Microbiomarker for Celiac Disease and a Related Product

Abstract

The present invention relates to the field of medicine and in particular to celiac disease (CD). Specifically the present invention relates to methods and means for detection of CD using novel microbiomarker, celiac gut index (CGI). The invention relates also to methods and means for treatment or prophylaxis of CD. The present invention provides a novel product comprising a gut microbiome altering agent that, when administered to an individual, improves the state of health of individuals suffering or susceptible to suffer from celiac disease and possibly reduces the likelihood of acquiring celiac disease. The product of the invention provides a natural and safe manner for the treatment of celiac disease. The present invention provides also a novel method for aiding diagnosis of CD by the specific gut health biomarker, Celiac Gut Index (GCI). In the method a probability of a subject having celiac disease is determined by measuring the relative abundances of one or more microbial taxa in a fecal sample from a subject; and the probability of the subject having CD is determined based on the measured abundances as celiac gut index (CGI).

Claims

1. A product comprising a gut microbiome altering agent that, when administered to an individual, increases a ratio of the total amount of Bifidobacteria and Faecalibacterium to GNPB (gram negative Proteobacteria).

2. The product according to claim 1, wherein the product includes at least one probiotic strain or a combination of strains that stimulate the growth of Faecalibacterium group bacteria and have an effect on villus growth and gut permeability in the intestinal tract.

3. The product according to claim 1, wherein the product includes Lactobacillus rhamnosus-, and Bifidobacterium lactis-strains and optionally prebiotics and/or a product stimulating Bifidobacteria and Faecalibacterium, or any combination of these.

4. The product according to claim 3, wherein the Lactobacillus rhamnosus-, and Bifidobacterium lactis-strains are Lactobacillus rhamnosus SP1 and Bifidobacterium lactis BLC1.

5. The product according to claim 3, wherein the prebiotic comprises fructo- and galacto-oligosaccharides, xylo-oligosaccharides, fibres or oat.

6. The product according to claim 1 for use in improving gut of health of individuals suffering or susceptible to suffer from celiac disease and for reducing the likelihood of acquiring celiac disease.

7. A method for aiding diagnosis of celiac disease in an individual, the method comprising: (a) determining a ratio of the total amount of Bifidobacteria and Faecalibacterium to gram-negative Proteobacteria (GNPB) in a fecal sample of an individual; and b) comparing the ratio determined in step a) to a reference value, wherein a decreased ratio of the total amount of Bifidobacteria and Faecalibacterium to gram-negative Proteobacteria (GNPB) compared to the reference value is indicative of celiac disease in said individual.

8. The method according to claim 7 for use in monitoring an individual's response to treatment of celiac disease.

9. A method for treatment of celiac disease, the method comprising administering an effective amount of a product increasing a ratio of the total amount of Bifidobacteria and Faecalibacterium to GNPB (gram negative Proteobacteria) to celiac disease patients or people suspected to have celiac disease.

10. The method of claim 9, wherein the product comprises a gut microbiome altering agent that, when administered to an individual, increases the ratio of the total amount of Bifidobacteria and Faecalibacterium to GNPB (gram negative Proteobacteria).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0019] As used herein the term celiac disease encompasses a spectrum of conditions caused by varying degrees of gluten sensitivity, including a severe form characterised by a flat small intestinal mucosa (hyperplastic villous atrophy) and other forms characterised by milder symptoms.

[0020] The individual used herein in the context of diagnosis or therapy is human. The individuals may have symptomatic or asymptomatic celiac disease or be suspected of having it. They may be on a gluten free diet. They may be susceptible to celiac disease, such as a genetic susceptibility.

[0021] Gut microbiota is an extremely complex ecosystem (over 1000 species, in total more than 10.sup.14 bacteria). A change in the percentual proportion of one bacteria group also changes the percentual proportions of other bacteria groups significant to health. For this reason, it is important to deal not only with the change in individual bacteria groups but also with the change in the whole system.

