WO2000022945A2 - Proteine pour le traitement ou la prevention d'un trouble de la voie gastro-intestinale - Google Patents

Proteine pour le traitement ou la prevention d'un trouble de la voie gastro-intestinale Download PDF

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Publication number
WO2000022945A2
WO2000022945A2 PCT/EP1999/007911 EP9907911W WO0022945A2 WO 2000022945 A2 WO2000022945 A2 WO 2000022945A2 EP 9907911 W EP9907911 W EP 9907911W WO 0022945 A2 WO0022945 A2 WO 0022945A2
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Prior art keywords
protein
milk
composition
serum
human
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PCT/EP1999/007911
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English (en)
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WO2000022945A3 (fr
Inventor
Karine Vidal
Anne Donnet
Eduardo Schiffrin
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Societe Des Produits Nestle S.A.
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Priority to AU64740/99A priority Critical patent/AU6474099A/en
Priority to CA002347069A priority patent/CA2347069A1/fr
Priority to EP99952603A priority patent/EP1123011A2/fr
Publication of WO2000022945A2 publication Critical patent/WO2000022945A2/fr
Publication of WO2000022945A3 publication Critical patent/WO2000022945A3/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • 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

Definitions

  • the present invention relates to a new isolated protein from mature human milk; a composition comprising it; a method for manufacture of the protein or the composition; use of the protein, a variant or fragment thereof in the manufacture of a medicament or nutritional product for the treatment or a gastro-intestinal (hereinafter GI) tract disorder; and a method of treatment or prevention of the disorder which comprises administering an effective amount of the protein or composition.
  • GI gastro-intestinal
  • AMP, CMP, GMP and UMP are taken to mean respectively the monophosphates of adenosine, cytidine, guanosine and uridine and their nucleotide equivalents which include polymeric RNA, ribo-nucleosides, ribonucleoside containing adducts and di-and triphosphate ribo-nucleotides.
  • infant formulae are generally designed to resemble human milk as closely as possible.
  • a plurality of constituents in human milk are bioactive and, because of synergies among the constituents, the inclusion of just one or a few of them in the infant formula may not produce the bioactivity observed in human milk.
  • bioactivity of the constituents may be affected by heat treatment for sterilisation and long term storage of the formula.
  • CD 14 is a myeloid cell-surface glycoprotein which acts as a receptor for bacterial lipopolysaccharide. It is well documented that monocyte/ macrophage activation by lipopolysaccharides via membrane CD 14 (mCD14) triggers the release of a variety of pro-inflammatory, immunoregulatory and cytotoxic molecules such as TNF- ⁇ , IL-1, IL-6, IL-8, oxygen radical products and nitric oxide. mCD14 lacks transmembrane and cytoplasmic domains. It is anchored to the cell membrane by a glycosyl-phosphatidylinositol linkage.
  • serum sCD14 soluble CD 14
  • serum sCD14 serum sCD14
  • serum sCD14 ⁇ 49 kDa
  • serum sCD14 ⁇ 55 kDa
  • the main source of serum sCD14 in normal human plasma is the monocyte.
  • Monocytes release the two isoforms of serum sCD14, ⁇ and ⁇ , into plasma.
  • the former is produced by limited proteolysis from membrane-bound CD 14 and the latter is directly derived from the intracellular compartment.
  • the sCD14 ⁇ :sCD14 ⁇ ratio in culture supernatant of normal monocytes is approximately 2:1.
  • serum sCD14 ⁇ levels are either similar to, or even higher than, those of serum sCD 14 ⁇ , suggesting that the amount of serum sCD14 ⁇ released in vivo is either lower or other cell types may contribute to the plasma pool.
  • serum sCD14 A substantial concentration of serum sCD14 is found in normal human plasma, 2-3 ⁇ g/ml. In sera of septic patients global concentrations of serum sCD14 are elevated, reaching around 4 ⁇ g/ml. It has been reported a correlation between high levels of serum sCD14 at the onset of septic shock and poor outcome in septic patients.
  • WO98/22580 discloses the presence of a protein in bovine colostral why which has some amino acid sequence similarity with human serum sCD14. It is speculated that this protein could be the bovine variant of CD 14.
  • Figure 7 of the document shows the differences between the amino acid sequences of bovine colostral whey CD 14, and the sequences of human serum CD 14 and mouse serum CD 14 which were known in the literature.
  • the document describes affinity purification of a CD 14 protein from human colostrum using a sepharose column having the monoclonal antibody 63D3 bound to it.
