HK1079661B - Use of lactobacillus reuteri strains for preparing composition of improving immune function in mammals - Google Patents

Description

Use of lactobacillus reuteri strains for the preparation of a composition for improving immune function in a mammal

Background

Technical Field

The present invention relates to improved methods of using Lactobacillus reuteri strains as immune enhancing agents, and selecting the most advantageous strains for this purpose.

Prior Art

The probiotic industry, which contains a wide variety of different gastrointestinal microorganisms, has been formulated primarily because of the increase in antibiotic-resistant pathogens. A wide variety of lactobacillus species, including strains of lactobacillus reuteri, have been used in probiotic formulations. Lactobacillus reuteri is a naturally occurring inhabitant of the gastrointestinal tract of animals, which is routinely found in the intestines of healthy animals. It is known to have antibacterial activity. See, for example, U.S. Pat. nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. When lactobacillus reuteri cells are grown under anaerobic conditions in the presence of glycerol, they produce an antibacterial substance known as a protein-free (β -hydroxy-propionaldehyde).

Immunomodulating activity is also associated with lactobacillus reuteri. See, for example, Wagner RD et al, "biotherapeutic effects of probiotics on mucocutaneous candidiasis in immunodeficient mice," infection immunity (infection immunity) 1997 Oct 65: 4165-72; however, there are differences in efficacy between strains and methods are needed to select the most effective strains, for example, methods are provided for selecting strains to complement CD4+ cells in the present conditions.

Furthermore, although lactobacillus reuteri is known to be useful as a generally advantageous probiotic, previous work has only recognized to some extent the importance of utilizing the best lactobacillus strains which neutralize toxins produced by these pathogens already present in the gastrointestinal tract. See, for example, El-Nezami HS et al, "removal of common fusarium toxins in glass tubes by lactobacilli and propionibacteria," Food additive pollution "(Food additive company) 2002 Jul 19: 680-6. Such toxin neutralization includes adhesion, and according to this invention, is reported to be important not only for ameliorating the direct effects caused by these pathogen-producing toxins, but also for reducing the general burden on the immune system.

There are many different causes of gastrointestinal problems caused by pathogenic microorganisms. For example, helicobacter pylori-induced gastric and duodenal ulcers, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma. Some pathogenic E.coli strains produce toxins, such as Vero Toxin (VT) produced by E.coli O157: H7, and antibiotics are less and less effective at preventing them.

It is therefore an object of the present invention to provide a method for improving immune function in mammals by using lactobacillus reuteri which has been tested to be effective for both CD4+ cell recruitment and toxin neutralization, and to provide a method for selecting such immuno-improved lactobacillus reuteri strains, and to provide products containing such strains. It is still another object of the present invention to provide a method for utilizing culture supernatants of lactobacillus reuteri strains effective for reducing the immune burden of these toxins.

Other objects and advantages will become more apparent from the following description and the appended claims.

Summary of The Invention

The present invention relates to the use of Lactobacillus reuteri strains as immune enhancing agents, and methods for improving immune function in mammals using Lactobacillus reuteri strains in products containing cells of such strains, as well as the products so referred to. These strains were selected to have good toxin binding and neutralizing effects; and has good CD4+ supplementing effect. Other objects and features of the present invention will become more fully apparent from the following description and appended claims.

Brief description of the drawings

FIG. 1 confirmation of prevention of vero cytotoxin (VT) and Gb₃The binding inhibition ability of the receptor in culture supernatants of lactobacillus reuteri by competitive ELISA. Each reaction was carried out as follows, coating plates with Gb₃Followed by ELISA using mAb against VT.

Comparison: VT + Trypsin Soybean soup (TSB)

VT + G: VT +250mM glycerol solution

VT + LRS: VT + Lactobacillus reuteri culture supernatant cultured in 250mM glycerol solution.

