NO161644B - PREPARATIONS FOR IN VITRO DIAGNOSTIC USE. - Google Patents

Description

The present invention relates to the use of preparations

ater for in vitro diagnosis of infections caused by uropathogenic E. coli bacteria.

Most bacterial infections occur due to

attack of bacteria on mucous membranes. For the establishment of such frictions, the bacteria's ability to adhere to the epithelial surface is

surfaces of greatest importance. The infectivity of gram-

negative bacteria (eg Escherichia coli) is thus directly related to the bacteria's ability to adhere to epithelial cells (reference 1). E. coli bacteria adhere more easily to vaginal epithelial cells from women who show a tendency to urinary tract infections compared to vaginal epithelial cells from healthy control women (reference 2). In connection with this, investigations both in vivo and in vitro have shown that a large number of bacteria adhere to periurethral epithelial cells from urinary tract infection-prone girls compared to the corresponding cells from healthy control girls (reference 3).

The importance of carbohydrate structures on cell surfaces

for biological recognition (ie receptors) has been strongly recognized recently. This also includes the bacteria's recognition (ie adhesion) of mammalian cells (reference 1). Studies have shown that the adhesion of several uropathogenic E. coli strains to periurethral cells is corre-

learned to the ability of specific agglutination only of human erythrocytes (ref. 4). From this, the conclusion can be drawn that certain substances, possibly of a carbohydrate nature, present on the surface of human erythrocytes are the receptors that are recognized by such urinary tract pathogens behind

teries.

An aim of the invention is to provide such

preparation or substance that can be used diagnostically.

An aim of the invention is to provide a method for the identification of receptor structures in natural biological material from mammals, including humans, in vitro

The invention relates in particular to receptor structures

for uropathogenic E. coli strains.

In connection with studies and experiments leading to the present invention, it has been found that the receptor for uropathogenic bacteria exposed on the surface of human erythrocytes contains a structural element with the minimum formula:

A particularly preferred structural element has the formula:

The active ingredient in the preparation

thus contains as active ingredient a structural element which appropriately has the formula:

In this formula, the symbol R denotes paranitrophenyl, methyl, 4-(N'-dodecylthioureido)-phenyl or ceramide which is bound in either the α- or 6-configuration.

Within this definition, the following compounds are of particular interest:

(p-nitrophenyl 4-O-a-g-galactopyranosyl~3-g-galactopyranoside); (p-nitrophenyl 4-O-(4-O-α-Q-galactopyranosyl)-3-D-galactopyranosyl-3-D-glucopyranoside);

(methyl 4-O-α-β;-galactopyranosyl-3-β:-galactopyranoside);

α-D-Galg-(1-4)-3-g-GalE~ (1-4 )-3-p-Glcp_-1-0-Me (methyl 4-0-(4-0-α-Q-galactopyranosyl)-3 -Q-galactopyranosyl-3-D-glucopyranoside); and

Ct-Q-Galg-(1-4)-3-g-Galp_-1-0-0-NHCSNH-(CH2)11~CH3 (4-(N<1->dodecylthioureido)-phenyl 4-0-a-g -galactopyranosyl-3-Q-galactopyranoside).

The relevant structural element with the minimum formula stated above is preferably arranged in the end position, i.e.

with the left end according to the formula in a freely exposed state.

The remaining preferred structural elements will appear

of the patent claims.

According to another aspect of the invention, there is

brought preparations that can be used e.g. for identification in-v i faith of receptor structures in natural biological material. The preparations contain one or more structural elements as indicated above, in at least a bivalent state and in covalent association. According to a suitable embodiment of the preparation, specified structural elements are covalently linked to a macromolecular

kylar carrier, possibly via a connecting arm. Synthetic or naturally occurring polypeptides or polysaccharides can be used as such macromolecular carriers.

Possible connecting arms between the structural element and the macromolecular carrier can be any of the following:

structural element macromolecular carrier, structural element macromolecular carrier, structural element macromolecular carrier, structural element macromolecular carrier, structural element aldonic acid

macromolecular carrier,

structural element - NH(CH„2 ) n - CO - NH - macromolecular carrier in which n can vary between 1 and 20.

Preferred structural elements in this type of preparation are specified in the subsequent patent claims.

The hemagglutination reaction of human erythrocytes caused by uropathogenic bacteria is due to interaction between acceptor structures (i.e. pili, syn. fimbriae, which are thread-like, rigid projections of a protein nature) on the surface of the bacterium and receptors on the surface of the erythrocytes containing the above-mentioned structural element.

