RESUMO
We have developed a new automated cell isolation system as one of the modules of automated cell sheet production system named Tissue-Factory (T-Factory). This system enables isolation of the target cells from tissue. Using this new system, we successfully isolated skeletal myoblast from skeletal muscle tissue. The cell isolation system makes us stably prepare cell suspension from each tissue automatically and safely. Isolation of skeletal myoblasts will contribute to labor-saving cell cultivation and operational stability, and lead further process in tissue engineering and regenerative medicine.
Assuntos
Automação , Músculo Esquelético/patologia , Medicina Regenerativa/instrumentação , Engenharia Tecidual/instrumentação , Animais , Biópsia , Separação Celular , Células Cultivadas , Desenho de Equipamento , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley , Medicina Regenerativa/métodos , Reprodutibilidade dos Testes , Engenharia Tecidual/métodosRESUMO
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.
Assuntos
DNA de Neoplasias/análise , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Ploidias , Adulto , Automação , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Feminino , Citometria de Fluxo , Glioma/diagnóstico , Glioma/patologia , Glioma/cirurgia , Humanos , Período Intraoperatório , Masculino , Kit de Reagentes para DiagnósticoRESUMO
Flow cytometry is well-known cell analysis method and useful to gain quantitative information from cells in blood, however, it is not widely used for solid tissues in clinical settings. This is partly because it takes a long time to prepare samples and the operation can be complicated. To resolve these problems, we developed a new automatic cell isolation system which consists of cell isolation unit and staining reagent kit specialized for flow cytometry. With this new system, cell isolation can be done more rapidly and easily. By using this method, we could determine optimum condition to disintegrate porcine colon tissue and stain cells stably in 6 minutes. This result indicates that our method can provide analysis data within 10 minutes. We also evaluated our method in colorectal cancer patients, and the result was promising. All the data suggests that this method can support and facilitate rapid diagnosis.
Assuntos
Separação Celular/instrumentação , Citometria de Fluxo/instrumentação , Análise de Injeção de Fluxo/instrumentação , Coloração e Rotulagem/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Indicadores e ReagentesRESUMO
This paper describes a cell culture monitoring system for regenerative medicine. To realize this monitoring system, a new culture vessel and a removable measurement unit were proposed. The measurement unit was installed in the culture vessel and it was used to measure important cell culture parameters (e.g., temperature, CO(2) level, and pH). Thus, the status of the culture could be monitored. In addition, we developed a novel noninvasive method based on spectrophotometry for measuring pH. This method is a non-contact method that permits noninvasive and contamination-free pH measurement. The spectroscopic pH measurements agreed well with pH measurements using an electrode. The error was within 0.02; thus, the new pH measurement method is sufficiently accurate for cell culture. This new system is expected to contribute to advances in tissue engineering and regenerative medicine.
Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Concentração de Íons de Hidrogênio , Fotometria/instrumentação , Telemetria/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We have studied noninvasive devices for measuring total hemoglobin and hemoglobin derivatives such as carboxyhemoglobin (COHb) and methemoglobin (MetHb). A calibration procedure needs to be developed to evaluate or calibrate these devices and pulse oximeters for clinical practice. However, people and animals are sometimes exposed to risk when they are used for calibration. In this paper, we propose a new in vitro calibration system for a pulse photometer. This system has a novel double-layer pulsation flow-cell that incorporates both venous and arterial blood flow. Using the calibration system, we are able to measure the in vitro pulsatile optical density ratio (Phivt). The measured Phivt agrees well with the in vivo pulsatile optical density ratio (Phivi). This system simulates an in vivo environment with high accuracy and enables safe calibration. Consequently, the calibration system is able to standardize the performance and accuracy of pulse photometry.
