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  • C12R2001/225—Lactobacillus

Definitions

• the invention relates to Lactobacillus salivarius strains and their use as probiotic bacteria in particular as immunomodulatory biotherapeutic agents.
• the defense mechanisms to protect the human gastrointestinal tract from colonization by intestinal bacteria are highly complex and involve both immunological and non- immunological aspects (1).
• Innate defense mechanisms include the low pH of the stomach, bile salts, peristalsis, mucin layers and anti-microbial compounds such as lysozyme (2).
• Immunological mechanisms include specialized lymphoid aggregates, underlying M cells, called peyers patches which are distributed throughout the small intestine and colon (3). Luminal antigens presented at these sites result in stimulation of appropriate T and B cell subsets with establishment of cytokine networks and secretion of antibodies into the gastrointestinal tract (4).
• antigen presentation may occur via epithelial cells to intraepithelial lymphocytes and to the underlying lamina limba immune cells (5). Therefore, the host invests substantially in immunological defense of the gastrointestinal tract.
• the gastrointestinal mucosa is the largest surface at which the host interacts with the external environment, specific control mechanisms must be in place to regulate immune responsiveness to the 100 tons of food which is handled by the gastrointestinal tract over an average lifetime.
• the gut is colonized by over 500 species of bacteria numbering 10 ⁇ -10 12 /g in the colon.
• these control mechanisms must be capable of distinguishing non-pathogenic adherent bacteria from invasive pathogens, which would cause significant damage to the host.
• the intestinal flora contributes to defense of the host by competing with newly ingested potentially pathogenic micro-organisms.
• Bacteria present in the human gastrointestinal tract can promote inflammation.
• Antigens associated with the normal flora usually lead to immunological tolerance and failure to achieve this tolerance is a major mechanism of mucosal inflammation (6).
• Evidence for this breakdown in tolerance includes an increase in antibody levels directed against the gut flora in patients with IBD.
• the present invention is directed towards Lactobacillus strains, which have been shown to have immunomodulatory effects, by modulating cytokine levels or by antagonizing and excluding pro-inflammatory micro-organisms from the gastrointestinal tract.
• a Lactobacillus salivarius strain selected from any one or more of AH102, AH103, AH105, AH109 and AH110 or a mutant or variant thereof.
• the mutant may be a genetically modified mutant.
• the variant may be a naturally occurring variant of Lactobacillus salivarius .
• Lactobacillus salivarius strains are in the form of viable cells.
• Lactobacillus salivarius strains are in the form of non- viable cells.
• the strains are in the form of a biologically pure culture.
• the Lactobacillus salivarius strains are isolated from resected and washed human gastrointestinal tract.
• the Lactobacillus salivarius strains are significantly immunomodulatory following oral consumption in humans.
• the invention also provides a formulation which comprises at least one Lactobacillus salivarius strain of the invention.
• the formulation may comprise two or more strains of Lactobacillus .
• the formulation includes another probiotic material.
• the formulation includes a prebiotic material.
• the formulation includes an ingestable carrier.
• the ingestable carrier may be a pharmaceutically acceptable carrier such as a capsule, tablet or powder.
• the ingestable carrier is a food product such as acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spreads, dressings or beverages.
• the formulation of the invention further comprises a protein and/or peptide, in particular proteins and/or peptides that are rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, mineral and/or trace element.
• Lactobacillus salivarius strains are present in the formulation at more than 10 6 cfu per gram of delivery system.
• the formulation includes any one or more of an adjuvant, a bacterial component, a drug entity or a biological compound.
• the formulation is for immunisation and vaccination protocols.
• the invention further provides Lactobacillus salivarius strains or a formulation of the invention for use as foodstuffs, as a medicament, for use in the prophylaxis and/or treatment of undesirable inflammatory activity, for use in the prophylaxis and/or treatment of undesirable gastrointestinal inflammatory activity such as inflammatory bowel disease such as Crohns disease or ulcerative colitis, irritable bowel syndrome, pouchitis, or post infection colitis, for use in the prophylaxis and/or treatment of gastrointestinal cancer (s), for use in the prophylaxis and/or treatment of systemic disease such as rheumatoid arthritis, for use in the prophylaxis and/or treatment of autoimmune disorders due to undesirable inflammatory activity, for use in the prophylaxis and/or treatment of cancer due to undesirable inflammatory activity, for use in the prophylaxis of cancer, for use in the prophylaxis and/or treatment of diarrhoeal disease
• the invention also provides Lactobacillus salivarius strains or a formulation of the invention for use in the preparation of an anti-inflammatory biotherapeutic agent for the prophylaxis and/or treatment of undesirable inflammatory activity or for use in the preparation of anti-inflammatory biotherapeutic agents for the prophylaxis and/or treatment of undesirable inflammatory activity.
