Feasibility of using microbiology diagnostic tests of moderate or high complexity at the point - of - care in a delivery suite.

Point-of-care testing (POCT) is one of the fastest growing sectors of laboratory diagnostics. Most tests in routine use are haematology or biochemistry tests that are of low complexity. Microbiology POCT has been constrained by a lack of tests that are both accurate and of low complexity. We describe our experience of the practical issues around using more complex POCT for detection of Group B streptococci (GBS) in swabs from labouring women. We evaluated two tests for their feasibility in POCT: an optical immune assay (Biostar OIA Strep B, Inverness Medical, Princetown, NJ) and a PCR (IDI-Strep B, Cepheid, Sunnyvale, CA), which have been categorised as being of moderate and high complexity, respectively. A total of 12 unqualified midwifery assistants (MA) were trained to undertake testing on the delivery suite. A systematic approach to the introduction and management of POC testing was used. Modelling showed that the probability of test results being available within a clinically useful timescale was high. However, in the clinical setting, we found it impossible to maintain reliable availability of trained testers. Implementation of more complex POC testing is technically feasible, but it is expensive, and may be difficult to achieve in a busy delivery suite.

Inhibition of scavenger receptor-mediated modified low-density lipoprotein endocytosis in cultured bovine aortic endothelial cells by the glycoprotein processing inhibitor castanospermine.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizidine) is a plant alkaloid that inhibits alpha-glucosidases, including the glycoprotein processing glucosidase I. When endothelial cells were grown for 48 h, or longer, in the presence of this alkaloid, they produced scavenger receptors for modified low-density lipoproteins (LDL) that had mostly Glc3Man7-9(GlcNAc)2 structures rather than the usual complex types of oligosaccharides. Furthermore, growth in the presence of castanospermine resulted in a substantial inhibition in degradation of endocytosed 125I-acetylated LDL, as well as a dose-dependent inhibition of 125I-acetylated LDL binding to these cells. Scatchard analysis of binding curves indicated that the diminished binding was due to a decrease in the number of scavenger receptor molecules at the cell surface rather than to a change in the affinity of the receptors for their ligand. Since castanospermine-treated cells had the same total number of cellular receptor molecules as did controls cells, it seemed likely that castanospermine caused an alteration in receptor targeting, rather than an inhibition in receptor synthesis or a stimulation in receptor degradation. Density gradient fractionation of cell homogenates showed that castanospermine-treated cells did have a much greater percentage of scavenger LDL receptor molecules in the endoplasmic reticulum-Golgi fraction and fewer receptors in the plasma membrane fraction, whereas normal cells showed the opposite distribution.

Pathophysiology of the atherogenic process.

Atherosclerosis is conceptually defined as the result of a multiplicity of interactive cascades among injurious stimuli and the healing responses of the arterial wall, occurring concurrently within a hyperlipidemic environment. In this discussion, the inflammatory nature of the disease is emphasized. Four aspects of the pathophysiology of atherogenesis are addressed: (1) The role(s) of fluid mechanical or hemodynamic stresses in the focal initiation and/or augmentation of lesions is discussed in terms of the influence of shear stress on endothelial cellular geometry, compliance, membrane anisotropy (r), low-density lipoprotein (LDL)-receptor expression, intracellular potential and replication; (2) mechanisms of blood monocyte recruitment to the arterial intima, including the roles of chemoattractants such as smooth muscle cell-derived chemotactic factor and oxidized LDL; (3) the alternate or "scavenger" receptor pathway of the macrophage and its pivotal roles in foam cell formation and plaque pathogenesis; and (4) the emerging significance of various lipoprotein modifications, and in particular, the oxidative modification of LDL, which facilitates the uptake of the cytotoxic oxidized LDL via the scavenger receptor, thus providing a non-down-regulating mechanism for foam cell formation and plaque development. Evidence indicates that the antioxidant drug probucol prevents the oxidative modification of LDL, thereby retarding atherogenesis independently of cholesterol reduction.

Castanospermine inhibits the function of the low-density lipoprotein receptor.

Castanospermine, a plant alkaloid that inhibits the glycoprotein processing enzyme glucosidase I, has been used to inhibit N-linked oligosaccharide modification, resulting in the production of glycoproteins having Glc3Man7-9(GlcNAc)2 oligosaccharides. This alkaloid caused a significant inhibition of LDL endocytosis in cultured primate smooth muscle cells and human skin fibroblasts. At an optimum concentration of 250 micrograms/mL, castanospermine caused a 40% decrease in cell surface receptor-mediated LDL binding at 4 degrees C, with no apparent change in affinity. Further, the inhibitor had no direct effect on LDL metabolism. This inhibition of LDL receptor expression and function occurred only when the drug was present during de novo receptor synthesis, i.e., during up-regulation. Although the number of cell surface LDL receptors was significantly reduced in the presence of castanospermine, the total number of receptors in the cell was only slightly reduced, indicating that castanospermine induced a redistribution rather than a reduction in the number of receptors. Similarly, subcellular fractionation studies confirmed that castanospermine treatment of fibroblasts results in an altered distribution of receptor activity compared with controls. These findings are consistent with the conclusion that the decrease in specific LDL binding to cells grown in the presence of castanospermine is due to intracellular redistribution of the LDL receptor so that more receptor remains in internal compartments as a result of a diminished rate of transport.

