Digest the Sugar, Kill the Parasite: A New Experimental Concept in Treating Alveolar Echinococcosis.

INTRODUCTION: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. METHODS: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or ß1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. RESULTS: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with ß1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac-tosidase. DISCUSSION: We raised our hypothesis that this "aborting" effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.

The effect of treatment with α-glycosidases from Bacteroides fragilis on the survival of rat erythrocytes in the circulation.

BACKGROUND: It has been demonstrated recently that α1,3-galactosidase from Bacteroides fragilis can efficiently convert human group B red blood cells (RBC) to group O cells. In addition, in vitro data indicated that the enzymatic conversion process did not affect the physiological or metabolic parameters of the RBC. The aim of this study was to investigate the lifespan of enzyme- treated RBC in vivo in the circulation. MATERIALS AND METHODS: This was an experimental, randomised study. The rat was selected as the experimental subject because it expresses α-1,3galactosyl on its RBC. The efficiency of Galα1,3Gal epitope removal from RBC treated with α1,3-galactosidase was tested before the transfusion experiment to track the survival of RBC in the circulation. The animals were divided into three groups and injected via the tail vein with native, mock-treated or enzyme-treated RBC labelled with fluorescein isothiocyanate. The survival rates of the fluorescently labelled RBC were monitored by flow cytometry. RESULTS: Flow cytometry showed that α-galactosidase (0.02 mg/mL for RBC with a haematocrit of 30%) efficiently removed Galα1,3Gal epitopes from rat erythrocytes, although small amounts of remaining Galα1,3Gal epitopes were still detected. The in vivo data demonstrated that the half-life of enzyme-treated RBC was a little shorter than that of native RBC. However, the 24-hour survival fractions of native, mock-treated and enzyme-treated RBC were virtually identical. Most importantly, the enzyme-treated RBC, like the native RBC, were still detectable 35 days after transfusion. DISCUSSION: Our results indicate that α-glycosidase treatment had little effect on the in vivo survival kinetics of RBC. These data add further support to the feasibility of translating enzymatic conversion technology into clinical practice.

Virulence attenuation of a UDP-galactose/N-acetylglucosamine beta1,4 galactosyltransferase expressing Leishmania donovani promastigote.

Protozoan parasites of the genus Leishmania are the causative agent of leishmaniasis, a disease whose manifestations in humans range from mild cutaneous lesions to fatal visceral infections. Human visceral leishmaniasis is caused by Leishmania donovani. Long-term culture in vitro leads to the attenuation of the parasite. This loss of parasite virulence is associated with the expression of a developmentally regulated UDP-Galactose/N-acetylglucosamine beta 1-4 galactosyltransferase and galactose terminal glycoconjugates as determined by their agglutination with the pea nut agglutinin (PNA). Thus, all promastigotes passaged for more than 11 times were 100% agglutinated with PNA, and represent a homogeneous population of avirulent parasites. Identical concentrations of PNA failed to agglutinate promastigotes passaged for < or =5 times. These PNA(-) promastigotes were virulent. Promastigotes passaged from 5 to 10 times showed a mixed population. The identity of populations defined by virulence and PNA agglutination was confirmed by isolating PNA(+) avirulent and PNA(-) virulent clones from the 7th passage promastigotes. Only the PNA(+) clones triggered macrophage microbicidal activity. The PNA(+) clones lacked lipophosphoglycan. Intravenous administration of [(14)C] galactose-labeled parasite in BALB/c mice resulted in rapid clearance of the parasite from blood with a concomitant accumulation in the liver. By enzymatic assay and RT-PCR we have shown the association of a UDP-Galactose/N-acetylglucosamine beta1,4 galactosyltransferase with only the attenuated clones. By immunofluorescence we demonstrated that the enzyme is located in the Golgi apparatus. By western blot analysis and SDS-PAGE of the affinity-purified protein, we have been able to identify a 29 KDa galactose terminal protein from the avirulent clones.

The heat-labile enterotoxin of Escherichia coli binds to polylactosaminoglycan-containing receptors in CaCo-2 human intestinal epithelial cells.