  • the document does not describe an isolated CD 14 variant purified from mature milk. Instead, colostrum is disclosed as the source of the protein.
  • CD 14 a new variant of CD 14 has now been identified. It has been isolated from mature human milk. Suprisingly, this new variant is not the same as serum CD14. Indeed, as described below, it differs from the known CD14 variants including the variants disclosed by WO98/22580 at least insofar as the new protein comprises no O- glycosylation. In stark contrast, the known CD 14 variants including human serum sCD14 have both O- and N- glycosylation. Furthermore, it has now been found that CD 14, a variant or fragment thereof retaining the bioactivity of CD 14, or a composition comprising it is effective in the treatment of GI tract disorders.
  • the invention provides an isolated protein having no O-glyclosylation and at least 70% homology of amino acid sequence with human serum CD 14.
  • the invention provides a method of production of the protein which comprises isolating it from mature milk.
  • the invention provides a composition which comprises the protein excluding mature milk.
  • the invention provides a method of production of the composition which comprises adding an embodiment of the protein according to the invention.
  • the invention provides use of a CD 14 variant or fragment that retains the bioactivity of CD 14 in the manufacture of a nutritional product or medicament for the treatment or prevention of a GI tract disorder.
  • the invention provides a method of treatment or prevention of a GI tract disorder which comprises administering an effective amount of a CD 14 variant or fragment thereof which retains the bioactivity of CD 14.
  • the new protein has the unique capacity of being capable of mediating bacterial interaction with intestinal surfaces. Furthermore, if the new variant is included in an infant formula, the formula closely resembles mature human milk in its protective capacity of the intestinal surface.
  • the amino acid sequence of an embodiment of the protein is at least about 90% homologous with the amino acid sequence of human serum CD 14, more preferably at least about 95% homologous, even more preferably it is substantially identical. In alternative embodiments the amino acid sequence preferably has these degrees of homology with the amino acid sequence of bovine or buffalo CD 14.
  • an embodiment of the protein has at least one N- glyclosylation site. Preferably it has a plurality of N- glycosylation sites, more preferably from about 1 to about 10. Even more preferably, an embodiment of the protein has from about 3 to about 5 N- glyclosylation sites, most preferably 4.
  • the presence of an embodiment of the protein is not revealed in a Western blot by the known commercially available anti-CD 14 monoclonal antibody MY4.
  • an embodiment of the protein is isolated from mature milk. More preferably it is isolated from mature human, bovine or buffalo milk. Most preferably the protein is isolated mature milk sCD14 (hereinafter referred to as mmsCD14). In alternative embodiments it is produced recombinantly by standard techniques.
  • an embodiment of the composition comprises an embodiment of the protein together with a physiologically acceptable carrier, adjuvant or diluent. More preferably it comprises a compound extracted from milk, even more preferably a plurality of compounds extracted from milk. Most preferably the composition is an infant formula or enteral composition.
  • an embodiment of the composition comprises a casein fraction and milk fat.
  • milk based products provide the advantage that they can preserve molecules from the proteolytic activity of the digestive tract.
  • the biological activity of the protein according to the invention takes place in the small intestine after the passage through the gastric environment.
  • an embodiment of the composition comprises a lipopolysaccharide binding protein (LBP), decay accelerating factor (DAF, CD55), bactericidal permeability increasing factor (BPI) or a mixture thereof.
  • LBP lipopolysaccharide binding protein
  • DAF decay accelerating factor
  • BPI bactericidal permeability increasing factor
  • Figure 1A illustrates a comparative SDS-PAGE pattern of sCD14 of mature human milk (several dilutions (from 1:6 to 1:100) and in normal human plasma serum (NP, 1:50), with a rabbit polyclonal antibody.
  • Figure IB illustrates the lack of milk CD 14 detection by an antibody specific for the C-terminus peptide of the ⁇ serum CD 14 molecule.
  • Figure 2 shows interleukine-8 release by undifferentiated HT29 cells following a 24h incubation with 100 ng/ml of E. Coli LPS in the presence of either 10% human serum (HS, pooled serum AB + ) or 1.7% human milk (HM, pooled mature human milk).
  • HS human serum
  • HM human milk
  • MY4 monoclonal anti-CD 14 antibody
  • IgG2b isotype control
  • Figure 3 shows interleukine-8 release by undifferentiated HT29 cells following a 24h incubation with 2.5 x 10 6 E. Coli in the presence of either 10% human serum (HS, pooled serum AB + ) or 1.7% mature human milk (HM, pooled breast milk).
