untitled Drug Target Insights 2007:2 221–228 221 ORIGINAL RESEARCH Correspondence: Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/. Effect of live Salmonella Ty21a in Dextran Sulfate Sodium-induced Colitis Gunnar Nysœter1, Kari Erichsen2, Anne Marita Milde3, Eva Colás4, Einar Kristoffersen5 and Arnold Berstad6 1Department of Medicine, Section for Gastroenterology, 2Children’s Clinic, 3Department of Biological and Medical Psychology, 4Innovest,5Section for Microbiology and Immunology, Haukeland University Hospital, Bergen, Norway, 6Institute of Medicine, University of Bergen, Norway. Abstract Background: Intestinal microbiota seems to play an essential role in the development of infl ammatory bowel diseases (IBD). We hypothesised that an oral vaccine based on live Salmonella typhi would be well tolerated and could even atten- uate dextran sulfate sodium (DSS) induced colitis in rats, an animal model of IBD. Methods: Nine male Wistar rats was used for an initial tolerance study, in which we used 3 dose-levels of Salmonella Ty21a, 0.5 × 109, 1 × 109, and 2 × 109CFU, each dose being tested in 3 rats. Four treatment groups consisting of 8 male Wistar rats per group: 1) control group given standard food and water, 2) control group given four daily administrations of Salmonella Ty21a 1 × 109 CFU, 3) water with 5% DSS the last 7 days, 4) four daily administrations of Salmonella Ty21a before water with 5% DSS the last 7 days. The Salmonella Ty21a was administered by gastric gavage on day 1, 3, 5 and 16, while DSS was given with the drinking water from day 15 to 22. The animals were sacrifi ced and colonic tissue removed for analysis 22 days after gavage of the fi rst vaccine dose. Results: The animals in the tolerance study got no signs of disease. In the treatment study, all animals receiving DSS had histologic indications of colitis, particularly in the distal part of the colon. Administration of Salmonella Ty21a had no signifi cant effect on crypt and infl ammation scores (p � 0.05). Conclusion: Gastric administration of live vaccine strain Salmonella Ty21a was well tolerated, but did not provide any signifi cant protection against development of DSS induced colitis in rats. Keywords: salmonella Ty21a, colitis, rats, infl ammatory bowel disease Introduction About 0.2% of the population in Scandinavia suffer from ulcerative colitis or Crohn’s disease, collec- tively called chronic infl ammatory bowel disease (IBD) (Fonager, Sorensen and Olsen, 1997; Lapidus 2006; Moum et al. 1996). But while the prevalence and cost of the disease is increasing, to fi nd the aetiology and causal treatment remain a huge challenge. Traditionally, IBD is considered an autoim- mune disease, and treatment with immunosuppressive drugs has had a prominent position. More recently, IBD has been ascribed to an inadequate mucosal immune response to the intestinal microbiota in genetically susceptible individuals. Consequently it might be better to stimulate the immune defence of the intestine rather than suppressing a secondary infl ammatory response. There is in fact mounting evidence that immune stimulation of the intestinal epithelium is one way to treat infl ammatory condi- tions in the gut. Thus, a study with granulocyte—macrophage colony-stimulating factor (GM-CSF), a myeloid growth factor, has shown promising results in Crohn’s disease (Korzenik et al. 2005). Several animal studies on experimental colitis have shown a protective or therapeutic effect from probiotics or vaccines (Boirivant et al. 2001; Fujiwara et al. 2003; Madsen et al. 2001; Ohman 2005; Osman et al. 2004). A similar effect on humans with IBD has presently not been shown, except pos- sibly for probiotics (Bibiloni et al. 2005; Kanauchi et al. 2003). The vaccine strain Salmonella Ty21a is of interest in this connection. Containing live bacilli it may theoretically combine the effects of probiotics and vaccines used in other studies. Extensive clinical use has proved that the vaccine is http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 222 Nysœter et al Drug Target Insights 2007:2 remarkably well tolerated, even by IBD-patients with moderate disease activity (Engels et al. 1998; Sands et al. 2004). One may speculate whether salmonella infections have some relation to IBD, considering the inverse prevalence of the two diseases. We even have reports from a few of our IBD-patients that the course of their IBD has become milder after they have been vaccinated against typhoid fever with this live vaccine. This has been a surprising observation as they were vaccinated before travelling to endemic areas and did not anticipate any effect on their chronic disease. Results from animal studies have limited value for the understanding of IBD. But as a fi rst step we wanted to see if experimental colitis could be infl uenced by live salmonella vaccine in a similar way that was previously found with probiotics and some other vaccines. We therefore studied whether administration of the live vaccine strain, Salmo- nella Ty21a would influence the induction of colitis by dextran sulphate sodium (DSS) in rats, a well-known model of ulcerative colitis (Kim and Berstad, 1992). Materials and Methods Animals and husbandry Forty-one male Wistar rats (Taconics Europe, Skensved, Denmark), 6 weeks old with mean weight of 147 g (95% CI 143.5–149.9), were housed indi- vidually in Makrolon III cages in an open system. They were kept under standard laboratory conditions with a temperature