Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin.

Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic Escherichia coli strains that produce Shiga toxins. Globotriaosylceramide (Gb3) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-alpha) upregulates Gb3 in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-alpha treatment enhanced Shiga toxin binding and sensitivity to toxin. This upregulation was specific for Gb3 species containing normal fatty acids (NFA). Central nervous system (CNS) pathology in HUS could involve cytokine-stimulated elevation of endothelial NFA-Gb3 levels. Differential expression of Gb3 species may be a critical determinant of Shiga toxin toxicity and of CNS involvement in HUS.

UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated.

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 microm). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra. Moreover, regardless of whether the enzyme came from encysting or non-encysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.

Oligosaccharides and glycoconjugates in human milk: their role in host defense.

Human milk contains an extremely high concentration of complex carbohydrates, especially oligosaccharides, the third most abundant solid constituent of human milk. The value of human milk nutrients to infants is now widely recognized, and a role for the secretory antibodies of human milk in the defense of the infant is generally accepted. However, a function for nonimmunoglobulin milk protective factors, many of them non-nutrients, in providing for the defense of the nursling is only now beginning to be appreciated. Prominent among postulated defense agents are the milk oligosaccharides and glycoconjugates. Their complex carbohydrate structures are thought to be assembled by the same enzymes, the glycosyltransferases, that synthesize the cell surface glycoconjugates often used as receptors by pathogens. Some milk oligosaccharides and glycoconjugates may protect the nursing infant by acting as receptor homologs, inhibiting the binding of enteropathogens to their host receptors. Ongoing research is linking specific carbohydrate structures with protection against specific pathogens. Current information regarding the composition, protective activities, and protective mechanisms of the milk glycolipids, glycoproteins, mucins, glycosaminoglycans, and oligosaccharides is reviewed.

In vitro accumulation of glucocerebroside in neuroblastoma cells: a model for study of Gaucher disease pathobiology.

Gaucher disease is the most common lysosomal glycosphingolipid storage disease; decreased activity of glucosylceramide beta-glucosidase (GCase) results in the accumulation of glucocerebroside (GlcCer) in macrophage-derived cells. The most devastating types of Gaucher disease also involve neuronopathology, thought to be mediated by intracellular GlcCer accumulation in the brain. In this study, we developed an in vitro neuronal cell model for accumulation of endogenous GlcCer to enable studies on the cellular basis for the neuronopathology of this disease. A human neuroblastoma cell line (SH-SY5Y) was selected because it produced appreciable GCase. When these cells were treated with conduritol B epoxide (CBE), a competitive, irreversible inhibitor of this enzyme, GCase levels fell precipitously, while other lysosomal hydrolase levels were unaffected. Relative to untreated control cells, the CBE-treated cells accumulated higher levels of GlcCer, but not other related glycolipids, over time. Thus, this in vitro system displayed many essential biological parameters relevant for studies on cellular events responsible for the neurologic damage that occurs in some types of Gaucher disease. This model should also be useful in investigations of the normal role of sphingolipids in neuronal cell function.

Oligosaccharides from human milk block binding and activity of the Escherichia coli heat-stable enterotoxin (STa) in T84 intestinal cells.

Enterotoxin-producing Escherichia coli are major causes of pediatric diarrhea in developing countries. The heat-stable enterotoxin of Escherichia coli (STa) causes diarrhea by virtue of its ability to bind to and stimulate intestinal membrane-bound guanylate cyclase, generating cyclic GMP (cGMP). Previous work showed that a fucosylated oligosaccharide fraction of human milk was able to protect suckling mice from the secretory effects of STa, but the mechanism of the protection could not be determined. Oligosaccharide fractions from human milk were tested for their ability to block the biochemical effects of STa in T84 cells, a human colon carcinoma line responsive to the toxin. Total and fucosylated oligosaccharide fractions were found to inhibit STa-stimulated guanylate cyclase activity in T84 cell membranes and cGMP production in intact T84 cells by 60-80%. In addition, the total oligosaccharide fraction and the fucosylated oligosaccharide fraction inhibited 125I-STa binding significantly (17% and 27% inhibition, respectively). These findings demonstrate the protective activity of human milk oligosaccharides against STa in a human-derived cell line and show that the biochemical step blocked by oligosaccharides is STa-mediated stimulation of guanylate cyclase. This represents a novel mechanism by which human milk oligosaccharides protect against diarrhea.

A human milk factor inhibits binding of human immunodeficiency virus to the CD4 receptor.

Perinatal transmission of human immunodeficiency virus (HIV) from infected mothers to their children occurs at rates reported as 20-50%. The role of breast feeding in perinatal transmission of viral infections has not been well established. We studied 34 milk and colostral samples obtained from HIV-seropositive and HIV-seronegative women to determine if they contained anti-HIV activity. We found that all the samples contained a factor that inhibited the binding of HIV epitope-specific MAb to recombinant CD4 receptor molecules. The titers of inhibitory activity ranged from 1:200 to 1:10,000 and did not differ between HIV-seropositive and HIV-seronegative mothers. This milk factor also inhibited the binding of gp120 to CD4. Neither human sera nor bovine milk exhibited appreciable inhibitory activity. Fractionation of human milk indicated that the inhibitory activity was confined to the macromolecular fraction; little activity was found in isolated milk lipids or oligosaccharides. Chromatographic procedures indicated that the active macromolecule has an isoelectric point of 9.3-9.6. The active material did not bind to concanavalin A; however, the activity was partially destroyed by chemical and enzymatic treatments that removed sulfated residues. The active material may thus be a sulfated protein, glycoprotein, mucin, or glycosaminoglycan that inhibits the binding of CD4 to HIV envelope glycoproteins. The role of this factor in the natural history of HIV infection in infants and children should be the subject of additional investigations.

