibodies were also detected in human plasma from 15 of 40 healthy volunteers. Specificity was closely similar to that of the monkey antibodies. Affinity-purified human homoreactive antibodies enhanced phagocytosis of platelets treated with the F(ab')2 hC4G1. Monkey plasma with high homo-reactive antibody activity was confirmed to decrease platelet count when administered together with F(ab')2 hC4G1 to a monkey with low antibody activity. These results suggest that F(ab')2 of humanized and human antibodies causes elimination of the corresponding antigens from the circulation by homo-reactive antibodies.