Assuntos
Análise de Injeção de Fluxo/instrumentação , Hemoglobinas/análise , Oximetria/instrumentação , Reologia/instrumentação , Análise Espectral/instrumentação , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Intrapancreatic accessory spleens are frequently confused with primary pancreatic tumors, and differentiation from neoplastic lesions is important to avoid an unnecessary laparotomy. We present three cases of intrapancreatic accessory spleen evaluated by computed tomographic arteriography (CTA) and discuss the characteristic findings. METHODS: CTA was performed, followed by digital subtraction angiography, with an injection of contrast material through a 4-F catheter placed in the celiac artery. Single-level dynamic CTA was also performed in two patients with a 30-s continuous scan in one breath-hold. RESULTS: CTA clearly demonstrated early inhomogeneous enhancement of the lesion, similar to the splenic parenchyma. On single-level dynamic CTA, inhomogeneous enhancement of the lesion in the early phase was diminished in the late phase. Multiplanar reformatted images obtained in two cases showed the deep cleft between the lesion and the pancreas, which suggested that the lesion was originally extrapancreatic. CONCLUSIONS: These two findings on CTA, inhomogeneous enhancement of the lesion and the deep cleft between the lesion and the pancreas, may help to confirm the diagnosis of an intrapancreatic accessory spleen.
Assuntos
Pâncreas/diagnóstico por imagem , Baço/anormalidades , Tomografia Computadorizada por Raios X , Adulto , Angiografia , Angiografia Digital , Diagnóstico Diferencial , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico por imagemRESUMO
BACKGROUND: Fluorescent in situ hybridization (FISH) has been shown to be one of the most reliable methods with which to estimate the status of the HER-2/neu (or c-erb B-2) oncogene at the DNA level. METHODS: To study interobserver reproducibility and to determine more clinically correlated criteria for HER-2/neu alterations, two observers independently estimated HER-2/neu DNA status. The correlation between the consensus HER-2/neu DNA status by FISH and HER-2/neu protein status detected by immunohistochemistry (IHC) using a polyclonal antibody was studied in 216 surgically resected breast carcinomas and 34 noncancerous tissues. RESULTS: According to the HER-2/CEP17 ratio and mean HER-2 copies per nucleus, agreement level of HER-2/neu amplification was shown to be nearly perfect between two observers (kappa statistic (kappa) = 0.94 and kappa = 0.84). Finally, 40 tumors (19%) were judged to have HER-2/neu DNA amplification, with 6 having low-level amplification (> or = 2 but < 3 folds) and 34 having high-level amplification (> or = 3 folds). One hundred seventy-six other tumors, including 3 tumors that only 1 of the observers determined to be low-level amplifiers, and 34 noncancerous tissues had no detected amplification. The DNA amplification status was concordant between invasive and intraductal components in 14 carcinomas. HER-2/neu protein overexpression of moderate (2+) or high (3+) intensity based on IHC was detected in 51 carcinomas (24%), and was 2+ in 20 carcinomas and 3+ in 31 carcinomas. The HER-2/CEP17 ratio of > or = 2 was concordant with IHC findings of 2+/3+ in 91% of carcinomas (195 of 215 carcinomas), with a sensitivity of 70% (35 of 50 carcinomas) and a specificity of 97% (160 of 165 carcinomas). High-level amplification was detected in 29 of 31 IHC 3+ cases (94%), but in only 5 of 20 IHC 2+ cases (25%) and 0 in 165 IHC 0/1+ cases. All 34 cases with high-level amplification showed an IHC score of 3+ (29 cases) or an IHC score of 2+ (5 cases), but only 1 case was found to have an IHC score of 3+ and the remainder were IHC 0/1+ in 6 low-amplification cases. The concordance rate of the high-level amplification with an IHC score of 3+ was 97% (208 of 215 cases), with a sensitivity of 94% (29 of 31 cases) and a specificity of 97% (179 of 184 cases). CONCLUSIONS: The results of the current study indicated that high-level HER-2/neu amplification and an IHC score of 3+ nearly optimally identified breast carcinomas with clinically and biologically significant HER-2/neu activation. Conversely, it was confirmed that careful interpretation of test results is required in the case of low-level amplification and/or an IHC score of 2+.