Effects of the alpha-mannosidase inhibitors, 1,4-dideoxy-1,4-imino-D-mannitol and swainsonine, on glycoprotein catabolism in cultured macrophages.

Thioglycollate-stimulated murine peritoneal macrophages were cultured for eight days in the presence of swainsonine, or 1,4-dideoxy-1,4-imino-D-mannitol (DIM), or both of these competitive alpha-mannosidase inhibitors together. Analysis of accumulated high-mannose oligosaccharides by reversed phase HPLC after perbenzoylation revealed that DIM- and DIM-plus swainsonine-treated macrophages contained larger amounts of Man7GlcNAc, Man8GlcNAc and Man9GlcNAc, while swainsonine-treated macrophages contained relatively more Man3GlcNAc and Man5GlcNAc. These results are consistent with the known inhibitory effects of DIM and swainsonine on Golgi mannosidases I and II, respectively, and on lysosomal alpha-mannosidase. Depletion of stored oligosaccharides to control values was complete within seven days of terminating swainsonine treatment.

Macrophages exposed in vitro to conduritol B epoxide resemble Gaucher cells.

In Gaucher disease the genetic lack of acid beta-glucosidase activity causes glucocerebroside to accumulate in the lysosomes of macrophage-derived cells, producing large characteristic Gaucher cells. The formation of Gaucher cells seems to be central to the pathobiology of this lysosomal storage disease. To develop a model simulating this process, cultured murine peritoneal macrophages were treated with conduritol B epoxide, a specific irreversible inhibitor of acid beta-glucosidase, for 6, 15, and 24 days. The conduritol B epoxide-treated macrophages accumulated glucocerebroside as a function of time, progressing to a fivefold elevation over control values after 24 days of treatment. Electron microscopy of the cells treated for 24 days reveals characteristics of Gaucher cells, including striations consisting of oriented fibrils. With conventional staining techniques, these fibrils have an appearance considered highly characteristic of Gaucher disease. Thus, macrophages treated with conduritol B epoxide are a useful model for studying the metabolic consequences and morphologic features associated with glucocerebroside accumulation in Gaucher cells.

Gaucher-like changes in human blood-derived macrophages induced by beta-glucocerebrosidase inhibition.

Human blood-derived macrophages were cultured in the presence of conduritol-B-epoxide, a specific inhibitor of beta-glucosidase, to induce changes resembling those occurring in the cells of patients with Gaucher's disease. After 24 hours of incubation, only 5% of the original beta-glucosidase activity remained; on removal of the inhibitor, the enzyme activity recovered almost fully to control levels after 5 days. After 30 days of incubation with conduritol-B-epoxide, the macrophages contained almost 10 times as much glucocerebroside as the untreated controls, and the cells displayed morphologic changes reminiscent of Gaucher's cells. This in vitro system may enable detailed studies on the pathogenetic mechanisms associated with glucocerebroside accumulation in human macrophages as well as on the turnover of the accumulated substrate and reversal of the morphologic abnormalities on removal of the inhibitor.

Lysosomal hydrolases in blood-derived macrophages of patients with I-cell disease.

Three lysosomal hydrolases, beta-galactosidase, beta-glucuronidase, and beta-N-acetylglucosaminidase, were examined in blood-derived macrophages and media of two patients with I-cell disease. The activities of the three enzymes were lower in I-cell macrophages than in normal controls. However, the media collected from these cells possessed higher activities than control media. beta-N-acetylglucosaminidase derived from media of I-cell macrophages was not endocytosed by fibroblasts from patients with Sandhoff's disease and was only partially endocytosed by I-cell macrophages. These findings indicate that blood-derived macrophages of patients with I-cell disease are affected. In addition, the data presented suggest the presence of two types of receptors in human blood-derived macrophages: mannose and mannose-6-phosphate.

beta-Glucosidase inhibition in murine peritoneal macrophages by conduritol-B-epoxide: an in vitro model of the Gaucher cell.

Murine peritoneal macrophages were cultured in the presence of conduritol-B-epoxide, a specific covalent inhibitor of beta-glucosidase. The inhibition was found to be dose and time dependent. Upon removal of the inhibitor from the culture medium, beta-glucosidase activity recovered to half maximum by 2.2 days. Treatment of macrophages with this inhibitor for 15 days did not affect cell viability, lysosomal enzyme release to the medium, or levels of intracellular lysosomal enzymes, other than beta-glucosidase activity. This inhibition results in the accumulation of glucocerebroside. In vitro studies on the pathobiology of such macrophages whose beta-glucosidase activity has been reduced may be useful toward understanding the pathogenesis of Gaucher disease.