• the strains of the invention act by antagonising and excluding proinflammatory micro-organisms from the gastrointestinal tract.
• the invention also provides Lactobacillus salivarius strains or a formulation of the invention for use in the preparation of anti-inflammatory biotherapeutic agents for reducing the levels of pro inflammatory cytokines such as IL8.
• the invention further provides Lactobacillus salivarius strains for use in the preparation of anti-inflammatory biotherapeutic agents for modulating cytokine levels such as IL-8, IL-10, IL-12, TNF or IFN ⁇ .
• the invention further provides Lactobacillus salivarius strains for use in the preparation of biotherapeutic agents for modifying the levels of IFN ⁇ .
• the strains are selected from any one of AH102, AH103 or AH105.
• the invention further provides Lactobacillus salivarius strains for use in the preparation of biotherapeutic agents for modifying the levels of IL-10.
• the invention further provides Lactobacillus salivarius strains for use in the preparation of biotherapeutic agents for modifying the levels of IL-12.
• the invention further provides Lactobacillus salivarius strains for use in the preparation of biotherapeutic agents for modifying the levels of IL-8.
• the invention further provides Lactobacillus salivarius AHllO for use in the preparation of biotherapeutic agents for modifying the levels of TNF ⁇ .
• the invention also provides for the use of Lactobacillus salivarius strains as anti- infective probiotic strains due to their ability to antagonise the growth of pathogenic species.
• the invention is therefore of major potential therapeutic value in the prophylaxis or treatment of dysregulated immune responses, such as undesirable inflammatory reactions, for example inflammatory bowel disease.
• the strains may be used as a panel of biotherapeutic agents from which a selection can be made for modifying the levels of IFN ⁇ , TNF ⁇ , IL-8, IL-10 and/or IL-12.
• the strains or formulations of the invention may be used in the prevention and/or treatment of inflammatory disorders, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancer (particularly of the gastrointestinal and immune systems), diarrhoeal disease, antibiotic associated diarrhoea, paediatric diarrhoea, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, coeliac disease, diabetes mellitus, organ transplantation, bacterial infections, viral infections, fungal infections, periodontal disease, urogenital disease, sexually transmitted disease, HIV infection, HIV replication, HIV associated diarrhoea, surgical associated trauma, surgical-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia, wound healing, ulcers, gut barrier function, allergy, asthma, respiratory disorders, circulatory disorders, coronary heart disease, anaemia, disorders of the blood coagulation system, renal disease, disorders of the central nervous system, hepati
• Lactobacillus strains are commensal microorganisms. They have been isolated from the microbial flora within the human gastrointestinal tract. The immune system within the gastrointestinal tract cannot have a pronounced reaction to members of this flora, as the resulting inflammatory activity would also destroy host cells and tissue function.
• Lactobacillus salivarius strain AH102 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) on April 20, 2000 and accorded the accession number NCIMB 41044.
• Lactobacillus salivarius strain AH103 was made at the NCIMB on April 20, 2000 and accorded the accession number NCIMB 41045.
• Lactobacillus salivarius strain AH105 was made at the NCIMB on April 20, 2000 and accorded the accession number NCIMB 41047.
• Lactobacillus salivarius strain AH109 was made at the NCIMB on March 22, 2001 and accorded the accession number NCIMB 41093.
• Lactobacillus salivarius strain AHllO was made at the NCIMB on March 22, 2001 and accorded the accession number NCIMB 41094.
• the Lactobacillus salivarius may be a genetically modified mutant or it may be a naturally occurring variant thereof.
• Lactobacillus salivarius is in the form of viable cells.
• the Lactobacillus salivarius is in the form of viable cells.
• Lactobacillus salivarius may be in the form of non- viable cells.
• Lactobacillus strain of the invention may be administered to animals (including humans) in an orally ingestible form in a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups.
• a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups.
• Suitable formulations may be prepared by methods commonly employed using conventional organic and inorganic additives.
• the amount of active ingredient in the medical composition may be at a level that will exercise the desired therapeutic effect. 3/010298
• the formulation may also include a bacterial component, a drug entity or a biological compound.
• a vaccine comprising any one or more of the strains of the invention may be prepared using any suitable known method and may include a pharmaceutically acceptable carrier or adjuvant.
• mutant, variant and genetically modified mutant include a strain of Lactobacillus salivarius whose genetic and/or phenotypic properties are altered compared to the parent strain.
• Lactobacillus salivarius includes the spontaneous alterations of targeted properties selectively isolated while deliberate alteration of parent strain properties may be accomplished by conventional genetic manipulation technologies, such as gene disruption, conjugative transfer, etc.
