An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

INTRODUÇÃO: A canulação da fístula arteriovenosa (FAV) pode ser realizada pelas técnicas de punção por área, rotatória ou em casa de botão (CB); entretanto, a técnica ideal ainda não foi estabelecida. OBJETIVO: Avaliar as dificuldades e complicações na introdução da técnica de punção em CB em FAV construída com veia nativa. MÉTODOS: 16 pacientes com idade de 57±14 anos, em hemodiálise há 63±38 meses foram submetidos à punção em CB. Na fase de formação do túnel (T), as punções foram feitas com agulha cortante (AC) e na fase de manutenção com agulha romba (AR). Nas duas fases, os pacientes foram avaliados para a intensidade da dor em uma escala de 0 a 10. RESULTADOS: O nº de sessões de HD para formação do T foi de 9,5 ± 1,5 e o número de sessões na fase de manutenção foi de 29,7 ± 0,8. Nas 152 HD para formação do T não ocorreram complicações significativas. Durante 475 HD com AR as complicações foram: resistência na punção (7,6%), punção com AC por opção do puncionador (5,7%), troca de AR para AC durante a punção (4,2%) e sangramento local durante a HD (0,8%). Um paciente necessitou antibioticoterapia. A mediana do índice de dor foi 4 na fase de formação do T e 2 na fase de manutenção. Os valores de Kt/V pré- e pós-alteração na técnica de punção não foram diferentes (1,48 ± 0,27 e 1,45 ± 0,23). CONCLUSÃO: A implantação da punção em CB com AR é tecnicamente fácil, apresenta poucas complicações, reduz a dor e não induz variação na dose de diálise.

INTRODUCTION: Cannulation of arteriovenous fistula (AVF) may be performed by the following techniques: area puncture, rope ladder, or buttonhole. The ideal technique has not yet been established. OBJECTIVE: To assess the complications and difficulties of introducing the buttonhole (BH) technique for cannulation of AVF created with a native vein in a dialysis unit. METHODS: Sixteen patients (mean age, 57 ± 14 years) undergoing hemodialysis for 63 ± 38 months were changed to BH AVF cannulation. In the phase of track formation cannulations were performed with sharp needles and, in the maintenance phase, with blunt needles. In both phases, patients were assessed for pain intensity on a 0 to 10 scale. RESULTS: The number of HD sessions required for the track formation was 9.5 ± 1.5 and the number of sessions during the maintenance phase was 29.7 ± 0.8 per patient. During the 152 HD for the track formation, no significant complications occurred. During the 475 HD sessions using the BH technique and a blunt needle, the complications were as follows: resistance to cannulation (7.6%); cannulation using a sharp needle due to cannulator choice (5.7%); change from a blunt to a sharp needle during cannulation (4.2%); and local bleeding (0.8%). One patient required antibiotic therapy. The median pain intensity reported by the patients was four during the track formation, and two during cannulation with a blunt needle. The Kt/V values before and after changing the cannulation technique did not differ (1.48 ± 0.27 and 1.48 ± 0.23). CONCLUSION: The introduction of the BH technique with a blunt needle is technically easy, has few complications, reduces pain, and does not induce change in dialysis dose.

Para determinar o papel de ExoU na citotoxicidade tardia de P. aeruginosa, células endoteliais (CE) foram expostas às cepas PA103, PA103deltaxoU e PA103::exsA por 1h e à gentamicina em meio de cultura. Após 24h, a viabilidade das CE infectadas com PA103 (33.7 ± 14.3%) foi inferior à de CE infectadas com PA103deltaexoU (77.7 ± 6.3%) e PA103::exsA (79.5 ± 23.3%). A citotoxicidade não dependeu da capacidade de interagir com as CE porque o percentual de células com bactérias associadas em culturas expostas a PA103 foi semelhante ao percentual em culturas expostas a PA103deltaexoU e inferior em culturas expostas a PA103::exsA. O tratamento das CE com citocalasina D reduziu a internalização de PA103, mas não interferiu em sua citotoxicidade.

To ascertain the role of ExoU in late P. aeruginosa cytotoxicity, endothelial cells (EC) were exposed to wild type PA103, PA103deltaexoU and PA103::exsA for 1h and to gentamicin in culture medium. After 24h, the viability of PA103-infected cells (33.7 ± 14.3%) was significantly lower than the viability of PA103deltaexoU- (77.7 ± 6.3%) or PA103::exsA- (79.5 ± 23.3%) infected EC. P. aeruginosa cytotoxicity did not depend on the bacterial ability to interact with EC because the percentage of cells with associated PA103 (35.9 ± 15.8%) was similar to the percentage in PA103deltaexoU- (34.2 ± 16.0%) and lower than the percentage in PA103::exsA-infected cultures (82.9 ± 18.9%). Cell treatment with cytochalasin D reduced the PA103 internalization by EC but did not interfere with its ability to kill host cells.