Patent Abstract:
the present invention relates to a microorganism , preferably a bacterial strain , preferably a non - pathogenic strain , preferably a non - invasive strain , preferably a food grade strain , preferably a gram - positive bacterial strain , delivering a trefoil peptide in vivo . preferably said trefoil peptide is tff1 . the present invention further relates to a method for the delivery of trefoil peptide to the gastro - intestinal tract comprising the administration of such a bacterial strain . the present invention also relates to a pharmaceutical composition comprising a trefoil peptide delivering bacterium as well as methods of treatment of acute gastro - intestinal inflammatory diseases comprising administration of said transformed bacterial strains , particularly for treating acute colitis , including but not limited to acute flare - ups of crohn &# 39 ; s disease and ulcerative colitis in humans , as well as for treating gastro - intestinal disorders of a similar nature in other animal species .

Detailed Description:
the present invention thus relates to the use of a microorganism as described above for the preparation of a medicament for treatment of gastric and / or intestinal diseases and / or disorders . the present invention also relates to the use of a microorganism as described above for the preparation of a medicament for treatment of acute gastro - intestinal inflammatory diseases , acute colitis , acute flare - ups of crohn &# 39 ; s diseases and ulcerative colitis , and for treatment of chronic and spontaneously recurring diseases of the gastro - intestinal tract comprising crohn &# 39 ; s disease ( enteritis regionalis ) and ulcerative colitis ( colitis ulcerosa ). according to another embodiment , the invention relates to the use of a microorganism as described above for the preparation of a medicament for inhibiting the formation of lesions caused by gastric and / or intestinal diseases and disorders . administration of the microorganism may be orally or by means of any other method known in the art allowing the microorganism to enter the desired places to be treated , such as e . g . anal , vaginal . the microorganism may be applied in a nutrient medium , i . e . a medium containing a substance or substances which sustain ( at least in vitro ) metabolic activity of the microorganism . such substances may sustain viability if not growth of the microorganism . such substances may include an energy source such as glucose , amino acids and so on . the individual to which the microorganism is administrated may be a human or an animal . in a therapeutic context , i . e . where the biological effect of delivery of the polypeptide to an individual is beneficial to that individual , administration is preferably in a ‘ therapeutically effective amount ’, this being sufficient to show benefit to the patient . such benefit may be at least amelioration of one symptom . the actual amount administered , and rate and time - course of administration , will depend on the aim of the administration , e . g . the biological effect sought in view of the nature and severity of the challenge and is the subject of routine optimisation . prescriptions of treatment , for example decisions on dosage etc , is within the responsibility of general practitioners and other medical doctors . a composition comprising microorganisms according to the present invention may be administered in accordance with the present invention alone or in combination with other treatments , either simultaneously or sequentially . according to another embodiment , the present invention relates to a method for producing a microorganism delivering a trefoil peptide in vivo as defined above comprising transforming a microorganism with a recombinant vector carrying a trefoil polypeptide coding sequence under the control of a suitable promoter and a suitable bacterial secretion signal sequence . said bacterial secretion signal sequence can be any sequence known in the art to perform said function . preferably , for l . lactis said secretion signal is the usp45 l . lactis secretion signal sequence . said promoter sequence can be any promoter allowing expression of said coding sequence in said microorganism . examples given in the examples section include the known inducible e . coli phage t7 promoter and the known constitutive p1 promoter of l . lactis . the present invention also relates to a recombinant vector comprising at least a part of a trefoil peptide coding