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WO1994018986A1 - Utilisation de glycosides oligosaccharidiques comme inhibiteurs de l'adherence bacterienne - Google Patents

Utilisation de glycosides oligosaccharidiques comme inhibiteurs de l'adherence bacterienne Download PDF

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Publication number
WO1994018986A1
WO1994018986A1 PCT/IB1994/000023 IB9400023W WO9418986A1 WO 1994018986 A1 WO1994018986 A1 WO 1994018986A1 IB 9400023 W IB9400023 W IB 9400023W WO 9418986 A1 WO9418986 A1 WO 9418986A1
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Prior art keywords
cells
bacterial cells
human
section
glycoside
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PCT/IB1994/000023
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English (en)
Inventor
Jan Staffan Normark
Per Falk
Thomas Boren
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Jan Staffan Normark
Per Falk
Thomas Boren
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Priority claimed from DK22293A external-priority patent/DK22293D0/da
Priority claimed from DK76093A external-priority patent/DK76093D0/da
Application filed by Jan Staffan Normark, Per Falk, Thomas Boren filed Critical Jan Staffan Normark
Priority to JP6518792A priority Critical patent/JPH08509467A/ja
Priority to AU60425/94A priority patent/AU6042594A/en
Priority to EP94906981A priority patent/EP0690717A1/fr
Publication of WO1994018986A1 publication Critical patent/WO1994018986A1/fr
Priority to NO953281A priority patent/NO953281D0/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the use of di- or oligo- saccharide glycosides containing at least one terminal L- fucose unit for the preparation of pharmaceutical composi ⁇ tions for the treatment or prophylaxis in humans of condi ⁇ tions involving infection by Helicobacter pylori in the human gastric mucosa, as well as a method of treating such condi- tions using the di- or oligosaccharide glycosides.
  • Helicobacter pylori is a microaerophilic spiral shaped orga ⁇ nism (originally assigned to the genus CampyloJbacter) which is found in the stomach and generally appears to have an exclusive habitat in the human gastrointestinal mucosa. It has been estimated that this bacterium infects the gastric mucosa of more than 60% of adult humans by the time they are 60 years old. Moreover, Helicob ⁇ cter pylori has been impli ⁇ cated as a contributing factor in a number of pathological conditions, including acute (type B) gastritis, gastric and duodenal ulcers, and gastric adenocarcinoma.
  • Tissue tropism of bacteria is partly governed by the ability of a bacterial strain to adjust to the local chemical envi ⁇ ronment in its specific habitat.
  • adherence is a necessary prerequisite for colonization in order to prevent removal from the new habitat, e.g. through peristalsis in the gastrointestinal tract.
  • bacteria adhere to pro- teins or glycoconjugates (glycosphingolipids, glycoproteins) on or in the vicinity of epithelial cell surfaces (mucus) , and a number of specific bacterial adhesin-protein and adhe- sin-carbohydrate interactions have been described in the literature.
  • Helicobacter pylori adheres or binds only to certain differentiated epithelial cells in the glandular epithelium which is composed of gas- trie units.
  • the gastric units in the glandular epithelium are lined with cells that have differentiated to perform various functions.
  • the upper pit region of the gastric unit (gastric pit) is lined with mucus-producing surface epithe ⁇ lial cells
  • the constricted mid-portion of the gas- trie unit (its isthmus) is composed of proliferating and non- proliferating immature cells, mucus neck cells and parietal cells
  • the lower portion of the gastric unit (the gland) may contain intrinsic factor- and pepsinogen-producing chief cells, acid-producing parietal cells, mucous neck and gland cells, and a variety of enteroendocrine cell types.
  • Helicobacter pylori binds solely to the mucus-producing surface epithelial cells in the upper pit region, i.e. the surface mucus cells.
  • the studies have shown that the binding of Heli - coJacter pylori to the mucus-producing surface epithelial cells does not involve interaction between bacterial adhesins and sialic acid-containing receptors but rather involves interaction with carbohydrate structures containing terminal units of L-fucose (also known as 6-deoxy-L-galactose) . Consequently, the present invention concerns in one aspect the use of a di- or oligosaccharide glycoside having at least one terminal L-fucose unit for the preparation of a pharma ⁇ ceutical composition for the treatment or prophylaxis in humans of conditions involving infection by Helicobacter pylori of the human gastric mucosa.
  • the invention concerns a method of treating and/or preventing diseases in humans caused by infection by Helicobacter pylori by admini ⁇ stering to a patient in need thereof an effective amount of a di- or oligosaccharide glycoside comprising at least one terminal L-fucose unit.
  • terminal L-fucose unit is intended to mean that the L-fucose unit or units in question, which is/are bound via the 1-position, is/are not itself/themselves glycosylated by other carbohydrate units or groups. This does not exclude, however, that a carbohydrate unit which is glycosylated by a L-fucose unit is itself glycosylated by other carbohydrate units or groups.
  • oligosaccharide is intended to mean that the saccharide portion of the glycoside comprises three or more carbohydrate units, such as between three and ten carbohydrate units.
  • the di- or oligosaccharide glycoside having at least one terminal L-fucose unit used according to the invention is capable of binding to adhesins present on the surface of Helicobacter pylori .
  • the most useful model system for de ⁇ termining the ability of the di- or oligosaccharide glycoside having at least one terminal L-fucose unit to inhibit the binding of cells of Helicobacter pylori to the gastric epithelium is an in vitro histological model involving the use of histological sections of human gastric tissue con ⁇ taining gastric epithelium.
  • a preferred embodiment of the use or the method of the invention is that in which the di- or oligosaccharide glycoside having a terminal L-fucose unit is one which is capable of inhibiting or substantially reducing the adhesion of cells of Helicobacter pylori to epithelial cells in a histological section of human gastric mucosa.
  • the di- or oligosaccharide glycoside employed in the use or the method of the invention is one which, when cells of Helicobacter pylori are preincubated with the di- or oligosaccharide glycoside in a concentration of up to 500 ⁇ g/ml, is capable of inhibiting the adhesion of said bacterial cells to epithelial cells of a histological section of gastric human mucosa by at least 50% compared to the adhesion of corresponding non-preincubated bacterial cells.
  • the histological section of human gastric mucosa is prepared by fixing a sample of non-diseased gastric human mucosa tissue with formalin, embedding the sample in paraffin, providing an approximately 5 ⁇ m section of the embedded sample, placing the section on a glass slide, deparaffinizing the section by washing with xylene and isopropanol, and incubating the section with a buffer consisting of bovine serum albumin and a non-ionic polyoxyethylene sorbitan monolaurate surfactant (such as Tween-20) , preferably at a concentration of about 0.2% and 0.05%, respectively, in phosphate-buffered saline, cf. also the example.