• Fig. 1 is a bar graph showing the number of adherent bacteria per 20 epithelial cells using human gastrointestinal epithelial cells, CaCo-2 and HT-29;
• Fig. 2 is a bar graph showing IFN ⁇ levels induced by PBMCs following co- incubation with Lactobacillus salivarius strains;
• Fig. 3 is a bar graph showing the effect on the levels of IL-10 (pg/ml) induced by PBMCs following co-incubation with Lactobacillus salivarius strains;
• Fig. 4 is a bar graph showing the effect of Lactobacillus salivarius strains on PBMC IL-12 (pg/ml) production; /010298
• Fig. 5 is a bar graph showing the effect of Lactobacillus salivarius strains on PBMC IL-8 (pg/ml) production.
• Fig. 6 is a bar graph showing the ability of Lactobacillus salivarius strains to modulate proinflammatory cytokine production measured in pg/ml.
• Lactobacillus salivarius strains AH102, AH103, AH105, AH109 and AHllO are not only acid and bile tolerant and adhere to human intestinal cell lines but also, surprisingly have immunomodulatory effects, by modulating cytokine levels or by antagonising and excluding pro-inflammatory or immunomodulatory microorganisms from the gastrointestinal tract.
• probiotic bacteria in the form of viable cells.
• non-viable cells such as killed cultures or compositions containing beneficial factors expressed by the probiotic bacteria. This could include thermally killed micro-organisms or micro-organisms killed by exposure to altered pH or subjection to pressure.
• non-viable cells product preparation is simpler, cells may be incorporated easily into pharmaceuticals and storage requirements are much less limited than viable cells.
• Lactobacillus casei YIT 9018 offers an example of the effective use of heat killed cells as a method for the treatment and/or prevention of tumour growth as described in US Patent No. US4347240.
• LPS lipopolysaccharide
• Interleukin-8 is one of the cytokines comprising the Macrophage Inflammatory protein family (MIP).
• MIP-1 and -2 families represent a group of proteins which are chemotactic factors for leukocytes and fibroblasts. This family of proteins are also called intercrines, as cells other than macrophages are capable of synthesizing them. These cells include T and B cells, fibroblasts, endothelial cells, keratinocytes, smooth muscle cells, synovial cells, neutrophils, chondrocytes, hepatocytes, platelets and tumour cells.
• MlP-l ⁇ , -l ⁇ , connective tissue activating protein (CTAP), platelet factor 4 (PF4) and IL-8 stimulate neutrophil chemotaxis.
• Monocyte chemotactic protein (MCP-1) and RANTES are chemotactic for monocytes, IL-8 for neutrophils and lymphocytes while PF4 and CTAP are chemotactic for fibroblasts. Roles other than chemotaxis have been described for some of these family members.
• MCP-1 stimulates monocyte cytostatic activity and superoxide anion release.
• CTAP and PF4 increase fibroblast proliferation
• IL-8 increases vascular permeability while MlP-l ⁇ and -l ⁇ are pyrogenic.
• IL-8 is intimately involved in inflammatory responses within the gastrointestinal tract. Stimulation of IL-8 (and other proinflammatory cytokines) could contribute to the development of gastrointestinal lesions therefore it is important that probiotic bacteria should not stimulate the production of this cytokine.
• IL-10 is produced by T cells, B cells, monocytes and macrophages. This cytokine augments the proliferation and differentiation of B cells into antibody secreting cells. IL-10 exhibits mostly anti-inflammatory activities. It up-regulates IL-1RA expression by monocytes and suppresses the majority of monocyte inflammatory activities. IL-10 inhibits monocyte production of cytokines, reactive oxygen and nitrogen intermediates, MHC class II expression, parasite killing and IL-10 production via a feed back mechanism (7). This cytokine has also been shown to block monocyte production of intestinal collagenase and type IV collagenase by interfering with a PGE2-cAMP dependant pathway and therefore may be an important regulator of the connective tissue destruction seen in chronic inflammatory diseases.
• IL-12 is a heterodimeric protein of 70 kD composed of two covalently linked chains of 35 kD and 40 kD. It is produced primarily by antigen presenting cells, such as macrophages, early in the inflammatory cascade. Intracellular bacteria stimulate the production of high levels of IL-12. It is a potent inducer of IFN ⁇ production and activator of natural killer cells.
• IL-12 is one of the key cytokines necessary for the generation of cell mediated, or Thl, immune responses primarily through its ability to prime cells for high IFN ⁇ production (8). IL-12 induces the production of IL-10 which feedback inhibits IL-12 production thus restricting uncontrolled cytokine production. TGF- ⁇ also down-regulates IL-12 production.