sequence under the control of a suitable promoter and a suitable secretion signal sequence . said recombinant vector can be used to deliver in vivo at least a part of a trefoil peptide sequence which can exert on healing effect on damaged areas of the mucosal surfaces . the present invention further relates to a recombinant vector as defined above , having a nucleotide sequence as represented by any of seq id nos 1 , 2 or 4 . the following examples merely serve to illustrate the present invention , and are not to be construed as limiting the invention in any way . all documents mentioned in this text are incorporated by reference . gm17 is m17 ( difco , detroit ) supplemented with 0 . 5 w / v % of glucose . m9 medium contains per liter : 6 g of na 2 hpo 4 , 3 g of kh 2 po 4 , 1 g of nh 4 cl , 0 . 5 g of nacl , 2 mmol of mgso 4 , 0 . 1 mmol of cacl 2 and 5 g of casitone ( difco ). m9b is m9 supplemented with 2 . 1 g of nahco 3 and 2 . 65 g of na 2 co 3 per liter . gm9b is m9b supplemented with 0 . 5 w / v % of glucose . lm9b is m9b supplemented with 0 . 5 w / v % of lactose . when appropriate the antibiotics , erythromycin ( er ) or chloramphenicol ( cm ), were added to the respective media at final concentrations of 5 □ g / ml each . the designation used to indicate the presence of antibiotic is , e . g . gm17er , lm9bcm and so on . solid media contained 1 . 2 % agar . dna modifying enzymes and restriction endonucleases were used under standard conditions and in the buffers recommended by the manufacturers . general molecular cloning techniques and the electrophoresis of dna and proteins were carried out according to standard procedures . l . lactis was transformed by electroporation of cells grown in the presence of glycin ( wells et al ., 1993a ). plasmid dna was routinely purified using the qiagen ® plasmid kit . the pcr reaction was carried out on a plasmid containing mtff1 cdna ( lefebvre , 1993 ) using the oligonucleotide primers mtff1s and mtff1a . the mtff1s primer corresponds to the first 18 nucleotides of the sense strand of mtff1 from the first nucleotide behind the signal sequence . the mtff1a primer is complementary to the last 26 nucleotides of the sense strand of mtff1 including the stop codon , and introduces an extra spei restriction site . pcr amplification was carried out using vent ™ dna polymerase ( new england biolabs , beverly , usa ) which gives a pcr product carrying blunt ends . the pcr mixture consisted of 2 units vent dna polymerase , 10 μl vent buffer ( thermopol ), 4 μl dxtp &# 39 ; s ( 0 . 5 mm maximum ), 5 μl ( 0 . 5 μm ) of each primer , 1 μl ( 50 ng ) template dna and 74 μl h 2 o . six reactions were set up differing in their final concentration of mgso 4 , adjusted to 0 , 1 , 2 , 3 , 4 and 5 mm respectively . pcr amplification cycles were : t 0 for 300 ″ at 94 ° c ., t 1 for 45 ″ at 94 ° c ., t 2 for 30 ″ at 60 ° c ., t 3 for 20 ″ at 72 ° c ., t 4 for 10 ″ at 20 ° c . these cycles t 1 until t 3 were carried out 30 times . pcr amplification with these primers rendered the gene for mature mtff1 lacking the signal sequence and including an additional spei restriction site . after checking by gel electrophoresis , the amplified fragment appeared as a band in the expected length range . the 5 ′ end of the mtff1 sequence contains two possible target sequences complementary to the forward primer . as a consequence two fragments of 202 base pairs and 208 base pairs respectively can be amplified from the mtff1 cdna by use of the mentioned primers . these fragments are not expected to be resolved by agarose gel electrophoresis . two different types of vectors were used as acceptors for the mtff1 trefoil peptide encoding pcr fragment . the primary structure of the two parental vectors — pt1nx , derived from ptrex1 ( wells and schofield , 1996 ), and plet2nx , derived from plet2n ( steidler et al ., 1995 )— contains the following common elements : a promoter ( t7 or p1 ), the l . lactis usp45 secretion signal sequence ( van asseldonk et al ., 1990 and european patent application published under no . 0 455 280 ), modified to contain a naei restriction site overlapping the sequence encoding the ultimate aa residue ( steidler et al ., 1995 ), and a downstream spei restriction site . pt1nx derived plasmids specify resistance to erythromycin ; plet2nx derived plasmids specify resistance to chloramphenicol . the pcr fragments were treated for 1 hour at 37 ° c . using 50 μl dna solution , 10 μl spei - buffer , 50 units spei , 10 units t4 polynucleotide kinase ( gibco brl , bethesda , usa ), 0 . 