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Genes erbB-2/genética , Hibridização in Situ Fluorescente/normas , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , DNA de Neoplasias/genética , Feminino , Humanos , Imuno-Histoquímica , Invasividade Neoplásica , Variações Dependentes do Observador , Prognóstico , Reprodutibilidade dos TestesRESUMO
BACKGROUND: We describe a 56-year-old woman admitted to the hospital with a diagnosis of acute myocardial infarction without an increase of serum creatine kinase (CK) activity during her clinical course. She died on the 11th hospital day, and the diagnosis was confirmed by autopsy. The patient had had no previous muscular symptoms. METHODS: Expression of the CK-muscle (CK-M) protein in cardiac tissue was examined by immunoblotting and immunochemical staining. CK-M mRNA expression was estimated by semiquantitative reverse transcription-PCR. Gene structure of CK-M was determined by Southern blotting and direct sequencing of 2251 bp. Existence of a point mutation in the CK-M gene was examined by restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) in the patient and in 108 controls. RESULTS: CK-M protein in the myocardial tissue of the patient was substantially lower (103 +/- 7 ng/mg protein) than in control myocardial tissue (35 800 +/- 2860 ng/mg protein). Immunoreactive CK-M in the patient tissue sample was 0.3% of the value for the control sample. CK-M mRNA was 53-fold less in the patient sample compared with the control. This very low expression of CK-M mRNA was considered to be the primary reason for CK-M deficiency. Direct sequencing revealed a point mutation at residue 54 in exon 2, which was specific for the patient. No other abnormalities were found in the CK-M gene of the patient. CONCLUSIONS: This report identifies a molecular abnormality in human CK deficiency and discusses the physiologic relevance of CK-M.
Assuntos
Creatina Quinase/genética , Isoenzimas/genética , Southern Blotting , Creatina Quinase/deficiência , Creatina Quinase Forma MM , Evolução Fatal , Feminino , Humanos , Imunoensaio , Immunoblotting , Isoenzimas/deficiência , Pessoa de Meia-Idade , Infarto do Miocárdio/diagnóstico , Miocárdio/enzimologia , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Tyrosine kinases are expressed in many tissues, particularly in the central nervous system, and regulate various cellular functions. We report here that a src family tyrosine kinase-specific inhibitor, PP2, enhances neurotransmitter release from PC12 cells and primary cultured neurons. PP2 enhances only Ca(2+)-dependent release; it does not affect basal release. These effects result from an enhancement of vesicular exocytosis and not from the reuptake or refilling of neurotransmitters because Ca(2+)-dependent secretion of an exogenously expressed reporter protein, the human growth hormone (hGH), is also enhanced by PP2. Overexpression of constitutive active v-src, but not of a kinase-inactive mutant, suppressed Ca(2+)-dependent release. In PP2-treated cells, Pyk2, paxillin, and some other proteins showed a decrease in tyrosine phosphorylation, and the enhancement of tyrosine phosphorylation of these proteins in response to Ca(2+) influx was also reduced. Electron and fluorescence microscopy showed that PP2 treatment induced morphological change and decreased phalloidin reactivity at the filopodium-like structures on the processes of PC12 cells. Interestingly, inhibition of actin polymerization with cytochalasin D and latrunculin A enhanced Ca(2+)-dependent, but not basal, release. It is possible that a src family tyrosine kinase, through the regulation of actin dynamics, has an inhibitory function to regulate neurotransmitter release.