Detailed description of the invention and preferred embodiments thereof

The invention relates to the use of Lactobacillus reuteri strains, which have good toxin binding and neutralizing effects; and has good CD4+ cell supplementing effect on the composition for improving the immune function of mammals. It also relates to products containing such Lactobacillus reuteri strains, and a method of improving immune function in mammals using such Lactobacillus reuteri strains. Furthermore, it provides a method for selecting such improved immunogenic Lactobacillus reuteri strains.

Cytokinins (cytokines) are immune system proteins, which are biological response modifiers. They coordinate the interaction of antibodies with the T cell immune system and amplify immune reactivity (10). Cytokinins include monokines synthesized by macrophages and lymphokines produced by activated T lymphocytes and Natural Killer (NK) cells. The CD4+ subclass in humans and mice is based on cytokinin production and effector functions. Th1 cells synthesize interferon-gamma (IFN-gamma), IL-2 and Tumor Necrosis Factor (TNF). They are mainly responsible for preventing cellular immunity of intracellular microorganisms and for delayed type hypersensitivity reactions. They affect immunoglobulin G2a (IgG2a) synthesis and antibody-dependent cell-mediated cytotoxicity. The INF-gamma they produce activates macrophages and leads to phagocytosis. Th-2 cells synthesize interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-9 (IL-9), interleukin-10 (IL-10), and interleukin-13 (IL-13). They induce IgE and G1 antibody responses,

included in the present invention are methods as evidenced by the various example procedures: the lactobacillus reuteri strain was administered, and the efficacy of this and the used strain in CD4+ cell supplementation and toxin neutralization was analyzed. The data show, for example, that a sheet-like preparation containing a lactobacillus reuteri strain gives similar levels of gastrointestinal transplantation as the directly administered cell culture. The lactobacillus reuteri strain transplantation is associated with the intake of lactobacillus reuteri, the wash time (the time required for the lactobacillus reuteri to drop to the pre-intake level) being at least 28 days after administration of the tablet, indicating that the invention is suitable for lactobacillus reuteri cell cultures and for formulating products containing such lactobacillus reuteri cultures.

The present invention preferably relates to the use of a product containing a lactobacillus reuteri strain as a disease-modifying preventive probiotic of the immune function of a mammal. Such products may be a variety of food products such as dietary supplements, candies, or tablets containing cells of such a selection strain.

The lactobacillus reuteri of the present invention can also be used for the preparation of various microbial medicines capable of producing toxins such as escherichia coli. Hemorrhagic escherichia coli and VT toxins can thus be treated.

According to the invention, both the cells of the Lactobacillus reuteri strain and the supernatant of its culture can be used for the preparation of prophylactic, probiotic and pharmaceutical compositions.

According to the invention, all lactobacillus reuteri with good CD4+ cell recruitment and/or good toxin adhesion can be used. The presence of CD4+ cells can be tested by using antibodies to CD4, for example, using immunohistochemistry (such as in example 5) or immunofluorescence. Toxin binding can be demonstrated by contacting lactobacillus reuteri cells or supernatant with a toxin and testing for differences in available toxins, as described in example 1.

The probiotic, prophylactic and pharmaceutical products according to the invention may contain additives and excipients suitable for nutritional or pharmaceutical use.

The features of the invention will become more apparent with the aid of the following examples, which, however, are not to be construed as limiting the invention.

Example 1 Vero toxin study

Three strains of lactic acid bacteria were used in this experiment, Lactobacillus reuteri ATCC 55730, Lactobacillus bulgaricus, strain LB12, (CHR, Horsholm, Denmark), and Lactobacillus casei, strain 01, (CHR, Horsholm, Denmark). Lactobacillus reuteri was cultured in MRS broth (plus 20mM glucose) and then cultured in an aerotropic fixed condition at 37 ℃ for 24-48 hours. In some cases, these initial cultures were followed by centrifugation at 2500rpm for 30 minutes, washed twice with Phosphate Buffered Saline (PBS) to remove medium components, suspended in 250mM glycerol solution, and then incubated at 37 ℃ for 6 hours under aerophilic fixation conditions. Lactobacillus bulgaricus and Lactobacillus casei were cultured for 24-48 hours at 37 ℃ under aerotropic fixation conditions with MRS plus 20ml glucose (without glycerol). Each test lactic acid bacterium was used, and then, adjusted to 2g/30ml (dry weight), centrifuged at 2500rpm for 30 minutes after the culture, the supernatant was taken out, its pH was adjusted to 7.0 with NaOH to inoculate vero cells (see below), and filtered through a 2.0 μm filter. The glycerol solution and MRS broth were adjusted to ph7.0, serving as a reference, and subsequently filtered through a 2.0 μm filter.