5 mm atp , adjusted to ph 7 . 5 , and 36 μl h 2 o . the vector pt1nx was digested for 1 hour at 37 ° c . using 10 á 20 μl purified dna , 10 μl naei buffer , 10 units naei , 50 units spei , 1 unit calf intestine alkaline phosphatase ( boehringer , mannheim , germany ) and 73 á 63 μl h 2 o . after 30 minutes incubation , 50 units of spei and 10 units of naei were again added to the mixture . the restriction enzymes were inactivated and extracted from the mixture by phenol / chloroform extraction . after restriction digestion , the mtff1 - derived band ( comprising a 195 bp and a 201 bp fragment as described before under “ pcr amplification of mouse tff1 ( mtff1 )”, and the vector parts were excised from the agarose gel . following ligation of the respective pcr fragments and the vector for 45 minutes at 16 ° c . using “ ready to go ” t4 dna ligase ( pharmacia biotech , uk ) recombinant plasmids were obtained containing the mtff1 cistron as an in - frame fusion to the usp45 secretion signal sequence under the control of the promoter . the plasmid pt1mtff1 ( fig1 b ), which contains the constitutive l . lactis p1 promoter , resulted from ligation of the purified naei - spei vector part of pt1nx and the spei cut and 5 ′ phosphorylated pcr fragment . the plasmid pl2 mtff1v1 ( fig1 a ), which contains the inducible e . coli phage t7 promoter , resulted from ligation of the purified naei - spei vector part of plet2n and the spei cut and 5 ′ phosphorylated pcr fragment . the t7 promoter can only be activated by the cognate t7 rna polymerase encoded by e . g . plasmid pilpol . this plasmid is present in l . lactis strain mg1820 [ pilpol ] ( wells et al ., 1993c ). for structural analysis plasmid pt1mtff1 was transformed into l . lactis strain mg1363 . the cells were grown on gm17er plates . colonies were grown in 2 . 5 ml gm17er and the plasmid was isolated . by means of an analytical digest , the restriction pattern of the pt1nx vector ( 2 μl dna ( pt1nx ), 20 units ecori , 50 units spei , 2 μl spei - buffer and 15 μl h 2 o ) and the isolated recombinant plasmid ( 5 μl dna , 20 units ecori , 50 units spei , 2 μl spei - buffer , 0 . 25 μl of a 10 μg / ml rnase a stock solution , 12 μl h 2 o ) were compared . the plasmids were cut with ecori and spei for 1 h at 37 ° c . in the reference plasmids , two linear fragments of 907 bp and 4999 bp are predicted . in pt1mtff 1 , two bands of 499 bp and 4999 bp are predicted . the sizes of the experimentally obtained fragments , as visualized by agarose gel electrophoresis and etbr staining , were consistent with the predicted lengths . from each recombinant plasmid , one positive culture was streaked out on gm17er plates to obtain isolated colonies . one colony was subsequently inoculated in 100 ml gm17er medium and grown to saturation . the cells were collected and the plasmids were purified . their physical structure was verified by restriction enzyme analysis and agarose gel electrophoresis . in addition , sequence analysis revealed that the mtff1 cistron had been ligated perfectly in frame with the usp45 secretion leader sequence . pt1 mtff1 contains a 208 bp insert which represents the complete coding sequence of mature mtff1 ( as described before under “ pcr amplification of mouse tff1 ( mtff1 )”). for structural analysis plasmids pl2mtff1v1 was transformed into strain mg1820 [ pilpol ]. the cells were grown on gm17cm plates . colonies were grown in 2 . 5 ml gm17cm and the plasmids were isolated . by means of an analytical digest , the restriction pattern of the plet2nx vector ( 2 μl dna ( plet2nx ), 20 units ecori , 50 units spei , 2 μl spei - buffer and 15 μl h 2 o ) and the isolated recombinant plasmid ( 5 μl dna , 20 units ecori , 50 units spei , 2 μl spei - buffer , 0 . 