Assuntos
Dopamina/metabolismo , Quinases da Família src/antagonistas & inibidores , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Cálcio/metabolismo , Tamanho Celular , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Quinase 2 de Adesão Focal , Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteína Oncogênica pp60(v-src)/genética , Células PC12 , Paxilina , Faloidina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Wistar , Tiazóis/metabolismo , Tiazolidinas , Tirosina/metabolismoRESUMO
BACKGROUND: We studied multiple myeloma oncogene 1/interferon-regulatory factor 4 (MUM1/IRF4) mRNA expression in various malignant human hematopoietic cell lines and normal leukocyte fractions. MATERIALS AND METHODS: A quantitative reverse transcription-polymerase chain reaction was used to assess expression and chromosomes were examined for anomalies by fluorescent in situ hybridization. RESULTS: Among 12 cell lines examined, mRNA transcripts were expressed only in B-lymphoblastic and myeloma cell lines. Myeloma cells and malignant cell lines derived from mature B cells expressed more transcript than cell lines derived from immature B cells. Transcript levels, however, showed no association with chromosomal translocations. Expression in B-cell fractions from healthy donors was much less than in the malignant cells. In addition, MUM1/IRF4 mRNA expressed in samples from patients with acute lymphoblastic leukemia derived from B cells but not T cells. CONCLUSION: Our results suggested that MUM1/IRF4 gene expression is related to stage of differentiation of malignant B cells and they indicated the possibility that the quantitative analysis of MUM1/IRF4 gene is a useful tool for detection of malignant B-cell proliferations in clinical laboratory tests.
Assuntos
Linfócitos B/metabolismo , Linfoma de Burkitt/genética , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Leucócitos/metabolismo , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Fatores de Transcrição/genética , Adolescente , Adulto , Linfoma de Burkitt/metabolismo , Diferenciação Celular , Criança , Cromossomos Humanos Par 6/genética , Proteínas de Ligação a DNA/biossíntese , Feminino , Humanos , Hibridização in Situ Fluorescente , Fatores Reguladores de Interferon , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Fatores de Transcrição/biossíntese , Células Tumorais Cultivadas/metabolismoRESUMO
A 16-year-old girl with short stature, short neck, shield chest, and cubitus valgus was studied. FISH analyses of her structurally altered X chromosome showed a der(X)- (wcpX+,TelXp/Yp++,SHOX++,STS++,KAL-, 37A12-,DXZ1+,XIST++,97L7++,300O13-,404F- 18-,417G15-,404F18-,140A-,TelXq/Yq-). These results, together with the high-resolution banding analysis, indicated her karyotype to be 46,X,der(X)(Xpter-->Xp22.3::Xq22.3--> cen-->Xq22.3::Xp22.3-->Xpter). The der(X) was an isochromosome, consisting of duplicated terminal short arms and duplicated proximal long arms. This in turn suggested that the chromosome was formed through pericentric inversion of an X chromosome, followed by isochromosome formation through sister chromatid exchange at Xp, close to the centromere. Replication R-banding analysis showed that the abnormal X chromosome was late replicating. Analysis of digestion patterns with a methylation-sensitive restriction endonuclease of the phosphoglycerate kinase 1 gene, located in Xq13.3, indicated that its inactivation patterns were completely skewed.
Assuntos
Nanismo/genética , Isocromossomos , Aberrações dos Cromossomos Sexuais , Cromossomo X , Adolescente , Bandeamento Cromossômico , Análise Citogenética , Mecanismo Genético de Compensação de Dose , Feminino , Humanos , Hibridização in Situ Fluorescente , CariotipagemRESUMO
Acute myelomonocytic leukemia (AMMoL) accompanied by monoclonal gammopathy is a rare condition, and its pathogenesis and the cytogenetic mechanism of such leukemogenesis have not been determined in detail. A case of AMMoL with eosinophilia accompanied by immunoglobulin G kappa monoclonal gammopathy is described. Immunophenotypic studies of the peripheral blood and bone marrow mononuclear cells revealed no evidence of abnormally proliferating cells of B-lineage. DNA analyses of bone marrow mononuclear cells containing leukemic cells revealed rearrangement of the kappa-light chain (Igkappa) gene and c-myc and c-jun proto-oncogenes. The intensities of the rearranged bands for these genes on Southern blot analysis suggested the existence of a major population of leukemic cells with rearranged Igkappa gene and minor population(s) of leukemic cells with rearranged c-myc and/or c-jun proto-oncogene(s) in the patient's bone marrow and indicated the occurrence of genetic evolutionary changes in leukemic cells in this patient before starting chemotherapy. These results suggest that these leukemic cells are the most likely candidate for immunoglobulin G kappa monoclonal protein production, and structural abnormalities of c-myc and c-jun proto-oncogenes may have contributed to the evolution of leukemic cells in this patient.
