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.

An assay for intercellular adhesive specificity.

A modification of an assay for intercellular adhesive specificity is described. The method involves the collection of radioactively labeled cells by aggregates of the same (isotypic aggregates) or different (heterotypic aggregates) types of tissue and determination of the number of cells collected by liquid scintillation counting. The use of (32)P to label the tissues permitted a much more rapid estimation of cell collection than was obtained previously. With the use of chick embryo neural retina, liver, forebrain, pectoral muscle, and heart ventricle tissue, it was shown that isotypic was always greater than heterotypic collection. Labeled neural retina cell collection by neural retina aggregates was studied as a function of time, cell suspension density, aggregate diameter, temperature, and aggregate number. Neural retina aggregates were treated with certain enzymes in an attempt to determine whether specific changes on the surface of the aggregates would interfere with labeled neural retina cell collection. Of the various proteases and glycosidases tested, only beta-galactosidase rendered the surface more nonspecific.

Group B erythrocytes enzymatically converted to group O survive normally in A, B, and O individuals.

With an alpha-galactosidase, B erythrocytes can be converted to blood group O under conditions that neither impair their viability in vitro nor affect their ability to survive normally after transfusion to individuals of groups O, A, and B. Such an approach has the potential for producing enzymatically converted group O cells for use in transfusion therapy. It should also be possible to convert A cells to group O by using the appropriate alpha-N-acetylgalactosaminidase.

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.

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.

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.

Milk intolerance in Manitoba Indian school children.

The breath H2 test was used to determine the prevalence of milk intolerance in Indian schoolchildren, 5 to 19 years old. One hundred four children were randomly assigned to one of six groups: 1) reconstituted skimmed milk powder (SMP); 2) SMP and sandwich; 3) SMP and lactase; 4) diluted (1:1) evaporated whole milk (EM); 5) EM and sandwich; and 6) EM and lactase. Samples of expired air were collected 2 hr after ingestion of 245 ml of milk. Five children had H2 levels suggestive of appreciable lactose malabsorption; two had symptoms. The mean H2 excretion levels of the subjects on SMP were significantly higher than those on EM. The group on SMP alone excreted significantly more H2 than any other group except those receiving SMP and a sandwich. The relatively low prevalence of milk intolerance in this populatin group should not preclude continuation and promotion of milk programs in their schools. However, preference should be given to the use of EM rather than SMP. The needs of children who cannot hydrolyze lactose must also be considered in the planning of food supplement programs.

Effects of dietary lactose and lactase preparation on the intestinal absorption of calcium and magnesium in normal infants.

Effects of dietary lactose and a lactase preparation on the intestinal absorption of calcium and magnesium were studied in 3 groups under 8 months of age; infants on a proprietary milk, infants on a lactose-free milk, and infants on a proprietary milk to which a lactase preparation was added. The intestinal absorption of both elements was reduced in infants on a lactose-free milk and enhanced in infants who were fed on a proprietary milk and given a lactase preparation.

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.

Porcine rotaviral infection of cell culture: effects of certain enzymes.

Porcine rotaviral infectivity for continuous porcine kidney (PK-15) cells was enhanced by incorporation of pancreatic endopeptidases into the cell culture maintenance medium. Marked enhancement of infectivity was induced by trypsin, whereas elestase and alpha-chymotrypsin enhanced infectivity to a lesser extent. Bacterial protease also induced some enhancement of porcine rotaviral infectivity. A synergistic enhancement of porcine rotaviral infectivity was noticed with trypsin and alpha-chymotrypsin combined. Porcine rotaviral infectivity was not affected by incorporation of alpha-amylase, alkaline phosphatase, beta-galactosidase, carboxypeptidase-A, deoxyribonuclease, enterokinase, lipase, or ribonuclease into the maintenance medium.

[Production and properties of a beta-galactosidase preparation from Alternaria tenuis]. / Poluchenie i svoistva preparata beta-galaktozidazy iz Alternaria tenuis

Different methods of the preparation of fungal beta-galactosidase from the 72-hour culture of Alternaria tenuis were tested: lyophilization of the culture liquid, precipitation with ethanol, acetone, ammonium sulphate. Optimal results were obtained with precipitation by 1.5 acetone volume. Studies of the properties of fungal beta-galactosidase demonstrated that the preparation retained its activity during 22 month storage at 5 degrees C. The fungal preparation had pH optimum at a more acidic zone (4.2 versus 6.9), was active in a wider pH range 2.8-5.7 and 6.2-7.5), had a much higher temperature optimum (65 degrees and 30 degrees) and better thermostability as compared with the yeast preparation. Data on other properties of the preparation are presented.

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.