25 μl of a 10 μg / ml rnase a stock solution , 12 μl h 2 o ) were compared . the recombinant plasmid was cut with ecori and spei for 1 h at 37 ° c . in the reference plasmids , two linear fragments of 907 bp and 4650 bp are predicted . in pl2 mtff1 , two bands of 499 bp and 4650 bp are predicted . the sizes of the experimentally obtained fragments , as visualized by agarose gel electrophoresis and etbr staining , were consistent with the predicted lengths . from the recombinant plasmid , one positive culture was streaked out on gm17cm plates to obtain isolated colonies . one colony was subsequently inoculated in 100 ml gm17cm medium and grown to saturation . the cells were collected and the plasmid was purified . its physical structure was verified by restriction enzyme analysis and agarose gel electrophoresis . in addition , sequence analysis revealed that the mtff1 cistron had been ligated in frame with the usp45 secretion leader sequence . the analysis further showed that pl2 mtff1v1 contains a 202 bp insert ( consequently missing the first two aminoterminal aa residues of mature mtff1 ; as described before under “ pcr amplification of mouse tff1 ( mtff1 )”). the sequences of the recombinant plasmids are given in fig2 a – 2 b and 3 a – 3 c . their complete sequences were compiled from the published sequences of the constituting parts . in addition , relevant sections of the sequences such as pcr fragments and ligation junction points were experimentally verified . l . lactis strains were transformed with the plasmids as constructed above . for transformation of the pt1mtff1 plasmid , l . lactis strain mg1363 ( gasson , 1983 ) was used . for transformation of the pl2mtff1v1 plasmid , l . lactis strain mg1820 ( pilpol ) ( maeda and gasson , 1986 ) was used . the expression of the proteins by these transformed l . lactis strains was detected by sds - page . to prepare culture supernatant fractions , the cells were grown for 20 hours at 28 ° c . in five ml gm17er medium for the pt1mtff1 plasmid or gm17cm medium for the pl2mtff1v1 plasmid . the cultures were diluted 1 / 100 in five ml of either gm17er or gm17cm medium and grown for 3 hours at 28 ° c . the cells were collected by centrifugation at 2800 rpm for 20 min and resuspended in five ml of the appropriate medium , i . e ., gm9ber for mg1363 cells or lm9bcm for mg1820 [ pilpol ] cells . after a further five hours of growth the cells were pelleted . the proteins present in the medium fractions were recovered by phenol extraction and ethanol precipitation . the proteins expressed in the culture supernatant fraction of a l . lactis mg1820 control strain compared to l . lactis mg1820 strains transformed with [ pilpol ; pl2 mtff1v1 ] and l . lactis mg1363 transformed with [ ptrex1 ; pt1mtff1 ] are shown in fig5 . this figure shows an extra protein band of the appropriate size ( indicated by the arrowhead ) in mg1820 [ pl2mtff1v1 ] and mg1363 [ pt1mtff1 ] when compared with the controls . as can be observed from this figure , the expression of the recombinant gene is quite low . this renders the observed in vivo result surprising since others use purified trefoil peptides in therapies for the repair of gastric and intestinal injury at dramatically higher levels ; e . g . tran et al . ( 1999 ) used daily intrarectal application of human recombinant ttf2 at levels of 2 . 5 mg / kg body weight for five days to obtain a reduction in the inflammatory index of experimentally installed colitis in rats ( intracolonic administration of dinitrobenzene sulphonic acid in alcohol ). transformants of l . lactis strains , mg1363 [ ptrex1 ], mg1363 [ pt1mtff1 ] were streaked on gm17er plates and grown overnight at 28 ° c . in each case a single colony was subsequently grown overnight at 28 ° c . in 15 ml gm17er medium . to this culture , 15 ml 100 % glycerol was added in order to preserve said cells at − 20 ° c . each day , the necessary amount of cells could be inoculated for treatment of mice . to this end the culture was diluted 1 / 200 into 10 ml gm17er medium . after minimum 20 hours of growth at 30 ° c ., the cells were collected by centrifugation for 15 min at 2800 rpm . the cells were then resuspended in 1 ml m9b without antibiotic . the effect of the trefoil peptides expressed from these l . lactis bacteria was tested out in mice suffering from acute colitis . twenty - one female balb / c mice received 5 % dss ( dextrane sodium sulphate ) dissolved in their drinking water during 7 days . in this manner , acute colitis was induced ( kojouharoff et al ., 1997 ). for therapeutic purposes these mice were orally inoculated daily by means of a gastric catheter using 100 μl bacterial suspension ( minimum 1 . 