Cellular mechanisms in the response of the arterial wall to injury and repair.

This synopsis emphasizes the inappropriateness of a "single stimulus-single response" approach in understanding the response of the arterial wall to injury and repair. The outcome of any injurious stimulus is a series of interactive cascades among the endogenous and exogenous cellular and non-cellular components of the arterial wall, and the cellular and non-cellular elements of the blood. Both genetic and hemodynamic factors can further influence this response. The more prominent of the cellular and non-cellular components have been discussed. These include: 1) the vascular endothelium, its dynamic interaction with macromolecules and formed elements of the blood, its role in the transport of plasma proteins, its influence on the function of arterial smooth muscle cells and the recruitment of blood-born monocytes; 2) the arterial smooth muscle cell, its role in the vasomotor function of the artery wall, arterial repair and reconstruction, metabolism of lipids and the secretion of cytokines regulating monocyte recruitment; 3) the mononuclear phagocyte, its role in arterial debridement, metabolism of modified LDLs, a precursor of the cholesteryl ester-rich foam cell, and the secretion of neutral hydrolases, bioactive lipids and cytokines; 4) lymphocytes, as mediators of the inflammatory response and possible autoimmune reactions; 5) platelets, their roles in hemostasis, thrombosis, atherogenesis, and the repair process and 6) plasma LDLs, their oxidative modification by cells of the vessel wall and their roles in the injury process. The interactive processes among arterial and circulating components in both injury and repair is emphasized.

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells.

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.

Low-density lipoprotein endocytosis. I. Influence of the multivalent ligand cationized ferritin on normal and receptor-negative human fibroblasts.

Based upon the observation that the multivalent ligand cationized ferritin (CF) alters the cell surface distribution of anionic domains and significantly enhances the adsorptive endocytosis of 125I-labeled human serum albumin, these studies were undertaken to probe the influence of CF on receptor-mediated low-density lipoprotein (LDL) endocytosis and the nature of the mechanisms involved. A brief 1-min exposure of normal receptor upregulated fibroblasts to CF (0.2 mg/ml) resulted in a significant decrease (P less than 0.001) in the subsequent internalization and degradation of 125I-LDL. Studies with receptor downregulated normal fibroblasts indicated that CF pretreatment did not measurably influence 125I-LDL internalization and only slightly inhibited its degradation (P less than 0.05). In contrast, CF pretreatment of FH receptor-negative mutant skin fibroblasts resulted in a modest but significant increase in both 125I-LDL internalization and degradation (P less than 0.05). Scatchard analyses of binding data indicated that CF-pretreated upregulated normal fibroblasts exhibit a single class of LDL binding sites with an affinity, Kd = 24.7 +/- 4.1 nM, almost 10-fold lower than the affinity of binding sites in untreated controls, Kd = 3.2 +/- 0.06 nM. Increasing either the concentration or the duration of CF exposure resulted in additional inhibition of LDL internalization and degradation associated primarily with a decrease in the number of LDL binding sites without any further change in binding affinity. Total cellular LDL receptor-mediated binding, measured using an octylglucoside solubilization-filtration assay, confirmed the CF-induced decrease in high-affinity LDL binding. Pulse-chase experiments showed that CF had no direct influence on LDL degradation, nor did it influence targeting of the LDL-containing endosome toward exocytosis. Further, restoration of LDL receptor function to control values after CF pretreatment required de novo protein synthesis. The normal feedback inhibition of HMG-CoA reductase activity was nearly abolished by CF pretreatment. Additionally, CF pretreatment was found to induce not only a redistribution of surface anionic sites, but also a very rapid internalization of surface components labeled with 4,4'-[3H]diisothiocyano-1,2..diphenylethane-2,2'-disulfonic acid. It is concluded that the inhibitory influence of CF on LDL endocytosis is mediated via a decrease in the affinity and in the number of functional LDL receptors.

Low-density lipoprotein endocytosis. II. Influence of the multivalent ligand cationized ferritin on acetylated low-density lipoprotein endocytosis in cultured cells.

