Patent Application
20080038228
The invention will now be further described only by way of example in which reference is made to the following examples and Figures:
The human colorectal carcinoma cell line Caco-2 was grown at 37° C. and 5% CO2 in serum-free Dulbeccos' MEM (Gibco) supplemented with 1 mM sodium pyruvate (Gibco) and 1× non-essential amino acids (Gibco) (DMEM). When grown without added bacteria, 20 Uml−1 penicillin (Gibco), 20 μgml−1 streptomycin (Gibco) and 0.5 μgml−1 amphotericin (Gibco) were added to the medium. Bifidobacterium sp. 420 was grown anaerobically for 48 hours at 37° C. in a BIF medium consisting of 10 gl−1 tryptic digest of casein peptone; 5 gl−1 meat extract; 5 gl−1 yeast extract; 10 gl−1 glucose; 3 gl−1 K2HPO4; 0.1% (volume/volume) Tween 80; 1% ascorbic acid and 0.05% cysteine-HCl; pH 6.8. 2 ml of logarithmic phase culture was centrifuged at 4000 rpm for 3 minutes. The cell pellet was suspended in 1.6 ml DMEM. The production of short chain organic acids by the bacterium was tested from fresh and conditioned bifidobacterium medium using gas chromatography.
To determine the effects of various treatments on the Cox-1/Cox-2 expression pattern, approximately 500 000 Caco-2 cells/well were seeded on 24-well cell culture plates. The cells were allowed to attach for 24 hours, after which the media were replaced by fresh media (total volume 1 ml/well) containing either no other added components than the ones described above (control treatments, with and without antibiotics) or antibiotic supplemented media with 5 mM sodium butyrate; 5 mM sodium propionate; 5 mM sodium acetate or 5 mM sodium lactate. In addition, wells were prepared that contained no antibiotics but a 0.2 μm anopore membrane tissue culture insert (Nunc, Denmark) into which 100 μl of the bacterial suspension described above was added.
After 24 hour exposure, media and culture inserts were discarded, cells were lysed and RNA was extracted using Qiagen's (Germany) RNEasy Mini Kit DNA was digested using the same manufacturer's RNase free DNase. Reverse transcription was performed using the High Capacity cDNA Archive Kit (Applied Biosystems, USA) according to the instructions provided by the manufacturer. Cox-1/Cox-2 expression patterns were determined by real-time quantitative TaqMan PCR (Holland et al., 1991 Proc. Natl. Acad. Sci. USA August 15; 88(16): 7276-80; and Livak and Scmittgen, 2001 Methods December; 25(4):402-8) using the default settings of an ABIPrism 7000 Sequence Detection instrument (Applied Biosystems).
Table 1 and
As can be seen in the
Inhibition of Cox-1 enzymatic activity can be detrimental; this is the main cause of the well-known side effects of non-steroidal anti-inflammatory drugs. It is likely that, in vivo, an equally detrimental condition is achieved in various situations where Cox-1 transcription level is decreased. In both of these situations—inhibition of Cox-1 enzymatic activity and decrease of Cox-1 transcription—an agent, such as a microorganism (or metabolite thereof) capable of modulating host cyclooxygenase expression, could be used to normalise Cox-1 expression level by increasing it so that sufficient Cox-1 enzymatic activity is achieved.
Table 2 shows the volatile fatty acid contents of fresh and filtered, conditioned bifidobacterial growth medium.
As can be seen in the table, Bifidobacterium sp. 420 produces acetate and lactate but only a minimal amount of butyrate, and no propionate. According to prior art, it is known that butyrate and propionate have a lowering effect on Cox-2 expression. In the present work it becomes evident that
We conclude that the distorted Cox-1/Cox-2 expression pattern seen in many disorders, including disorders of the intestine, can be corrected without any known side-effects using a microorganism and/or a microbial suspension (for example comprising at least one metabolite of the microorganism) capable of affecting the expression pattern. By modifying the expression pattern using microorganisms or a microbial suspension thereof (for example comprising at least one metabolite of the microorganism) the status of the cells, tissues, organs or organisms can be shifted from a tumourigenic and carcinogenic state to anti-tumourigenic and anti-carcinogenic.
