Patent Application: US-10559005-A

Abstract:
a lactobacillus salivarius strain , ah102 , ah103 , ah105 , ah109 or ah110 or mutants or variants thereof are useful in the prophylaxis and / or treatment of inflammatory activity especially undesirable gastrointestinal inflammatory activity , such as inflammatory bowel disease or irritable bowel syndrome .

Description:
we have found that lactobacillus salivarius strains ah102 , ah103 , ah105 , ah109 and ah110 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 micro - organisms from the gastrointestinal tract . the general use of probiotic bacteria is in the form of viable cells . however , it can also be extended to 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 . with 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 u . s . pat . no . 4 , 347 , 240 . it is unknown whether intact bacteria are required to exert an immunomodulatory effect or if individual active components of the invention can be utilized alone . proinflammatory components of certain bacterial strains have been identified . the proinflammatory effects of gram - negative bacteria are mediated by lipopolysaccharide ( lps ). lps alone induces a proinflammatory network , partially due to lps binding to the cd14 receptor on monocytes . it is assumed that components of probiotic bacteria possess immunomodulatory activity , due to the effects of the whole cell . upon isolation of these components , pharmaceutical grade manipulation is anticipated . interleukin - 8 ( il - 8 ) is one of the cytokines comprising the macrophage inflammatory protein family ( mip ). the 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 . mip - 1α , - 1β , 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 mip - 1α and - 1β 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 ifnγ production and activator of natural killer cells . il - 12 is one of the key cytokines necessary for the generation of cell mediated , or th1 , immune responses primarily through its ability to prime cells for high ifnγ production ( 8 ). il - 12 induces the production of il - 10 which feedback inhibits il - 12 production thus restricting uncontrolled cytokine production . tgf - β 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 th1 associated inflammatory disorders , such as multiple sclerosis ( 9 ). interferon - gamma ( ifnγ ) 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 . ifnγ also amplifies lipopolysaccharide ( lps ) induction of monocytes and macrophages by increasing cytokine production ( 10 ), increased reactive intermediate release , phagocytosis and cytotoxicity . ifnγ 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 . ifnγ may also have anti - inflammatory effects . this cytokine inhibits phospholipase a 2 , thereby decreasing monocyte production of pge2 and collagenase ( 11 ). ifnγ may also modulate monocyte and macrophage receptor expression for tgfβ , tnfα 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 . tnfα 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 tnfα . tnfα is synthesised as a prohormone and following processing the mature 17 . 5 kda species can be observed . purified tnfα 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 tnfα . a soluble receptor seems to function as a tnfα inhibitor ( 12 ) while two membrane bound forms have been identified with molecular sizes of 60 and 80 kda respectively . local tnfα production at inflammatory sites can be induced with endotoxin and the glucocorticoid dexamethasone inhibits cytokine production ( 13 ). tnfα production results in the stimulation of many cell types . significant anti - viral effects could be observed in tnfα treated cell lines ( 14 ) and the ifns synergise with tnfα 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 . tnfα stimulates neutrophil adherence , phagocytosis , degranulation ( 15 ), reactive oxygen intermediate production and may influence cellular migration . leucocyte synthesis of gm - csf , tgfβ , il - 1 , il - 6 , pge2 and tnfα itself can all be stimulated upon tnfα administration ( 16 , 17 ). programmed cell death ( apoptosis ) can be delayed in monocytes ( 18 ) while effects on fibroblasts include the promotion of chemotaxis and il - 6 , pge 2 and collagenase synthesis . while local tnfα production promotes wound healing and immune responses , the dis - regulated systemic release of tnfα 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 . characterisation of bacteria isolated from resected and washed human gastrointestinal tract . demonstration of probiotic traits appendices and sections of the large and small intestine of the human gastrointestinal tract ( g . i . t .) obtained during reconstructive surgery , were screened for probiotic bacterial strains . all samples were stored immediately after surgery at − 80 ° c . in sterile containers . frozen tissues were thawed , weighed and placed in cysteinated ( 0 . 