This structural element is present preferentially on the surface of human erythrocytes as part of glycosphingolipid-trihexosyl-ceramide with the formula

corresponding to blood group antigen p k. A similar structural element is present on human erythrocytes also as part of glycosphingolipid corresponding to blood group antigen P: The above-mentioned glycosphingolipids present on human erythrocytes are also found on the surface of many other cells including epithelial cells. Of particular interest in this connection is the fact that glycosphingolipid-biosyl-ceramide of formula

containing the structural element identified above, is abundantly present in normal human renal tissue.

The fact that human urinary tract epithelial cells from individuals with the p phenotype (i.e. individuals lacking the glyco-sphingolipids P, P^ and P ) exhibit significantly lower binding of urinary tract pathogenic E. coli bacteria strongly suggests that the above-mentioned structural elements contained in the preparations has an influence on the bacteria's adhering capacity in the urinary tract.

Further evidence that the structural element

constitutes a significant part of the receptor in human cell surfaces is that of the substance

ability to inhibit the agglutination of human erythrocytes

k k

of P phenotype, i.e. of P^, , or P2 phenotypes.

Examples

The invention will be further described in the following examples.

Hemagglutination test

The strains of bacteria used in the experiments are listed

in the subsequent table 1, and of the strains shown there, all but strain T1 were isolated from children with acute pyelo-nephritis. The 20 faecal strains of E. coli were obtained from 20 healthy individuals, and one strain, H10407, obtained from D. J. Evans (Evans, D. G. , Evans, J. D. J. and Tjoa, W. (1977) Infect Immun. 18, 330-337) was also investigated.

Erythrocytes were obtained from cattle, guinea pigs, adults and umbilical cord blood. Erythrocytes from individuals of p, Vel(-) and Kp(b-) phenotypes were obtained from the blood bank at the university hospital in Umeå, and erythrocytes from a P-^ donor were obtained from Dr. H. Nevanlinna (Helsinki Red Cross Blood Central).

Blood samples were collected in 1% (w/v) sodium citrate or in acid citrate dextrose (ACD), and after washing 4 times in phosphate buffered saline (PBS), pH 7.2, the erythrocytes were suspended in PBS to 3% and applied on the same day. The hemagglutination tests were performed in the conventional manner (Kallenius, G. and Mollby, R. (1979). FEMS Microbiol. Lett. 5, 295-299).

Briefly, the bacteria were grown overnight on CFA agar (Evans, D. G., Evans, J. D. J. and

Tjoa, W. (1977). Infect. Immune. 1É5, 330-337) and suspended

in PBS to 5 x 10 9 bacteria per ml. Serial dilutions were made in PBS using 50 µl diluent in microtiter plates (Linbro Sc. Comp. Inc., Hamden, Connecticut). To each well (well) was added 50 ul PBS and

50/ul of the erythrocyte suspension to be examined. The hemagglutination titer was then determined after incubation at 4°C for 1 hour as the reciprocal of the highest dilution that produced visible agglutination of the erythrocytes. In all cases, parallel experiments were performed with 40 mM Q-mannose.

The results of these hemagglutination experiments are shown

in the following Table 1, from which it is clear that pyelonephritic E. coli strains all gave hemagglutination with human erythrocytes. In contrast, little or no agglutination was obtained using the faecal strains (Table 1).

For the hemagglutination inhibition tests, serial dilutions of the relevant inhibitor (active ingredient) (50 µl/well) and an equal volume of the bacterial suspension diluted to twice the lowest hemagglutination concentration were used. After 30 seconds of mixing, aliquots (50 µl) of the test erythrocytes were added to each well, and the inhibitory effect was then noted.

Example 1

Preparation of oligosaccharides containing the structural element α-Q-Galp-(.1-4)-g-Gal

A. α-Q-Galp-(1-4) -$-g-Galp-(1-4)-g-Glucitol

Erythrocytes from drawn blood of blood groups A, B, AB

and 0 became the light, and the membranes were isolated by centrifugation. In this way, approx. 10 g of membranes from 1 liter of blood. The membranes were pretreated by the procedure of Dodge, J. T., Mitchell, C., and Hanahan, D. J.

(1963), Arch. Biochem. Biophys. 100, 119-130, i.e. homogenized, suspended in water and lyophilized.

To 50 g of the lyophilized material, 1.25 1 of trifluoroacetic anhydride and 1.25 1 of trifluoroacetic acid were added, and the reaction mixture was heated to approx. 100°C in a tube of acid-proof steel at approx. 4 atmospheres overpressure in a period of approx. 4 8 hours.