Vero cells (Vero cells, ATCC-CCL81) were cultured in minimal medium (MEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) at 25g/ml gentamicin (Sigm)a) At 37 ℃ in 10% CO₂The culture was performed in an incubator for 48 hours, and used after confirming monolayer formation.

Escherichia coli O157: H7(ATCC 43894) which secreted VT1 and VT2 was used, after which it was inoculated into Tryptic Soy Broth (TSB), cultured for 24 hours while stirring in a shaking incubator at 37 ℃ and centrifuged at 2500rpm for 30 minutes, and the culture supernatant was filtered.

500Vero cell (2X 10)⁵Cells/ml) were seeded onto 96-well culture plates and incubated at 37 ℃ in 10% CO₂The incubator was inoculated for 48 hours to confirm monolayer formation, after which, experiments were carried out by using the following treatments: a: VT only (positive reference); b: TSB (E.coli O157: H7 medium); c: MRS soup (pilot lactobacillus); d: glycerol solution (lactobacillus reuteri medium); e: VT + MRS decoction; f: VT + glycerol solution; g: VT + lactobacillus reuteri glycerol solution culture supernatant; h: VT + lactobacillus bulgaricus MRS broth culture supernatant; i: VT + lactobacillus bulgaricus culture supernatant; and J: VT + lactobacillus casei culture supernatant. Treatments B, C and D were used to determine whether each broth itself could cause cytotoxicity to Vero cells; and E and F are used to determine whether the medium in which the lactic acid bacteria are tested has its own neutralizing capacity against VT. The culture supernatants of each of the test lactic acid bacteria in G, H and J were serially diluted 2X, after which the culture supernatants and VT of each diluted test lactic acid bacteria were combined as follows: 400ml +100 ml; 300ml +200 ml; 200ml +300 ml; and 100ml +400ml, after which, at 37 ℃ in 10% CO₂The cells were incubated in an incubator for 48 hours to determine the presence of cytopathic effect (CPE).

300mM neutralized semicarbazide hydrochloride (Sigma), which inhibits the production of no protein, was added to a culture solution of Lactobacillus reuteri in 250mM glycerol solution to inhibit the production of no protein, and the supernatant was collected as described above. Mu.l of Vero cells were seeded in 96-well plates and incubated at 37 ℃ in 10% CO₂Cultured in an incubator for 24 hours to examine whether or not a monolayer is formed, and thereafter, the following was performedAnd (3) treatment: a: VT only; b: VT +25mM glycerol solution; c: VT + lactobacillus reuteri glycerol solution culture supernatant; and D: VT + glycerol solution culture supernatant of lactobacillus reuteri cultured after treatment without proteic inhibitors (as described above). At B, C and D, each culture of Lactobacillus reuteri treated with glycerol solution and without proteobacterial inhibitor, or without treatment, was combined with VT as follows: 20+ 80; 30+ 70; 40+ 60; 50+ 50; 60+ 40; 70+ 30; and 80+ 20. mu.l, after which they were incubated at 37 ℃ for 18 hours in a 10% CO2 incubator to examine whether a cytopathic effect was present. After microscopic observation, the culture broth was stained with crystal violet, and their o.d (optical density) values were read at 490 nm.