• IL-4 and IL-13 can have stimulatory or inhibitory effects on IL-12 production. Inhibition of IL-12 in vivo may have some therapeutic value in the treatment of Thl associated inflammatory disorders, such as multiple sclerosis (9).
• Interferon-gamma (IFN ⁇ )D is primarily a product of activated T lymphocytes and due to variable glycosylation it can be found ranging from 20 to 25 kDa in size. This cytokine synergizes with other cytokines resulting in a more potent stimulation of monocytes, macrophages, neutrophils and endothelial cells. IFN ⁇ also amplifies lipopolysacchari.de (LPS) induction of monocytes and macrophages by increasing cytokine production (10), increased reactive intermediate release, phagocytosis and cytotoxicity.
• LPS lipopolysacchari.de
• IFN ⁇ induces, or enhances the expression of major histocompatibility complex class II (MHC class II) antigens on monocytic cells and cells of epithelial, endothelial and connective tissue origin. This allows for greater presentation of antigen to the immune system from cells within inflamed tissues.
• IFN ⁇ may also have anti-inflammatory effects. This cytokine inhibits phospholipase A 2 , thereby decreasing monocyte production of PGE2 and collagenase (11). IFN ⁇ may also modulate monocyte and macrophage receptor expression for TGF ⁇ , TNF ⁇ and C5a (11) thereby contributing to the anti-inflammatory nature of this cytokine. Probiotic stimulation of this cytokine would have variable effects in vivo depending on the current inflammatory state of the host, stimulation of other cytokines and the route of administration.
• MHC class II major histocompatibility complex class II
• TNF ⁇ is a proinflammatory cytokine which mediates many of the local and systemic effects seen during an inflammatory response.
• This cytokine is primarily a monocyte or macrophage derived product but other cell types including lymphocytes, neutrophils, NK cells, mast cells, astrocytes, epithelial cells endothelial cells and smooth muscle cells can also synthesise TNF ⁇ .
• TNF ⁇ is synthesised as a prohormone and following processing the mature 17.5 kDa species can be observed.
• Purified TNF ⁇ has been observed as dimers, trimers and pentamers with the trimeric form postulated to be the active form in vivo. Three receptors have been identified for TNF ⁇ .
• a soluble receptor seems to function as a TNF ⁇ inhibitor (12) while two membrane bound forms have been identified with molecular sizes of 60 and 80 kDa respectively.
• Local TNF ⁇ production at inflammatory sites can be induced with endotoxin and the glucocorticoid dexamethasone inhibits cytokine production (13).
• TNF ⁇ production results in the stimulation of many cell types. Significant anti-viral effects could be observed in TNF ⁇ treated cell lines (14) and the IFNs synergise with TNF ⁇ enhancing this effect.
• Endothelial cells are stimulated to produce procoagulant activity, expression of adhesion molecules, IL-1, hematopoitic growth factors, platelet activating factor (PAF) and arachidonic acid metabolites.
• TNF ⁇ O stimulates neutrophil adherence, phagocytosis, degranulation (15), reactive oxygen intermediate production and may influence cellular migration.
• Leucocyte synthesis of GM-CSF, TGF ⁇ , IL-1, IL-6, PGE2 and TNF ⁇ itself can all be stimulated upon TNF ⁇ administration (16, 17).
• Programmed cell death (apoptosis) can be delayed in monocytes (18) while effects on fibroblasts include the promotion of chemotaxis and
• TNF ⁇ IL-6, PGE2 and collagenase synthesis. While local TNF ⁇ production promotes wound healing and immune responses, the dis-regulated systemic release of TNF ⁇ can be severely toxic with effects such as cachexia, fever and acute phase protein production being observed (19).
• the invention will be more clearly understood from the following examples.
• Example 1 Characterisation of bacteria isolated from resected and washed human gastrointestinal tract. Demonstration of probiotic traits.
• Frozen tissues were thawed, weighed and placed in cysteinated (0.05%) one quarter strength Ringers ' solution. The sample was gently shaken to remove loosely adhering microorganisms (termed -wash 'W'). Following transfer to a second volume of
• sample was vortexed for 7 mins to remove tightly adhering bacteria (termed -sample 'S ') .
• samples 356, 176 and A were also homogenized in a Braun blender (termed -homogenate ⁇ ').
