An in vitro model for toxin-mediated vascular leak syndrome: ricin toxin A chain increases the permeability of human endothelial cell monolayers

Vascular leak syndrome (VLS) is the dose-limiting toxicity observed in clinical trials of immunotoxins containing ricin toxin A chain (RTA). RTA itself is thought to cause VLS by damaging vascular endothelial cells, but the exact mechanism remains unclear. This is partially due to the paucity of appropriate models. To study VLS, we developed an in vitro model in which human umbilical vein-derived endothelial cells were first grown to confluence on microporous supports and then cultured under low pressure in the presence or absence of RTA. Endothelial cell barrier function was assessed by measuring the volume of fluid that passed through each monolayer per unit time. We found that RTA significantly increased monolayer permeability at times and concentrations consistent with the onset of VLS in patients treated with RTA-based immunotoxins. Scanning electron microscopy showed that intercellular gaps formed in endothelial monolayers exposed to RTA. Intercellular gap formation followed endothelial cell death caused by the enzymatic activity of RTA. We conclude that RTA is directly toxic to endothelial cells in vitro and speculate that this contributes to VLS in vivo.     

The interaction of peripheral blood leukocytes with alpha1-acid glycoprotein, its carbohydrate chains and neoglycoconjugates

The interaction of human peripheral blood leukocytes with alpha 1-acid glycoprotein (AGP), its glycoforms as well as neoglyco-conjugates representing carbohydrate chains of AGP or its fragments was studied by flow cytometry. It was shown that the main target cells for AGP as well as for conjugates of its carbohydrate chains with polyacrylamide (PAA) are monocytes and polymorphonuclear leukocytes but not lymphocytes. The interaction of AGP with monocytes and granulocytes are mediated by its carbohydrate chains: the binding of AGP with cells was inhibited by AGP, AGP oligosaccharides as well as conjugates of oligosaccharides and its fragments with PAA. The data obtained show the existence of monocyte (and granulocyte) receptors which interact with complex type sialooligosaccharides of AGP.     

Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.     

Myxomas of the mandible and maxilla

Two cases of congenital absence of a cervical vertebral pedicle are reported. This condition includes hypoplasia of the ipsilateral posterior arch and may predispose to spinal cord injury. The radiographic and computed tomography (CT) findings are reviewed, and the importance of the ipsilateral oblique radiographic view and of CT in diagnosis is stressed.     

A method for studying the biomechanical load response of the (in vitro) lumbar spine under dynamic flexion-shear loads

A method was developed to study the biomechanical response of the lumbar motion segment (Functional Spinal Unit, FSU) under a dynamic (transient) load in flexion. In order to inflict flexion-distraction types of injuries (lap seat-belt injuries) different load pulses were transferred to the specimen by means of a padded pendulum. The load response of the specimen was measured with a force and moment transducer. The flexion angulation and displacements were determined by means of high-speed photography. Two series of tests were made with ten specimens in each and with two different load pulses: one moderate load pulse (peak acceleration 5 g, rise time 30 ms, duration 150 ms) and one severe load pulse (peak acceleration 12 g, rise time 15 ms, duration 250 ms). The results showed that the moderate load pulse caused residual permanent deformations at a mean bending moment of 140 Nm and a mean shear force of 430 N at a mean flexion angulation of 14 degrees. The severe load pulse caused evident signs of failure of the segments at a mean bending moment of 185 Nm and a mean shear force of 600 N at a mean flexion angulation of 19 degrees. Significant correlations were found between the load response and the size of the specimen, as well as between the load response and the bone mineral content (BMC) in the two adjacent vertebrae. Comparisons with lumbar spine response to static flexion-shear loading indicated that the specimens could withstand higher bending moments before injury occurred during dynamic loading, but the deformations at injury tended to be smaller for dynamic loading.