RESUMO
BACKGROUND: The Epstein-Barr virus (EBV) was used as an agent of B lymphocyte proliferation for subsequent diagnosis of lysosomal storage disease. Due to the constant handling of long-preserved samples in our cell bank, we decided to observe the behavior and then compare cultured and frozen samples for at least one year's cryopreservation. METHODS: Twenty-five samples from healthy individuals were used to assess the possible changes in activity of enzymes ß-galactosidase, ß-glucosidase, α-iduronidase, α-galactosidase, and α-glucosidase. Transmission electron microscopy was used to confirm cell transformation of B lymphocytes into EBV-infected cells, generating lymphoblastoid cell lines. RESULTS: Transmission electron microscopy findings confirmed previous reports in the literature that is, significant and evident morphological changes in the nucleus occur after day 12 and the consequent cell transformation into EBV-infected cells. After thawing and subsequent treatment with the five enzymes utilized, we observed no significant changes in samples cryopreserved for more than one year, as compared to samples cultured for 12 days.
Assuntos
Linfócitos B/enzimologia , Linfócitos B/virologia , Criopreservação , Hidrolases/metabolismo , Lisossomos/enzimologia , Linfócitos B/metabolismo , Linhagem Celular , Transformação Celular Viral , Herpesvirus Humano 4/metabolismo , Humanos , Iduronidase/metabolismo , Ativação Linfocitária , alfa-Galactosidase/metabolismo , alfa-Glucosidases/metabolismo , beta-Galactosidase/metabolismo , beta-Glucosidase/metabolismoRESUMO
OBJECTIVE: The Epstein-Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B-lymphocytes. For this reason, EBV is used in conservation of human B-lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1-gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I. MATERIALS AND METHODS: Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes beta-galactosidase, beta-glucosidase, alpha-iduronidase, alpha-galactosidase and alpha-glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC. RESULTS: We observed some significant alterations in enzymatic activity of non-cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days. CONCLUSIONS: However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.
Assuntos
Linfócitos B , Criopreservação , Herpesvirus Humano 4 , Doenças por Armazenamento dos Lisossomos/diagnóstico , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Estudos de Casos e Controles , Linhagem Celular , Doença de Fabry/diagnóstico , Doença de Fabry/enzimologia , Estudos de Viabilidade , Gangliosidose GM1/diagnóstico , Gangliosidose GM1/enzimologia , Doença de Gaucher/diagnóstico , Doença de Gaucher/enzimologia , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/enzimologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/metabolismo , Humanos , Iduronidase/imunologia , Iduronidase/metabolismo , Imuno-Histoquímica , Ativação Linfocitária/imunologia , Doenças por Armazenamento dos Lisossomos/enzimologia , Lisossomos/enzimologia , Lisossomos/imunologia , Lisossomos/virologia , Mucopolissacaridose I/diagnóstico , Mucopolissacaridose I/enzimologia , alfa-Galactosidase/imunologia , alfa-Galactosidase/metabolismo , alfa-Glucosidases/imunologia , alfa-Glucosidases/metabolismo , beta-Galactosidase/imunologia , beta-Galactosidase/metabolismo , beta-Glucosidase/imunologia , beta-Glucosidase/metabolismoRESUMO
AIM: Amalcomp is a technique that combines composite resin to amalgam in restorative procedures to improve esthetics and minimize the negative effects of polymerization on dental tissues. The objective of this in vitro study was to measure the diametral compressive bond strength between Fill Magic composite (Vigodent) versus Permite (DFL) or Velvalloy (SS White) amalgams in different oxidation stages. METHODS: Twenty-four cylinders of each amalgam brand were fabricated using a Teflon matrix and divided into 3 groups according to the immersion period in artificial saliva for oxidation: A (1 day), B (7 days) and C (30 days). After immersion, the amalgam cylinders were bonded to the composite specimens using the Scotch Bond Multi Use Plus (3M) bonding system. Diametral compression assays were then carried out in an EMIC-MEM 2000 universal testing machine set to 0.5 mm/min. Statistical analysis was performed using ANOVA and Tukey's test. RESULTS: The mean recorded strength (MPa) for each oxidation group was: A=9.71, B=8.21 and C=6.98 (A>B = C; P<0.01). Permite (7.24) provided significantly less adhesion to the composite than Velvalloy (9.36; P<0.05). There was no interaction between the factors alloy, resin and time. CONCLUSIONS: Under the conditions of this study, the less oxidized amalgam showed the greatest diametral compressive strength values.
Assuntos
Resinas Compostas/química , Ligas Dentárias/química , Amálgama Dentário/química , Colagem Dentária , Força Compressiva , Cobre/química , Teste de Materiais , Oxirredução , Saliva Artificial/química , Prata/química , Fatores de TempoRESUMO
Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B lymphoblastoid cell lines (LCLs). These LCLs have been widely used for the diagnostic of several genetic metabolic disorders. However, up to now, efficiency of LCL generation has been based on misleading subjective analysis. In this study, quantitative analyses have been performed to indicate efficiency of B-cell transformation to measuring human lysosomal acid hydrolases associated with: GM1-gangliosidosis type I, Gaucher disease and mucopolysaccharidosis type I. Peripheral blood mononuclear cells were isolated from 13 subjects, and LCLs were produced by culturing them with EBV for 12 days. Activities of the enzymes beta-galactosidase, beta-glucosidase and alpha-iduronidase were measured before and after cryopreservation in liquid nitrogen for 30 days. Efficiency of the B-cell transformation was screened every 4 days by the enumeration of cell proliferation, cell counts and changes in granularity estimated by flow cytometry. We observed the generation of 13 LCLs. Cell transformation was confirmed by the gradual increase of cellular clusters, cell size and granularity. In addition, we determined that the activity of the enzymes mentioned above did not change following cryopreservation. These data suggest that our enumerative approach for screening of EBV-LCLs is efficient for the enzymatic determination of human lysosomal acid hydrolases and may thus replace misleading subjective analyses.