  • a buffer consisting of bovine serum albumin and a non-ionic polyoxyethylene sorbitan monolaurate surfactant (such as Tween-20) , preferably at a concentration of about 0.2% and 0.05%, respectively, in phosphate-buffered saline, c
  • the bacterial cells are labelled in some manner to enable detection of the bacterial cells at specific sites on the section.
  • the labelling may conceivably be performed according to any of the well-known methods for labelling live bacterial cells or bacterial cells located on a microscopic sample, such as staining by means of a dye (such as a dye specific to e.g.
  • the presently preferred labelling principle is fluorescence-labelling, in particular labelling by means of fluorescein isothiocyanate.
  • This may advantageously be carried out by treating a suspension of cells of Helicobacter pylori in buffer containing sodium chloride and sodium car ⁇ bonate, preferably at a concentration of about 0.15 M and 0.1 M, respectively, at about pH 9.0 with fluorescein isothiocya- nate at a concentration of about 0.1 mg/ml, incubating the bacterial cell suspension for 1 hour at room temperature and separating the bacterial cells by centrifugation followed by washing of the bacterial cells with phosphate buffered saline at about pH 7.5 containing a non-ionic polyoxyethylene sorbitan monolaurate surfactant, preferably at a concentra ⁇ tion of about 0.05%.
  • the bacteria are typically subjected to preincubation with the glycoside prior to bring ⁇ ing the bacteria into contact with the tissue section to allow them to adhere to the surface of epithelial cells.
  • One useful way of performing the preincubation of the bacterial cells is by adding the di- or oligosaccharide glycoside in a concentration of up to 500 ⁇ g/ml to a suspension of the bacterial cells in a buffer consisting of bovine serum albu ⁇ min and of a non-ionic polyoxyethylene sorbitan monolaurate surfactant, preferably at a concentration of about 0.2% and 0.05%, respectively, in phosphate-buffered saline at about pH 7.5 for a period of about 2 hours at room temperature, sepa ⁇ rating the bacterial cells by centrifugation, and washing the cells in the same buffer.
  • the adhesion testing may be carried out using cells of the Helicobacter pylori strains NCTC 11637, NCTC 11638, WV229 or P466 (cf. the example below).
  • the conditions involving gastrointestinal infection by Heli - coJbacter pylori comprise for example chronic active (type B) gastritis, gastric ulcers, duodenal ulcers, gastric adenocarcinoma, and gastric lymphoma.
  • the oligosaccharide contains two terminal fucose units as defined above.
  • the di- or oligosaccharide glycoside to be employed is a glycoprotein.
  • interesting examples of compounds to be employed are human K -casein, human colostrum IgA, and bovine submaxillary gland mucin.
  • the terminal tetra- saccharide at the non-reducing end of the oligosaccharide chain is Lewis b-tetrasaccharide, i.e. Fuc ⁇ l-2Gal3l-3 (FuCQU-4)GlcNACj8l- .
  • the di- or oligosaccharide glycosides may be contained in any appropriate amount in a pharmaceutical composition, and are generally contained in an amount between 0.01% and 99%, or between 0.1% and 99% by weight of the total weight of the composition.
  • compositions containing the active in ⁇ gredients may suitably be formulated so that they provide doses within these ranges either as single dosage units or as multiple dosage units.
  • the active di- or oligosaccharide glycosides used according to the invention as well as compositions containing the glycosides may typically be administered to a human patient in amounts corresponding to from about l mg per day to about 50 g per day, preferably from about 10 mg per day to about 5 g per day, of the active glycoside ingredient.
  • the dosage of a di- or oligosaccharide glycoside depends in general on the choice of administration route, the particular disease to be treated and the severity of same, and also whether the disease is to be treated or prevented, as well as the age and weight of the person to be treated.
  • the pharmaceutical composition may preferably be administered by the oral route, by the parenteral route or by the rectal route.
  • Human milk was kept frozen at -20°C until used. The milk was thawed and skimmed milk obtained by centrifugation at 15,000 x g for 45 min. The fat layer at the top of the tube was carefully removed while the soluble fraction and the pellet were pooled. The pool was acidified with HC1 to pH 4.3 and CaCl 2 was added to a concentration of 60 mM. Following incu ⁇ bation at room temperature for l h, the precipitated casein fraction was collected by centrifugation at 18,000 x g for 90 min.
  • the casein fraction was dissolved in 20 mM ethanolamine, 6 M urea, at pH 9.5. To remove any remaining fat, this aqueous fraction was extracted three times with 4 volumes of hexane. The aqueous phase was thereafter dialysed against distilled water and lyophilized.
  • the lyophilized proteins were dis ⁇ solved in 50 mM imidazole-HCl, 0.5% SDS, 0.5% 2-mercaptoetha- nol at pH 7.0 and chromatographed on a Sephadex G-200 column (1.6 x 120 cm, Pharmacia-LKB, Uppsala, Sweden), equilibrated with 50 mM imidazole-HCl, 0.5% SDS, 0.5% 2-mercaptoethanol at pH 7.0 at a temperature of 37°C.
  • K -Casein-containing frac ⁇ tions which were detected by Schiff staining were pooled, dialysed against 40% methanol in distilled water for 3 h and lyophilized to reduce the volume.
  • the protein was redissolved in 50 mM imidazole-HCl, 0.5% SDS, 0.5% 2-mercaptoethanol at pH 7.0 and a second time on the Sephadex G-200 column under the same conditions as above. Human K -casein-containing fractions completely free of ⁇ -casein were collected, dialysed against 40% methanol and lyophilized.
  • Bovine mature milk and colostrum collected within 24 h of parturition were obtained from Agrisera AB (Tvar-iback, Sweden) . Both were defatted and the respective casein frac- tion was prepared as described above for the purification of human milk with the exception that no delipidation with hexane was necessary.
  • the casein pellet was dissolved in 10 mM imidazole-HCl, 3.3 M urea, 10 mM 2-mercaptoethanol, at pH 7.0 and applied to a Q-Sepharose column (2.6 x 15 cm, Pharma- cia LKB, Sweden) . After washing, the column was eluted with a linear gradient of 0.1-0.5 M NaCl in the same buffer.
  • Bovine K -casein-containing fractions were dialysed against water and lyophilized. The identification of bovine caseins was con ⁇ firmed by chymosin cleavage.
