RESUMO
Paired plasma and cerebrospinal fluid (CSF) specimens drawn from 15 HIV-infected patients with neurological disease before and after a median 6-week duration of highly active antiretroviral therapy (HAART) were studied to assess the short-term virological response of CSF and whether this can be predicted on the basis of baseline resistance mutations. After treatment, the median plasma and CSF viral load (VL) decreased by, respectively, 2.08 log10 (p = 0.0001) and 0.91 log10 copies/ml (p = 0.007) in comparison with baseline. A plasma virological response was observed in all but one patient, whereas the posttreatment CSF VL increased, remained unchanged, or decreased at a substantial lower rate than in plasma of six "CSF non/slow responders" (40%). Direct sequencing of baseline specimens showed that none of these patients had reverse transcriptase (RT) or primary protease resistance mutations in the CSF alone, but two had RT mutations conferring high-level resistance to drugs included in the HAART regimen in both CSF and plasma. The other four patients had no RT or primary protease resistance mutations. There was no significant difference in the nucleotide diversity of the CSF and plasma RT sequences, baseline plasma or CSF VL, the CSF-to-plasma VL ratio, the number of CSF cells, the CD4+ cell counts, or the history of antiretroviral treatment between the CSF non-slow responders and the other patients. During this short-term follow-up and despite a plasma response, a significant proportion of HAART-treated patients with neurological symptoms showed a slow or absent CSF response. Most of these cases were not associated with the presence of resistant HIV strains in the CSF.
Assuntos
Líquido Cefalorraquidiano/virologia , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Infecções por HIV/virologia , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Adulto , Terapia Antirretroviral de Alta Atividade , Sequência de Bases , Viroses do Sistema Nervoso Central/virologia , Feminino , Seguimentos , Genótipo , Infecções por HIV/sangue , Infecções por HIV/líquido cefalorraquidiano , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Valor Preditivo dos Testes , Alinhamento de Sequência , Carga Viral , Viremia/tratamento farmacológicoRESUMO
Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (less than 2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times (less than 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (greater than 10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mM H2O2 resulted in a time-dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.
Assuntos
Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Relação Dose-Resposta a Droga , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Peróxido de Hidrogênio/administração & dosagem , Cinética , L-Lactato Desidrogenase/metabolismo , Microscopia de PolarizaçãoRESUMO
Amniotic fluid analysis of microvillar enzymes, including alkaline phosphatase (ALP) total activity and ALP isoenzymes, has been widely experimented with and used for the prenatal diagnosis of cystic fibrosis in the second trimester of gestation. Since the development of cystic fibrosis molecular analysis, interest in these biochemical tests has been maintained for those instances in which the pregnancy is not fully informative by restriction fragment length polymorphism analysis or DNA is not available from the index-affected child. However, recommended biochemical protocols do not provide clear-cut diagnostic results in a minority of cases. We have tested the reliability of cystic fibrosis biochemical prediction by ALP high-resolution electrophoresis and ALP kinetic studies after inactivation by urea. With this approach, all the amniotic fluid samples that had not been unambiguously classified as affected or unaffected by standard microvillar enzymes analysis were definitely categorized. The proposed method seems to improve the diagnostic accuracy in pregnancies with a one in four risk of resulting in a child with cystic fibrosis.
Assuntos
Fosfatase Alcalina/análise , Líquido Amniótico/enzimologia , Fibrose Cística/diagnóstico , Isoenzimas/análise , Diagnóstico Pré-Natal , Fosfatase Alcalina/antagonistas & inibidores , Ensaios Enzimáticos Clínicos , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Isoenzimas/antagonistas & inibidores , Cinética , Gravidez , Ureia/farmacologiaRESUMO
The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (n = 15) or other central nervous system conditions (n = 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions.
Assuntos
Líquido Cefalorraquidiano/virologia , Citomegalovirus/isolamento & purificação , DNA Viral/análise , Encefalite Viral/diagnóstico , Kit de Reagentes para Diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/virologia , Antígenos Virais/análise , Citomegalovirus/genética , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/virologia , Encefalite Viral/virologia , Humanos , Reação em Cadeia da Polimerase/métodos , Replicação de Sequência Autossustentável , Sensibilidade e EspecificidadeRESUMO
An attempt has been made to understand whether 50 Hz electric and magnetic fields (EMFs) are involved in producing bioeffects by exposing human erythrocytes in vitro. The study evaluated some key glycolytic enzymes, glucose consumption, lactate production, energy charge, 2,3-diphosphoglycerate, and reduced glutathione levels, all of which are biochemical parameters significant to erythrocyte function. Cells exposed to individual or superimposed EMFs have not shown any significant difference compared with the controls.
Assuntos
Campos Eletromagnéticos/efeitos adversos , Eritrócitos/efeitos da radiação , Glicólise/efeitos da radiação , 2,3-Difosfoglicerato , Nucleotídeos de Adenina/sangue , Adulto , Glicemia/metabolismo , Ácidos Difosfoglicéricos/sangue , Metabolismo Energético/efeitos da radiação , Eritrócitos/metabolismo , Glutationa/sangue , Humanos , Técnicas In Vitro , Lactatos/sangue , Ácido Láctico , Pessoa de Meia-Idade , OxirreduçãoRESUMO
The use of highly active anti-retroviral therapy in patients with HIV-related progressive multifocal leukoencephalopathy is associated with increased survival and disease stabilization. However, approximately half of the patients receive no benefit from these treatments. In a group of HIV-infected patients with histologically or virologically confirmed PML, we recognized two distinct patterns of response, i.e., long survivors versus nonresponders, but could not identify any factors at baseline predictive of PML outcome. In addition, the use of cidofovir did not substantially affect survival. However, the survival rate was higher during the first years of HAART, i.e., 1996-1997, with better outcomes observed in patients receiving a protease inhibitor-containing regimen either irregularly or after a switch from a 2-nucleoside reverse transcriptase inhibitor combination. In contrast, PML outcome was frequently poor in both HAART-naive and -experienced patients who responded promptly to anti-HIV therapy in terms of CD4 increase and viral load decrease. In addition, in a number of patients, PML onset was temporally associated with immune reconstitution. It may be that, in some patients, rapid immune reconstitution due to HAART paradoxically worsens the course of PML. Gradual reversal of immune deficiency might be associated with better outcome.