Two bifidobacteria (Bifidobacterium sp. 420 and Bifidobacterium longum 913) were cultured as described in Experiment 1. Lactobacillus acidophilus 770, Escherichia coli (ATCC 1175) and Salmonella enteritidis were grown aerobically at 37° C. in MRS broth (Becton Dickinson, USA; lactobacillus) or in tryptic soy broth (TSB, LAB M, England; E. coli and S. enteritidis). Bacterial growth was stopped by chilling confluent cultures on ice, after which cell densities were determined by flow cytometry (FACS Calibur, Becton Dickinson). Culture supernatants were filtered through 0.22 μm sterile filter units (Mllipore, USA). The short chain organic acid contents of the supernatants and of fresh BIF, MRS and TSB media were determined using gas chromatography.
Filtered bacterial culture supernatants were diluted 1/10 in serum-free Dulbeccos' MEM (Gibco) supplemented with 1 mM sodium pyruvate (Gibco) and 1× non-essential amino acids (Gibco). The effects of soluble microbial metabolites on Cox-1/Cox-2 expression pattern were then determined using the Caco-2 cell-based exposure test described in Experiment 1. Two kinds of controls were included in the experiment: a base-level control (hereafter called control) where Caco-2 cells were only given DMEM; and three bacterial growth medium controls where caco-2 cells were given 10% unused BIF, MRS or TSB medium diluted in DMEM. The latter controls were included in the experiment to confirm that any potential changes seen in Cox-1/Cox-2 expression pattern would not be caused by some component of the bacterial growth media.
Table 3 and
Bifidobecterium sp. 420
B. longum
L. acidophilus
E. coli
S. enteritidis
aControl = Caco-2 cells maintained in serum-free Dulbecco's MEM with 1 X non-essential amino acids and 1 mM sodium pyruvate (DMEM); Fresh BIF, MRS or TSB medium = 10% fresh, unused bacterial culture media in DMEM.
Fresh bacterial culture media had little effect on Cox expression profile, although TSB medium caused an approximately 2-fold induction of Cox-2 in relation to the control treatment Within 22 hours, metabolites produced by bifidobacteria caused a 2.5 (Bifidobacterium sp. 420) and 3.2 (B. longum)-fold increase in Cox-1 expression with a simultaneous decrease (0.5 and 0.6-fold for B. sp. 420 and B. longum, respectively) in Cox-2 expression. L. acidophilus had a different effect: Cox-1 and Cox-2 expression levels, compared to the untreated control, were 1.7 and 1.3. These changes were hardly distinguishable from those caused by fresh MRS medium (1.6 and 1.0 for Cox-1 and Cox-2, respectively), for which reason it is questionable whether L. acidophilus had any effect at all. E. coli and S. enteritidis caused no significant change in Cox-1 expression either but a slight up-regulation of Cox-2 (1.6 and 1.5, respectively)—this induction, however, was less than that caused by fresh TSB medium, so based on this, the metabolites produced by these two bacterial species probably do not have a significant effect on Cox expression pattern.
Based on these results, it can be concluded that the ability to modulate host cyclooxygenase expression profile is not common to all probiotic and non-probiotic microorganisms. Only specific microorganisms are capable of producing such an effect. In this experiment, it has been clearly shown that the bifidobacteria that were tested caused an anti-tumourigenic and anti-inflammatory effect similar to that obtained using butyrate or propionate (Experiment 1). L. acidophilus and E. coli, both of which are used as probiotics, did not have such an effect. Neither did S. enteritidis, which is a pathogenic organism.
Of course, a skilled person following the teachings in the present application would have the ability to screen probiotic and non-probiotic microorganisms to identify specific microorganisms, additional to the ones specifically taught herein, capable of producing the claimed effect. In particular, the skilled person could screen microorganisms using the “Caco-2 cell-based exposure assay” taught above. Microorganisms which cause an increase in Cox-1 expression level and/or increase the Cox-1/Cox-2 ratio compared with an untreated control, may be microorganisms which can be used in accordance with the present invention.