Using Gb₃(erythrocytetraglycosylceramide) (Sigma) 96-well plates were coated with Gb₃Blocked by 5% Bovine Serum Albumin (BSA) and reacted with Lactobacillus reuteri culture supernatant that had been cultured in VT or VT +250mM glycerol solution. Thereafter, horseradish peroxidase (HRP) -conjugated goat anti-aging mouse IgG was added thereto using a single cell antibody (mAb) against VT as a primary antibody, and after that, O-phenylenediamine was cultured and its optical density (o.d.) was observed at 490nm by an ELISA reader. This experiment confirmed the inhibition of Lactobacillus reuteri and the Gb₃The presence of substances that interact between receptors.

From VT between 250mM glycerol solution and VT between coating Gb₃As a result of the interaction between the culture supernatant of lactobacillus reuteri cultured in a post glycerol solution, it was confirmed that the combination of VT and the culture supernatant of lactobacillus reuteri produced a low o.d. value at a very significant level, as compared to the case of VT alone, as shown in fig. 1. This indicates that the presence of a substance in the culture supernatant of lactobacillus reuteri capable of inhibiting VT binding to the Gb3 receptor can be detected indirectly.

96 well culture plates were coated with Lactobacillus reuteri culture supernatant, which was incubated in VT/VT +250mM glycerol solution, blocked by 3% (BSA) and reacted with VT. Thereafter, horseradish peroxidase (HRP) -conjugated goat anti-aging mouse IgG (H + L) was added thereto using a single cell antibody against VT as a primary antibody, after which O-phenylenediamine was cultured and its optical density (o.d.) was observed at 490nm by an ELISA reader. This experiment confirmed the presence of VT interacting substances in lactobacillus reuteri supernatant.

EXAMPLE 2 study of the neutralization of the secretion of Vero cytotoxins (VT) I and II by lactic acid bacteria from Escherichia coli O157: H7

No cytopathic effect was observed when TSB, MRS broth and glycerol solution were added to Vero cells. In addition, when VT and MRS broth/glycerol solutions were added to Vero cells, CPE was observed in Vero cells, indicating that the culture broth itself lacked neutralizing ability against VT.

When the culture supernatant of each test lactic acid bacterium was adjusted to pH7.0, filtered and combined with VT, the results shown in Table 1 were obtained. For Lactobacillus bulgaricus and Lactobacillus casei CPE was present over the whole concentration range, however, for Lactobacillus reuteri CPE was not present in many glycerol supernatants, with the exception of the ratio of test lactic acid bacteria to VT of 4: 1, in which case very low CPE was present. Thus, there was a discernible neutralization of anti-VT in culture supernatants cultured in 250mM glycerol solution. For cultures in MRS broth (+20mM glucose), CPE appeared at all concentrations.

TABLE 1 comparison of Vero cell potency against cytopathic Effect (CPE) by Vero cytotoxin in culture supernatants of Lactobacillus reuteri, Lactobacillus bulgaricus and Lactobacillus casei

Lactic acid bacteria	Lactic acid bacteria culture supernatant	VT(μl)	Serial dilution (x2) of lactic acid bacteria culture supernatant
								0	1	2	3	4
			Lactobacillus reuteri (G)*	400	100	-	-						-	+a	+a
300	200	-						-	+a	+	+
				200	300	-	+a					+	+	+
100	400	+a						+a	+	+	+
				Lactobacillus reuteri (MRS)**	400	100	+a					+	+	+	+
300	200	+	+					+	+	+
					200	300	+				+	+	+	+
100	400	+	+					+	+	+
					Lactobacillus bulgaricus	400	100				+	+	+	+	+
300	200	+	+	+				+	+
						200	300			+	+	+	+	+
100	400	+	+	+				+	+
						Lactobacillus casei	400			100	+	+	+	+	+
300	200	+	+	+	+			+
							200		300	+	+	+	+	+
100	400	+	+	+	+			+

^*: the culture supernatant is obtained by culturing Lactobacillus reuteri in 250mM glycerol solution

^**: the culture supernatant is obtained by culturing Lactobacillus reuteri in MRS soup (with 20mM glucose)

-: without CPE (cytopathic effect)

+：CPE

a: light CPE

Student tests were performed using the results of ELISA and binding assays comparing VT to lactobacillus reuteri culture supernatants using the Microcal Origin 6.1(Microcal Software, inc.