05 %) one quarter strength ringers &# 39 ; solution . the sample was gently shaken to remove loosely adhering microorganisms ( termed — wash ‘ w ’). following transfer to a second volume of ringer &# 39 ; s solution , the sample was vortexed for 7 mins to remove tightly adhering bacteria ( termed — sample ‘ s ’). in order to isolate tissue embedded bacteria , samples 356 , 176 and a were also homogenized in a braun blender ( termed — homogenate ‘ h ’). the solutions were serially diluted and spread - plated ( 100 μl ) on the following 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 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 . seven tissue sections taken from the g . i . t . were screened for the presence of strains belonging to the lactobacillus genera . there was some variation between tissue samples . table 1 below shows the bacterial count of the tissue samples expressed as colony forming units per gram ( cfu / ml ) of tissue . ( nd = not determined ) samples a ( ileum ) and 316 ( appendix ) had the lowest counts with approximately 10 2 cells isolated per gram of tissue . in comparison , greater 10 3 cfu / g tissue were recovered from the other samples . similar numbers of bacteria were isolated during the ‘ wash ’ and ‘ sample ’ steps with slightly higher counts in the ‘ sample ’ solutions of 433 ( ileal - caecal ). metabolism of the carbohydrate glucose and the subsequent organic acid end - products were examined using an lkb bromma , aminex hpx - 87h high performance liquid chromatography column . 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 , 10 mm 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 μl 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 β - 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 . approximately fifteen hundred 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 . thirty eight strains were characterised in more detail — 13 isolates from 433 ; 4 from 423 ; 8 from 312 ; 9 from 356 ; 3 from 176 and 1 from 316 . all thirty eight isolates tested negative both for nitrate reduction and production of indole from tryptophan . growth at different temperatures , concentrations of nacl and gelatin hydrolysis are recorded in table 2 below . the api 50chl ( biomerieux sa , france ) system was used to tentatively identify the 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 &# 39 ; 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 . 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 , 000 g for 10 mins . the pelleted cells were washed and resuspended in 50 mm phosphate buffer , ph 6 . 8 to the same optical density . the strips were inoculated in accordance with the manufacturer &# 39 ; s instructions , incubated for 4 h at 37 ° c . and colour development recorded . the enzyme activity profiles of the 5 strains ah102 , ah103 , ah105 , ah 109 , ah110 are presented in table 4 below . none of the strains exhibited lipase , trypsin , α - chymotrypsin , α - glucuronidase , α - mannosidase or α - fucosidase activities . antibiotic sensitivity profiles of the isolates were determined using the ‘ disc susceptibility ’ assay . cultures were grown up in the appropriate broth medium for 24 - 48 h spread - plated ( 100 μl ) 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 1 mm or greater were seen . antibiotics of human clinical importance were used to ascertain the sensitivity profiles of each of the 5 lactobacillus strains . each of the lactobacilli tested was sensitive to ampicillin , amoxacillin , ceftaxime , ceftriaxone , ciprofloxacin , cephradine , rifampicin and chloramphenicol . the antibiotic sensitivities ( ug / ml ) of lactobacillus salivarius ah102 , ah103 , ah105 , ah109 and ah110 are given in table 5 below . 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 μm and 0 . 2 μm filters and divided into 40 ml aliquots which were stored at 4 ° c . and − 20 ° c . the ph and pepsin activity of the samples were measured prior to experimental use . pepsin activity was measured using the quantitative haemoglobulin assay . briefly , aliquots of gastric juice ( 1 ml ) were added to 5 ml of substrate ( 0 . 7 m urea , 0 . 4 % ( w / v ) bovine haemoglobulin ( sigma chemical co ., 0 . 25 m kcl - hcl 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 ( whatman , no . 113 ), centrifuged at 14 , 000 g for 15 min and absorbance at 280 nm was measured . one unit of 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 . to determine whether growth of the lactobacillus strains occurred at low ph values equivalent to those found in the stomach , overnight cultures were inoculated ( 1 %) into fresh mrs broth adjusted to ph4 . 0 , 3 . 0 , 2 . 0 and 1 . 0 using 1n hcl . at regular intervals aliquots ( 1 . 5 ml ) were removed , optical density at 600 nm ( od600 ) was measured and colony forming units per ml ( cfu / ml ) calculated using the plate count method . growth was monitored over a 24 - 48 h period . survival of the strains at low ph in vitro was investigated using two assays : ( a ) cells were harvested from fresh overnight cultures , washed twice in phosphate buffer ( ph 6 . 