The E. coli type I heat-labile enterotoxin (LT-I) shares considerable functional, structural, and immunological homology with cholera toxin (CT). Although the ganglioside GM1 is the sole receptor for CT, LT-I also appears to utilize additional, unique receptors on intestinal cells not recognized by CT. We characterized this second class of LT-I receptors using the human intestinal epithelial cell line, CaCo-2. CaCo-2 cells bound 8-fold more LT-I than CT, and some of these additional LT-I receptors appeared to be functional, as CT-B only partially inhibited LT-I activity at concentrations that completely inhibited CT activity. Membranes from unlabeled or [3H]galactose-labeled cells were incubated with toxin B subunits and extracted with Triton X-100, and the solubilized toxin B-receptor complexes were immunoabsorbed with anti-B bound to protein A-Sepharose. When organic extracts of the complexes were separated by thin-layer chromatography and overlayed with [125I]toxin, both toxins were found to bind only GM1. Separation of the complexes from [3H]galactose-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a series of galactoproteins specifically recognized by LT-I but not by CT. Similar proteins were detected on Western blots probed with [125I]toxin. LT-I activity on intact cells and binding to membranes and the above galactoproteins were enhanced by neuraminidase treatment even in the presence of CT-B. beta-1,4-Galactosidase and endo-beta-1,4-galactosidase, but not beta-1,3-galactosidase, significantly reduced LT-I binding. LT-I binding to fetuin and transferrin exhibited a similar glycosidase sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adherence of pathogenic and non-pathogenic Entamoeba histolytica strains to neutrophils.

The adherence of polymorphonuclear leukocytes (PMNs) to eight pathogenic and nine nonpathogenic strains of Entamoeba histolytica was examined. No difference between pathogenic and nonpathogenic strains was found. The addition of different carbohydrates confirmed the importance of the 170-kDa lectin of E. histolytica in binding to PMNs, corroborated by the finding that treatment of PMNs with galactosidase inhibited adherence. Inhibition of the microfilament system of E. histolytica using cytochalasin B resulted in a loss of adherence to PMNs. Inhibition of the microtubule system using nocodazole did not affect adherence. Preincubation of the trophozoites with serum resulted in enhanced adherence, but the serum factor responsible for this effect could not be identified. Fibronectin, vitronectin, integrins (CD11/CD18 molecules), complement, and mannose-binding protein did not seem to mediate adherence between E. histolytica and PMNs. In summary, these results indicate that defective adherence mechanisms are not a common feature of nonpathogenic E. histolytica strains.

Mannose 6-phosphate-containing peptides activate phospholipase C in proximal tubular basolateral membranes from canine kidney.

To ascertain whether mannose 6-phosphate-containing peptides that bind to the insulin-like growth factor II (IGF II)/mannose 6-phosphate receptor activate phospholipase C, we determined the effect of proliferin, transforming growth factor-beta 1 (TGF-beta 1) precursor, and beta-galactosidase on production of inositol trisphosphate (Ins-P3) in basolateral membranes isolated from the renal proximal tubule of dogs. Both proliferin and TGF-beta 1 precursor stimulated Ins-P3 production in a concentration-dependent manner. Maximal production was stimulated by approximately 10(-13) M of each peptide. beta-Galactosidase had no effect on Ins-P3 generation. Neither proliferin nor TGF-beta 1 precursor potentiated IGF II-stimulated Ins-P3 production. Mannose 6-phosphate itself had no effect on Ins-P3 generation. However, mannose 6-phosphate potentiated production stimulated by 10(-11) M proliferin or 10(-11) M TGF-beta 1 precursor while inhibiting production stimulated by 10(-14) M of either peptide. Addition of anti-mannose 6-phosphate receptor antibodies to basolateral membranes abolished proliferin and TGF-beta 1 precursor-stimulated Ins-P3 generation. We conclude that, in addition to IGF II, mannose 6-phosphate-containing ligands for the IGF II/mannose 6-phosphate receptor activate basolateral membrane phospholipase C. Such activation could reflect a common mechanism for signal transduction by these peptides mediated via the IGF II/mannose 6-phosphate receptor.