  • tissue samples of adult human esopha ⁇ gus, stomach, duodenum, colon, kidney, cervix, endometrium and midbrain were obtained from the surgical pathology and autopsy files of the Department of pathology at Washington University.
  • Dawley rats, and 6-12 week old FVB/N mice were removed after sacrifice by cervical dislocation and regionally dissected as described in Sweetser, D. A., Birkenmeier, E. H. , Hoppe, P. C, McKeel, D. W. , and Gordon, J. I., Genes Devel . 2 (1988), 1318-1332.
  • Stomachs from adult dogs were obtained from the Department of Surgery, St. Louis University Medical Center. All tissues were fixed in 10% formalin or in a solution of picric acid/formaldehyde/glacial acetic acid (15:5:1; Bouin's fluid) and subsequently embedded in paraffin (see Sweetser, D. A., Birkenmeier, E. H. , Hoppe, P. C.
  • Sections with a thickness of 5 ⁇ m placed on glass slides were prepared and used for hematoxylin and eosin staining (to identify the cell types present in gastric units, and to verify that the tissue samples had no pathologic changes) and/or for sub ⁇ sequent adherence, histochemical and/or immunocytochemical assays.
  • NCTC 11637 and NCTC 11638 are reference strains isolated in 1982 from patients with active chronic gastritis (see Warren, J. R. , and Marshall, B., Lancet 1 (1983), 1273- 1275) .
  • Strain WV229 was recovered from a patient with gastric ulcer (see Westblom, T. U. , Madan, E., Subik, M. A., Buriex, D. E., and Midkiff, B. R. , Scand. J. Gastroenterol . 27
  • pylori were grown at 37°C on Brucella agar supplemented with 10% bovine blood and 1% IsoVitalex (Becton Dickinson Microbiology Systems, Cockeyville, MD) under microaerophilic conditions (5% 0 2 , 10% C0 2 , 85% N 2 ) and 98% humidity.
  • Five days after inoculation about 1 ⁇ l of bacteria were removed with a sterile loop from a plate and were resuspended by gentle pipetting in 1 ml of 0.15 M NaCl/0.1 M sodium carbonate with a pH of 9.0.
  • the intensity of FITC- labelling of all bacterial strains was similar as judged by inspection of comparable numbers of organisms by fluorescence microscopy. Aliqouts of 100 ⁇ l were taken from the final suspensions with an optial density of 1.00 O.D./ml, measured by A 600n ⁇ n , and utilized immediately or stored at -20°C until use. No differences in binding patterns were observed between bacteria labelled and used fresh and bacteria that were frozen and thawed once before use.
  • Human K -casein and human colostrum secretory IgA were also incubated with 100 mU of bovine kidney ⁇ !-L-fucosi- dase or Vibrio colerae neuraminidase (Boehringer-Mannheim, Germany) for 2 h at 37°C prior to incubation with bacteria.
  • K -Casein incubated in phosphate buffered saline at pH 7.4 served as control.
  • Glycosidase treated samples and the con ⁇ trol were all incubated at 80°C for 20 min to inactivate the enzymes before the K -casein was incubated with bacteria.
  • IgA incubated in PBS served as control.
  • Glycosidase treated samples and the control were all incubated at 80°C for 20 min to inactivate the enzymes before the IgA was incubated with bacteria.
  • DIG-conjugated lectins were detected with peroxidase- conjugated monoclonal mouse anti-DIG antibody (500 mU per- oxidase/ml, dissolved in blocking buffer 1) by washing the tissue three times in PBS, overlaying the secondary antibody on the tissue for 1 h at room temperature, followed by stan- dard H 2 0 2 /DAB detection.
  • Fucosylated blood group antigens were detected by first treating the sections with mouse monoclonal antibodies directed against the histo-blood group antigens A, B, and H (Dakopatts A/S, Glostrup, Denmark) (final concentration of 1 ⁇ g protein/ml, dissolved in blocking buffer 1), or Le a and Le b (Immucor, Norcross, GA) (final concentrations 10 ⁇ g protein/ml, dissolved in blocking buffer 1) .
  • Antigen-antibody complexes were detected by visualizing them with a second overlay of FITC-, TRITC- or HRP-conjugated (fluorescein-, rhodamine- or peroxidase-conjugated) rabbit anti-mouse immu- noglobulins (Dakopatts A/S, Glostrup, Denmark) (final concen- tration 30 ⁇ g protein/ml, dissolved in blocking buffer 1) and studying the sections under a fluorescence microscope.
  • Strains WV229 and P466 did not adhere to the squamous epithe ⁇ lium of human esophagus but did adhere under the reaction conditions employed to esophageal submucosal glands and their ducts, and to duodenal villus-associated enterocytes (see Figure 2D) .
  • a weak binding to enterocytes situated in the colonic homolog of small intestinal villi - the surface epi ⁇ thelial cell cuff which surrounds each crypt orifice was also observed.
  • strains WV229, P466 and M019 grown and labelled with FITC as described above, were incubated with sections of rat and mouse gastrointestinal tracts as well as with stomach from dogs. Strain M019 did not bind in detectable amount to tissue sections prepared from any of these three species. As in the human stomach, strains WV229 and P466 bound to the gastric pit region in the rat but not to any other differentiated epithelial cell population located in the zymogenic or pure mucous zones (see Figure 2F) .
  • the stratified squamous epithelium of the rat esophagus and forestomach produced positive results in the in si tu adherence assay, while the small intestinal epithelium was negative with the exception of Brunner's glands.
  • the forestomach of the mouse showed high-density binding with adherent bacteria, while the remainder of the stomach, i.e. the zymogenic, mucoparietal and pure mucous zones (see Lee, E. R., Trasler, J., Dwivedi, S., and leBlond, C. P., Am. J. Anat.
  • bovine submaxillary gland mucin which is a rich source of both fucosylated and sialylated carbohydrates (see Savage, A. V. , D'Arcy, S. M. T. , and Donoghue, C. M. , Biochem. J. 279 (1991), 95-103 and 33), completely inhibited binding of bacteria to surface mucous cells (see Figure 1A,B) and to duodenal, villus-associated, epithelial cells.
  • Human ⁇ -casein and human colostrum secretory IgA were also potent inhibitors: 250 ⁇ g/ml of human K -casein (see Figure 3A) and 15 ⁇ g/ml of human colostrum secretory IgA, respectively, fully inhibited adherence of Helicobacter pylori .