After this treatment, the reaction mixture was cooled and evaporated to dryness, and a dark to black colored residue was obtained. To this residue was added

700 ml of methanol, and the mixture was evaporated to dryness. The residue was then diluted with a 50% aqueous solution of acetic acid (1000 ml) and allowed to stand at room temperature for approx. 18 hours. The reaction mixture was filtered with a glass filter and evaporated to dryness.

The residue obtained was partitioned between water and diethyl ether. The ether phase was washed 4 times with water, and the combined aqueous washings were washed 4 times with diethyl ether. The aqueous solution obtained was yellow and contained the released oligosaccharides.

The oligosaccharides were purified by gel chromatography followed by preparative paper chromatography, and the purification procedure was followed by gas chromatography-direct mass spectrometry.

The oligosaccharide described above was found in hemagglutinase ion inhibitory tests to be effective in inhibiting the hemagglutination induced by pyelonephritogenic E. coli strains. This fact shows that the oligosaccharide prevents interaction between the bacteria and the receptors in human erythrocytes.

B. The inhibition test according to A was repeated, but using a disaccharide derivative of formula

The compound was synthesized as follows: The octaacetate of 4-0-a-g-Galp_-a,p-g-Gal^ (ref. 14) was treated with hydrogen bromide in glacial acetic acid. The acetobromo disaccharide derivative formed was treated with sodium p-nitrophenoxide (ref. 15). After purification on silica gel, the product was de-O-acetylated with sodium methoxide in methanol during formation

of the connection.

This disaccharide derivative was found to inhibit the hemagglutination reaction in the same manner as shown under section A.

C. The inhibition test according to A was repeated using the saccharide derivative of the formula

as an inhibitor.

This compound was synthesized as follows: The undecaacetate of 4-0-a-g-Galp_-3-g-Galp-a , 3-D~Glcp_

(ref. 16) was treated with hydrogen bromide in glacial acetic acid. The acetobromotrisaccharide derivative formed was treated with sodium p-nitrophenoxide (ref. 15). After purification on silica gel, the product was de-O-acetylated with sodium methoxide in methanol to form the compound.

This saccharide derivative was also found to inhibit the hemagglutination reaction.

D. The inhibition test described above was repeated using a disaccharide derivative of formula

This compound was synthesized as follows:

The acetobromo-disaccharide derivative (described under B)

was treated with silver oxide in methanol to form the methylglycoside (ref. 17). After purification on silica gel, the product was de-O-acetylated with sodium methoxide in methanol to form the compound.

The saccharide derivative was found to inhibit the hemagglutination reaction as described above.

E. The inhibition test described above was repeated using a saccharide derivative of formula

This compound was synthesized as described in reference no. 16 and was found to inhibit the hemagglutinase ion reaction.

Example 2

Preparation of preparations containing the structural element in at least a bivalent state linked to a macromolecular carrier

I. The preparation was prepared from natural oligosaccharides containing a free reducing end sugar

rest. In the manufacturing sequences schematically shown below, X symbolizes the structural element according to the invention, while the abbreviation MMB relates to macromolecule carriers.

a. The following reaction sequences illustrate the preparation of a preparation according to the invention containing the active structural element covalently bound to a macromolecular carrier via a linking arm.

b. The reactions were carried out according to Ia as above, but the p-aminophenethylamine compound was not converted to the corresponding phenylisothiocyanate compound, but was instead diazotized, and the diazonium derivative was then reacted with free primary aliphatic amines on the macromolecular support (ref. 5 , 6).

f. Preparation of preparations containing the active element as defined could also be carried out by incorporating synthetic glycolipids (see below) into liposomes or erythrocytes or other lipophilic carriers.

Example 3

a. Characterization of the receptor of MRHA^um

In this context, MRHA means "mannose-resistant hemagglutination of human erythrocytes".

Treatment of human erythrocytes with α-galacto-sidase reduces their hemagglutination. This is an indication that α-γ-galactopyranoside groups in an end position are of particular importance in the receptor structure.

The para-nitro derivatives and methyl derivatives of the di- and trisaccharides, i.e. compounds according to example IB, 1C,

ID and 1E, were found to be effective in inhibiting MRHA^^ with different pyelonephritogenic strains of E. coli and with blood from different donors. This indicates the fact that the glucose molecule is of minor importance, as no difference between the di- and trisaccharides with regard to inhibitory capacity was observed.

Erythrocytes from guinea pigs and from p-individuals that normally react with P-fimbriae were coated with purified trihexosyl-ceramide (THC). These erythrocytes were then reacted with MRHAj^^ with P-fimbriae-containing E. coli. Thus, THC acts as a receptor for hemagglutination. The test was performed both with commercial THC (Supelco, U.S.A.) and with THC prepared from pig intestines by A. Lundblad and S. Svensson, Lund, Sweden.