Beta-galactosidase decreases the binding affinity of the insulin-like-growth-factor-II/mannose-6-phosphate receptor for insulin-like-growth-factor II.

The insulin-like growth-factor-II/mannose-6-phosphate (IGF-II/Man6P) receptor binds two classes of ligands, insulin-like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, beta-galactosidase, to modulate the binding of 125I-IGF-II to the receptor. beta-Galactosidase purified from bovine testis was fractionated on a DEAF-Sephacel ion-exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125I-IGF-II to the IGF-II/Man6P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture, exhibited greater potency in inhibiting the binding of 125I-IGF-II to the receptor. A pool of these fractions from the DEAE-Sephacel column inhibited 125I-IGF-II binding to pure receptor by 80% with the concentration required for half-maximal inhibition being 25 nM. The inhibition of binding by beta-galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzymatic activity of beta-galactosidase with D-galactonic acid gamma-lactone did not affect the ability of beta-galactosidase to inhibit the binding of 125I-IGF-II to the receptor. Scatchard analysis of IGF-II binding to pure receptor in the presence and absence of beta-galactosidase showed that beta-galactosidase decreased the binding affinity for IGF-II (Kd 0.26 nM versus 1.0 nM in the presence of 57 nM beta-galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 125I-IGF-II to the IGF-II/Man6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF-II/Man6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF-II-Sepharose column all exhibited stimulation of 125I-IGF-II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125I-IGF-II binding by Man6P. We conclude that beta-galactosidase decreases the binding affinity of the IGF-II/Man-6-P receptor for IGF-II by binding with high affinity to the Man6P-recognition site.

Effect of treatment with phospholipase A2, colchicine, and beta-galactosidase on agglutination of rat hepatocytes.

The effects of phospholipase A2, colchicine, and beta-galactosidase on concanavalin A-induced agglutination of viable hepatocytes isolated from normal and diabetic rats are reported. Phospholipase A2 (0.92 microgram/mL), colchicine (400 micrograms/mL), and beta-galactosidase (300 micrograms/mL) treatments caused a significant increase in the agglutination rate of hepatocytes. These findings suggest that phospholipase A2 treatment resulted in the unshielding of lectin receptors. Colchicine treatment seemed to release those receptors from cellular restraints which tend to separate and/or direct them. The promoting effect of beta-galactosidase could be attributed to a decrease in repulsive forces due to a reduction in net negative charge density after removal of N-acetylneuraminic acid residues. Normal rat hepatocytes seem to be richer in galactosides, phospholipids, and the microfilament-microtubule network than their diabetic counterparts.

Lactose digestion and clinical tolerance to milk, lactose-prehydrolyzed milk and enzyme-added milk: a study in undernourished continuously enteral-fed patients.

We investigated: (1) the capacity to digest and tolerate the lactose administered by continuous infusion of intact milk to undernourished tube-fed patients, and (2) the effectiveness of lactose-prehydrolyzed milk, and of the addition of exogenous lactase to milk at infusion time, to reduce lactose maldigestion and increase clinical tolerance. Carbohydrate digestion was evaluated in 10 subjects with the hydrogen breath analysis test during 8 hr of observation. Lactose intolerance was determined by evaluation of subject's symptoms. With the infusion of intact milk (IM), none of the subjects were able to efficiently digest the lactose infused (5.6 +/- 0.35 g/hr, mean +/- SEM) and 86% of them experienced major symptoms of intolerance. With the infusion of lactose-prehydrolyzed milk (HM) and enzyme-added milk (EM) there was a highly significant reduction in lactose maldigestion. More importantly, major symptoms were present in only 10% of subjects with EM, and were completely eliminated with HM. Lactose maldigestion and intolerance represent a major limitation for the application of milk-based polymeric formula for liquid diets in undernourished subjects. The use of exogenous beta-galactosidases represents an alternative to avoid such reactions.

The quantitative discrimination of corneal type I, but not skeletal type II, keratan sulfate in glycosaminoglycan mixtures by using a combination of dimethylmethylene blue and endo-beta-D-galactosidase digestion.