10 8 cells ) from day 1 until day 7 of the dss treatment . as indicated six mice were inoculated with mg1363 [ ptrex1 ] cells , six mice were inoculated with mg1363 [ pt1mtff1 ] cells and three mice were not inoculated ( dss control ). on day 8 after the induction of colitis , the mice were sacrificed and examined immunologically and histologically . immunological testing of the sera showed that the treated mice did not show an immune response towards the expressed proteins . serum was taken from the mice which were bled at day 8 . this serum was analysed via western blotting to check whether it contained antibodies against the proteins present in the medium fractions of the l . lactis cells . the medium fractions used were derived from the l . lactis strains mg1363 [ ptrex1 ] and mg1363 [ pt1mtff1 ]. an equivalent of 1 ml of concentrated ( phenol extraction and ethanol precipitation ) medium fractions were analysed by sds - polyacrylamide ( 20 %) gel electrophoresis . after blotting to nitrocellulose filters , the filters were incubated for 1 hour with the serum solutions of the 4 groups of mice . the serum was diluted 500 times in 20 ml nitrocellulose blocking buffer blotto ®: 100 ml 10 × pbs , 150 ml 1m nacl , 2 ml triton x - 100 ®, 25 g fat - free milkpowder , water up to a total volume of 1 liter ). as a secondary antibody , sheep anti - mouse igg coupled to horseradish peroxidase ( hrp ) was used . using the 500 times diluted serum , no signal was detected . histological analysis was performed on colons of the treated mice . the colons were cut in the length direction and divided in three equal portions : the distal ( nearest to the anus ), middle and proximal parts . these colon parts were analysed histologically after an overnight fixation in 3 . 7 % formaldehyde ( in pbs ), followed by paraffin embedding , ensuring upright positioning of the tissue samples in the paraffin blocks . of each tissue sample , three parallel 3 μm thick longitudinal sections , evenly spaced over the sample , were made . these crossections were coloured with hematoxylin / eosin . histological analysis was performed in a blind fashion , meaning that the labels on the slides were covered before scoring the sections . slides carrying sections obtained from the several groups of mice were randomized before microscopic examination . each slide was then assigned a histological score ( ranging from 0 to 5 ) according to the symptomatic description as defined in table 1 . for each mouse and for each colon part , the average score of the three sections was calculated . in the distal and middle parts of the colon , the inflammation consisting of epithelial damage and infiltration were the most pronounced . in the proximal part , almost no inflammation could be observed . the average histological score was calculated for both the distal and the middle colon part per group of animals . the final histological sum score is the sum of the two separate scores ( sum score = score of epithelial damage + score of infiltration ) and is a measure for the degree of the inflammation . the histological sum scores of the distal colon part for each of the groups of mice is shown in fig6 a – 6c . from the histological scores for the distal part of the colon as set out in fig6 a – 6c , it could be concluded that there is a clear decrease of inflammation upon inoculation of mice with l . lactis cells producing trefoil peptides . mice having received [ pt1mtff1 ] transformed l . lactis cells show a significant reduction of the inflammation of more than 65 %. as can be seen from fig6 a – 6c , the inflammatory infiltration and the epithelial damage in the distal part of the colon are significantly decreased following inoculation with recombinant l . lactis strains which secrete mtff1 polypeptide these results were confirmed in a separate experiment which was conducted equally , including larger groups ( group size = 10 ) and more control groups . fig7 shows histological scores ( obtained as described above ) of healthy control mice ( control ) and of mice which received dss as described , either left untreated ( dss ) or treated ( as described above ) with mg1363 , mg1363 [ pt1trex1 ] or mg1363 [ pt1mtff1 ] as indicated . the experiment shows a clear and significant decrease in the intestinal inflammation in the group of mice treated with mg1363 [ pt1mtff1 ] the latter experiment was also evaluated by determining the levels of interleukin - 1β ( il - 1β ) and interferon - γ ( ifn - γ ), both pro - inflammatory cytokines well known to the skilled . mice ( n = 10 ) were inoculated with the strains indicated as described . control = healthy mice , dss = mice receiving 5 % dss in the drinking water without any treatment . the colon was prepared out and areas with equal surface were isolated by means of a punch ( ø = 4 mm ). the tissue samples of each group were overlayed with 500 μl rpmi + 10 % fetal calf serum and incubated overnight at 37 ° c . the