RESUMO
A 38-year-old female patient with systemic lupus erythematosus presented with pulmonary infiltrates and hypoxemia for several months following immunodepleting autologous hematopoietic stem cell transplantation. She was treated for influenza, which was isolated repeatedly from oropharynx and bronchoalveolar lavage (BAL) fluids, and later empirically for lupus pneumonitis, but died 6 months after transplant. Autopsy findings failed to show influenza in the lungs or lupus pneumonitis. A novel generic polymerase chain reaction (PCR)-based assay using degenerate primers identified human coronavirus (CoV) HKU1 RNA in BAL fluid at autopsy. CoV was confirmed by virus-specific PCRs of lung tissue at autopsy. Electron microscopy showed viral particles consistent with CoV HKU1 in lung tissue both at autopsy and from a previous biopsy. Although human CoV HKU1 infection is not usually severe, in highly immunocompromised patients, it can be associated with fatal pneumonia.
Assuntos
Infecções por Coronavirus/virologia , Coronavirus/classificação , Coronavirus/isolamento & purificação , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Pulmão/virologia , Pneumonia Viral/virologia , Adulto , Autopsia , Biópsia , Coronavirus/genética , Infecções por Coronavirus/diagnóstico , Evolução Fatal , Feminino , Humanos , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase/métodosRESUMO
BACKGROUND: A key assumption in the analysis of microarray data is that the quantified signal intensities are linearly related to the expression levels of the corresponding genes. To test this assumption, we experimentally examined the relationship between signal and expression for the two types of microarrays we most commonly encounter: radioactively labeled cDNAs on nylon membranes and fluorescently labeled cDNAs on glass slides. RESULTS: We uncovered two sources of nonlinearity. The first, which led to discrepancies in analysis affecting the fluorescent signals, was signal quenching associated with excessive dye concentrations. The second, affecting the radioactive signals, was a nonlinear transformation of the raw data introduced by the scanner. Correction for this transformation was made by some, but not all, image-quantification software packages. CONCLUSIONS: The second type of nonlinearity is more troublesome, because it could not have been predicted a priori. Both types of nonlinearities were detected by simple dilution series, which we recommend as a quality-control step.
Assuntos
Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Calibragem , Fluorescência , Controle de Qualidade , Radioatividade , Padrões de Referência , Transdução de Sinais , SoftwareRESUMO
MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/toxicidade , Daunorrubicina/farmacocinética , Resistência a Múltiplos Medicamentos , Proteínas de Neoplasias , Transcrição Gênica , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Primers do DNA , Feminino , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Software , Transfecção , Células Tumorais CultivadasRESUMO
We have developed an antibody detection enzyme-linked immunosorbent assay (ELISA) for the identification of animals infected by feline immunodeficiency virus (FIV). The ELISA solid-phase antigen consists of recombinant FIV gag proteins expressed in bacteria. The proteins are purified from bacterial lysates as insoluble inclusion bodies. In the case of bacterially expressed p24gag, it is shown that all of the linear, sequential epitopes presented by viral p24 during infection are retained. Purified preparations can be substituted for solid-phase whole virus in the IDEXX PetChektm immunoassay. The antibody ELISA duplicates the sensitivity and specificity of the whole virus based PetChek plate assay.