Interactions of the basic multivalent ligand cationized ferritin (CF) with cultured cells markedly alter their endocytic function. In this study, the influence of CF treatment on the binding, internalization, and degradation of chemically modified (acetylated) low-density lipoproteins (Ac-LDL) was examined in aortic smooth muscle cells (SMC); and in normal and FH mutant LDL receptor-negative human skin fibroblasts, which lack the Ac-LDL (scavenger) receptor; and in vascular endothelial cells, which normally express the receptor. Although CF treatment of all three cell types at 37 degrees C resulted in the induction of Pronasesensitive, high-capacity, high-affinity binding (Kd = 12.0 +/- 2.0 nM at 4 degrees C) of labeled Ac-LDL, which at 37 degrees C was accompanied by significant internalization and degradation, these processes were not receptor-mediated. CF-induced high-affinity binding was inhibited by unlabeled Ac-LDL, fucoidan, carrageenan, and dextran sulfate but was unaffected by native LDL and albumin and only partially inhibited by acetylated albumin. However, analysis of membrane preparations of the cells for "scavenger" receptor protein by solid-phase filtration assay and Western blotting identified the receptor in endothelial cells and in granuloma (positive control) macrophages, but not in either CF-treated or untreated SMC. In addition, studies with both glutaraldehyde-fixed cells and CF bound to culture dishes indicated that Ac-LDL avidly binds to CF. Further, ultrastructural studies using colloidal gold-conjugated Ac-LDL showed Ac-LDL preferentially binding to CF aggregates on the cell surface. Thus, these studies indicate that treatment of cells with CF induces an endocytic process which, although remarkably similar to the scavenger pathway, is mediated by Ac-LDL binding to membrane-associated CF. These observations have implications in terms of mechanisms that might regulate the endocytosis of modified low-density lipoproteins.

Thrombosis and the development of atherosclerosis: Rokitansky revisited.

In this article we have reviewed the evidence that implicates the organization and incorporation of mural thrombi as a significant component of atherosclerotic plaque growth in man. It has been emphasized that there is little or no evidence for a pathogenic role for thrombosis in plaque initiation or for the development of fatty streaks. We have suggested that the rapidly progressive category of atherosclerosis in man, as described by DeBakey, may well reflect a heightened propensity for mural or occlusive thrombosis in these patients. A broad spectrum of experimental studies examining the role of thrombosis in atherogenesis has been critically reviewed. These studies have established that experimental thrombi can become transformed into arterial fibrofatty plaques having many of the morphologic features of atherosclerosis. We have provided evidence, however, that the evolution of thrombi to fibrofatty lesions is dependent on the initial composition of the thrombi and that thrombi with a paucity of platelets and consisting predominantly of fibrin result only in fibrous intimal thickenings. The presence of hypercholesterolemia has been shown to influence the transformation of experimental thrombi. In particular, it slows the rate of thrombolysis, enhances the lipid content of the fibrofatty plaques, increases the numbers of macrophage-derived foam cells, and the frequency and extent of lesion calcification. Detailed lipid compositional studies of organizing thrombi in normolipidemic animals have shown that their lipid composition does not evolve toward the profile characteristic of atherosclerotic lesions and that the macrophage uptake of interstitial lipoproteins is probably a necessary component for the full biochemical development of the lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Monocyte-macrophage participation in atherogenesis: inflammatory components of pathogenesis.

This article has addressed the roles of the monocyte-macrophage in atherogenesis and factors influencing monocyte recruitment to the intima. The diversity of the secretory products of the macrophage and their putative participatory roles in pathogenesis have been reviewed and discussed. Additionally, we have presented summary data on the monocyte chemoattractant peptide SMC-CF and on the differentiation of the monocyte-derived foam cell. Discussion has centered on the concept of atherosclerosis as an inflammatory process.

Interaction of tritium-labeled H2DIDS (4,4'-diisothiocyano-1,2,diphenyl ethane-2,2'disulfonic acid) with the Ehrlich mouse ascites tumor cell.

The experiments reported in this paper were undertaken to explore the interaction of tritiated H2DIDS (4,4'-diisothiocyano-1,2,diphenyl ethane-2,2'-disulfonic acid) with Ehrlich ascites tumor cells. Addition of (3H)H2DIDS to tumor cell suspension at 21 degrees C, pH 7.3, resulted in: (i) rapid reversible binding which increased with time and (ii) inhibition of sulfate transport. Tightly bound H2DIDS i.e., reagent not removed by cell washing, also increased with time. Binding of 0.02 nmol H2DIDS/mg dry mass or less did not affect sulfate transport, but, at greater than 0.02 nmol and up to 0.15 nmol the relationship between tight binding and inhibition of transport is linear. The fact that H2DIDS could bind to the cell and yet not affect anion transport suggests that binding sites exist unrelated to those concerned with the regulation of anion permeability. Support for this is the observation that H2DIDS is spontaneously released from cells even after extensive washings by a temperature-sensitive process. The most important source of released H2DIDS is the cell surface coat which labels rapidly (within 1 min) and is then spontaneously released into the medium. A second source is derived from H2DIDS that slowly entered the cells. Consequently, at least four modes of interaction exist between H2DIDS and ascites tumor cells. These include both reversible and irreversible binding to membrane components which regulate anion permeability, irreversible binding to cell surface proteins or glycocalyx, and finally incorporation of H2DIDS into the intracellular phase.