  • HS human serum
  • HM 1.7% mature human milk
  • MY4 monoclonal anti-CD 14 antibody
  • This invention is based upon the finding that mature milk comprises an unknown variant of CD 14.
  • the new molecule has a high homology at the amino acid sequence with the serum sCD14 molecule, however it has a different gel mobility that either of the two serum soluble forms, is the secretory product of the mammary gland epithelial cell, has a different glycosylation pattern with regard to the known forms, and has a unique capacity for mediating bacterial interaction with intestinal surfaces.
  • the invention relates to the use of the new form of soluble CD 14 molecule, present in human, bovine and buffalo milk, in infant formula, clinical nutrition and animal feed.
  • the amino acid sequence including the N- and C-terminus ends of the mature human milk derived molecule is substantially identical to that of serum CD 14 showing that milk and serum CD 14 are highly homologous.
  • N- and C-terminal sequence analysis shows that milk CD 14 is not posttranslationally truncated and corresponds therefore to the ⁇ form found in serum. But, whereas the ⁇ -form has now been found to migrate on SDS-PAGE with about 55KDa, milk CD14 migrates on SDS-PAGE only with about 48 KDa. This difference indicates that mature human milk and serum CD 14 have different glycosylation patterns. Indeed, whereas CD14 has been reported to be N- and O- glycosylated, deglycosylation assays with milk CD 14 showed no O- glycosylation.
  • N-glycosylation was confirmed by LC-MS analysis of N-glycosidase F treated mmsCD14 in that the resulting molecular mass corresponded to the theoretical molecular mass based on the published amino acid sequence.
  • the main form of mature human milk sCD14 is therefore different from blood sCD14 that was reported to be N- and O-glycosylated.
  • the MEM-18 mAb and a control isotype antibody (mouse IgGl, BectonDickinson) were immobilised in 10 mM Na-acetate at pH 5 on a dual well carboxymethyl dextran matrix of an IAsys cuvette using standard EDC [1-ethyl- 3-(3-dimethyl-aminopropyl) carbodiimide-HCl] and NHS (N- hydroxysuccinimide) coupling chemistry and subsequent blocking with ethanolamine.
  • Running buffer was PBS/Tween 20.
  • lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide binding protein and serum sCD14.
  • LPS induces IL-8 secretion by IEC in the presence of human serum. This effect is maximum at an LPS concentration of 100 ng/ml.
  • mmsCD14 has now been tested for its capacity to mediate LPS effects on IEC. Different mature human milk concentrations were assayed in IEC serum-free culture media containing a fixed dose of LPS (100 ng/ml). IL- 8 production increased with increasing concentrations of human milk (0-10%).
  • mmsCD14 The contribution of mmsCD14 to stimulation of HT-29 cells by gram-negative non-pathogenic Escherichia coli has now been tested.
  • An increasing quantity of IL-8 secretion has been observed with increasing concentrations of bacteria in the range of 1 XIO ⁇ , to 5 X10 ⁇ CFU per ml in the presence of 2% human milk.
  • mmsCD14 is more efficient than serum sCD14 in its stimulatory function of intestinal epithelial cells by bacteria or lipopolysaccharide.
  • An embodiment of the enteral composition of the invention preferably comprises an effective amount of mmsCD14, and preferably contains at least about 25 ⁇ g/ml of mmsCD14.
  • the other components of the enteral composition are those conventionally added to infant formulae or enteral products and may be at least one of those described below.
  • the mmsCD14 may be any form of mmsCD14, but is preferably the form found in mature human milk and recognised by a polyclonal rabbit antibody. This form has a molecular mass of approximately 48 KDa, an electrophoretic mobility faster than the serum alpha form and cannot be recognised by the commercially available anti-CD 14 antibody MY4.
  • the mmsCD14 may be extracted from bovine, buffalo, goat or sheep milk. In addition, it can be produced by recombinant microorganisms; for example recombinant fungi or yeast.
  • the supplementation of baby formula with this mmsCD14 has physiological benefit during the baby's neonatal period. This period is characterised by bacterial challenge due to bacterial colonisation and antigen challenge that could lead to inflammation, septic conditions or an allergic reaction.
  • the invention includes the use of a CD 14 variant or fragment thereof having CD 14 activity of mediating interaction of bacteria with an intestinal surface in the manufacture of a composition for the treatment or prevention of a disorder of the gastro-intestinal tract of a mammal.
