Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome.
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The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin-producing Escherichia coli infection. The endothelial cell is believed to play an important role in the pathogenesis of HUS. Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1. Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding. Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days. The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide. Glycolipid extracts of TNF-alpha-treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells. We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer. These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS. (+info)
Forssman synthetase expression results in diminished shiga toxin susceptibility: a role for glycolipids in determining host-microbe interactions.
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Forssman glycolipid (FG), the product of Forssman synthetase (FS), is widely expressed among nonprimate mammalian species. Here, we describe a molecular and genetic relationship between FG expression and Shiga toxin (Stx) susceptibility. We have isolated the FS cDNA from human, canine, and murine cells. Whereas the murine and canine FS genes express a functional enzyme, the human FS cDNA was found to express a protein that lacks FS activity, despite a high degree of sequence identity with the enzymatically active murine and canine FS genes. In order to examine the relationship between FG expression and Stx susceptibility, Vero cells were transfected with the three FS orthologues or a vector control. Complementation with the human FS cDNA had no effect on Stx susceptibility, whereas stable expression of the canine and murine FS resulted in markedly decreased susceptibility to toxin. Among individual cells, an inverse correlation between FG expression and Stx binding was demonstrated. Moreover, only strongly FG-reactive cells were capable of growing in the presence of Stx. These cells were found to have high levels of FG expression and a correspondingly diminished GbO(3) content. We conclude that expression of a functionally active FS modifies Stx receptor glycolipids to FG and results in markedly decreased susceptibility to toxin. We speculate that inactivation of the FS gene during primate evolution may account, at least in part, for the marked susceptibility of human cells to Stx. (+info)
Role of multiple drug resistance protein 1 in neutral but not acidic glycosphingolipid biosynthesis.
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Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis. (+info)
Oral therapeutic agents with highly clustered globotriose for treatment of Shiga toxigenic Escherichia coli infections.
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Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections. (+info)
A novel ceramide trihexoside from the eggs of the sea urchin, Hemicentrotus pulcherrimus.
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Glucosylceramide (Glc beta 1-1Cer) and a novel ceramide trihexoside (Gal beta 1-6Gal beta 1-6Glc beta 1-1Cer) were purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, chromic acid oxidation, enzymatic hydrolysis, enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ceramide trihexoside has a novel carbohydrate structure, and its core structure, Gal beta 1-6Glc, is also novel. The ceramide moieties of these glycolipids are almost identical. Two fatty acids, 22:1 and 22h:1, constitute more than 80% of the total acids. Long-chain bases are all phytosphingosine, approximately 90% of which is n-t18:0. The finding of melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) from the eggs of another sea urchin species [Kubo, H. et al. (1988) J. Biochem. 104, 755-760] and the present finding of the novel ceramide trihexoside suggest that there are a variety of unique sugar structures in sea urchin glycosphingolipids. (+info)
Bovine ileal intraepithelial lymphocytes represent target cells for Shiga toxin 1 from Escherichia coli.
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The discovery that bovine peripheral lymphocytes are sensitive to Stx1 identified a possible mechanism for the persistence of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) in the bovine reservoir host. If intraepithelial lymphocytes (IEL) are also sensitive to Stx1, the idea that Stx1 affects inflammation in the bovine intestine is highly attractive. To prove this hypothesis, ileal IEL (iIEL) were prepared from adult cattle, characterized by flow cytometry, and subjected to functional assays in the presence and absence of purified Stx1. We found that 14.9% of all iIEL expressed Gb(3)/CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb(3)/CD77(+) cells were activated CD3(+) CD6(+) CD8 alpha(+) T cells, whereas only some CD4(+) T cells and B cells expressed Gb(3)/CD77. However, Stx1 blocked the mitogen-induced transformation to enlarged blast cells within all subpopulations to a similar extent and significantly reduced the percentage of Gb(3)/CD77(+) cells. Although Stx1 did not affect the natural killer cell activity of iIEL, the toxin accelerated the synthesis of interleukin-4 (IL-4) mRNA and reduced the amount of IL-8 mRNA in bovine iIEL cultures. Because the intestinal system comprises a rich network of interactions between different types of cells and any dysfunction may influence the course of intestinal infections, this demonstration that Stx1 can target bovine IEL may be highly relevant for our understanding of the interplay between STEC and its reservoir host. (+info)
Monitoring the 3-year efficacy of enzyme replacement therapy in fabry disease by repeated skin biopsies.
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The earliest clinical signs of Fabry disease often manifest as dermatologic disturbances such as angiokeratomata, hypohidrosis, acroparesthesias, and impaired thermal and vibration detection. These disturbances are caused by cellular globotriaosylceramide accumulation in the skin due to deficient lysosomal alpha-galactosidase A activity. In this histologic study, we analyzed pre- and post-treatment dermatologic biopsies from 58 Fabry patients enrolled in a 5 mo, Phase 3 double-blind, randomized, placebo-controlled trial followed by a 30 mo open label extension study of recombinant human alpha-galactosidase A (r-halphaGalA), administered i.v. at 1 mg per kg every 2 wk. Baseline evaluations revealed globotriaosylceramide in multiple dermal cell types (vascular endothelial cells, vascular smooth muscle cells, perineurium). Five months of r-halphaGalA treatment in the Phase 3 trial resulted in complete clearance of globotriaosylceramide from the superficial capillary endothelium in all treatment patients and in only 1 (3%) placebo patient (p<0.001). The placebo group achieved similar results after 6 mo of r-halphaGalA in the open label trial. The capillary endothelium remained free of globotriaosylceramide for up to 30 mo into the extension study among 39 of 40 (98%) patients who underwent biopsies. Globotriaosylceramide clearance from deep vascular endothelial cells was similarly robust. Vascular smooth muscle cells and perineurium demonstrated moderate clearance. These findings suggest that long-term treatment with r-halphaGalA may halt the progression of pathology and prevent the dermatologic disturbances in Fabry patients, and that periodic dermal biopsies can serve as a reliable monitor of sustained efficacy. (+info)
Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity.
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In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3. (+info)