Responses of human intestinal microvascular endothelial cells to Shiga toxins 1 and 2 and pathogenesis of hemorrhagic colitis.
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Endothelial damage is characteristic of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). Because Stx-mediated endothelial cell damage at the site of infection may lead to the characteristic hemorrhagic colitis of STEC infection, we compared the effects of Stx1 and Stx2 on primary and transformed human intestinal microvascular endothelial cells (HIMEC) to those on macrovascular endothelial cells from human saphenous vein (HSVEC). Adhesion molecule, interleukin-8 (IL-8), and Stx receptor expression, the effects of cytokine activation and Stx toxins on these responses, and Stx1 and Stx2 binding kinetics and bioactivity were measured. Adhesion molecule and IL-8 expression increased in activated HIMEC, but these responses were blunted in the presence of toxin, especially in the presence of Stx1. In contrast to HSVEC, unstimulated HIMEC constitutively expressed Stx receptor at high levels, bound large amounts of toxin, were highly sensitive to toxin, and were not further sensitized by cytokines. Although the binding capacities of HIMEC for Stx1 and Stx2 were comparable, the binding affinity of Stx1 to HIMEC was 50-fold greater than that of Stx2. Nonetheless, Stx2 was more toxic to HIMEC than an equivalent amount of Stx1. The decreased binding affinity and increased toxicity for HIMEC of Stx2 compared to those of Stx1 may be relevant to the preponderance of Stx2-producing STEC involved in the pathogenesis of hemorrhagic colitis and its systemic complications. The differences between primary and transformed HIMEC in these responses were negligible. We conclude that transformed HIMEC lines could represent a simple physiologically relevant model to study the role of Stx in the pathogenesis of hemorrhagic colitis. (+info)
Interaction of Shiga toxins with human brain microvascular endothelial cells: cytokines as sensitizing agents.
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Neurologic abnormalities are among the most serious extraintestinal complications of infection with Shiga toxin (Stx)-producing bacteria. Histopathologic examination of tissues from patients with extraintestinal sequelae suggested that Stxs damage endothelial cells. It is shown here that human brain microvascular endothelial cells (HBMECs) are relatively resistant to purified Stxs (50% cytotoxic doses [CD50s] >/=10 microgram/mL). Pretreatment of HBMECs with tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, n-butyric acid, or a cAMP analogue resulted in a 103- to 104-fold decrease in CD50 values and a 2- to 4-fold increase in fluoresceinated Stx binding to HBMECs. Treatment of HBMECs with lipopolysaccharides did not significantly alter cytotoxicity or toxin binding. TNF-alpha and IL-1beta treatment was associated with the increased HBMEC expression of the toxin-binding glycolipid globotriaosylceramide. HBMECs did not produce IL-1beta and produced only trace amounts of TNF-alpha when stimulated with purified Stx1 in vitro. (+info)
Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence from CD34(+) hematopoietic stem cells: implications for ex vivo tumor purging and autologous stem cell transplantation.
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The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma patients. (+info)
Monoclonal antibody to Shiga toxin 2 which blocks receptor binding and neutralizes cytotoxicity.
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A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of Escherichia coli O157:H7. MAb VTm1.1 belonged to the immunoglobulin G1 subclass and had a kappa light chain, and it could neutralize the cytotoxic activity of Stx2 and variants derived from patient strains but not that of variants derived from animals. MAb VTm1.1 was shown to bind to the B subunit of these neutralized Stx2s by Western blotting. Comparison of B-subunit amino acid sequences and reactivities to these Stxs suggested six amino acids (Ser30, Ser53, Glu56, Gln65, Asn68, and Asp69) that were candidates for the MAb VTm1.1 epitope. Consequently, five Stx2 mutants (S30N, S53N, E56H, Q65K, and N68Ter) were prepared by site-directed mutagenesis to determine which residue is essential for the epitope. All of these mutants showed cytotoxicity almost equal to that of the wild-type Stx2. Of the five Stx2 mutants, only E56H could not be neutralized by MAb VTm1.1. Western blot analysis also showed that MAb VTm1.1 could not bind to the E56H B subunit. These results indicated that Glu56 is an important residue recognized by MAb VTm1. 1. Immunofluorescence analysis further indicated that MAb VTm1.1 inhibits the binding of Stx2 to its receptors. MAb VTm1.1 could be a useful therapeutic agent for Shiga toxin-producing E. coli infection. (+info)
Glycolipid receptors for verotoxin and Helicobacter pylori: role in pathology.