The quantitation of individual glycosaminoglycans in mixtures of polyanions using the dimethylmethylene blue (DMB) method described by R. W. Farndale, D. J. Buttle, and A. J. Barrett (1986, Biochim. Biophys. Acta 883, 173) is dependent on enzymatic hydrolysis by specific polysaccharidases. While using this method to examine the keratan sulfate (KS) of the intervertebral disc we found that digestion with commercially available keratanase decreased binding to DMB by less than 30%, whereas corneal KS was reduced by 85%. However, by preincubating the KS fractions with endo-beta-D-galactosidase prior to keratanase treatment the corneal KS could be completely digested and disc KS digestion increased to 60%. It is suggested that the resistance of the disc KS to these digestive procedures arises from branching and/or sites of multisulfation on the polysaccharide chain. Agarose gel electrophoresis and compositional analyses of the keratan sulfates supported such an interpretation.

Hemoglobin binding to deglycosylated haptoglobin.

The carbohydrate portion of polymeric haptoglobin was gradually removed by exoglycosidases in order to investigate its role in complex formation between haptoglobin and hemoglobin. Total removal of sialic acid diminished the haptoglobin-hemoglobin complex formation 15%. Removal of about 25% of the galactose residues from asialohaptoglobin, i.e., about 40% of the total weight of the carbohydrate moiety, totally inhibited the ability of haptoglobin to form complex with hemoglobin and react with haptoglobin-specific antibodies. Liberation of further galactose residues resulted in slow precipitation of the protein. Removal of a similar part of the carbohydrate moiety from haptoglobin-hemoglobin complex did not liberate hemoglobin from it, and the complex reacted with haptoglobin antibodies. The combined data indicate that the carbohydrate portion is essential for the functionally active form of polymeric haptoglobin to complex with hemoglobin, but it hardly has any direct role in the binding event, and other factors are responsible for the stability of the complex.

Effect of various conditions on the formation of oligosaccharides in milk treated with beta-galactosidase.

Fresh pasteurized milk was inoculated with beta-galactosidase from two different microbial sources at three levels and analyzed for oligosaccharides by high performance liquid chromatography. Five oligosaccharides were observed in the course of hydrolysis. However, only two of these showed measurable increases in concentration. Rate of oligosaccharide formation during hydrolysis was influenced by source and amount of enzymes, reaction time, and temperature. Maximum concentrations of the two oligosaccharides occurred between .5 and 3 d at 7 degrees C and between 1 and 3 d at 1 degrees C, depending on the level of the enzyme used. The higher level of enzyme formed a higher concentration of oligosaccharides than the low level, except at 37 degrees C. After maximum concentration was reached, the two oligosaccharides decreased following hydrolysis, but rate of decrease was dependent upon time, temperature, and enzyme concentration. Hydrolysis of the oligosaccharide was faster at 7 degrees C and at the higher enzyme concentration. Maximum concentration of oligosaccharides was .374 g/100 g milk but was negligible after 7 d, except when the low level of enzyme was used. The total concentration of the oligosaccharides formed in enzyme-treated milk was insignificant.

Diverse effects of the MalE-LacZ hybrid protein on Escherichia coli cell physiology.

The hybrid protein between the periplasmic maltose-binding protein and the cytoplasmic beta-galactosidase (the MalE-LacZ hybrid protein) was previously shown to block the export of envelope proteins when synthesized in large amounts. Now we show that the hybrid protein exerts another major effect on the cell, that is, induction of the heat shock proteins. This latter effect was dependent on the htpR gene product but independent of the function of the signal sequence on the hybrid protein. On the other hand, the previously reported induction of the SecA protein by the hybrid protein was independent of htpR and may be caused by the reduced protein export ability of the cell. The functional htpR gene is essential for viability of the cell in which the basal level of the hybrid protein is synthesized, whereas in the absence of the hybrid protein htpR is dispensable at low temperature. These results indicate that the hybrid protein somehow generates a signal or stress that is similar to what the cell experiences at elevated temperatures.