  • the disorder is selected from the group which comprises inflammatory bowel disease, Chrone's disease, ulcerative cholitis, coeliac disease, intestinal bacterial overgrowth, chronic hepatitis, necrotising enterocolitis, neonotal sepsis, infectious diarrhoea, disbalance of the intestinal microflora, allergic reactions to food and bacterial translocation from the gut to other compartments of the body. Mammals most likely to develop these disorders are premature, mal-nourished, immuno- depressed or mammals under trauma conditions.
  • the CD 14 variant or fragment thereof is an embodiment of the protein according to the invention.
  • An embodiment of a composition according to the invention preferably comprises about 1.8 to about 4.5 g protein/100 kcal, preferably about 1.8 to about 3.6 g/100 kcal.
  • the protein may be any suitable protein such as cow's milk protein, casein, whey, soy protein, egg protein, pea protein or a mixture thereof.
  • the protein may be in the form of a salt, such as a caseinate. It may be an isolate or concentrate.
  • the protein may be in intact or hydrolysed form. Alternatively or in addition, free amino acids may be used.
  • a mixture of the whey : casein mass ratio is 60:40.
  • Whey protein can be prepared to have reduced allergenicity using conventional techniques such as described in U.S. Patent 5039532. For example, it can be prepared by electrodialysis or ultrafiltration.
  • An embodiment of a composition according to the invention preferably comprises about 7g to about 14g/100kcal of carbohydrate or provide 40 to about 60% of calories as carbohydrate.
  • the carbohydrate can be supplied in a conventional form including simple form or complex form.
  • Simple carbohydrates include lactose, maltose, sucrose and corn syrup solids.
  • Complex carbohydrates include starches and maltodextrins. Starch may be precooked or pregelitanised.
  • An embodiment of a composition according to the invention preferably comprises about 3.3 to 6.5 g/100 kcal of fat.
  • the fat can be supplied in a suitable form including saturated fat, monounsurated fat (MUFA), polyunsaturated fat (PUFA) or a mixture thereof.
  • MUFA monounsurated fat
  • PUFA polyunsaturated fat
  • fat is provided as about 1/3 saturated fat, about 1/3 MUFA and about 1/3 PUFA.
  • Saturated fat includes butyric, valeric, caproic, capylic, decanoic, lauric, myristic, palmitic, steraic, arachidic, behenic and lignoceric fatty acids.
  • MUFAs include palmitoleic, oleic, elaidic, vaccenic and erucic fatty acids.
  • Preferred PUFAs are C18, C20 or C22 ⁇ -3 or C18, C20 or C22 ⁇ -6 polyunsaturated fatty acids. Most preferred are the C20 or the C22 ⁇ -3 or C20 or C22 ⁇ -6 polyunsaturated fatty acids. These include not only arachidonic acid (ARA) but also eicosapentaenoic acid (EPA) and docoshexaenoic acid (DHA).
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docoshexaenoic acid
  • More than one PUFA can be added.
  • two or more PUFAs may be from a different source, they can be added either separately or together during preparation of the composition according to the invention.
  • fish oil containing DHA may be mixed with one or more microbial oils containing another PUFA (e.g. ARA).
  • another PUFA e.g. ARA
  • An embodiment of a composition according to the invention preferably comprises a carbohydrate : lipid weight ratio which is greater than 60:40.
  • the ratio of carbohydrate to lipid is between 65:35 and 90:10. More preferably still it is between 70:30 and 85:15.
  • An embodiment of a composition according to the invention preferably comprises a nutritionally acceptable quantity of a minerals or vitamin selected from the group which comprises calcium, phosphorus, potassium, sodium, chloride, magnesium, iron, copper, zinc, manganese, iodine, selenium, vitamin A, vitamin D, vitamin E, vitamin Kl, vitamin Bl, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, niacin, folic acid, biotin, choline, inositol or a mixture thereof.
  • a minerals or vitamin selected from the group which comprises calcium, phosphorus, potassium, sodium, chloride, magnesium, iron, copper, zinc, manganese, iodine, selenium, vitamin A, vitamin D, vitamin E, vitamin Kl, vitamin Bl, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, niacin, folic acid, biotin, choline, inositol or a mixture thereof.
  • compositions according to the invention preferably comprises an additional nutritional factor selected from taurine, carnitine, etc.
  • the composition may contain at least about 7.5 mmoles carnitine /100 kcal.
  • composition according to the invention preferably comprises one or more of AMP, CMP, GMP or UMP.
  • An embodiment of a composition according to the invention preferably comprises a pharmaceutically acceptable filler, color additive, taste modifier, non-antagonistic antibiotic, pharmaceutical, medicament or mixture thereof.