• agar media RCM (reinforced clostridia media) and RCM adjusted to pH 5.5 using acetic acid; TPY (trypticase, peptone and yeast extract); MRS (deMann, Rogosa and Sharpe); ROG (acetate medium (SL) of Rogosa); LLA (liver-lactose agar of Lapiere); BHI (brain heart infusion agar); LBS (Lactobacillus selective agar) and TSAYE (tryptone soya sugar supplemented with 0.6% yeast extract). TPY and MRS supplemented with propionic acid was also used. All agar media was supplied by RCM (reinforced clostridia media) and RCM adjusted to pH 5.5 using acetic acid; TPY (trypticase, peptone and yeast extract); MRS (deMann, Rogosa and Sharpe); ROG (acetate medium (SL) of Rogosa); LLA (liver-
• Oxoid Chemicals with the exception of TPY agar. Plates were incubated in anaerobic jars (BBL, Oxoid) using CO 2 generating kits (Anaerocult A, Merck) for 2-5 days at 37°C. Gram positive, catalase negative rod-shaped or bifurcated/pleomorphic bacteria isolates were streaked for purity on to complex non-selective media (MRS and TPY).
• Isolates were routinely cultivated in MRS or TPY medium unless otherwise stated at 37°C under anaerobic conditions. Presumptive Lactobacillus were stocked in 40% glycerol and stored at -20°C and -80°C.
• TPYP 0 >9.0 x 10 3 >6.0 x 10 3 >3.0 x 10 4 0 1.9 x lO 2 2.8 x 10 2
• ROG 0 >9.0 x 10 3 >6.0 x 10 3 7.7 x 10 2 3.8 x 10 2 9.7 x 10 1 4.0 x 10 1
• RCM ND ND ND >3.0 x 10 4 ND >1.0 x 10 4 ND
• the column was maintained at 60°C with a flow rate of 0.6 ml/min (constant pressure).
• the HPLC buffer used was 0.01 N H 2 SO 4 . Prior to analysis, the column was calibrated using 10 mM citrate, lOmM glucose, 20 mM lactate and 10 mM acetate as standards. Cultures were propagated in modified MRS broth for 1-2 days at 37°C anaerobically. Following centrifugation for 10 min at 14,000 g, the supernatant was diluted 1:5 with HPLC buffer and 200 ⁇ l was analysed in the HPLC. All supernatants were analysed in duplicate.
• Biochemical and physiological traits of the bacterial isolates were determined to aid identification. Nitrate reduction, indole formation and expression of ⁇ -galactosidase activity were assayed. Growth at both 15°C and 45°C, growth in the presence of increasing concentrations of NaCl up to 5.0% and protease activity on gelatin were determined. Growth characteristics of the strains in litmus milk were also assessed.
• catalase negative bacterial isolates from different samples were chosen and characterised in terms of their Gram reaction, cell size and morphology, growth at 15°C and 45°C and fermentation end-products from glucose (data not shown). Greater than sixty percent of the isolates tested were Gram positive, homofermentative cocci (HOMO-) arranged either in tetrads, chains or bunches. Eighteen percent of the isolates were Gram negative rods and heterofermentative coccobacilli (HETERO-) . The remaining isolates (twenty two percent) were predominantly homofermentative coccobacilli.
• HOMO- homofermentative cocci
• HETERO- heterofermentative coccobacilli
• API 50CHL BioMerieux SA, France
• Lactobacillus species by their carbohydrate fermentation profiles. Overnight MRS cultures were harvested by centrifugation and resuspended in the suspension medium provided with the kit. API strips were inoculated and analysed (after 24 and 48 h) according to the manufacturers ' instructions. Identity of the Lactobacillus sp. was then checked by SDS-Polyacrylamide gel electrophoresis analysis (SDS-PAGE) of total cell protein (Bruno Pot, University of Ghent, Belgium, personal communication). Finally, 16s RNA analysis and ribotyping were used to confirm strain identity.
• SDS-PAGE SDS-Polyacrylamide gel electrophoresis analysis
• the API 50CHL allowed rapid identification of the Lactobacillus isolates. Analysis of total cell protein of the Lactobacillus sp. (Bruno Pot, personal communication) by SDS-PAGE, 16s RNA analysis and ribotyping revealed further information on the specific species. Table 3 below shows the identification of the 5 Lactobacillus strains by four different techniques.
• the API ZYM system (BioMerieux, France) was used for semi-quantitative measurement of constitutive enzymes produced by Lactobacillus isolates.
• Bacterial cells from the late logarithmic growth phase were harvested by centrifugation at 14,000g for 10 mins. The pelleted cells were washed and resuspended in 50mM phosphate buffer, pH 6.8 to the same optical density. The strips were inoculated in accordance with the manufacturer's instructions, incubated for 4 h at 37°C and colour development recorded.