Cyclooxygenases (Cox) 1 and 2 play important roles in gastrointestinal health; chronic overexpression of Cox-2 is associated with inflammatory and cancerous disease, whereas Cox-1 is expressed constitutively and its inhibition results in gastrointestinal malfunction. We set up a standardised cell culture-based screening assay for investigation of the effects of food components on intestinal epithelial gene expression profiles. In this model, we studied the effects of two probiotic bacterial strains (Bifidobacterium sp. 420 and Lactobacillus acidophilus) on the expression levels of the Cox genes in the enterocyte-like Caco-2 cells. Bifidobacterial metabolites shifted the Cox-1/Cox-2 ratio by increasing the amount of Cox-1 transcription 2.5-fold while simultaneously decreasing the amount of Cox-2 mRNA 0.5-fold. L. acidophilus had no effect on the amounts of Cox-1 or Cox-2. The beneficial effect of Bifidobacterium sp. 420 on cyclooxygenase expression was not mediated by butyrate, since these two bacteria did not produce butyrate. This is the first piece of evidence showing a direct relationship between a probiotic microorganism and host cyclooxygenase expression profile. The ability of a nutraceutical to induce such health-promoting transcriptional changes may provide an important criterion in the selection of novel anti-inflammatory and anticarcinogenic functional food ingredients.
A previously described rat model was used to study indomethacin-induced gastrointestinal damage (see Meddings and Gibbons, Gastroenterology 1998; 114:83-92). Briefly, male Wistar rats were used and ten rats were included in each treatment group. Five different treatments were included: control group with no indomethacin treatment, indomethacin control group with no intervention treatment, indomethacin with 108 Bifidobacterium per rat, indomethacin with 1010 Bifidobacterium per rat, and indomethacin with betaine (200 mg/rat). A single dose of indomethacin (10 mg/kg) was given p.o. preceded by a seven-day intervention with or without Bifidobacterium or betaine. In addition, a mixture of sucrose, lactulose, mannitol and sucralose was given p.o to measure gastrointestinal (GI) permeability. Urine samples were obtained at 16 hours after the challenge with indomethacin and prior to sacrificing the animals. Mucosa of the stomach and the intestine were examined by a pathologist and tissue samples of healthy-appearing mucosa for RNA isolation were collected from stomach, proximal and distal small intestine, caecum and proximal and distal colon. RNA was isolated and expression of cyclooxygenase-1 and -2 (COX-1 and COX-2, respectively) were determined by QPCR.
No mortality and no differences in the weight gain during the experiment were observed. A clear effect by indomethacin challenge was observed in the rat intestinal tissue. Indomethacin induced ulceration mainly to fundic stomach, but also to pyloric stomach. No other gross pathological changes were observed in the intestine (
Based on these results it can be concluded that the in vitro findings of reduction of inflammatory responses obtained from the Caco-2 cell model by bifidobacteria were conformed in a rat model. Not all compounds with effects on cyclooxygenase expression can reduce gastrointestinal side effects of the nonsteroid anti-inflammatory drugs. Betaine had no effect on the permeability caused by the indomethacin challenge. However, the damage area was somewhat reduced. It may be that the protective effect caused by betaine is mediated by a different mechanism to the one mediated by bifidobacteria. Lack of effect of the intervention by bifidobacteria in the expression profile of cox-1 or cox-2 in the intestinal tissue not exhibiting clear damage indicates that the effect is limited to compromised areas in the mucosa This feature widens the safe use of the tested bifidobacteria also in healthy individuals with no evident parallel challenge e.g. by nonsteroid drugs.
Male Wistar rats as described previously in Experiment 3 above are split into the following treatment groups: a control, an indomethacin challenge group as a control, and treatment groups 3-5, which receive intervention treatment by live Bifidobacterium (1010 daily dose per animal) and betaine (200 mg of daily dose per animal) either separately or in combination. Damage is determined from the sacrificed rats only.
Preliminary investigations suggest that no mortality or differences between weight gain in the different treatment groups is observed. The clear protective effect by Bifidobacterium is repeatedly demonstrated by reduced area of damage in the gastrointestinal tract. In addition, the betaine treatment tends to decrease the damage area as observed previously. The combination of betaine with bifidobacteria exhibits a synergistic effect and damage area not significantly different from the baseline control group. The synergy between the two ingredients probably results in the different mechanism mediating the protective effect as indicated by the different effect on the permeability of the mucosa in the Experiment 2. It is concluded that combined use of betaine and certain live bifidobacteria may give additional protection against intestinal inflammatory challenges when compared with use of live bifidobacteria alone, i.e., without betaine.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0323039.8 | Oct 2003 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB04/03273 | 9/24/2004 | WO | 00 | 2/7/2007 |