  • An embodiment of a composition according to the invention is preferably in a form suitable for ingestion orally or for administration by enteral tube feeding. More preferably it is an oral formulation.
  • a composition of the present invention is preferably formulated to provide about 600 to about 800 kcals/litre, more preferably about 670 kcals/litre.
  • the total volume per dose is about 50 top about 200 ml preferably about 100 to about 150 ml.
  • the composition is preferably a solid, in which case it is preferably dried, and optimally in the form of a powder. Preferably, it is miscible or dispersible in an aqueous liquid, such as water.
  • composition is a liquid which is ready for use, or a concentrated liquid which can be diluted before use, preferably with water.
  • An embodiment of a composition according to the invention is preferably prepared by a method which comprises the following steps; (1) Standardising pasteurised milk (skimmed, evaporated or whole milk) by the addition of whey protein concentrate, minerals, water-soluble vitamins, trace elements and carbohydrates at a high temperature, for example 60°C, (2) mixing vegetable oil, oil-soluble emulsifier, at least one oil-soluble vitamin and antioxidant at high temperature, for example 60°C, (3) adding the oil mixture obtained from (2) to the standardised milk obtained from (1) with sufficient agitation to allow mixing, (4) homogenising the mixture obtained in (3) in two stages at high temperature and pressure, for example 60°C at 150 and then 30 bar, (5) cooling the emulsion obtained under (4) to a low temperature, for example 5°C, (6) adding water- soluble vitamins, minerals and trace elements to the cooled emulsion if desired, (7) sterilising the emulsion obtained under (6) on-line at ultra high temperature (UHT) and/or in an appropriate container to obtain a formula in the form
  • the enteral composition may be supplemented with an embodiment of the protein according to the invention by adding it to the oil during processing.
  • adding the protein at such an early stage can have disadvantages because it may be degraded. In the light of this, alternatively it is added at a later stage. This provides the advantage that exposure of the protein to unfavourable conditions is minimised.
  • the protein is added after drying.
  • the protein can be added in a variety of forms. It may be added in solution, for example in a lipid and/or an oil composition.
  • the oil may contain solely the protein or it may contain a number of other ingredients. If a solid composition is used, the protein may be encapsulated in capsules or it maybe in a powdered form.
  • the starting oil phase does not contain any PUFAs. Preferably they are added later, preferably after drying.
  • Test 1 Ion exchange chromatography for purification of mmsCD14 from mature milk (bovine, buffalo, goat or sheep) and supernatant of CD14 cDNA transfected cells.
  • mmsCD14 The purification of mmsCD14 from milk or culture supernatant of transfectant cells was performed by ion exchange chromatography. Diluted milk samples or conditioned medium of CD 14 cDNA transfectant cells were applied to a Mono QlO/10 column equilibrated with 20mM ethanolamine pH 9.5 . After washing, the column was subjected to a linear gradient of NaCl (0-500mM) in the equilibrating buffer. Fractions are collected and the mmsCD14 content was determined by ELISA. Further characterisation by reverse phase HPLC was carried out to confirm the purity of the protein. The selected fractions were pooled , desalted with PBS, and kept aliquoted at -70°C until further use. The purified material was routinely characterised by N-terminal sequencing, amino acid analysis, SDS-Page, and mass spectrometry.
  • the human intestinal epithelial cells HT29 (CD14-negative) were stimulated with varying concentrations of LPS (0.1 to lOOOng/ml) in the presence or absence of human AB serum (10%). After 24h culture at 37°C, culture supernatants were collected and tested for IL-8 release by ELISA. The levels of IL-8 were compared with those detected by stimulating the HT29 with the same amounts of LPS in the absence of serum and the presence of human breast milk (1.7%).
  • the anti-CD14 specific mAb MY4 (20 ⁇ g/ml), which binds to the same epitope as LPS on the sCD14 molecule, was used to block the stimulation mediated by the human milk or serum.
  • Test 3 mmsCD14 quantitative determination in human milk
  • Concentration of mmsCD14 in human milk was determined by a human CD 14- specific ELISA test (IBL, Germany). Samples taken in the first days of lactation had a higher amount of sCD14 than the overall average: 75.4 ⁇ 19.1 ⁇ g/ml compared to 52.9 ⁇ 24.0 ⁇ g/ml. This is thought to be at least partially due to leakage of serum sCD14 into the colostrum.