5 ) and resuspended in mrs broth adjusted to ph 3 . 5 , 3 . 0 , 2 . 5 , and 2 . 0 ( with 1n hcl ) to a final concentration of approximately 10 8 cfu / ml for the lactobacilli . cells were incubated at 37 ° c . and survival measured at intervals of 5 , 30 , 60 and 120 min using the plate count method . ( b ) the 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 . to determine the ability of the lactobacilli to survive passage through the stomach , an ex - vivo study was performed using human gastric juice . cells from fresh overnight cultures were harvested , washed twice in buffer ( ph 6 . 5 ) and resuspended in human gastric juice to a final concentration of 10 6 - 10 8 cfu / ml , depending on the strain . survival was monitored over a 30 - 60 min incubation period at 37 ° c . the experiment was performed using gastric juice at ph ˜ 1 . 2 ( unadjusted ) and ph 2 . 0 and 2 . 5 . each of the lactobacillus strains tested grew normally at ph 6 . 8 and ph 4 . 5 reaching stationary phase after 8 h with a doubling time of 80 - 100 min . at ph 3 . 5 growth was restricted with doubling times increasing to 6 - 8 h . no growth was observed at ph 2 . 5 or lower , therefore , survival of the strains at low ph was examined . 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 ). to determine the ability of the lactobacillus strains to survive conditions encountered in the human stomach , viability of each of the 5 strains was tested in human gastric juice at ph 1 . 2 and ph 2 . 5 , as shown in table 6 below . the survival is expressed at log10 cfu / ml ( nd = not determined ). fresh cultures were streaked onto mrs agar plates supplemented with bovine bile ( b - 8381 , sigma chemical co . ltd ., poole ) at concentrations of 0 . 3 , 1 . 0 , 1 . 5 , 5 . 0 and 7 . 5 % ( w / v ) and porcine bile ( b - 863 1 , sigma chemical co . ltd ., poole ) at concentrations of 0 . 3 , 0 . 5 , 1 . 0 , 1 . 5 , 5 . 0 and 7 . 5 % ( w / v ). plates were incubated at 37 ° c . under anaerobic conditions and growth was recorded after 24 - 48 h . 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 . human gall - bladder bile possesses a bile acid concentration of 50 - 100 mm and dilution in the small intestine lowers this concentration to 5 - 10 mm . furthermore , under physiological conditions , bile acids are found as sodium salts . therefore , cultures were screened for growth on mrs agar plates containing the sodium salt of each of the following bile acids ( sigma chemical co . ltd ., poole ): ( a ) conjugated form : taurocholic acid ( tca ); glycocholic acid ( gca ); taurodeoxycholic acid ( tdca ); glycodeoxycholic acid ( gdca ); taurochenodeoxycholic acid ( tcdca ) and glycochenodeoxycholic acid ( gcdca ); ( b ) deconjugated form : lithocholic acid ( lca ); chenodeoxycholic acid ( cdca ); deoxycholic acid ( dca ) and cholic acid ( ca ). for each bile acid concentrations of 1 , 3 and 5 mm were used . growth was recorded after 24 and 48 h anaerobic incubation . both a qualitative ( agar plate ) and a quantitative ( hplc ) assay were used to determine deconjugation activity . plate assay : all the cultures were streaked on mrs agar plates supplemented with ( a ) 0 . 3 % ( w / v ) porcine bile , ( b ) 3 mm tdca or ( c ) 3 mm gdca . deconjugation was observed as an opaque precipitate surrounding the colonies . high performance liquid chromatography ( hplc ): analysis of in vitro deconjugation of human bile was performed using hplc . briefly , overnight cultures were inoculated ( 5 %) into mrs broth supplemented with 0 . 3 % ( v / v ) human bile and were incubated anaerobically at 37 ° c . at various time intervals over a 24 h period , samples ( 1 ml ) were removed and centrifuged at 14 , 000 rpm for 10 min . undiluted cell - free supernatant ( 30 μl ) was then analysed by hplc . lactobacillus salivarius ah102 , ah103 , ah105 , ah109 and ah110 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 . regardless of the bile resistance profiles in the presence of both bovine and porcine bile , 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 . growth in the presence of deconjugated bile acids was also tested . each lactobacillus strain was resistant to concentrations of 5 mm lca . growth in the presence of ca was also tested . as shown in table 9 below , three of the 5 strains , ah102 , ah105 and ah109 grew in the presence of 1 mm ca . none of the strains grew in the presence of 1 mm cdca . ( data not shown ). antimicrobial activity was detected using the deferred method ( 21 ). 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 ( 100μl ) onto mrs / tpy agar medium . after overnight incubation , plates with distinct colonies were overlayed 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 . inhibition due to bacteriophage activity was excluded by flipping the inoculated mrs / tpy agar plates upside down and overlaying with the indicator . bacteriophage cannot diffuse through agar . lactobacillus salivarius ah102 , ah103 , ah105 , ah109 and ah110 were screened for inhibitory activity using ls . innocua , l . fermentum kld , p . fluorescens and e . coli as indicator microorganisms . when the test strains were inoculated on unbuffered mrs , inhibition of the four indicators was observed . 