  • Human K -casein oligosaccharides are fucosylated via an Q!l-»4- linkage to N-acetylglucosamine and differs in this respect from bovine K -casein.
  • Human colostrum secretory IgA carries a highly varied set of N- and O-linked oligosaccharides. Thus, it is therefore interesting that only the human - - casein and human colostrum secretory IgA were able to prevent the cell-lineage specific attachment of Helicobacter pylori to human gastric surface mucous cells.
  • K -casein from bovine mature milk and colostrum did not inhibit binding of Helicobacter pylori at concentrations as high as 1000 ⁇ g/ml, and human serum IgA did not inhibit binding at concentrations as high as 100 ⁇ g/ml.
  • Pretreatment of sections of human stomach with proteinase K also produced a marked decrease in the binding of the two Helicobacter pylori strains P466 and WV229.
  • Maackia amurensis agglutinin which is specific for NeuAc ⁇ .2 ⁇ 3Galj3l ⁇ 4GlcNAc epitopes (on sialylated N/O-glycans and glycosphingolipids) (see Knibbs, R. N., Goldstein, I. J., Ratcliffe, R. M. , and Shi ⁇ buya, N., J. Biol . Chem. 266 (1991), 83-88), and Sambucus nigra agglutinin which recognizes NeuAc ⁇ .2 ⁇ 6Gal/GalNAc struc ⁇ tures (see Shibuya, N.
  • MAA Maackia amurensis agglutinin
  • the cholera toxin B subunit recognizes internally linked sialic acid in glycoproteins and glycosphingolipids, specifically GalNAc ⁇ l ⁇ 4 (NeuAc ⁇ 2 ⁇ 3)Gal- ⁇ -structures (see
  • the "mild" meta-periodate oxidation reaction conditions employed above had no effect on UEAl binding (see Figure 1J) .
  • This treatment also had no effect on Helicobacter pylori binding (see Figure 1K,L) .
  • Harsher cleavage conditions i.e. reducing the pH to 4.5, increasing the incubation time to 1 h; and raising the incubation temperature to 20°C) were required to ablate UEAl binding and the binding of the monoclonal blood group H antibody to human surface mucous cells. These conditions also resulted in loss of cholera toxin B subunit binding and loss of adherence of Helicobacter pylori strains WV229 and P466.
  • Figure 1 shows the characterization of a putative Helico ⁇ bacter pylori adhesin receptor as a glycoprotein that lacks sialyl-lactose.
  • Fig. IA and IB Sections of human stomach containing gastric units with zymogenic glands were incubated with FITC-labelled Helicobacter pylori strain WV229 (IA) or with bacteria that had been treated with a 0.5% solution of bovine submaxillary gland mucin (IB) . The mucin prepa ⁇ ration produces marked reductions in binding.
  • Fig. 1C and ID Section of human stomach containing members of the surface mucous, mucous neck, parietal and chief epithelial cell lineages were incubated with digoxigenin-labelled Maackia amurensis agglutinin (MAA, 1C) or Sambucus nigra agglutinin (SNA, ID) . DIG-conju ⁇ gated lectins were detected with a peroxidase-conjugated monoclonal mouse anti-DIG antibody preparation. None of the epithelial cell lineages produces a signal above background when incubated with these lectins that recog ⁇ nize sialic acid-containing carbohydrate epitopes. Con ⁇ trol experiments employing the monoclonal antibody alone produced no staining (data not shown) .
  • Fig. IE Adjacent section of human stomach incubated with rhodamine-conjugated cholera toxin B subunit. Mucus- producing cells located in the isthmus or upper portion of the glandular domain of gastric units react with this toxin which recognizes internally linked sialic acids in glycoproteins and glycosphingolipids. The subunit does not bind to surface mucous cells located in the pit.
  • Fig. 1F-1H Sections of human stomach were incubated with FITC-labelled Helicobacter pylori strain P466 (IF) and a mouse monoclonal antibody directed against the fucosy- lated blood group antigen H (visualized with rhodamine- conjugated rabbit anti-mouse IgG in Fig. 1G) .
  • Fig. 1H is a double exposure showing that surface mucous cells co- express the bacterial adhesin receptor and the blood group antigen.
  • IF monoclonal antibody
  • Similar reductions in binding were obtained using mouse monoclonal antibodies directed against fucosylated blood group antigens B and Le b (data not shown) .
  • Non-immune mouse IgG failed to produce this effect (data not shown) .
  • Fig. II A section of human stomach was incubated with FITC-conjugated ⁇ lex europaeus agglutinin (UEAl) which recognized ⁇ -L-fucose. The lectin binds to surface mucous cells.
  • Fig. 1J Pretreatment of a section of human stomach with sodium meta-periodate, pH 5.5 at 0°C for 10 min produces no appreciable reduction in binding of UEAl.
  • Fig. IK and IL The binding of Helicobacter pylori to pit mucous cells (IK) was also unaffected by the sodium meta- periodate pretreatment (IL) .
  • Figure 2 shows the in situ assay for binding of Helicobacter pylori to gut epithelial cell lineages.
  • Fig. 2A Section of human stomach stained with hematox- ylin and eosin showing the uppermost portion of gastric units in the zymogenic zone. Surface mucous (M) and parietal (P) cells are indicated.
  • Fig. 2B and 2C Sections of stomach of an adult human were incubated with FITC-labelled Helicobacter pylori strains WV229 (2B) and M019 (2C) after the bacteria had been grown on blood agar for 5 days under microaerophilic conditions.
  • Strain WV229 which was recovered from a patient with gastric ulcer disease, is associated with surface mucous cells located in the upper pit of gastric unit and their associated luminal surfaces.
  • Strain M019 which was isolated from a healthy carrier, is not bound to any cell lineage.
  • Fig. 2D and 2E Incubation of strain WV229 with section of duodenum (2D) and colon (2E) from an adult human.
  • the FITC-labelled bacterial preparation used for staining sections of stomach also stains villus-associated epi ⁇ thelial cells (including enterocytes) and very weakly colonocytes located in the upper portion of colonic crypts and their surface epithelial cuffs. Less differen- tiated, proliferating and non-proliferating, cells located in small and large intestinal crypts did not contain adherent bacteria. Cell populations located in the mesenchymal compartment were also negative.
  • Incuba- tion of section of human stomach, intestine and colon with strains NCTC 11637, NCTC 11638 and P466 produced results comparable to those shown in Fig. 2B, 2D and 2E (data not shown) .