  • the human astrocytoma cell line U373 (CD 14 negative) was stimulated with varying concentrations of lipopolysaccharides, lpg/ml to lOng/ml, in the presence or absence of human AB serum (10% or 1%) and soluble recombinant human CD14 (3 ug/ml). After 48h culture at 37°C, culture supernatants were collected and tested for IL-6 release by ELISA. The levels of IL-6 were compared with those detected by stimulating the U373 with the same amounts of lipopolysaccharides in the absence of serum and the presence of 1% human milk.
  • the anti-CD 14 specific mAbs MEM-18 and MY4 (15ug/ml), which bind to the same epitope as lipopolysaccharides on the sCD14 molecule, was used to block stimulation mediated by human milk or serum.
  • lipopolysaccharides alone nor 0.5% or 1% human milk alone are able to induce significant levels of IL-6.
  • lipopolysaccharides were capable of inducing substantial amounts of IL-6 in the presence of mature human milk (0.5 and 1%). This effect was abrogated by two CD14-specific monoclonal, antibodies , MEM-18 and MY4, but not by an irrelevant monoclonal antibody (iMab MOPC-21). MEM-18 and MY4 were also able to block the release of IL-6 induced by lipopolysaccharides in the presence of 10 AB human serum, albeit partially, suggesting that other mechanisms of IL-6 release independent of CD 14 may operate in human serum but not in mature milk.
  • mature milk-derived sCD14 is biologically active and capable of mediating cell activation by lipopolysaccharides. Differences in the biological activities are observed between serum sCD14 and mature milk-derived sCD14.
  • the contribution of mmsCD14 on the stimulation of intestinal epithelial cells by Gram-negative non-pathogenic bacteria was tested.
  • HT29 cells were stimulated with varying amounts of E. Coli (1 x 10 3 to 5 x 10 6 CFU per ml) in the presence or absence mature human breast milk (1.7%). After 24h culture at 37°C, culture supernatants were collected and tested for IL-8 release by ELISA. The levels of IL-8 were compared with those detected by stimulating the HT29 with the same amounts of LPS in the absence of milk and the presence of human AB serum (10%).
  • E. Coli alone was unable to induce significant levels of IL- 8.
  • Incubation of E.Coli in the presence of either human serum or human milk was capable of inducing substantial amounts of IL-8. This effect was abrogated by CD14-specific monoclonal antibodies MY4, but not by the irrelevant antibody (IgG2b).
  • Test 5 Production of mmsCD14 from natural sources.
  • mmsCD 14 Small scale purification of mmsCD 14 was performed by immunoaffinity chromatography using a purified anti-sCD14 monoclonal antibody, MY4 (Coulter Immunotech, USA). Briefly, a diluted mature milk sample was applied to an anti-CD 14-Sepharose 4B matrix. After washing, the column was eluted with 100 raM Glycine.HCl, pH 2.5. Fractions are collected and neutralized. The mmsCD 14 content of each fraction was determined by an anti-sCD 14 ELISA test (IBL, Hamburg, Germany), and the purity was analysed by SDS-PAGE and silver staining. The selected fractions were pooled and kept in -20°C until further use.
  • MY4 Coulter Immunotech, USA
  • Polyclonal antibodies against mmsCD14 were obtained by immunisation of rabbits with the antigen-adjuvant mixture. Briefly, an emulsion composed of 0.5 mg/ml purified mmsCD14 and 2 ml complete Freund adjuvant (Sigma, St Louis, MO) with insoluble Mycobacterium tuberculosis bacilli was injected into multiple intamuscular sites. The animal was bled 14 days following the first immunisation. Booster immunisation using incomplete Freund adjuvant (Sigma) was performed 6 weeks after priming immunisation and with intervals of 2-3 weeks thereafter. Bleedings were performed 10-14 days after immunisation. The antibody titers were performed by ELISA.
  • the formula has the composition (per 100 g of powder) which is described in the table I below.
  • the formula was reconstituted by mixing 142g of powder to 900mL of water to give IL of ready-to- drink preparation.
  • the composition given above can vary to accommodate for local directives concerning the amounts of specific ingredients.
  • Other trace elements e.g. selenium, chromium, molybdenum, fluoride
  • Nucleosides and/or nucleotides can also be present.
  • the formula has the composition (per 100 g of powder) which is described in the table II below.
  • the formula was reconstituted by mixing 132g of powder to 900mL of water to give IL of ready-to-drink preparation.
  • the composition given above can vary to accommodate for local directives concerning the amounts of specific ingredients.