Assuntos
Eosinofilia/complicações , Leucemia Mielomonocítica Aguda/complicações , Paraproteinemias/complicações , Southern Blotting , Células da Medula Óssea/química , Divisão Celular , Pré-Escolar , DNA/análise , Rearranjo Gênico , Genes fos , Genes jun , Genes myc , Humanos , Imunoglobulina G , Cadeias kappa de Imunoglobulina/genética , Imunofenotipagem , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Aguda/genética , Leucócitos Mononucleares/química , Leucócitos Mononucleares/imunologia , Masculino , Paraproteinemias/genética , Proto-Oncogene MasRESUMO
The transcriptional start site of the human cholecystokinin (CCK)-A receptor gene was determined by the Capsite Hunting method. Two sequence changes were detected, a G to T change in nucleotide -128, and an A to G change in nucleotide -81. The homozygote (T/T, G/G) was detected in 25 of 1296 individuals (1.9%) in the cohort study. This polymorphism showed a significantly higher percent body fat and higher levels of serum insulin and leptin, compared with wild type and heterozygotes. Our study provided the possibility that polymorphism in the promoter region of the CCK-A receptor gene may be one of genetic factors affecting fat deposition.
Assuntos
Tecido Adiposo , Polimorfismo Genético , Regiões Promotoras Genéticas , Receptores da Colecistocinina/genética , Adulto , Idoso , Sequência de Bases , Estudos de Coortes , Feminino , Genótipo , Humanos , Insulina/sangue , Leptina/sangue , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Obesidade/genética , Polimorfismo de Fragmento de Restrição , Receptor de Colecistocinina ARESUMO
The Ser149Arg mutation of peripheral myelin protein 22 (PMP22) was found in a 19-year-old woman with a sporadic case of Dejerine-Sottas disease. The patient showed delayed motor development. She walked for the first time with support at the age of 2 years. Scoliosis developed at age 4 years. Her walking ability was best at age 11. Thereafter, she showed progressive muscle weakness and sensory disturbances in the distal extremities. At the age of 18 years, the use of a wheelchair became necessary. Motor and sensory nerve conduction studies showed absent motor and sensory responses on electrical stimulation of the limb nerves. A sural nerve biopsy specimen showed marked decreases in the numbers of both large and small myelinated fibers, abundant onion-bulb formation, and hypomyelination. Electron microscopic observation revealed the presence of demyelinated axons and myelin sheaths disproportionately thin relative to axon diameter. That this was a de novo mutation was established by parentage testing and PMP22 gene analysis of the parents. The mutation seems to be novel and dominant.
Assuntos
Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , Proteínas da Mielina/genética , Nervos Periféricos/patologia , Nervos Periféricos/fisiopatologia , Adulto , Biópsia , Feminino , Neuropatia Hereditária Motora e Sensorial/patologia , Humanos , Microscopia Eletrônica , Nervos Periféricos/ultraestrutura , Nervo Sural/patologia , Nervo Sural/fisiopatologia , Nervo Sural/ultraestruturaRESUMO
This study was undertaken to characterize the clinical, electrophysiologic, and histopathologic features of five presumably unrelated Japanese patients with Charcot-Marie-Tooth (CMT) disease type 1B and Arg98His substitution of Po protein and, in particular, to correlate Arg98His substitution to the ultrastructural abnormalities of the myelin sheath. Systematic morphometric studies of the sural nerve, where the CMT type 1B gene abnormality is expressed, have not been performed, especially on the basis of the type of mutation causing CMT type 1B. Electrophysiologic evaluation of limb nerves and morphometric analysis of sural nerves obtained at biopsy were performed. Ultrastructural myelin abnormalities were precisely examined. Clinical symptoms appeared from the second to the fifth decade. All probands presented with gait disturbance. Motor and sensory conduction velocities in the median and ulnar nerves ranged from 10 to 30 m/s. Segmental demyelination and remyelination and marked loss of myelinated fibers were the main findings. On electron microscopy, widening between major dense lines was found between the paired intraperiod lines, where the extramembranous portion of the Po protein resides. This widening is probably directly related to Arg98His substitution. Focal uncompaction of major dense lines coexisted with this widening. This uncompaction, which directly decreases the number of myelin lamellae, may be a secondary effect of Arg98His substitution on the intramembranous domain of Po protein. In conclusion, myelin changes at both extracellular and cytoplasmic appositions of Schwann cell membranes were found in association with Arg98His substitution of Po protein. This study contributes to a better understanding of myelin abnormalities in patients with CMT type 1B and Arg98His or other similar extramembranous amino acid substitutions of Po protein.