Example 3 administration of Lactobacillus reuteri to test subjects

In this example, two chews, each containing 1X 10 chews, were administered to a subject twice daily⁸CFU (colony Forming Unit) of Lactobacillus reuteri (SD 2112: ATCC 55730) for a total daily dose of 4X 10⁸CFU lactobacillus reuteri. All other excipients used for the chewable tablets are known and follow the international pharmacopoeia. The study was performed in two parts: the duration of the gastroscopy of the upper gastrointestinal tract was studied, and the duration of the ileoscopy of the distal small intestine was studied (as will be described in detail below). The exclusion criteria were: two weeks of antibiotics were administered before and during the study; three weeks of probiotic administration prior to and during the study, ongoing treatment with gastrointestinal related medications, and severe organic disease requiring routine treatment (e.g., cancer). For the patient treatment protocol, was approved by the Danish national Committee and was in compliance with the Helsinki statement. The study was conducted in Denmark.

Wilcoxon signed rank test was used to compare symptoms, blood test values, fecal Lactobacillus content, and tissue differences before and after ingestion of Lactobacillus reuteri. P < 0.05 was considered significant.

All subjects completed the study. During gastroscopy, 10 healthy volunteers were studied, all older than 18 years of age, with normal eating habits. Gastroduodenal examinations were performed after overnight day 0 before intake of lactobacillus reuteri, and on day 28 at the end of the study. Biopsies taken from the body and gastric cavity of the stomach and from the third part of the duodenum were both used for lactobacillus reuteri cultures and histological examinations. The average size of the biopsy was 33 mg. The biopsy was immediately placed in PBS (2: 1vol/wt sample), stored on ice and transferred to the Daiid biosystems Inc. laboratory, Lund, Sweden for analysis of the number of Lactobacillus reuteri. The time from tissue isolation to analysis was within 36 hours.

During the ileoscopy, 9 test subjects with ileostomy after colectomy due to ulcerative colitis (3 subjects) or crohn's disease (6 subjects) were studied. In all subjects, there was no evidence of inflammation in the small intestine. Retroenteroscopy was performed on day 0 and day 28. Live tissue examination was taken within 20cm of the distal small intestine as described above for lactobacillus reuteri cultures and other lactobacilli and for histological examination.

Example 4 analysis of microorganisms

Fecal samples were collected from each subject prior to intake of lactobacillus reuteri (day 0) and at the end of the study (day 28). The stool of the enteroscopy subject (explained below) is taken from the stoma. On day 42 (14 days after cessation of loehrlic acid intake), fecal samples were collected from 7 volunteers during gastroscopy and 4 volunteers during ileoscopy. Samples (not less than 5g) were collected in sterile containers and immediately placed in a refrigerator. Within 24 hours, 20ml (1: 5, wt/vol) of 0.1% peptone water was added. The sample was homogenized and aliquots were dispensed into cryovials immediately prior to freezing at-70 ℃. The samples (frozen on dry ice) were transported to the geolife goddess ltd laboratory for analysis of total lactobacilli and lactobacillus reuteri. The samples were thawed, diluted and spread on MRS-3 agar containing vancomycin (50mg/l) for Lactobacillus reuteri and LBS agar plates (KEBOLABAB, Lund, Sweden) for total Lactobacillus count. MRS-3 is a modified MRS agar (KEBOLAB AB, Lund, Sweden) containing 2% sodium acetate (wt/vol). LBS agar is preferably prepared by the preparation method by adding 1.32ml glacial acetic acid/l. Agar plates were incubated anaerobically at 37 ℃ for 48 hours in anaerobic jars using BBL air bags (Gas packs). DNA from selected isolates of this study (see below) was analyzed by PCR using a bacterial barcode repPROTM DNA fingerprinting kit (BacterialBarCodes, Inc., Houston, TX) and by using Bionumerics software (Applied Maths BVBA, site-Martens-latex, Belgium).