  • Fig. 2F A section from the zymogenic zone of stomach from an adult Sprague-Dawley rat was incubated with FITC- labelled strain WV299 grown for 5 days on blood agar under microaerophilic conditions.
  • Helicobacter pylori associated with rat surface mucous cells but not with cell lineages associated with the isthmus or glandular domains of rat gastric units.
  • Figure 3 shows the evaluation of the inhibitory activity of various K -caseins on the attachment of Helicobacter pylori to the human gastric mucosa in situ.
  • Fig. 3A Helicobacter pylori strain P466 preincubated with human K -casein (250 ⁇ g/ml) prior to overlay on section of human stomach.
  • Fig. 3B Helicobacter pylori strain P466 preincubated with bovine colostrum K -casein (1000 ⁇ g/ml) prior to overlay on section of human stomach.
  • Fig. 3C Pretreatment of human ⁇ -casein with oj-L-fuco- sidase prior to overlay on section of human stomach. Overlays were done on adjacent sections from the same paraffin embedded tissue.
  • Example 1 The labelling of the bacteria was performed as described in Example 1 and the bacterial strains used were the H. pylori strain P466.
  • Proteins which were separated on SDS-PAGEs were transferred by a semidry Multiphore Nova Blot system onto nitrocellulose for 1.5 hour. Proteins were stained with Ponceau S solution
  • BB2 blocking buffer 2
  • TBS Tris buffered saline
  • Blocking reagent Boehringer Mann ⁇ heim, Indianapolis, IN.
  • ImM MnCl 2 ImM MgCl 2
  • ImM CaCl 2 0.05% polyoxyethylene sorbitan monolaurate
  • Anti-Le a CBM-LAl
  • anti-Le b CBM-LBl
  • anti-Le x 672/7E3
  • anti-Le b ' y 64/4D8
  • anti-H-2 92FR A2
  • anti-type 1 chain LNT
  • lectins were used: Biotin-labelled ⁇ lex europaeus type 1, Lotus tetragronoloJus, and Anguilla anguilla recognizing the H-anti- gen (Sigma, St. Louis, MO); digoxigenin (DIG) -labelled Aleu- ria aurantia recognizing branched Fuco!l-6GlcNAc in glycocon- jugates (Boehringer Mannheim) . Blots were incubated with antibodies or lectins in concentrations of 5-10 ⁇ g/ml in BB2 for 6 hours followed by 7 washes in TBS.
  • DIG digoxigenin
  • Monoclonal anti ⁇ bodies were detected by alkaline phosphatase (AP) -conjugated goat anti-mouse antibodies (Boehringer Mannheim) .
  • Digoxigenin or biotin labelled lectins were detected with AP-conjugated sheep anti-DIG or anti-biotin Fab fragments (Boehringer Mann ⁇ heim) , respectively.
  • Blots were washed 5x in TBS, once in 0.1M Tris-HCl pH 9.5, 0.1M NaCl, 5 mM MgCl 2 (APBIII) and developed with BCIP/NBT.
  • Anguilla anguilla lectin was biotin labelled using a bio- tin-NHS kit from Calbiochem Immunochemicals, La Jolla, CA.
  • the degree of biotinylation was determined by the shift in molecular weight on SDS-PAGE.
  • 50 ⁇ g of secretory IgA was incubated in 100 ⁇ l 0.1M phosphate buffer, pH 8.0, 10 mM j8-MSH, 20 mM EDTA and 6 units of PNGase F (Boehringer Mannheim) (native conditions) or 50 ⁇ g of secretory IgA was first incubated in 0.2% SDS, 10 mM jS-MSH for 3 min at 100°C and then diluted with 0.1M phosphate buffer pH, 8.0 to 100 ⁇ l (denatured conditions). n-Octylglu- coside (0.5%) was added to the denatured sample to reduce the interference of the PNGase F enzyme with SDS.
  • glycoconjugates to inhibit the bacterial adherence to human stomach in si tu was analyzed by preincu- bation of 200 ⁇ l of labelled bacterial suspension in BBl for 1.5 hour at room temperature with the glycoconjugates listed below. The bacteria were washed once, resuspended in 200 ⁇ l of BBl, and added to the sections. Adherence was compared to sections incubated with non-inhibited bacteria.
  • the Hl-HSA was also obtained from Accurate Chemical & Scientific Corporation. All oligosaccharides for neoglycoconjugate preparations were purified by HPLC and structurally identified and characte ⁇ rized and shown by the manufacturer to be more than 95% pure with NMR-spectroscopy.
  • neoglycoproteins were verified by immunoblots using monoclonal antibody K21 recognizing LNT (non-fucosylated precursor chain) , anti-H-1 (17-206) (the H-antigen on a lactoseries type 1 chain) , anti-H-2 (92FR A2) (the H-antigen on a lactoseries type 2 chain) , anti-Le a (CBM-LAl) (monofucosylated type 1 chain) , (anti-Le b CBM-LBl) (difucosylated type 1 chain) , anti-Le b (T218) (a clone with less cross-reactivity in our hands) , anti-Le x (630/7H1) (monofucosylated type 2 chain) , anti-Le y (672/7E3) (difucosylated type 2 chain), anti-Le b ' y (64/4D8) (recognizing difucosylated type 1 and 2 chains) and the H- anti- anti-
  • glycoconjugates to inhibit bacterial adherence in si tu was analyzed by preincubating labelled bacteria for 3.5 hours at room temperature with the following oligosaccha ⁇ rides diluted in BBl: Lacto-N-fucopentaose I (H-l) , lacto-N- fucopentaose II (Le a ) , lacto-N-difucohexaose I (Le b ) , lacto- N-fucopentaose III (Le x ) , lacto-difucotetraose, 2'-fucosyl- lactose, 3-fucosyllactose (all from IsoSep AB) , H-disacchari- de (Accurate Chemical & Scientific Corporation) , Fuco-l-4-GlcNAc ⁇ O-TMSE (Symbicom AB, Umei, Sweden) , and L-fu ⁇ cose (S)
  • SDS-PAGE was performed as described above, with 20 ⁇ g each of unfractionated human colostrum, goat milk, cow milk or 1 ⁇ g each of the different neoglycoconjugates. After transfer to nitrocellulose, the filters were incubated with BB2 over ⁇ night, and washed 2x in TBS. Bacterial suspension (0.1 OD 600 ) was added (0.2 ml/cm2 in BB2) and incubated overnight in room temperature in rolling bottles. Filters were washed 6x5 min in TBS and rabbit antiserum was added 1:100 and incubated for 1 hour. Blots were washed 5x in TBS.