  • Other trace elements e.g. selenium, chromium, molybdenum, fluoride
  • Nucleosides and/or nucleotides can also be present.
  • a formula for infants, from 5 months of age, is prepared.
  • the formula has the composition (per liter of ready to use preparation) which is described in the table III below.
  • composition given above can be varied to accommodate for local directives concerning the amounts of specific ingredients.
  • Other trace elements e.g. selenium, chromium, molybdenum, fluoride
  • Nucleosides and/or nucleotides can also be present.
  • a formula for infants, from 5 months of age, containing partly hydrolyzed protein for low allergenicity, in powder form was prepared.
  • the formula has the composition (per lOOg of powder) which is described in the followed table IV.
  • the formula was reconstituted by mixing 138g of powder to 900mL of water to give IL of ready-to-drink preparation.
  • the composition given above can be varied to accommodate for local directives concerning the amounts of specific ingredients permitted.
  • Other trace elements e.g. selenium, chromium, molybdenum, fluoride
  • Nucleosides and/or nucleotides can also be present.
  • a formula that can be administered to an infant suffering from bovine milk allergy, comprising ulfrafiltered/microfiltered extensively hydrolyzed protein, in powder form was prepared.
  • the formula had the composition (per 100 g of powder) which is described in the following table V.
  • the formula was reconstituted by mixing 150g of powder to 900ml of water to give 11 of a ready-to-drink preparation.
  • the composition can be varied to accommodate local directives concerning the amounts of specific ingredients permitted.
  • Other trace elements e.g. selenium
  • Nucleosides and/or nucleotides can also be present.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Nutrition Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une nouvelle protéine isolée exempte de O-glycosylation et présentant au moins 70 % d'homologie avec la séquence d'acide aminé du sérum humain CD14. Par ailleurs, cette invention comprend d'une part une composition renfermant une quantité efficace de la protéine précitée, et d'autre part l'utilisation d'un variant de CD14 dans le traitement ou la prévention d'un trouble de la voie gastro-intestinale chez un mammifère. Plus particulièrement, le trouble est compris dans le groupe constitué par les maladies intestinales inflammatoires, la maladie de Chrone, les colites ulcéreuses, les maladies coeliaques, la prolifération de bactéries intestinales, l'hépatite chronique, l'entérocolite nécrosante néonatale, la septicémie néonatale, les diarrhées infectieuses, le déséquilibre de la microflore intestinale, et les réactions allergiques dues au passage des aliments et des bactéries de l'intestin à d'autres parties du corps.
PCT/EP1999/007911 1998-10-20 1999-10-15 Proteine pour le traitement ou la prevention d'un trouble de la voie gastro-intestinale WO2000022945A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU64740/99A AU6474099A (en) 1998-10-20 1999-10-15 Protein for treatment or prevention of a gi tract disorder
CA002347069A CA2347069A1 (fr) 1998-10-20 1999-10-15 Proteine pour le traitement ou la prevention d'un trouble de la voie gastro-intestinale
EP99952603A EP1123011A2 (fr) 1998-10-20 1999-10-15 Proteine pour le traitement ou la prevention d'un trouble de la voie gastro-intestinale

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP98203501.6 1998-10-20
EP98203501 1998-10-20

Publications (2)

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WO2000022945A2 true WO2000022945A2 (fr) 2000-04-27
WO2000022945A3 WO2000022945A3 (fr) 2000-07-27

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EP (1) EP1123011A2 (fr)
AU (1) AU6474099A (fr)
CA (1) CA2347069A1 (fr)
WO (1) WO2000022945A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004112512A1 (fr) * 2003-06-23 2004-12-29 Nestec S.A. Supplementation en acides amines pour un microbiote sain de l'ecosysteme
ES2232273A1 (es) * 2003-03-21 2005-05-16 Jose Manuel Fernandez-Real Lemos Nuevas aplicaciones terapeuticas de la glucoproteina cd14s.