supernatant was collected and titrated for cytokine content by elisa . the amount of il - 1β and ifn - γ in the respective tissues is shown in fig8 a – 8b . the results show a clear reduction in these pro - inflammatory cytokines in groups of mice treated with mg1363 [ pt1mtff1 ]. for the expression of mtff1 form pichia pastoris we constructed the plasmid ppicmtff1 ( fig1 c ). for this , the mtff1 gene was pcr amplified as described ( pcr amplification of mouse tff1 ). this fragment was ligated in the opened naei restriction site of a derivative of ppic9 ( invitrogen ). the ligation mixture is transformed to e . coli mc1061 and correctly assembled clones were identified by restriction analysys and dna sequencing ( sequence as in fig4 a – 4c ). in the resulting plasmid ppicmtff1 , the mtff1 sequence is fused in frame with the sacharomuces cerevisiae α - mating factor prepro secretion signal the plasmid ppicmff1 was transferred to pichia pastoris gst115 by a method as described in logghe ( 1995 ) and positive clones , which had the mtff1 unit integrated in the his4 locus , were selected by pcr identification . these positive clones were induced with methanol and screened for expression by protein analysis of culture supernatant and one clone which showed , when compared to the negative control ( negative ), a particularly high expression of an extra band at 6 . 5 kda ( gst115 :: ppicmtff1 ) was retained for further work ( fig9 , indicated by arrowhead ). the extra protein band was identified as mtff1 by protein sequencing . the expression procedure was optimised scaled up and optimised to a 16 l culture and mtff1 was purified from the culture supernatant . for this , methanol induced gst115 :: ppicmtff1 supernatans was concentrated by tangential filtration ( millipore proflux m12 , cut off 3000 da ) and was dialysed to ph 7 . 4 in a 0 . 02 m phosphate buffer . mtff1 was purified from this concentrate on an ion - exchange column ( q - column of biorad ). the proteins were eluted form the column by an isocrational salt gradient . the resultant mtff1 was more than 99 % pure and was further concentrated . the final preparation contains less than 160 ng lps / ml this amount of lps is within acceptable limits and the ps2 protein can be used in future experiments . following analysis on a size exclusion column of purified mtff1 ( superdex 75 ®; pharmacia ) we conclude that 7 . 5 % of the mtff1 is in the monomeric form , and 92 . 5 % is in the dimeric form ( fig1 a ). this was confirmed by reducing versus non reducing sds - page of the purified mtff1 ( fig1 b ). a well know feature of tff1 protein is that after administration of the protein to caco - 2 cell monolayers it significantly lowers the surface expression of e - cadherine ( liu et al ., 1997 ). we showed a lowering of 10 % of the e - cadherine surface expression after the above described preparation of mtff1 was administred to caco - 2 monolayers . for induction of acute colitis mice received 6 % dextran sulfate sodium ( dss , mw 40 000 ) dissolved in drinking water for 7 days ( kojouharoff et al ., 1997 ). mice used for experiments were age - matched and had received dss treatment simultaneously . for therapeutic purposes , mice were treated daily with 50 μg mtff1 in 200 μl pbs before dss administration from day − 7 to 0 ( pre - treatment groups ), during dss administration from day 0 to 7 ( during - treatment groups ) and after dss administration from day 7 to 14 ( post - treatment groups ). to study different routes to deliver mtff1 , mice were treated by intraperitoneal ( i . p .) injection , intragastric inoculation and rectal administration in each setup . mice were killed on day 8 after receiving drinking water without dss for one day ( pre - treatment and during - treatment groups ) and on day 14 after receiving drinking water without dss for seven days ( post - treatment groups ). non - treated control groups with dss in drinking water were killed on day 8 and day 14 . all groups consisted of 9 mice . results are represented in fig1 and clearly show that in no treatment regime any statistically significant improvement can be observed . this renders the described invention surprising since a clear improvement has been observed ( fig6 a – 6c and 7 ). this means that the delivery of tff1 through l . lactis makes an essential contribution to the observed therapeutic effect . babyatsky m . w ., de beaumont m ., thim l ., podolsky d . k . 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