  • Bacterial bindings were then incubated with either AP-conjugated antibody diluted l:2000x for l hour, washed 5x in BB2, washed once in APBIII and developed with BCIP/NBT or incubated with gold-labelled antibody, washed 5x in BB2, washed 4x in ddH 2 0, and developed with silver enhancement IntenSE BL (Amersham, Arlington Heights, IL.) .
  • Monoclonal antibodies anti-Le a (CBM-LAl) and anti-Le b (CBM-LBl) were used to detect the presence of the correspon ⁇ ding antigens in the glycolipids with secondary antibodies and development as described in Materials and Methods in the section entitled Immunoblot Analysis.
  • Fuco!l-6GlcNAc in glycoconjugates also identified common fuco- sylated structures. These observations taken together indi ⁇ cate that terminal Q.-1-2 linked fucose presented in the H-antigen or ⁇ -1.6 linked fucose does not by itself consti ⁇ tute the H. pylori receptor. Monoclonal antibodies against the Lewis blood group antigens Le a , Le b , Le and Le y recog ⁇ nized the presence of Le x and Le y in the heavy chains of both IgA types while Le b was restricted to the secretory component of secretory IgA.
  • Le a could not be found in either of the two IgA types but was detected in a 120-150 kD glycoprotein co- purified with the secretory IgA. This glycoprotein in addi ⁇ tion presents the Le b antigen of equal intensity to the secretory component corresponding to a 10-fold higher Le b /- protein ratio.
  • Human secretory IgA was applied to a FPLC-Superose 6 column o and the dominating secretory IgA component was separated from the minor 120-150 kD glycoproteins. The peaks were identified by SDS-PAGE and pooled separately. The purified fractions were then analyzed for H. pylori inhibitory properties. The two components did not mediate adherence inhibitory activity related to their respective protein content but rather reflected the Le b content, since 15 ⁇ g/ml of secretory IgA was needed for an efficient bacterial inhibition as described previously (PNAS) compared to 2 ⁇ g/ml of the 120-150 kD protein.
  • PNAS efficient bacterial inhibition
  • the unfractionated secretory IgA preparation was digested by the N-chain releasing enzyme PNGase F in denatured and native conditions. During native conditions the digestion was very limited and only a minor part of the carbohydrate content was removed, while after SDS-denaturation the oligosaccharide chains of the secretory component were readily released as visualized by a decrease of molecular weight in SDS-PAGE and the absence of Le b antigens as detected by immunoblots. Neither the Le a nor the Le b content of the 120-150 kD glyco ⁇ protein was, however, affected by the PNGase F treatment indicating the presentation of these blood group antigens in 0-linked carbohydrate chains on this protein.
  • neoglycoproteins having natural HPLC-purified or synthesized carbohydrate chains characterized by NMR- spectroscopy chemically attached to serum albumin were ob ⁇ tained: Lacto-N-tetraose (LNT) -HSA, lacto-N-neotetraose (LNnT) -HSA, lacto-N-fucopentaose I (H-l)-HSA, lacto-N-fuco ⁇ pentaose II (Le a ) -HSA, lacto-N-fucopentaose III (Le x ) -HSA, lacto-N-difucohexaose I (Le b ) -HSA, Le y -tetrasaccharide (Le y ) -HSA, lacto-N-neofucopentaose I (H-2)-BSA, GlcNAc/3l-4 (F
  • neoglyco ⁇ proteins were verified by immunoblots using monoclonal anti ⁇ bodies. The results confirmed a high degree of purity of the oligosaccharide chains of the neoglycoproteins since all antibodies except the anti-Le b monoclonal antibodies selec- tively stained the corresponding neoglycoconjugate.
  • the anti-Le b (CBM-LBl) clone cross-reacted with the H-l-HSA and to a lesser extend with the Le y -HSA.
  • bacteria were preincubated as described above with 20 ⁇ g/ml of neoglycoconjugates, and the inhibitory activity was compared to 20 ⁇ g/ml of the previous ⁇ ly described secretory IgA.
  • the Le b neoglycoprotein was the only glycoconjugate that could totally eliminate H. pylori binding at this concentration although a reduction in binding could be observed with the H-1-neoglycoconjugate.
  • Le -HSA, Le y -HSA or GlcNAcJl4(Fuc ⁇ -1-6)GlcNAc/3-BSA (data not shown) .
  • the levels of Leb-glycoconjugate and secretory IgA which resulted in almost complete reduction of bacterial binding was estimated to 5 ⁇ g/ml and 20 ⁇ g/ml, respectively, although partial inhibitory effects could be demonstrated at concentrations below 1 ⁇ g/ml for the former.
  • bacteria were preincubated with series of 0.25 mM, 2.5 mM and 25 mM concentrations of the H-l, Le a , Le b , Le x , and Le y oligosaccharides.
  • the H-l, Le b , and Le y oligosaccharides reduced bacterial binding at a concentration of 2.5 mM and almost eliminated binding at a concentration of 25 mM.
  • Le a and Le x oligosaccharides did not exhibit binding inhibition at a concentration of 25 mM.
  • the H-disaccharide and 2' -fucosyllactose reduced bacterial binding by 50-75% at a concentration of 20 mM.
  • the Fuco.l-4GlcNAc-?0-TMSE and 3-fu ⁇ cosyllactose preparations reduced bacterial binding at a concentration of 50 M.
  • the monosaccharide ⁇ -L-fucose was inactive even at a concentration of 175 mM.
  • Human colostrum, goat milk, and cow milk were separated on SDS-PAGE and transferred on to nitrocellulose and the filters were incubated with bacteria. Binding to the immobilized glycoproteins was detected to a 90 kD protein in case of human colostrum while the goat and cow milk samples did not show any binding.
  • the series of neoglycoproteins were run on SDS-PAGE and transferred on to nitrocellulose.
  • the filters were incubated with bacteria and binding to the immobilized glycoproteins was detected with gold-conjugated antibodies.
  • the gold-con ⁇ jugated antibody signal was amplified by silver enhancement. Specific binding of H. pylori to the Le b -HSA and weak binding to the H-l-HSA could be demonstrated.
  • the panel of glycolipids was chromatographed on HPTLC-plates, incubated with bacteria and binding to the immobilized glyco ⁇ lipids was detected with AP-conjugated antibodies. No binding of H. pylori could be detected to any glycolipid even after extensive development of the chromatogram. Le a and Le b antigens were detected in the respective glycolipids by the corresponding monoclonal antibodies.