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 . 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 22 mm 2 glass coverslips , which were placed in corning tissue culture dishes , at a concentration of 4 × 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 ( 2 ml ) and 2 ml of 18 h lb . suspension containing ˜ 10 9 cfu / ml were added to each dish and cells were incubated for 2 h at 37 ° c . in a humidified atmosphere containing 5 % co 2 . after incubation , the monolayers were washed 5 times with pbs , fixed in methanol ( bdh laboratory supplies , poole , uk ) for 3 min , gram stained ( gram stain set , merck ) and examined microscopically under oil immersion . for each glass coverslip monolayer the number of adherent bacteria per 20 epithelial cells was counted in 10 microscopic fields . the mean and standard error of adherent bacteria per 20 epithelial cells was calculated . each adhesion assay was carried out in duplicate . in a second method , after washing 5 times in pbs , adhering bacteria were removed by vortexing the monolayers rigorously in cold sterile h 2 o . bacterial cells were enumerated by serial dilution in quarter strength ringer &# 39 ; s solution ( oxoid ) and incubation on mrs ( lactobacilli ). each of the 5 lactobacillus strains , ah102 , ah103 , ah105 , ah109 and ah110 adhered to gastrointestinal epithelial cells ( fig1 ). these probiotic strains would be suitable as vaccine / drug delivery vehicles as they adhere to the gastrointestinal epithelium and therefore interact with the relevant host tissue . determination of the effect of each of the lactobacillus strains on pbmc cytokine production peripheral blood mononuclear cells were isolated from healthy donors ( n = 19 ) by density gradient centrifugation . pbmcs were stimulated with the probiotic bacterial strains for a 72 hour period at 37 ° c . at this time culture supernatants were collected , centrifuged , aliquoted and stored at − 70 ° c until being assessed for il - 8 , il - 10 , il - 12 and ifnγ levels using elisas ( boehringer mannheim ). ah102 , ah103 and ah105 stimulated production of ifnγ from pbmcs ( fig2 ). ah102 , ah103 , ah109 and ah110 co - incubation did not significantly alter il - 10 levels ( fig3 ). stimulation with ah105 significantly reduced secretion . of il - 10 by pbmcs . ah102 , ah105 , ah109 and ah110 co - incubation significantly upregulated il - 12 production by pbmcs ( fig4 ). ah103 had no significant effect on il - 12 production . none of the 5 lactobacillus strains stimulated il - 8 production in vitro , from pbmcs isolated from healthy donors . indeed , in each case , il - 8 levels were reduced ( fig5 ). determination of cytokine levels in an epithelial / pbmc co - culture model following incubation with ah103 and ah110 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 . to this end , human caco - 2 epithelial cells were seeded at 5 × 10 5 cells / ml on the apical surface of 25 mm transwell inserts with a pore size of 3 □ 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 . when the epithelial cells were fully differentiated , human peripheral blood mononuclear cells ( pbmcs ) were isolated by density gradient centrifugation . 1 × 10 6 washed pbmcs was incubated basolaterally to the epithelial cells and cultured with 1 × 10 7 probiotic bacteria . controls contained media alone . no direct cell - cell contact between pbmcs and epithelial cells was possible in this model system and cellular communication was mediated solely by soluble factors . following 72 hours of incubation with ah103 or ah110 , cell culture supernatants were removed , aliquoted and stored at − 70 ° c . tnfα and il - 8 extracellular cytokine levels were measured using standard elisa kits ( r & amp ; d . systems ). tnfα levels and il - 8 levels were measured , in duplicate , using pbmcs from 3 healthy volunteers . following incubation of epithelial cell - pbmc co - cultures with probiotic bacteria , tnfα and il - 8 cytokine levels were examined by elisas ( fig6 ). ah103 significantly reduced the level of il - 8 released by these cells . ah110 reduced the levels of tnfα 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 . these 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 &# 39 ; 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 , osteoporosis , endocrine disorders , epidermal disorders , psoriasis and acne vulgaris . the effects on cytokine production are specific for each of the probiotic strains examined . thus 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 . tnfα is a pivotal proinflammatory cytokine as it initiates a cascade of cytokines and biological effects resulting in the inflammatory state . therefore , agents which inhibit tnfα are currently being used for the treatment of inflammatory