Influence of patient position on withdrawal forces during removal of lumbar extradural catheters.

We have investigated the force required to remove lumbar extradural catheters from 88 parturients to determine the effects of patient positioning at removal, relative to the position at insertion. Parturients were allocated randomly to one of four groups: LS (lateral insertion, sitting withdrawal), LL (lateral insertion, flexed lateral withdrawal), SL (sitting insertion, lateral withdrawal) or SS (sitting insertion, sitting withdrawal). In both positions, the lumbar spine was kept maximally flexed. The force required to remove the catheter was measured at withdrawal. We found that the withdrawal force was influenced by the relationship between the position at removal and that at insertion, and we recommend that for ease of removal, patients should be placed in the same position as they were at the time of insertion. Compared with all other groups, the withdrawal force in patients in group LS was significantly greater (P < 0.05).

Thyroxine targets different pathways of internalization of type II iodothyronine 5'-deiodinase in astrocytes.

In the brain, thyroid hormone dynamically regulates levels of the short-lived plasma membrane protein, type II iodothyronine 5'-deiodinase. In cultured astrocytes, thyroxine modulates deiodinase levels by activating cytoskeletal-plasma membrane interactions that increase the rate of inactivation of the enzyme. Here we characterized the effects of these thyroxine-dependent cytoskeletal interactions upon the route of internalization of the deiodinase by following the intracellular transit of the affinity-labeled substrate-binding subunit of the deiodinase (p29). Thyroxine rapidly induced the inactivation of the deiodinase and initiated the binding of p29 to F-actin. By 40 min, > 75% of the p29 had been transported to an endosomal pool, which was followed by dissociation of the F-actin-p29 complex. There was no significant accumulation of p29 in the dense lysosomes seen in the presence of thyroxine. In the absence of thyroxine, p29 was internalized and transported to the dense lysosomes at a rate parallel to the inactivation rate of the deiodinase (t1/2 0.75 and 0.64 h, respectively) without involvement with the microfilaments. These data demonstrate that thyroxine targets type II iodothyronine 5'-deiodinase to an endosomal pool by activating specific protein-F-actin interactions involved in microfilament-mediated intracellular protein trafficking.

Decomposition of alpha-hydroxyaryl ketones and characterization of some unusual products.

Alpha-Hydroxyaryl ketones such as 2-hydroxypropiophenone and 1-(2,4-difluorophenyl)-2-hydroxy-1-propanone, the key intermediates in the preparation of antifungal agents, decompose into oxidized, rearranged, and condensed products. These products were isolated and characterized. The possible mechanisms for the formation of the products are discussed.

Inhibition and killing of fungi by the polyamine oxidase-polyamine system. Antifungal activity of the PAO-polyamine system.

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.

Paradoxical role of capsule in murine bronchoalveolar macrophage-mediated killing of Cryptococcus neoformans.

Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.

Differential stimulation of murine resident peritoneal cells by selectively opsonized encapsulated and acapsular Cryptococcus neoformans.

Stimulation of murine resident peritoneal cells (RPCs) by encapsulated strain 52 and acapsular strain 602 of Cryptococcus neoformans was compared. Fresh serum was required for fungistasis of both strains. Encapsulated organisms were killed only if the RPCs were activated with gamma interferon (IFN-gamma) or if the organisms were opsonized with anticapsular immunoglobulin G (IgG). In contrast, acapsular organisms were killed by unactivated RPCs, with enhanced killing seen if the cells were activated with IFN-gamma. Except for unopsonized strain 52, all organisms of both strains were phagocytosed. The respiratory burst was stimulated in unactivated and IFN-gamma-activated RPCs by encapsulated strain 52 only if organisms were opsonized with both IgG and serum. In contrast, the burst was stimulated by acapsular strain 602, with or without opsonization. Only unopsonized strain 602 stimulated significant lysosomal enzyme release. Nitrite synthesis by unactivated RPCs was seen only if strain 52 was opsonized with anticapsular IgG or if strain 602 was opsonized with serum. If RPCs were activated with IFN-gamma, then serum-opsonized strain 52 was also able to stimulate nitrite synthesis. Thus, RPC killing, phagocytosis, respiratory burst, lysosomal enzyme release, and nitrite synthesis following challenge by both unopsonized and opsonized with serum or anticapsular IgG strains 52 and 602 varied according to the surface properties of the organisms, the state of activation of the RPCs, and the particular RPC event studied. However, stimulation of nitrite synthesis was the only RPC event which correlated with killing of both strains.

A rapid fluorescent assay to distinguish attached from phagocytized yeast particles.

In studies of phagocytosis and its consequences for cell activation, it is important to distinguish those particulate stimuli which are completely ingested and internalized from those which are only attached to phagocytic surfaces. Ingestion can be profoundly influenced by both the type of opsonins on the surface of the stimulus and the expression and activation of receptors on the phagocytes for these opsonins. We report a new fluorescent assay which facilitates rapid and reproducible quantitation of attached versus fully internalized live or dead yeast particles by phagocytes. The assay employs the fluorescent dye diaethanol (Uvitex 2B) which stains chitin on the cell wall of fungi and is excluded from live phagocytes. The diaethanol assay and a standard, previously published methylene blue dye exclusion assay yielded comparable results using human neutrophils or monocytes incubated with heat-killed, serum-opsonized Candida albicans. The diaethanol assay proved useful in distinguishing differences in effects of various opsonins, as human neutrophils selectively opsonized with either pooled human serum (PHS), IgG (heat-inactivated PHS) or complement (IgG-depleted PHS) completely internalized 69.5%, 91.3% and 52.5% of cell-associated zymosan particles respectively. Finally, the new assay permitted comparisons of differing macrophage populations, as resident murine peritoneal macrophages internalized only 10.6% of complement-opsonized, cell-associated zymosan particles compared with 41.7% when the macrophages were elicited with thioglycolate. The assay should prove useful to investigators studying both fungal phagocytosis and killing, as well as to those performing general studies of receptor expression, regulation and function.