Synthetic disaccharide was converted to the corresponding p-isothiocyanotophenyl derivative and was reacted with dodecylamine to give the synthetic glycolipid according to the formula

This compound was synthesized as follows: The compound according to Example IB was hydrogenated using Adam's catalyst (PtO 2 ). The p-aminophenylglycoside thus formed was converted to the corresponding p-isothio-cyanatophenylglycoside by treatment with thiophosgene, mainly as described in ref. 18. This glycoside was then reacted with dodecyl-1-amine, and the compound formed was purified on silica gel. This synthetic glycolipid was used in a corresponding coating test, and a positive result was obtained. This test indicates that the results obtained above with preparations made from biological material were not affected by contaminants present in the preparations.

Adhesion tests with uroepithelial cells

Uroepithelial cells in morning urine from p-phenotype individuals were compared in parallel tests with cells from P-individuals with respect to their ability to adhere three strains of pyelonephritogenic E. coli with P. fimbriae. In the same way as the erythrocytes, the β-cells bound significantly smaller amounts of bacteria (p < 0.02). As these individuals lack the receptor structure on their epithelial cells, this also confirms the fact that the structure in question acts as a receptor also for adhesion to urinary epithelium.

The adhesion to uroepithelial cells (of P type) of pyelonephritogenic E. coli was effectively inhibited by the synthetic disaccharide derivative (the compound of Example ID). This proves that the disaccharide structure is a receptor structure involved in adhesion to epithelial cells.

Adhesion to the same cells was increased approx. 3 times by adding the para-nitro-phenyl derivative of the disaccharide (the compound according to Example 1C), when the lipophilic para-nitrophenyl group was associated with the lipophilic membrane of the cell resulting in coating. This shows that adhesion to epithelial cells is also obtained by coating the cells with a synthetic receptor substance.

Example 4

Production of antibodies with specificity against the relevant structural element

a. Production of monoclonal antibodies by hybridoma technique.

I. Balb/c mice were immunized with a preparation according to example 2 or 3. The spleen from the hyperimmunized animal was harvested, and a cell suspension was prepared by mechanical pulverization of the tissue. After gradient centrifugation to form a pure cell preparation, the cells were fused using polyethylene glycol (PEG average molecular weight 1500) with established B-myeloma cell lines from Balb/c mice according to known techniques. After cloning the hybridoma cells that developed the desired antibody, the cells were propagated on a large scale. The culture medium supernatants were harvested and the antibodies purified by conventional means. The so-called enzyme-linked immunosorbent assay (ELISA) was used for identification of the anti-substance developing clones (ref. 11).

II. Mammals were immunized with an oligosaccharide-

protein or polymer preparation according to example 2 or 3. Antibodies were isolated from the hyperimmune serum of the mammal and purified by conventional techniques.

Example 5

Test for the identification of bacteria with an acceptor structure that exhibits specificity against the relevant structural element a. Bacteria were incubated with human erythrocytes of the P phenotype. Human erythrocytes of p phenotype were used as negative control cells. Positive hemagglutination of Pf but not of p erythrocytes confirmed the fact that the bacterium in question exhibits acceptor structures. The test was carried out according to the previously described hemagglutination technique.

b. E. coli bacteria were incubated with a preparation in which the relevant structural element was covalently or in some other way in multivalent form bound to a specific matrix according to example 2 or 3. The incubation was carried out on preparation slides for approx. 15 minutes, and the preparation was then studied under a light microscope. In the case of a positive reaction, the particles were found to be completely covered by bacteria that adhere to the receptor structures in the particles. With a negative reaction, the particles are completely free of bound bacteria. c. E. coli bacteria were mixed with a preparation according to example 2 or 3 directly on a slide, and a positive reaction resulted in an agglutination reaction visible to the naked eye. The receptor structures described here resulted in a positive reaction.

Example 6

Purification of acceptor structures of the bacteria (pili,

sight, fimbriae)

A preparation according to example 2 or 3 was arranged in the form of a column. E. coli bacteria were treated mechanically which released the filamentous projections (pili). A slurry of the released pili was then passed through the column, the acceptor structures being held in the column by interaction with the receptor structures in the column. After rinsing the column, the acceptor structures retained therein could then be eluted by addition of the disaccharide derivative (described under ID) and were obtained in purified form. Purified pili can be used in vaccines or for antibody analyzes in e.g. body fluids, such as urine or breast milk.

In the present description, the abbreviations that have been used in the structural formulas have the following meanings:

Galp_ = galactopyranosyl

Glcp_ = glucopyranosyl

GlcNAcp_ = 2-acetamido-2-deoxy-glucapyranosyl

Cer. = ceramide

It should be noted that the invention is not limited to the embodiments described in the examples, but that many modifications are possible within the framework of the invention.