• Antibiotic sensitivity profiles of the isolates were determined using the 'disc susceptibility' assay. Cultures were grown up in the appropriate broth medium for 24- 48h spread-plated (lOO ⁇ l) onto agar media and discs containing known concentrations of the antibiotics were placed onto the agar. Strains were examined for antibiotic sensitivity after 1-2 days incubation at 37°C under anaerobic conditions. Strains were considered sensitive if zones of inhibition of 1mm or greater were seen.
• Human gastric juice was obtained from healthy subjects by aspiration through a nasogastric tube (Mercy Hospital, Cork, Ireland). It was immediately centrifuged at 13,000 g for 30 min to remove all solid particles, sterilised through 0.45 ⁇ m and 0.2 ⁇ m filters and divided into 40 ml aliquots which were stored at 4°C and -20°C.
• Pepsin activity was measured using the quantitative haemoglobulin assay. Briefly, aliquots of gastric juice (1ml) were added to 5 ml of substrate (0.7 M urea, 0.4% (w/v) bovine haemoglobulin (Sigma Chemical Co., 0.25 M KC1-HC1 buffer, pH 2.0) and incubated at 25°C. Samples were removed at 0, 2, 4, 6, 8, 10, 20 and 30 min intervals. Reactions were terminated by the addition of 5% trichloracetic acid (TCA) and allowed to stand for 30 min without agitation. Assay mixtures were then filtered
• pepsin enzyme activity was defined as the amount of enzyme required to cause an increase of 0.001 units of A 280 nm per minute at pH 2.0 measured as TCA-soluble products using haemoglobulin as substrate.
• Lactobacillus strains were propagated in buffered MRS broth (pH 6.0) daily for a 5 day period. The cells were harvested, washed and resuspended in pH adjusted MRS broth and survival measured over a 2 h period using the plate count method.
• Each of the strains was generally resistant to pH values 3.5, 3.0, and 2.5, with
• Lactobacillus salivarius AH102 and AH105 also exhibiting resistance at pH 2.0 (data not shown).
• Bile samples isolated from several human gall-bladders, were stored at -80°C before use. For experimental work, bile samples were thawed, pooled and sterilised at 80°C for 10 min. Bile acid composition of human bile was determined using reverse-phase High Performance Liquid Chromatography (HPLC) in combination with a pulsed amperometric detector according to the method of Dekker et al. (20). Human bile was added to MRS/TPY agar medium at a concentration of 0.3% (v/v). Freshly streaked cultures were examined for growth after 24 and 48 h.
• HPLC High Performance Liquid Chromatography
• TCA taurocholic acid
• GCA glycocholic acid
• GDC A glycodeoxycholic acid
• TCDCA taurochenodeoxycholic acid
• GCDCA glycochenodeoxycholic acid
• HPLC High Performance Liquid Chromatography
• Lactobacillus salivarius AH102, AH103, AH105, AH109 and AHllO were capable of growth (bile acid resistance) on three sources of bile used. It was observed that resistance to bovine bile was much higher than to porcine bile. Each of the Lactobacillus strains tested was resistant to concentrations up to and including 5.0% bovine bile (data not shown).
• Porcine bile was more inhibitory as shown in Table 7 below.
• each of the Lactobacillus salivarius strains grew to confluence at the physiological concentration of 0.3% (v/v) human bile (data not shown).
• Each of the Lactobacillus strains when analysed specifically for its resistance to individual bile acids, grew well in the presence of taurine conjugated bile acids. Isolates from each of Lactobacillus strain grew to confluence on agar medium containing up to and including 5 mM of taurine conjugates TCA, TDCA and TCDCA. Of the glycine conjugates tested, GCDCA was the most inhibitory. GDCA was less inhibitory and GCA was the least inhibitory of the three glycine conjugates. Each of the Lactobacillus strains grew on agar medium supplemented with 5 mM GCA. This is shown in Table 8 below.
• Indicators used in the initial screening were L. innocua, L. fermentum KLD, P. flourescens and E. coli V157. Briefly, the lactobacilli (MRS) were incubated for 12-16 h and 36-48 h, respectively. Ten-fold serial dilutions were spread-plated (lOO ⁇ l) onto MRS/TPY agar medium. After overnight incubation, plates with distinct colonies were overlay ed with the indicator bacterium. The indicator lawn was prepared by inoculating a molten overlay with 2% (v/v) of an overnight indicator culture which was poured over the surface of the inoculated MRS plates. The plates were re-incubated overnight under conditions suitable for growth of the indicator bacterium. Indicator cultures with inhibition zones greater than 1 mm in radius were considered sensitive to the test bacterium.
• Lactobacillus salivarius AH102, AH103, AH105, AH109 and AHllO were screened for inhibitory activity using Ls. innocua, L. fermentum KLD, P. fluorescens and E. coli as indicator microorganisms.