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Eukaryotic cell surface glycolipids can act as both the primary interface between bacteria and their host and secondly as a targeting mechanism for bacterial virulence factors. The former is characterized by redundancy in adhesin-receptor interactions and the latter by a higher affinity, more restrictive glycolipid binding specificity for targeting. Interactions of verotoxin with its glycolipid receptor globotriaosylceramide and Helicobacter pylori binding to a variety of different glycolipids, which can be environmentally regulated, provide examples of these differing modes of glycolipid receptor function. Verotoxins are involved in endothelial targeting in the microangiopathies of hemorrhagic colitis and hemolytic uremic syndrome (HUS). The highly restricted binding specificity and crystal structure of the verotoxin B subunit have allowed theoretical modeling of the Gb3 binding site of the verotoxin B subunit pentamer which provides an approach to intervention. Studies of the role of glycolipid function in verotoxin-induced disease have concentrated on the distribution of Gb3 and its ability to mediate the internalization of the toxin within the target cell. The distribution of Gb3 within the renal glomerulus plays a central role in defining the age-related etiology of HUS following gastrointestinal infection with VT producing Escherichia coli. H. pylori, on the other hand, instigates a less distinct but more complex disseminated gastric inflammation. Studies on the role of glycolipid receptors in H. pylori infection have been bogged down in establishing the importance of each binding specificity defined. In addition, the physiological condition of the organism within the various binding assays has not been extensively considered, such that spurious non-physiological interactions may have been elucidated. The identification and cloning of a Le(b) binding adhesin and the identification of cell surface hsp70 as a mediator of sulfoglycolipid binding under stress conditions may now allow a more molecular approach to define the role of glycolipid recognition in this infection. (+info)
Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains.
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Shiga toxin (Stx) is an enterotoxin produced by Shigella dysenteriae serotype 1 and enterohemorrhagic Escherichia coli, which binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. We previously demonstrated that Stx induced apoptosis in human renal tubular cell line ACHN cells (Taguchi, T., Uchida, H., Kiyokawa, N., Mori, T., Sato, N., Horie, H., Takeda, T and Fujimoto, J. (1998) Kidney Int. 53, 1681-1688). To study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from ACHN cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomains (DIM). Gb3 was only recovered in DIM and was associated with Src family kinase Yes. Phosphorylation of tyrosine residues of proteins in the DIM fraction increased by 10 min and returned to the resting level by 30 min after the addition of Stx. Since the kinase activity of Yes changed with the same kinetics, Yes was thought to be responsible for the hyperphosphorylation observed in DIM proteins. Unexpectedly, however, all of the Yes kinase activity was obtained in the high density, detergent-soluble fraction. Yes was assumed to be activated and show increased Triton X-100 solubility in the early phase of retrograde endocytosis of Stx-Gb3 complex. Since Yes activation by the Stx addition was suppressed by filipin pretreatment, Gb3-enriched microdomains containing cholesterol were deeply involved in Stx signal transduction. (+info)
Role of glycosphingolipids in HIV-1 entry: requirement of globotriosylceramide (Gb3) in CD4/CXCR4-dependent fusion.
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We have recently shown that addition of human erythrocyte glycosphingolipids (GSL) to non-human CD4+ or GSL-depleted human CD4+ cells rendered those cells susceptible to gp120-gp41-mediated cell fusion (Puri et al., BBRC, 1998). One GSL fraction (Fraction 3) isolated from human erythrocyte GSL mixture exhibited the highest recovery of fusion following incorporation into CD4+ non-human and GSL-depleted HeLa-CD4 cells (HeLa-CD4/GSL-). Structural analysis of Fraction 3 showed that this GSL had identical head group as the known GSL, Gal(alpha1-->4)Gal(beta1-->4)Glc-Ceramide (Gb3) (Puri et al., PNAS, 1998). Here we report that presence of Gb3 in CD4+/CXCR4+ cells but not CD4+/CXCR4 cells allows fusion with HIV-1Lai-envelope glycoprotein expressing cells (TF228). Therefore, Gb3 functions in conjunction with HIV-1 co-receptor, CXCR4 to promote fusion. We propose that Gb3 functions by recruiting CD4 and/or CXCR4 at the fusion site through structurally specific interactions. (+info)
Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease.
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Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb(3); also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified alpha-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered alpha-gal A, (ii) to assess the pharmacokinetics of i.v.-administered alpha-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb(3). alpha-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified alpha-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of alpha-gal A, nine of the 10 patients had significantly reduced Gb(3) levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of alpha-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb(3) burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder. (+info)