[0022] In the method of the present invention the probability of a subject having celiac disease (CD) is determined by a method wherein a sample is obtained from a subject; the relative abundances of one or more microbial taxa in the sample are measured; and the probability of the subject having CD is determined based on the measured relative abundances of one or multiple microbial taxa in the sample. Specifically, in the present invention, the composition of the bacterial system of gut is represented by a simple and understandable Celiac Gut Index (CGI) and the index is utilized in a method for aiding diagnosis of celiac disease. The CGI index is calculated from the percentual proportions of three bacteria groups and genera significant to gut health, i.e. by dividing the sum of the amount of Bifidobacteria and Faecalibacterium in a sample by the amount of gram-negative Proteobacteria (GNPB). The change in the ratio of the total amount of Bifidobacteria and Faecalibacterium to the amount of GNPB in the intestines correlates negatively with celiac disease. The expression correlates negatively means that when one variable increases, another one decreases. Thus, the higher the relative value of the CGI in a subject is, the lower is the likelihood of having celiac disease. In other words, low CGI is a specific microbiomarker for dysbiotic gut microbiota composition and an indication of celiac disease. CGI is low when the level of potentially inflammatory bacteria, GNPB, is high compared to amounts of gut protective groups of bacteria, Bifidobacteria and Faecalibacterium. In an embodiment of this invention, the CGI index value above 15 represents a healthy phenotype and the value below 10, in turn, represents a phenotype with celiac disease.

[0023] The CGI index is determined indirectly by analysing the microbiota in the intestinal contents. Normally a fecal sample of the subject is examined to quantitatively determine the Bifidobacteria, Faecalibacterium and GNPB and/or total bacteria by methods known per se. Preferably, the proportion of these bacteria in the total bacteria is determined by a method based on 16S rRNA hybridisation, DNA staining and flow cytometry (FCM-FISH), which allows different gut bacteria groups to be determined rapidly and reliably. The relative abundance of different gut bacteria groups can also be measured using techniques based on DNA sequencing, quantitative PCR, DNA microarray, or any other suitable method. In one embodiment of the invention the gut microbiota composition, i.e. the CGI index, is determined from a non-invasive fecal sample by flow cytometry-FISH methodology.

[0024] All of the above mentioned bacteria groups belong, in the light of present knowledge to the commensal human gut microbiota. Bifidobacteria and Faecalibacterium are both known to have positive effect on the condition of gut villus. Bifidobacteria are gram-positive, immobile anaerobic bacteria that appear in the digestive tract (and belong to the Bifidobacteriaceae family and particularly to the Bifidobacterium genus). Faecalibacterium refers herein to Faecalibacterium prausnitzii, which is the most abundant bacterium in the human intestinal microbiota of healthy adults, representing more than 10% of the total bacterial population. F. prausnitzii are gram-negative anaerobic bacteria, which belong to the Clostridium leptum group (Clostridium cluster IV), belonging to phylum Firmicutes. Proteobacteria (gram-negative proteobacteria, GNPB) are a major group (phylum) of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter, and many other notable genera. In excessive amounts proteobacteria may potentially cause inflammation in the gut.

[0025] The CGI index can be used for monitoring a change in the composition of the gut bacteria for instance when a person has started a gluten-free diet by taking samples at different points of dietary treatment. By means of the CGI index, a customized probiotic and/or prebiotic intervention aiming at alleviating symptoms of celiac disease is possible. CGI index may serve as a preliminary microbiological biomarker i.e. microbiomarker for celiac disease in addition to genetic marker (HLA) tests from blood. The CGI index may be determined before invasive biopsies are taken from patients.

[0026] Changes in diet can induce significant changes in the gut microbiota composition. The present invention provides a novel product containing micro-organisms that improves the state of health of individuals suffering or susceptible to suffer from disorders related to the ingestion of gluten, especially celiac disease and reduces the likelihood of acquiring celiac disease. The product acts on the gut microflora and changes the relative proportion of Bifidobacteria and Faecalibacterium and Proteobacteria (GNPB) in the intestines and, in particular, the ratio of Bifidobacteria and Faecalibacterium to GNPB. The relative proportion of e.g. bifidobacteria refers to the ratio of e.g. Bifidobacteria to other bacteria in the intestines, normally to the total bacteria. The product according to the invention may also have a decreasing effect on the relative proportion of Proteobacteria (GNPB) to the total bacteria in the intestines.