At the start of the study and prior to administration of the study product, noneOne subject had detectable lactobacillus reuteri in the stool during gastroscopy (table 2). After 28 days of intake of Lactobacillus reuteri, all subjects had Lactobacillus reuteri in the feces in the range of 1.0X 10²-3.5×10⁵CFU/g (number of colony forming units per gram of fecal material) averages 4.0X 10⁴CFU/g, indicating a significant increase in viable Lactobacillus reuteri in feces due to administration of the study product. Selection of 5 subjects required stool samples to be voided at day 42 and day 56 (i.e., at day 28 after administration of the study product). In these subjects, the lactobacillus reuteri remained in the feces for four weeks after ingestion (table 2).

TABLE 2 Lactobacillus reuteri in feces recovery

Gastroscopic object

Object	1	2	3	4	5	6	7	8	9	10	Average	SD
													% consistency of consumed tablets	10089	10493	10493	8677	9383	9282	8172	10493	10291	9888	9686	87
CFU Lactobacillus reuteri/g feces
													0 day, 28 days, 42 days, and 56 days	nd5.0E+03**	Nd1.0E+031.4E+03*	nd3.8E+041.5E+031.3E+03	Nd3.5E+05**	nd1.0E+02**	nd1.0E+021.2E+032.2E+03	nd4.1E+031.6E+031.0E+03	nd2.0E+026.0E+026.0E+02	nd1.0E+023.0E+02*	nd1.9E+035.0E+021.0E+03	nd4.0E+041.0E+031.2E+03	1.1E+055.3E+026.0E+02

Retroenteroscopy subject

Object	11	12	13	14	15	16	17	18	19	Average	SD
												% consistency of consumed tablets	9282	11098	112100	11098	9888	11098	112100	112100	112100	10896	77
CFU Lactobacillus reuteri/g ileostomy faecal
												0 day, 28 days, 42 days, and 56 days	ndnd**	NdNd1.0E+031.0E+03	ndnd**	Nd6,8E+036.0E+028.0E+02	nd1.0E+021.9E+03*	nd3.0E+026.0E+022.0E+02	nd5.0E+02**	5.00E+044.0E+04**	1.00E+021.0E+02**	nd8.0E+031.0E+036.7E+02	1.6E+046.1E+024.2E+02

nd is not detected (< 1.0 × 10)²CFU/g fecal matter);^*indicating that the subject did not give a sample of stool (ileostomy faeces) which was taken at 28 days before supplementation with lactobacillus reuteri (day 0). Results are expressed as Colony Forming Units (CFU)/g wet weight fecal material.

Example 5 histology

To evaluate all local immune responses, changes in the numbers of B-lymphocytes, T-lymphocytes and macrophages were determined. Initial period biopsies and 28-day biopsies were fixed with formalin and embedded in paraffin. Thereafter, 4 μm sections were excised and histochemically and immunohistochemically stained by using standard techniques (hematoxylin, van gieson, PeriodicAcidSciff-amylase preparation). Primary antibodies obtained from DAKO, Glostrup, Denmark are: CD20 (B-lymphocytes), CD3, CD4+, CD8 (T-lymphocytes), CD68 (histiocytes), helicobacter, and Ki-67 (proliferation)A marker). Immunohistochemical staining was at DAKO TechMate^TM500 to obtain a uniform staining.

The biopsy was evaluated by a pathologist. Tissue lesions were graded according to Madsen et al (Gastroenterol.1999; 116: 1107-. This hierarchy represents a total number of four criteria: ulcers, epithelial hyperplasia, monocyte counts, and neutrophils in the sheet itself. The number of CD20, CD3, CD4+, CD8 and CD68 positive cells in the slices themselves were semi-quantitatively evaluated based on immunohistochemical staining. After three months, the initial period and the 28 day biopsy were mixed and evaluated by the same pathologist. All correlations between positive staining from the initial period and the 28 day biopsy were calculated for each evaluation and compared.