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteína P0 da Mielina/genética , Fibras Nervosas Mielinizadas/patologia , Adulto , Arginina , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Feminino , Histidina , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Fibras Nervosas Mielinizadas/ultraestrutura , Condução Nervosa/fisiologia , Linhagem , Mutação Puntual/genéticaRESUMO
To assess the abnormal T-cell expansion in chronic active Epstein-Barr virus infection (CAEBV), T-cell antigen receptor (TCR) repertoire was analyzed in four patients with the disease. All fulfilled the diagnostic criteria of CAEBV, presenting with fever, hepatosplenomegaly, cytopenia, abnormal high titers of anti EBV-antibodies, and positive EBV genome of unknown cause. Southern blotting probed with EBV-terminal repeats and TCR Cbeta gene indicated clonal expansion of the infected cells in 3 and 2 patients, respectively. The number of CD4+ HLA-DR+ cells appreciably increased in patients 1 (59%) and 2 (24%), who had a coronary aneurysm and central nervous system involvement, respectively. TCR gene expression examined by the inverse polymerase chain reaction methods revealed that Vbeta gene usages were preferential in all patients (Vbeta7 and Vbeta12: patient 1, Vbeta4: patient 2, Vbeta13: patients 3 and 4), compared with those in healthy controls. Valpha18 gene expression was remarkably high in patients 1 and 2. Moreover, Jbeta gene expression was skewing in the reigning Vbeta clones in all patients. Vbeta4-Jbeta1.5 and Vbeta13-Jbeta1.5 genes were clonally expressed in patients 2 and 4, respectively. These results suggest that CAEBV is associated with the restricted diversity of T-cells, which may stem from the sustained expansion of oligoclonal T-cells possibly driven by conventional viral antigens, but not, superantigens. Although the study is limited by the small number of patients, the unbalanced T-cell repertoire might contribute to the evolution of T-lymphoproliferative disease, otherwise, imply the innate defective immunity to EBV in CAEBV patients.
Assuntos
Infecções por Vírus Epstein-Barr/patologia , Transtornos Linfoproliferativos/virologia , Linfócitos T/patologia , Adolescente , Anticorpos Antivirais/sangue , Southern Blotting , Criança , Doença Crônica , DNA/análise , DNA Viral/análise , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Rearranjo Gênico do Linfócito T , Hepatomegalia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Masculino , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/genética , Esplenomegalia , Linfócitos T/imunologiaRESUMO
OBJECTIVE: To investigate the genetic contribution for myocardial infarction. METHODS: We investigated common polymorphisms of apolipoprotein E gene and angiotensin converting enzyme (ACE) gene in Japanese population. Subjects were 422 healthy people and 254 patients with myocardial infarction. We evaluated the 287 base pair (bp) insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene and a polymorphism in the apolipoprotein E gene by using the polymerase chain reaction. RESULTS: The ACE genotype prevalences for II, ID, and DD were 36.2, 46.1, and 17.7%, respectively, among the myocardial infarction patients. The prevalence of the D allele of the ACE gene among the myocardial infarction patients (0.593) exceeded that among the healthy controls (0.407). The prevalences of the epsilon 2, epsilon 3, and epsilon 4 alleles of the apolipoprotein E genotype among healthy controls were 0.024, 0.882, and 0.094, and those among survivors of myocardial infarction were 0.024, 0.834, and 0.142, respectively. Myocardial infarction patients had an excessive prevalence of the apolipoprotein E epsilon 4 allele (P < 0.05). Multiple regression analysis demonstrated that the independent risk factors for developing myocardial infarction were age, DD genotype of ACE gene, and apolipoprotein E epsilon 4 allele. Stenotic coronary vessels in myocardial infarction patients did not differ significantly among the patients with various ACE and apolipoprotein E genotypes in the present study. CONCLUSIONS: Among the Japanese, apolipoprotein E epsilon 4 carriers and subjects with ACE DD genotype are at an increased risk of myocardial infarction.