[0027] The product of the present invention contains living active bacteria in a concentrated, freeze-dried form. The product includes at least one probiotic strain or combination of strains that stimulate the growth of Faecalibacterium group bacteria and has an effect on villus growth and gut permeability in the intestinal tract. Preferably the product includes selected probiotic lactic acid bacteria, such as Lactobacillus rhamnosus-, and Bifidobacterium lactis-strains. Bifidobacteria lactis are preferably selected from strains Bifidobacterium animalis subsp. lactis Bb-12, Bifidobacterium lactis BLC1 and Lactobacillus acidophilus. According to one embodiment of the invention the product includes two live, bacterial strains Lactobacillus rhamnosus SP1 (DSM 21690) and Bifidobacterium lactis BLC1 (DSM 17741, LGM23512), both 1000 millions/dose. The product of the invention may also be any combination of above mentioned Bifidobacteria, Lactobacilli, prebiotic, probiotic or a product stimulating Bifidobacteria.

[0028] The daily dose of the bacteria is preferably 100-1000 mil bacteria. The dose may be adjusted based on the personal CGI. An increase in CGI indicates improved gut health. Higher CGI in the end of intervention indicates that the amounts so-called beneficial bacteria (Bifidobacteria and Faecalibacterium) and potentially inflammatory bacteria (GNPB) are more balanced.

[0029] The product of the invention may be in the form of a food composition, pharmaceutical composition, nutraceutical, or supplement. The product of the invention may be administered mixed in food or drink, for example, or separately in the form of a tablets, capsules, microcapsules, powders, solutions, pastes, etc. Food composition may be any kind of food (functional, conventional and novel), food supplement, formula for nutritional purposes, or nutraceutical and it may contain any suitable additives and excipients. The product in the form of a pharmaceutical composition may be used in treatment or prevention of celiac disease.

[0030] The increase of the amount of Faecalibacterium is enhanced by using in combination with probiotics some prebiotic compound.Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Prebiotics are indigestible food ingredients that have a beneficial effect on the intestinal tract. A prebiotic through their selective metabolism is a component which is usually a carbohydrate (an oligo- or polysaccharide) and which has a selective promoting effect on the growth or activity of one or more bacterial strains in the colon. A prebiotic is preferably a fructo- or galacto-oligosaccharide, fibre, particularly cereal fibre, such as bglucan of oat, pure oat, polydextrose, special sugar, such as isomaltulose, or for example a fatty acid, such as omega-3 fatty acid, or any mixture of these. Preferably pure oat is used. In an unpublished animal trial it was shown that feed supplemented with dehulled oat increased the amount of Faecalibacterium by 74 percentages in piglet feces after the 3-week intervention (GutGuide Oy, unpublished results). This result suggests that pure oat may be suitable liquid fiber source for celiac patients and could serve as an efficient prebiotic combined with probiotic strain(s).

[0031] The product stimulating Bifidobacteria may be a product containing propionic acid bacteria, such as Propionibacterium freudenreichii, Propionibacterium shermanii, and/or Lactobacilli, such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei or Lactobacillus lactis. The bacterial strains of the product can be combined with other microorganisms and bioactive compounds to improve their protective and metabolic properties.

[0032] The present invention relates further to a method for treatment or prevention of CD in an individual by administering said individual an effective amount of a composition comprising a gut microbiome altering agent that, when administered to an individual, increases the CGI. An effective amount of a composition increasing the CGI refers to an amount sufficient for changing the relative proportion of Bifidobacteria and Faecalibacterium and GNPB in the intestines and, in particular, increasing the ratio of Bifidobacteria and Faecalibacterium to GNPB in the gut. Preferably, the composition to be administered is the product of the present invention. The term treatment may refer to both therapeutic treatment and prophylactic treatment or preventative measures, wherein the goal of the treatment is to slow the disease process or even stop it, or prevent CD.