  • Example 1 Attachment of Helicobacter pylori to human gastric epithelium using an in situ adherence assay was shown in Example 1 to be inhibited by human colostrum secretory IgA, a molecule carry ⁇ ing a highly variable set of N- and 0-linked oligosaccharides while serum IgA was devoid of such inhibitory properties.
  • This inhibitory activity of secretory IgA could be markedly reduced by o;-L-fucosidase treatment of the secretory IgA.
  • the efforts to delineate the fucosidase sensitive receptor struc ⁇ ture for H. pylori have focused on the distribution of fucose residues in the secretory IgA molecule.
  • Free secretory com- ponent has previously been described to present the Le x -anti ⁇ gen and GlcNAc3l-4 (Fucc-1-6)GlcNAc3 (Mizoguchi/Kobata, 1982) and unusual fucosylated carbohydrate structures such as Fuc ⁇ l-3Fuc and Gal ⁇ l4 (Fuco!l-6)GlcNAc (Purkayastha/Lamm, 1979)
  • the Le a -blood group antigen could not be found in either of the two IgA types, but was detected in a 120-150 kD glycoprotein co-purified with the secretory IgA.
  • Size-fractionation chromatography of the secretory IgA prepa ⁇ ration separated the secretory IgA from the 120-150 kD pro- teins.
  • This high molecular weight glycoprotein in addition presents the Le b -antigen of equal intensity to the secretory component, corresponding to a 10-fold higher Le b to protein ratio.
  • Le b -antigen as the only fuco ⁇ sylated carbohydrate structure unique for both the secretory IgA molecule, as well as for fractions with H. pylori adhe ⁇ rence inhibitory activity.
  • the carbohydrate chains of the secretory component have been reported to be exclusively N-linked. Subsequent analysis of the distribution of the Le b antigens of the secretory IgA and the 120-150 kD proteins revealed that these antigens can be presented on both N- and 0-linked carbohydrate chains, since only the Le b -presenting carbohydrate chains of the secretory component were released by PNGase F even after SDS-denatura- tion of the glycoprotein for maximum processivity of the glycosidase.
  • Le b -blood group antigen can be presented on a multitude of complex carbohydrate chains in glycoproteins, in addition to glycolipids, and variations in receptor presentation can probably affect their efficiencies as functional receptors in the human gastric epithelium (Gl, PNAS) .
  • neoglycoproteins In order to analyze the fine-detailed inhibitory properties of glycoproteins carrying defined fucosylated structures, a library of neoglycoproteins was analyzed for their inhibitory properties in the in situ adherence assay.
  • the neoglycoprote- in conjugate exclusively carrying the Le b -blood group antigen was found to be the only structure which, at a concentration of 1 ⁇ g/ml, could interfere with bacterial adherence, while the Le a -, Le x - and Le y -neoglycoproteins at a concentration of 100 ⁇ g/ml did not display any inhibitory activities.
  • the Fuc ⁇ l-4GlcNAcS ⁇ -TMSE oligosaccharide chain surprisingly inhibits at somewhat higher concentrations, emphasizing the importance of a ter ⁇ minal fucose residue, although the specific linkage might not be crucial and subsequently the monofucosylated 3-fucosyl ⁇ lactose (short Le x ) with a Fuco;l-3-terminal reduces bacterial binding at high concentrations.
  • the terminal Fuco;l-2Gal-linkage preferable on an full-length type 1 chain is the most efficient struc ⁇ ture of a receptor analogue for H. pylori , but this configu ⁇ ration can be minimized to the H-disaccharide while still retaining inhibitory activity.
  • Le b - ntigen could also be the H. pylori receptor structure of the host target tissue in addition to the receptor structure of natural soluble receptor analogues or clearance factors, such as colostrum.
  • the interaction of H. pylori with soluble Le b -containing glycoproteins could nevertheless be skewed when it comes to immobilized receptors presented on cell surfaces.
  • two solid phase assay systems were used: bacterial overlay to neoglycoproteins separated on Western blots and bacterial overlays to glycolipids separated on HPTLC-plates.
  • the bacterial Western blot overlays demon ⁇ strated a specific interaction with the Le b -neoglycoprotein and in addition some weak binding to the H-antigen on type 1 chains (LNF1) .

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Abstract

Helicobacter pylori est impliqué comme facteur contribuant à un certain nombre d'états pathologiques tels que la gastrite aiguë (type B), les ulcères gastrique et duodénal, l'adénocarcinome gastrique, et le lymphome gastrique. On a prévu l'utilisation de glycosides di- ou oligosaccharidiques renfermant au moins une unité L-fucose terminale, dans la préparation de compositions pharmaceutiques destinées au traitement ou à la prévention, chez l'homme, d'états impliquant une infection par Helicobacter pylori dans la tunique muqueuse de l'estomac, ainsi qu'un procédé de traitement de ces états à l'aide des glycosides di- ou oligosaccharidiques, procédé consistant à administrer à un malade atteint une dose efficace d'un glycoside di- ou oligosaccharidique comportant au moins une unité de L-fucose terminale.