Predominantly single B-lymphocytes were found in the stomach (corpus and antrum) of two subjects at day 0 and two subjects at day 28. One subject had scattered cells in the stomach (lumen) for 28 days. 4 subjects had single cells in the duodenum at day 0, and 8 subjects had predominant single cells in the duodenum at day 28. 8 subjects had predominant single cells in the stomach (corpus and antrum) on day 0, CD3, CD4+ and CD8 (T-lymphocytes), and 9 subjects had predominant single cells in the ventricles (corpus and antrum) on day 28 (Table 3). Scattered cells were found in the duodenum of 8 subjects on day 0, and predominantly scattered T-lymphocytes were found in the duodenum of 7 subjects on day 28.

Predominant single tissue cells were found in the stomach (corpus and antrum) of 9 subjects on day 0 and 5 subjects on day 28. The predominant scattered cells were found in the duodenum of 6 subjects (not the same six subjects) on days 0 and 28.

After the subjects received lactobacillus reuteri, there was a statistically significant decrease in the number of tissue cells (CD68) in the biopsy, from bulk (p 0.025) and lumen (p 0.046), and a significantly increased number of B-cells (CD20) (p 0.046; table 3) in the duodenum. No significant change in T lymphocyte numbers was observed due to the intake of Lactobacillus reuteri. The agreement between the two studies (first, not blind, and second, blind analysis of tissue samples) was 76% (table 3). For somatic and luminal tissue cells (CD68), the identity was only 40% and 65%, respectively. The duodenal B-cell identity (CD20) was 60%. Thus, there is some uncertainty in these findings. The consistency of the CD3, CD4+, and CD8 assays was high (up to 90%; Table 3).

During ileoscopy, all biopsies were histologically normal and helicobacter negative. Ki-positive cells were normal in all biopsies. Predominantly single B-lymphocytes were found in 6 subjects and scattered cells were found in 2 subjects on day 0. Single cells were observed in all subjects at day 28. Predominantly dispersed CD3 cells (B-lymphocytes) were found in 7 subjects on both day 0 and day 28. Predominant dispersed CD4+ cells were found in 7 subjects on day 0 and a predominant contiguous population of cells was found in 7 subjects on day 28. Predominant dispersed CD8 cells were found in 7 subjects on day 0 and 9 subjects on day 28 (table 3). Predominantly dispersed tissue cells were found in 7 subjects on both day 0 and day 28.

After 28 days of supplementation with lactobacillus reuteri, a significantly higher number of CD4+ lymphocytes were present (p ═ 0.046; table 3). No significant change in B-lymphocyte or histiocyte numbers was found due to administration of Lactobacillus reuteri. The agreement between these two studies was generally high for all cell types tested and 78% for the CD4+ results, indicating good reliability of these data (table 3). All helicobacter stains were negative.

Table 3 histological evaluation from ileal biopsy during retroenteroscopy

Object	Histological score at day 0	B-cells	T-cells			Tissue cell	28 day histological score	B-cells				Tissue cell
													CD20	CD3	CD4	CD8	CD68	CD20	CD3	CD4	CD8	CD68
		11	0	X	XX	XX		XX	XX	0	X	X											XXX	XX	XX
12	0						XX						XXX	XXX	XX	XX	0	X	XX	XXX	XX	XX
		13	0	-	XX	XX		XX	XX	0	X	XX											XXX	XX	X
14	0						X						XX	XX	XX	XX	0	X	X	XXX	XX	XX
		15	0	XX	XXX	XX		XX	XX	0	X	XX											XXX	XX	X
16	0						X						XX	XX	XX	XX	0	X	XX	XXX	XX	XX
		17	0	X	XX	XX		X	XX	0	X	XX											XX	XX	XX
18	0						X						XX	XX	X	X	0	X	XX	XX	XX	XX
		19	0	X	XX	XXX		XX	X	0	X	XX											XXX	XX	XX

Biopsies are taken within 20cm of the distal small intestine and fixed and sectioned for histological examination by staining specific cell types using immunohistochemistry (see description).