References

1. Jones, G.W. (1977) in Microbial Interactions (Reisaig, K.L. ed.), Receptors and Recognition.

Looking. B, Volume 3, pp. 139-176, Chapman and Hall, London.

2. Fowler, J.E. & Stamey, T. (1977). J. Turmoil. 1, 472-476. 3. Kallenius, G. & Winberg, J. (1978). Lancet. ii, 540-543. 4. Kallenius, G. & Mollby, R. (1979). FEMS Microbiol.

Easy. 5, 295-299. 5. Svenson, S.B. and A.A. Lindberg (1979), J. Immunol.

Meth. 25, 323.

6. Zopf, D. et al (1978) Immunol. Meth. enzymol. L,

part C, 163.

7. Lemieux et al (1975) JACS 97:14, 4076.

8. Lonngren, J. et al (1976) Arch. Biochem. Biophys.

175, 661. 9. Svenson, S.B. and A.A. Lindberg (1978) J. Immunol. 120, 1750.

10. Gray, G.R. (1978). In: Meth. enzymol. L, Part C, 155.

11. Svenson S.B. and K. Larsen (1977), J. Immunol. Meth. 17, 249. 12. J.K.N. Jonen and W.W. Reid, J. Chem. Soc. (1955) 1890. 13. D.M. Marcus, N. Naiki and S.K. Customer. Pro. Nat. Acad.

Pollock. USA 7_3 (1976), 3263-3267. 14. Cox, D.D., Metzner, E.K., Reist, E.J. A new synthesis of 4-0-a-D-galactopyranosyl-D-galactopyranose.

Carbohydrate Research 62, 245-252 (1978).

15. Sham, R., Bahl, 0. Reaction of tetraacetyl a-D-hexopyranoside bromides with sodium nitrophenoxide in PNF. Formation of p-nitrophenyl hexopyranosides ....

Carbohydrate Research 74, 105-116 (1979).

16. Cox, D.D., Metzner, K., Reist, E.J. The synthesis of methyl 4-0-(4-0-a-g-galactopyranosyl-p-B-galactopyranosyl)-3-0-glycopyranoside: The methyl 3-glycoside of the trisaccharide related to Fabry's disease. Carbohydrate Research 63, 139-147 (1978).

17. Koenigs, W., Knorr, E.

Berichte 34, 957- (1901).

18. McBroora, C.R., Samanen, C.H., Goldstein, I.J.

in: Methods in Enzymology, Volume 28B, ed.

V. Ginsburg, p. 212. Academic Press, New York (1972).

Claims (10)

1. Use of a preparation containing an active ingredient consisting of the structural element, preferably in the final position: for in vitro diagnosis of infections caused by uropathogenic Escherichia coli bacteria. 2. Use according to claim 1 of a preparation containing the structural element of formula in which R is paranitrophenyl, methyl, 4-(N'-dodecylthioureido)-phenyl or ceramide which is bound in either the a- or 3-configuration, or wherein R is as above indicated. 3. Use according to claim 1 or 2 of preparation which contains the structure element 4. Use according to any of claims 1-3 of a preparation containing the structural element in at least a bivalent state and in covalent association. 5. Use according to claim 4 of a preparation containing the structural element covalently bound to a macromolecular carrier. 6. Use according to claim 4 of a preparation containing the structural element covalently bound to a macromolecular carrier via a connecting arm. 7. Use according to claim 5 or 6 of a preparation containing as macromolecular carrier a synthetic or naturally occurring polypeptide or polysaccharide. 8. Use according to claims 5 - 7 of a preparation where the connecting arm between the structural element and the macromolecular carrier consists of structural element macromolecular carrier, structural element macromolecular carrier, structural element macromolecular carrier/ structural element macromolecular carrier, structural element aldonic acid macromolecular carrier, or structural element -NH(CH2)n~CO-NH - macromolecular carrier, where n can vary between 1 and 20. 9. Use according to any one of claims 1 - 3 of a preparation containing the structural element in a bivalent state. 10. Use according to claim 9 of a preparation containing the structural element α-Q-Galp_-(1-4)-6-Q-Galp-(1-4)-3-D-Glcp-1-O-Ceramide α-D-Galp_-(1 -4) -B-p-Galp_-(1 -4) -B-Q-GlcNAcp-(1 -3) -S-Q-Galp_-(1 - 4) - B-Q-Glcp-I-O-Ceramide, or α-D-Galp_-(1-4)-tf-Q-Galp-1 -O-Ceramide.