Effects of enzymes and protein modifying reagents on the binding of 3H-prostaglandin E2 to porcine oxyntic mucosa in vitro.

In the present study we have investigated the macromolecular nature of porcine oxyntic mucosal PGE2 binding sites and the involvement of specific functional groups in the binding interaction. Incubation of oxyntic mucosal membranes with DNAse or RNAse did not influence binding. Phospholipase A2 was strongly inhibitory while phospholipases C and D exerted variable effects. Trypsinization of the membranes also reduced binding and this reduction was prevented by addition of soybean trypsin inhibitor. Neuraminidase and beta-galactosidase treatments resulted in variable increases in binding activity. The increase in binding was due to an increase in binding affinity and/or binding site concentration. Protein modifying reagents acetic anhydride, N-ethylmaleimide and mercaptoethanol all reduced binding. These results suggest the importance of protein, lipid and carbohydrate components of the membrane in the binding interaction between PGE2 and its binding site. The ability of mercaptoethanol and N-ethylmaleimide to reduce binding suggest the involvement of both sulphydryl and disulphide groups in the PGE2 binding reaction.

Pathogenesis of shigella diarrhea: evidence for an N-linked glycoprotein shigella toxin receptor and receptor modulation by beta-galactosidase.

Pathogenic mechanisms in infectious diseases often involve specific receptor-ligand interactions of cells and soluble molecules. To further elucidate structure-function relations for shigella toxin receptors, we studied binding of purified 125I-labeled toxin and biologic response under various conditions in an experimental model using HeLa cells. Response to toxin was reversibly inhibited by treatment of cells with trypsin or tunicamycin, an inhibitor of glycoprotein synthesis that also significantly inhibited toxin binding, a result indicating that the receptor is an N-linked glycoprotein. Removal of terminal beta-linked galactose from the HeLa cell surface with beta-galactosidase increased toxin binding and activity, and it also potentiated the effects of lysozyme and wheat-germ agglutinin, which recognize oligomeric beta 1----4-linked N-acetyl-D-glucosamine and inhibit toxin activity as well. Incubation of cells with beta-N-acetylglucosaminidase, which cleaves terminal beta-linked N-acetyl-D-glucosamine, inhibited toxin activity. Effects of beta-galactosidase were reversed by readdition of galactose to cell-surface oligosaccharide acceptors. The data demonstrate that alterations of a single sugar on cell-surface glycoproteins may have a dramatic effect on receptor activity and indicate that shigella toxin is a sugar-binding protein with specificity for beta 1----4-linked N-acetyl-D-glucosamine.

Preferential lectin binding to specific layers of rat oral epithelium and modification by enzyme pretreatment.

Three fluorescein-labeled lectins were shown to bind differentially to cell surfaces in different epithelial layers of rat oral mucosa regardless of the age or the site of origin of the tissue. Griffonia simplicifolia (GS-1-B4), specific for alpha-D-galactosyl end groups, labeled basal cells only; Ulex europeus (Ulex 1) specific for alpha-L-fucosyl groups labeled spinous cells; and Bandeiraea simplicifolia (BSII), specific for N-acetyl-D-glucosamine, labeled cornified cells. Pretreatment of sections with alpha-galactosidase completely abolished the staining of basal cells by GS-1-B4, but had no effect on the staining of spinous cells by Ulex 1. In contrast, alpha-fucosidase abolished the staining of spinous cells by Ulex 1 and caused staining of both basal and spinous cells by GS-1-B4. Neuraminidase and chondroitinase ABC produced results similar to one another, with staining of basal cells by GS-1-B4 and labeling of both basal and spinous cells with Ulex 1. beta-galactosidase, beta-glucuronidase, and testicular hyaluronidase did not affect the staining pattern of GS-1-B4 or Ulex 1, whereas chymotrypsin completely abolished any staining with either lectin. The results demonstrate a complex arrangement of cell surface carbohydrates in the epithelium of rat oral mucosa. The findings indicate a possible simplification in the spatial arrangements of cell surface carbohydrates during the differentiation of basal to spinous cells.

Action of beta-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions.

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.