"-", no cells were detected; "X" is a single cell; "XX" refers to dispersed cells or cell aggregates; "XXX" refers to multiple contiguous cell populations. Histological scores (1-10) demarcate the degree of tissue damage.

Statistical analysis: wilcoxon Signed Rank Test. The samples give nominal fractions: -, X, XX and XXX, which are set to 1, 2, 3 and 4, respectively.

Example 6 biochemistry of blood

Blood samples were taken at day 0 and day 28 and analyzed for hemoglobin, hematocrit, platelets, lueccoytes, C-reactive protein, potassium, sodium, creatinine, b-urea, p-glucose, cholesterol, HDL (high density lipoprotein), LDL (low density lipoprotein), VLDL (very low density lipoprotein), trigyceries, total bilirubin, urate, ALAT, alkaline phosphatase, and lactate.

Before and after intake of lactobacillus reuteri, many blood tests are normal. There were some outliers in blood variables, however, no clinically significant abnormalities were found and no systemic changes were observed after treatment.

Example 7 DNA fingerprinting

DNA fingerprinting analysis was performed on selected Lactobacillus reuteri isolates from this study. Thus, fecal isolates taken from 3 subjects who had consumed 28 days of Lactobacillus reuteri, as well as one isolate derived from a duodenal biopsy and one isolate derived from an ileal biopsy, both of which were taken 0 days prior to administration of Lactobacillus reuteri. All isolates were found to have 98% group similarity to each other. All these isolates showed 97% similarity to strain SD2112 incorporated into the tablets.

Example 8 formulation of products to improve immune function in humans

In this example, lactobacillus reuteri SD2112, ATCC 55730 was selected for addition to standard yogurt by using the method described above for toxin neutralization and CD4+ supplementation. The lactobacillus reuteri strain was grown and lyophilized using standard methods used in the dairy industry for the cultivation of lactobacillus. The yoghurt was used by people as a method to improve their immune function using traditional yoghurt cultures at 10E +7 CFU/g yoghurt, followed by adding this culture to the fermented milk described previously.

While the invention has been described with reference to specific embodiments, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of the invention.

Claims (9)

1. Use of lactobacillus reuteri strains, thereof

a. Has good toxin binding and neutralizing effects; it is confirmed by contacting lactobacillus reuteri cells or supernatant with a toxin and a test for available toxin differences, by an antibody test; and

b. has good CD4+ supplementation, which is tested by using antibodies to CD4, using immunohistochemistry or immunofluorescence, for the preparation of compositions for improving immune function in mammals.

2. A product comprising a strain having at least the features of claim 1.

3. The product of claim 2, wherein the product is formulated as a food product comprising cells of the selected strain.

4. The product of claim 2, wherein said product is formulated as a tablet containing cells of said selected strain.

5. The product of claim 2, wherein the product is formulated as an additive comprising cells of the selected strain.

6. The product of claim 2, wherein the product is formulated as a confection containing cells of the selected strain.

7. The product of claim 2, wherein the product is formulated as a medicament containing cells of the selected strain.

8. Use of culture supernatant of lactobacillus reuteri ATCC 55730 for the preparation of a product according to claim 2 for neutralizing bacterial toxins.

9. Use of a lactobacillus reuteri strain for the manufacture of a product according to claim 2 for improving immune function in a mammal, comprising: use of such strains, they

a. Has good toxin binding and neutralizing effects; it is confirmed by contacting lactobacillus reuteri cells or supernatant with a toxin and a test for available toxin differences, by an antibody test; and

b. has good CD4+ supplementation, which was tested by using antibodies to CD4, using immunohistochemistry or immunofluorescence.