diseases , e . g . infliximab . pro - inflammatory cytokines are thought to play a major role in the pathogenesis of many inflammatory diseases , including inflammatory bowel disease ( ibd ). current therapies for treating ibd are aimed at reducing the levels of these pro - inflammatory cytokines , including il - 8 and tnfα . such therapies may also play a significant role in the treatment of systemic inflammatory diseases such as rheumatoid arthritis . irritable bowel syndrome ( ibs ) is a common gastrointestinal disorder , affecting up to 15 - 20 % of the population at some stage during their life . the most frequent symptoms include abdominal pain , bowel habit disturbance , manifested by diarrhoea or constipation , flatulence , and abdominal distension . there are no simple tests to confirm diagnosis , and if no other organic disorders can be found for these symptoms , the diagnosis is usually ibs . patients suffering from ibs represent as many as 25 - 50 % of patients seen by gastroenterologists . many factors are thought to be involved in onset of symptoms including e . g . bout of gastroenteritis , abdominal or pelvic surgery , disturbances in the intestinal bacterial flora , perhaps due to antibiotic intake , and emotional stress . compared with the general population , ibs sufferers may have a significantly reduced quality of life , are more likely to be absent from work , and use more healthcare resources . there are no effective medical treatments and to date , 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 . 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 . the 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 . the production of multifunctional cytokines across a wide spectrum of tumour types suggests that significant inflammatory responses are ongoing in patients with cancer . it is currently unclear what protective effect this response has against the growth and development of tumour cells in vivo . however , these inflammatory responses could adversely affect the tumour - bearing host . complex cytokine interactions are involved in the regulation of cytokine production and cell proliferation within tumour and normal tissues ( 26 , 27 ). it has long been recognized that weight loss ( cachexia ) is the single most common cause of death in patients with cancer and initial malnutrition indicates a poor prognosis . for a tumour to grow and spread it must induce the formation of new blood vessels and degrade the extracellular matrix . 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 . due to the anti - inflammatory properties of lactobacillus salivarius these bacterial strains they may reduce the rate of malignant cell transformation . furthermore , 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 ). in general , species of lactohacillus 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 . the majority of pathogenic organisms gain entry via mucosal surfaces . efficient vaccination of these sites protects against invasion by a particular infectious agent . oral vaccination strategies have concentrated , to date , on the use of attenuated live pathogenic organisms or purified encapsulated antigens ( 29 ). probiotic bacteria , engineered to produce antigens from an infectious agent , in vivo , may provide an attractive alternative as these bacteria are considered to be safe for human consumption ( gras status ). murine studies have demonstrated that consumption of probiotic bacteria expressing foreign antigens can elicit protective immune responses . the gene encoding tetanus toxin fragment c ( ttfc ) was expressed in lactococcus lactis and mice were immunized via the oral route . this system was able to induce antibody titers significantly high enough to protect the mice from lethal toxin challenge . in addition to antigen presentation , live bacterial vectors can produce bioactive compounds , such as immunostimulatory cytokines , in vivo . l . lactis secreting bioactive human il - 2 or il - 6 and ttfc induced 10 - 15 fold higher serum igg titres in mice immunized intranasally ( 30 ). however , with this particular bacterial strain , the total iga level was not increased by coexpression with these cytokines . other bacterial strains , such as 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 ). thus 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 . the introduction of probiotic organisms is accomplished by the ingestion of the micro - organism 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 . it will be appreciated that 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 . in addition , 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 . the invention is not limited to the embodiments herein before described which may be varied in detail . 1 . mccracken v . j . and gaskins h . r . probiotics and the immune system . in : probiotics a critical review , tannock , g w ( ed ), horizon scientific press , uk . 1999 , p . 85 - 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