PCT/IB1994/000023 1993-02-26 1994-02-25 Utilisation de glycosides oligosaccharidiques comme inhibiteurs de l'adherence bacterienne WO1994018986A1 (fr)

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WO1998031241A1 (fr) * 1997-01-16 1998-07-23 N.V. Nutricia Melange d'hydrates de carbone
EP1010434A3 (fr) * 1998-12-11 2000-11-08 Ghen Corporation Inhibiteur de formation de colonies de Hélicobacter pylori
US6204431B1 (en) 1994-03-09 2001-03-20 Abbott Laboratories Transgenic non-human mammals expressing heterologous glycosyltransferase DNA sequences produce oligosaccharides and glycoproteins in their milk
WO2001060346A3 (fr) * 2000-02-17 2002-01-17 American Home Prod Formulation nutritionnelle contenant des substances prebiotiques
WO2003002127A1 (fr) * 2001-06-29 2003-01-09 Glykos Finland Oy Utilisation d'au moins une substance glyco-inhibitrice
WO2003028738A3 (fr) * 2001-09-25 2003-08-21 Nutricia Nv Glucides anti-infectieux
US6630452B2 (en) 2000-02-17 2003-10-07 Wyeth Nutritional formulation containing prebiotic substances
US20120171165A1 (en) * 2010-12-31 2012-07-05 Abbott Laboratories Human milk oligosaccharides to promote growth of beneficial bacteria
EP2415475A4 (fr) * 2009-03-30 2012-09-05 Dikovskiy Aleksander Vladimirovich Composition pharmaceutique à inhibiteur de la pompe à protons et prébiotique pour le traitement des ulcères de l'estomac ou du duodénum
EP2451462A4 (fr) * 2009-07-06 2012-12-12 Childrens Hosp Medical Center Inhibition de l'inflammation par des oligosaccharides du lait
US9132142B2 (en) 2007-09-07 2015-09-15 The General Hospital Corporation Use of secretor, Lewis and sialyl antigen levels in clinical samples as predictors of risk for disease
US9164099B2 (en) 2002-09-12 2015-10-20 Life Technologies Corporation Site-specific labeling of affinity tags in fusion proteins
US10626460B2 (en) 2013-02-21 2020-04-21 Children's Hospital Medical Center Use of glycans and glycosyltransferases for diagnosing/monitoring inflammatory bowel disease
CN111436617A (zh) * 2020-05-12 2020-07-24 西北大学 基于牛血清白蛋白的岩藻糖拟糖蛋白在抑制惰性凝集杆菌方面的用途及制备方法
US10857167B2 (en) 2015-04-28 2020-12-08 Children's Hospital Medical Center Use of oligosaccharide compositions to enhance weight gain

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EP2692740A1 (fr) * 2012-07-30 2014-02-05 Le Centre National De La Recherche Scientifique Compositions de glycane, leurs procédés de préparation et leurs utilisations en tant que médicament

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US4935406A (en) * 1988-09-20 1990-06-19 Marion Laboratories, Inc. Use of bismuth (phosph/sulf)ated saccharides against Camplyobacter-associated gastrointestinal disorders
US5116821A (en) * 1990-11-20 1992-05-26 The Procter & Gamble Company Sulfated glyceroglucolipids as inhibitors of bacterial adherence
WO1994003184A1 (fr) * 1992-07-31 1994-02-17 Neose Pharmaceuticals, Inc. Compositions destinees a traiter et inhiber les ulceres gastriques et duodenaux

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4935406A (en) * 1988-09-20 1990-06-19 Marion Laboratories, Inc. Use of bismuth (phosph/sulf)ated saccharides against Camplyobacter-associated gastrointestinal disorders
US5116821A (en) * 1990-11-20 1992-05-26 The Procter & Gamble Company Sulfated glyceroglucolipids as inhibitors of bacterial adherence
WO1994003184A1 (fr) * 1992-07-31 1994-02-17 Neose Pharmaceuticals, Inc. Compositions destinees a traiter et inhiber les ulceres gastriques et duodenaux

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6204431B1 (en) 1994-03-09 2001-03-20 Abbott Laboratories Transgenic non-human mammals expressing heterologous glycosyltransferase DNA sequences produce oligosaccharides and glycoproteins in their milk
US6576251B1 (en) 1997-01-16 2003-06-10 N. V. Nutricia Carbohydrate mixture
WO1998031241A1 (fr) * 1997-01-16 1998-07-23 N.V. Nutricia Melange d'hydrates de carbone
EP1010434A3 (fr) * 1998-12-11 2000-11-08 Ghen Corporation Inhibiteur de formation de colonies de Hélicobacter pylori
US6630452B2 (en) 2000-02-17 2003-10-07 Wyeth Nutritional formulation containing prebiotic substances
WO2001060346A3 (fr) * 2000-02-17 2002-01-17 American Home Prod Formulation nutritionnelle contenant des substances prebiotiques
WO2003002127A1 (fr) * 2001-06-29 2003-01-09 Glykos Finland Oy Utilisation d'au moins une substance glyco-inhibitrice
WO2003028738A3 (fr) * 2001-09-25 2003-08-21 Nutricia Nv Glucides anti-infectieux
EP1970065A1 (fr) * 2001-09-25 2008-09-17 N.V. Nutricia Hydrates de carbone anti-infectieux
US9164099B2 (en) 2002-09-12 2015-10-20 Life Technologies Corporation Site-specific labeling of affinity tags in fusion proteins
US9132143B2 (en) 2007-09-07 2015-09-15 The General Hospital Corporation Use of secretor, lewis and sialyl antigen levels in clinical samples as predictors of risk for disease
US9132142B2 (en) 2007-09-07 2015-09-15 The General Hospital Corporation Use of secretor, Lewis and sialyl antigen levels in clinical samples as predictors of risk for disease
EP2415475A4 (fr) * 2009-03-30 2012-09-05 Dikovskiy Aleksander Vladimirovich Composition pharmaceutique à inhibiteur de la pompe à protons et prébiotique pour le traitement des ulcères de l'estomac ou du duodénum
US9034847B2 (en) 2009-07-06 2015-05-19 Children's Hospital Medical Center Inhibiting inflammation with milk oligosaccharides
EP2451462A4 (fr) * 2009-07-06 2012-12-12 Childrens Hosp Medical Center Inhibition de l'inflammation par des oligosaccharides du lait
US10098903B2 (en) 2009-07-06 2018-10-16 Children's Hospital Medical Center Inhibiting inflammation with milk oligosaccharides
US11058697B2 (en) 2009-07-06 2021-07-13 Children's Hospital Medical Center Inhibiting inflammation with milk oligosaccharides
US11324765B2 (en) 2009-07-06 2022-05-10 Children's Hospital Medical Center Inhibiting inflammation with milk oligosaccharides
US12171773B2 (en) 2009-07-06 2024-12-24 Children's Hospital Medical Center Inhibiting inflammation with milk oligosaccharides
US20120171165A1 (en) * 2010-12-31 2012-07-05 Abbott Laboratories Human milk oligosaccharides to promote growth of beneficial bacteria
US11337990B2 (en) * 2010-12-31 2022-05-24 Abbott Laboratories Human milk oligosaccharides to promote growth of beneficial bacteria
US10626460B2 (en) 2013-02-21 2020-04-21 Children's Hospital Medical Center Use of glycans and glycosyltransferases for diagnosing/monitoring inflammatory bowel disease
US10857167B2 (en) 2015-04-28 2020-12-08 Children's Hospital Medical Center Use of oligosaccharide compositions to enhance weight gain
CN111436617A (zh) * 2020-05-12 2020-07-24 西北大学 基于牛血清白蛋白的岩藻糖拟糖蛋白在抑制惰性凝集杆菌方面的用途及制备方法
CN111436617B (zh) * 2020-05-12 2022-12-23 西北大学 基于牛血清白蛋白的岩藻糖拟糖蛋白在抑制惰性凝集杆菌方面的用途及制备方法

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