• Ls. innocua L. fermentum KLD
• P. fluorescens P. fluorescens
• E. coli indicator microorganisms.
• Zones ranging in size from 1 mm to 5 mm were measured. Inhibition of Ls. innocua by each of the lactobacilli produced the largest zones.
• Example 2 Adhesion of probiotic bacteria to gastrointestinal epithelial cells.
• the adhesion of the probiotic strains was carried out using a modified version of a previously described method (22).
• the monolayers of HT-29 and Caco-2 cells were prepared on sterile 22mm glass coverslips, which were placed in Corning tissue culture dishes, at a concentration of 4 X 10 4 cells/ml. Cells were fed fresh medium every 2 days. After ⁇ 10 days, and differentiation of the monolayer had occurred, the monolayers were washed twice with Phosphate Buffered Saline (PBS). Antibiotic- free DMEM (2ml) and 2ml of ⁇ 18 Lb.
• PBS Phosphate Buffered Saline
• Example 3 Determination of the effect of each of the Lactobacillus strains on PBMC cytokine production.
• AH102, AH103 and AH105 stimulated production of IFN ⁇ from PBMCs (Fig. 2).
• AH102, AH103, AH109 and AHllO co-incubation did not significantly alter IL-10 levels (Fig. 3). Stimulation with AH105 significantly reduced secretion of IL-10 by PBMCs.
• Example 4 Determination of cytokine levels in an epithelial/PBMC co-culture model following incubation with AH103 and AHllO.
• the appropriate in vitro model with physiological relevance to the intestinal tract is a culture system incorporating epithelial cells, T cells, B cells, monocytes and the bacterial strains.
• human Caco-2 epithelial cells were seeded at 5x10 5 cells/ml on the apical surface of 25 mm transwell inserts with a pore size of 3D m (Costar). These cells were cultured for four weeks in RPMI 1640, supplemented with 10% foetal calf serum, glutamine, penicillin and streptomycin, at 37°C in a 5% CO 2 environment. Culture media was changed every 3 days.
• PBMCs peripheral blood mononuclear cells
• TNF ⁇ and IL-8 extracellular cytokine levels were measured using standard ELISA kits (R&D Systems). TNF ⁇ levels and
• IL-8 levels were measured, in duplicate, using PBMCs from 3 healthy volunteers.
• TNF ⁇ and IL-8 cytokine levels were examined by ELISAs (Fig. 6).
• AH103 significantly reduced the level of IL-8 released by these cells.
• AHllO reduced the levels of TNF ⁇ and IL-8 released by these cells.
• the human immune system plays a significant role in the aetiology and pathology of a vast range of human diseases. Hyper and hypo-immune responsiveness results in, or is a component of, the majority of disease states.
• One family of biological entities, termed cytokines, are particularly important to the control of immune processes. Pertubances of these delicate cytokine networks are being increasingly associated with many diseases.
• diseases include but are not limited to inflammatory disorders, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancer (particularly those of the gastrointestinal and immune systems), diarrhoeal disease, antibiotic associated diarrhoea, paediatric diarrhoea, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, coeliac disease, diabetes mellitus, organ transplantation, bacterial infections, viral infections, fungal infections, periodontal disease, urogenital disease, sexually transmitted disease, HIV infection, HIV replication, HIV associated diarrhoea, surgical associated trauma, surgical-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia, wound healing, ulcers, gut barrier function, allergy, asthma, respiratory disorders, circulatory disorders, coronary heart disease, anaemia, disorders of the blood coagulation system, renal disease, disorders of the central nervous system, hepatic disease, ischaemia, nutritional disorders, osteop
• cytokine production is specific for each of the probiotic strains examined.
• specific probiotic strains may be selected for normalising an exclusive cytokine imbalance particular for a specific disease type.
• Customisation of disease specific therapies can be accomplished using a selection of the probiotic strains listed above.
• the enteric flora is important to the development and proper function of the intestinal immune system. In the absence of an enteric flora, the intestinal immune system is underdeveloped, as demonstrated in germ free animal models, and certain functional parameters are diminished, such as macrophage phagocytic ability and immunoglobulin production (23). The importance of the gut flora in stimulating non- damaging immune responses is becoming more evident. The increase in incidence and severity of allergies in the western world has been linked with an increase in hygiene and sanitation, concomitant with a decrease in the number and range of infectious challenges encountered by the host. This lack of immune stimulation may allow the host to react to non-pathogenic, but antigenic, agents resulting in allergy or autoimmunity. Deliberate consumption of a series of non-pathogenic immunomodulatory bacteria would provide the host with the necessary and appropriate educational stimuli for proper development and control of immune function.