EXAMPLES

[0033] Fecal samples were collected from 34 adult Finns that have elicited a celiac diagnose (3 men, 31 women) and were already on gluten-free diet. Two samples, pre and post intervention, were collected from each participant. Pre samples were collected before the consumption of the test product and post samples after 40 day use of the test product.

[0034] The gut microbiota composition analysed from the pre samples were compared to the reference database i.e. database of gut microbiota composition of healthy Finns without any gastrointestinal disorders or diseases.

[0035] The test product included two live, bacterial strains Lactobacillus rhamnosus SP1 (DSM 21690) and Bifidobacterium lactis BLC1(DSM 17741, LGM23512), both 1000 millions/dose. The bacterials strains were obtained from Sacco Ltd, Italia. In addition, a minimal amount (1 g) of fructo-oligosaccharide, (FOS) were included in the product in order to support beneficial probiotic events in the gut. All subjects consumed the product i.e. each subject served as his/her own control.

[0036] After the 40-day-period of consumption the post samples were collected and the gut microbiota composition were analysed by using a method based on whole cell, 16S rRNA in situ hybridisation, DNA staining and flow cytometry (Vaahtovuo J et al. J Microbiol Methods. 2005; 63:276-286). Briefly, bacteria from fecal samples were isolated from debris and fixed prior the in situ hybridisation. The following gut bacterial groups or genera were determined from them: Bifidobacteria, Faecalibacterium and GNPB including for example Escherichia coli. All of the above bacteria groups belong, in the light of present knowledge to the commensal human gut microbiota. The percentage proportions (of total bacteria amount) of bacteria groups were determined from the samples. Celiac Gut Index (CGI) was counted for each person by dividing the sum of percentages of Bifidobacteria and Faecalibacterium by the percentage of GNPB.

[0037] The statistical analysis has been done by IBM Statistics-program by using Student's paired t-test (Ce vs. normal and pre-post analysis).

Results

[0038] Comparison of the gut microbiota composition of celiacs compared to reference data revealed significant differences in all the bacterial groups/genera analysed (Table 1). In addition, there is a clear significant difference in CGI between the groups (Table 1). Low CGI could serve as a new specific biomarker for dysbiotic, unbalanced gut microbiota composition that may refer to celiac disease. In celiac patients the level of potentially inflammatory bacteria (GNPB) is high compared to amounts of gut protective groups of bacteria.

TABLE-US-00001 TABLE 1 The comparison of the gut microbiota composition between celiacs pre samples and reference data (healthy adults). Reference Celiacs data P Bifidobacteria (%) 4.7 7.4 0.003 Faecalibacterium (%) 5.3 10.0 <0.001 GNPB (%) 2.1 0.9 <0.001 CGI.sup. 6.6 21.8 <0.001 CGI = Celiac Gut Index, GNPB = gram-negative proteobacteria .sup.CGI was calculated for each individual as described i.e. value in the Table 1 is average of those values.

[0039] The test product had clear, beneficial effect on the gut microbiota composition of the celiacs after the 40 days intervention. The amount of beneficial bacterial group Faecalibacterium increased significantly, and the amount of potentially, inflammatory, enteric bacteria decreased (Table 2). The overall gut microbiota composition described by the significant increase in CGI (Table 2). The significant increase of CGI indicates that improved gut health was obtained after the consumption of test product. Higher CGI in the end of intervention indicates that the amounts so-called beneficial bacteria (Bifidobacteria and Faecalibacterium) and potentially inflammatory are more balanced.

TABLE-US-00002 TABLE 2 Effects of the intervention (test product for 40 days) on gut microbiota composition Percentual Pre Post change p Bifidobacteria (%) 4.7 5.1 7.8 >0.05 Faecalibacterium 5.3 6.7 26.4 0.010 (%) GNPB (%) 2.1 1.4 33.3 <0.001 CGI 6.6 10.3 56.1 0.002

[0040] It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.

REFERENCES

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