Assuntos
Apolipoproteínas E/genética , Infarto do Miocárdio/genética , Peptidil Dipeptidase A/genética , Polimorfismo Genético/genética , Idoso , Alelos , Sequência de Bases , Feminino , Genótipo , Humanos , Íntrons/genética , Japão , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/etnologia , Reação em Cadeia da Polimerase/métodos , Fatores de RiscoRESUMO
To explore the clinical significance of p53 in the pathogenesis of adrenal neoplasms, we investigated the incidence of p53 gene mutations in functioning human adrenal tumours using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen p53 exons 4 to 9. We examined 29 adrenocortical adenomas (primary aldosteronism, n=17; Cushing's syndrome, n=12, all benign), and 33 phaeochromocytomas (benign solitary, n=18; benign multiple, n=5; malignant, n=10) in Japanese and Chinese patients. PCR-SSCP did not show any abnormal band-shifts in any of the adrenocortical adenoma and benign solitary phaeochromocytoma tissues. In contrast, six phaeochromocytoma tissues (two cases benign multiple, four cases malignant) showed PCR-SSCP band-shifts. Subsequent DNA sequencing analysis of the shifted bands revealed six cases with nine mutations or intronic sequence alterations: three cases contained sequence alterations within intronic regions, three cases with silent mutation (sequence alteration in codon without amino acid alteration), and three cases contained missense mutations (one case each in exons 5, 6 and 9). Immunohistochemical staining demonstrated that two of three cases with missense mutations and one case with an intronic sequence alteration over-expressed p53 protein in tumour cell nuclei. We observed no association between p53 gene mutation and p21/WAF1/Cip-1 expression. The relatively high incidence of p53 gene mutations or intronic sequence alteration in multiple and malignant phaeochromocytomas, but not in benign solitary cases, suggests that p53 mutation could play some role in the pathogenesis of multiple and/or malignant phaeochromocytomas.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Genes p53 , Feocromocitoma/genética , Adenoma/química , Adenoma/genética , Neoplasias das Glândulas Suprarrenais/química , Adulto , Éxons , Humanos , Imuno-Histoquímica , Masculino , Mutação , Feocromocitoma/química , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/análiseRESUMO
PURPOSE: A patient with aggressive chronic active Epstein-Barr virus (CAEBV) infection is described whose disease activity subsided after interferon (IFN)-alpha therapy. PATIENT AND METHODS: The patient had intermittent fever, cytopenia, liver dysfunction, hepatosplenomegaly, abnormal titers of EBV-associated antibodies, and positive EBV genomes. RESULTS: Despite repeated trials of the antiviral agents prednisolone and gamma-globulin, his condition deteriorated. The administration of IFN-alpha (1 x 10(5) U/kg subcutaneously 3 times per week) led to a dramatic clinical improvement. During the IFN-alpha therapy, the rearrangement bands of T-cell antigen receptor genes disappeared assessed by Southern blotting with a decrease in the number of activated T cells, although the EBV-genome remained evident. CONCLUSIONS: These observations suggest that IFN-alpha is useful in managing CAEBV, possibly restraining the clonal development of T-lymphoproliferative disease (LPD) and EBV-associated B-LPD, although it does not eradicate the proliferation of EBV.