• Inflammation is the term used to describe the local accumulation of fluid, plasma proteins and white blood cells at a site that has sustained physical damage, infection or where there is an ongoing immune response. Control of the inflammatory response is exerted on a number of levels (24).
• the controlling factors include cytokines, hormones (e.g. hydrocortisone), prostaglandins, reactive intermediates and leukotrienes.
• Cytokines are low molecular weight biologically active proteins that are involved in the generation and control of immunological and inflammatory responses, while also regulating development, tissue repair and haematopoiesis. They provide a means of communication between leukocytes themselves and also with other cell types. Most cytokines are pleiotrophic and express multiple biologically overlapping activities.
• Cytokine cascades and networks control the inflammatory response rather than the action of a particular cytokine on a particular cell type (25). Waning of the inflammatory response results in lower concentrations of the appropriate activating signals and other inflammatory mediators leading to the cessation of the inflammatory response.
• TNF ⁇ is a pivotal proinflammatory cytokine as it initiates a cascade of cytokines and biological effects resulting in the inflammatory state. Therefore, agents which inhibit TNF ⁇ are currently being used for the treatment of inflammatory diseases, e.g. infliximab.
• IBD inflammatory bowel disease
• Current therapies for treating IBD are aimed at reducing the levels of these pro-inflammatory cytokines, including IL-8 and TNF ⁇ .
• Such therapies may also play a significant role in the treatment of systemic inflammatory diseases such as rheumatoid arthritis.
• IBS Irritable bowel syndrome
• IBS IBS
• IBS sufferers may have a significantly reduced quality of life, are more likely to be absent from work, and use more healthcare resources.
• recommended therapies have included antispasmodic agents, anti-diarrhoeal agents, dietary fibre supplements, drugs that modify the threshold of colonic visceral perception, analgesics and anti-depressants.
• strains of the invention While each of the strains of the invention have unique properties with regard to cytokine modulation and microbial antagonism profiles, it should be expected that specific strains can be chosen for use in specific disease states based on these properties. It also should be anticipated that combinations of strains from this panel with appropriate cytokine modulating properties and anti-microbial properties will enhance therapeutic efficacy.
• strains of the present invention may have potential application in the treatment of a range of inflammatory diseases, particularly if used in combination with other anti- inflammatory therapies, such as non-steroid anti-inflammatory drugs (NSAIDs) or Infliximab.
• NSAIDs non-steroid anti-inflammatory drugs
• Infliximab Infliximab
• the inflammatory response may have significant roles to play in the above mechanisms, thus contributing to the decline of the host and progression of the tumour.
• intestinal bacteria can produce, from dietary compounds, substances with genotoxic, carcinogenic and tumour-promoting activity and gut bacteria can activate pro-carcinogens to DNA reactive agents (28).
• species of Lactobacillus have low activities of xenobiotic metabolizing enzymes compared to other populations within the gut such as bacteroides, eubacteria and clostridia. Therefore, increasing the number of Lactobacillus bacteria in the gut could beneficially modify the levels of these enzymes.
• TTFC tetanus toxin fragment C
• Streptococcus gordonii are also being examined for their usefulness as mucosal vaccines.
• Recombinant S. gordonii colonizing the murine oral and vaginal cavities induced both mucosal and systemic antibody responses to antigens expressed by this bacterial (31).
• oral immunization using probiotic bacteria as vectors would not only protect the host from infection, but may replace the immunological stimuli that the pathogen would normally elicit thus contributing to the immunological education of the host.
• probiotic organisms The introduction of probiotic organisms is accomplished by the ingestion of the microorganism in a suitable carrier. It would be advantageous to provide a medium that would promote the growth of these probiotic strains in the large bowel.
• the addition of one or more oligosaccharides, polysaccharides, or other prebiotics enhances the growth of lactic acid bacteria in the gastrointestinal tract.
• Prebiotics refers to any non- viable food component that is specifically fermented in the colon by indigenous bacteria thought to be of positive value, e.g. bifidobacteria, lactobacilli. Types of prebiotics may include those that contain fructose, xylose, soya, galactose, glucose and mannose.
• the combined administration of a probiotic strain with one or more prebiotic compounds may enhance the growth of the administered probiotic in vivo resulting in a more pronounced health benefit, and is termed synbiotic.
• the probiotic strains may be administered prophylactically or as a method of treatment either on its own or with other probiotic and/or prebiotic materials as described above.
• the bacteria may be used as part of a prophylactic or treatment regime using other active materials such as those used for treating inflammation or other disorders especially those with an immunological involvement.
• Such combinations may be administered in a single formulation or as separate formulations administered at the same or different times and using the same or different routes of administration.
• IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J Exp Med 1991 Oct l;174(4):915-24.

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