WO2009095916A2 (fr) * 2008-01-29 2009-08-06 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Cd14 et peptides de celui-ci pour la protection de cellules contre la mort cellulaire
GB2433889B (en) * 2004-10-14 2010-02-17 Adventures Plus Pty Ltd Treatment of gastrointestinal and other disorders with a buffered admixture of vitamins, minerals and amino acids
WO2014152985A1 (fr) * 2013-03-14 2014-09-25 Children's Hospital Medical Center Biomarqueurs de l'entérocolite nécrosante avant terme
JPWO2014104305A1 (ja) * 2012-12-28 2017-01-19 持田製薬株式会社 プレセプシン測定による炎症性腸疾患の診断

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WO1998022580A2 (fr) * 1996-11-18 1998-05-28 The Wellesley Hospital Foundation Proteine immunotrope (cd14) associee a la lactation bovine, gene codant celle-ci et application de cette proteine dans une activation de lymphocytes b
WO1999061468A2 (fr) * 1998-05-27 1999-12-02 Gemma Biotechnology Ltd. INDUCTION DE PROTEINES ET DE PEPTIDES ANTIBIOTIQUES AU MOYEN DE LA PROTEINE sCD14/LAIT (PROTEINE IMMUNOTROPHIQUE ASSOCIEE A LA LACTATION)

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WO1992004908A1 (fr) * 1990-09-14 1992-04-02 Imtox Privatinstitut Für Immunbiologische Forschung Gmbh Medicament contenant du cd14
WO1998022580A2 (fr) * 1996-11-18 1998-05-28 The Wellesley Hospital Foundation Proteine immunotrope (cd14) associee a la lactation bovine, gene codant celle-ci et application de cette proteine dans une activation de lymphocytes b
WO1999061468A2 (fr) * 1998-05-27 1999-12-02 Gemma Biotechnology Ltd. INDUCTION DE PROTEINES ET DE PEPTIDES ANTIBIOTIQUES AU MOYEN DE LA PROTEINE sCD14/LAIT (PROTEINE IMMUNOTROPHIQUE ASSOCIEE A LA LACTATION)

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STELTER F. ET AL.: "The myeloid differentiation antigen CD14 is N- and O-glycosylated. Contribution of N-linked glycosylation to different soluble CD14 isoforms" EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 236, no. 2, March 1996 (1996-03), pages 457-464, XP000905218 *
WANG Y ET AL: "DETECTION AND IDENTIFICATION OF SOLUBLE CD14 IN BOVINE MILK" MOLECULAR BIOLOGY OF THE CELL,US,BETHESDA, MD, vol. 8, 1 November 1997 (1997-11-01), page 85A XP002062360 ISSN: 1059-1524 *
YANG Z ET AL: "SOLUBLE CD14 AND LIPOPOLYSACCHARIDE-BINDING PROTEIN FROM BOVINE SERUM ENABLE BACTERIAL LIPOPOLYSACCHARIDE-MEDIATED CYTOTOXICITY AND ACTIVATION OF BOVINE VASCULAR ENDOTHELIAL CELLS IN VITRO" JOURNAL OF LEUKOCYTE BIOLOGY,US,FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL, vol. 59, no. 2, 1 February 1996 (1996-02-01), pages 241-247, XP002062361 ISSN: 0741-5400 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2232273A1 (es) * 2003-03-21 2005-05-16 Jose Manuel Fernandez-Real Lemos Nuevas aplicaciones terapeuticas de la glucoproteina cd14s.
WO2004112512A1 (fr) * 2003-06-23 2004-12-29 Nestec S.A. Supplementation en acides amines pour un microbiote sain de l'ecosysteme
EP2382874A1 (fr) * 2003-06-23 2011-11-02 Nestec S.A. Supplémentation d'acides aminés pour un écosystème microbiotique sain
US9271958B2 (en) 2003-06-23 2016-03-01 Nestec S.A. Amino acid supplementation for a healthy microbiota ecosystem
US9884033B2 (en) 2003-06-23 2018-02-06 Nestec S.A. Amino acid supplementation for a healthy microbiota ecosystem
GB2433889B (en) * 2004-10-14 2010-02-17 Adventures Plus Pty Ltd Treatment of gastrointestinal and other disorders with a buffered admixture of vitamins, minerals and amino acids
WO2009095916A2 (fr) * 2008-01-29 2009-08-06 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Cd14 et peptides de celui-ci pour la protection de cellules contre la mort cellulaire
WO2009095916A3 (fr) * 2008-01-29 2009-11-26 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Cd14 et peptides de celui-ci pour la protection de cellules contre la mort cellulaire
JPWO2014104305A1 (ja) * 2012-12-28 2017-01-19 持田製薬株式会社 プレセプシン測定による炎症性腸疾患の診断
WO2014152985A1 (fr) * 2013-03-14 2014-09-25 Children's Hospital Medical Center Biomarqueurs de l'entérocolite nécrosante avant terme

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EP1123011A2 (fr) 2001-08-16
CA2347069A1 (fr) 2000-04-27
AU6474099A (en) 2000-05-08

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