of 21 ± 1 °C, dark/light cycles of 12/12 hours, relative humidity of 55% ± 5%, and 20 air changes per hour. Access to food, SDS RM1 (E) (Scanbur BK AS, Nittedal, Norway) was ad libitum. Tap water was given ad libitum if not oth- erwise stated. The Norwegian Animal Research Authority approved the protocol. Induction of colitis Acute colitis was induced by 50 g/L of DSS (MW 44000; TdB Consultancy AB, Uppsala, Sweden) given in distilled drinking water for 7 days. Test substance Salmonella serovar Ty21a (Vivotif®; Berna, Como Italy), 2 × 109 colony forming units (CFU) freeze dried live bacilli per capsule. The content of one capsule was dissolved in 2 mL NaCl 0.9% imme- diately before use. Antibody response Serum salmonella antibodies were measured semi- quantitatively by bacteria agglutination using the Widal reaction with O-antigens (SIFIN Gmbh, Berlin, Germany). Experimental Protocol After 7 days of acclimatisation, 9 animals were divided at random into 3 groups of 3 animals for the dose-tolerance study, while the remaining 32 animals waited for the colitis study starting one week later. Dose-tolerance study The 9 animals were given Ty21a; one group with 0.5 × 109, one with 1 × 109, and one with 2 × 109 CFU. Each dose was given three times at two days interval. Six days after the fi rst dose the animals were sacrifi ced and blood culture was taken. We registered general condition, consistency and blood content of the stool daily. Treatment study The animals were divided into the following four groups of 8 rats in each group: (1) Control. (2) Ty21a alone. (3) DSS alone. (4) DSS + Ty21a. Rats in the groups 2 and 4 got 1 × 109 CFU Salmo- nella Ty21a in 1 mL saline 0.9% through a metal- lic oral tube on day 1, 3, 5 and 16. In group 3 and 4 drinking water was replaced by DSS 5% in dis- tilled water from day 15 to 21. Group 1 remained on normal food and drinking water during the whole test period. The rats were weighed at start of treatment with Ty21a (day 1), after 7 and 14 days, and then daily until sacrifi ce (day 22). On day 22 the animals were anaesthetised by sub- cutaneous injection of a combination of phen- antyl citrate + fl uanisone (Hypnorm; Jansen Pharmaceutica, Beerse, Belgium) and mid- azolam (Dormicum; Roche, Oslo, Norway). (Phenantyl citrate 0.079 mg/mL, fl uanisone 2.5 mg/mL, midazolam 1.25 mg/mL given at dose 0.2 mL/100g.) Thoracotomy, cardiac puncture and exsanguina- tion were performed. The colon was taken out. 223 Effect of Salmonella Ty21a on experimental colitis Drug Target Insights 2007:2 Sample collection and analyses Stool was observed daily from day 15 to 21 for the presence of diarrhoea or blood. We used a guyac method (Haemofec. Med-Kjemi A/S, Asker, Norway). Granulocyte marker protein (GMP), the rat equivalent to human calprotectin, was measured in stool (Kristinson et al. 2002; Milde et al. 2003). Samples for analysis were collected on day 1, 7 and 21. They were stored at –20 °C until analysis for GMP. On exsanguination blood was drawn for blood culture and for analysis of salmonella antibodies. Tissue preparation and histologic examination Colon from the colocecal junction to the anal verge was removed. The length of the colon was recorded. The colon was rinsed with phosphate-buffered saline, opened longitudinally, and divided into one proximal and one distal segment, which were fi xed in 10% formalin and embedded in paraffi n. Eight pieces per segment were stained with hematoxylin and eosin and specimens coded and randomised before microscopic examination. Crypt and infl ammatory scores were determined according to a validated scoring system (Carrier et al. 2001). Crypt injury was scored as follows: grade 0, intact crypts; grade 1, loss of the bottom third of crypts; grade 2, loss of the bottom two thirds of crypts; grade 3, loss of entire crypt with the surface epithe- lium remaining intact; grade 4, loss of entire crypt and surface epithelium. The severity of infl ammation was scored as follows: grade 0, normal; grade 1, focal infl ammatory cell infi ltration; grade 2, infl ammatory cell infi ltration, gland drop out, and crypt abscess. Both scores include a measure of involvement as follows: grade1, 1% to 25%; grade 2, 26% to 50%; grade 3, 51% to 75%; grade 4, 76% to 100%. The score was the product of either the crypt or infl am- mation grade by the involvement grade. Statistical analysis Data were analysed using the GraphPad Prism version 4 (GraphPad Software, San Diego, Calif.) statistical software package. Results are presented as mean ± SEM. Differences between means were evaluated with 1-way Anova and Bonferroni post- test for selected pairs of columns. Differences between means and 95% confi dence intervals (CIs) are given if not otherwise stated. p values less than 0.05 were considered statistically signifi cant. Results Tolerance study All the animals were well during the test period. None got diarrhoea or bloody stools. Blood cul- tures were all negative. Treatment study All the animals appeared to be at good condition until sacrifi ce. DSS Intake Total intake of DSS-containing water was similar in the DSS alone (161.0 ± 8.1mL) and the DSS + Ty21a groups (168 ± 8.4 mL; p = 0.45). There was no signifi cant difference of drinking volume in the four groups. Weight change All rats gained weight without signifi cant differences between groups until DSS treatment was started on day 15. During the following period until day 22 both groups on DSS had signifi cantly less weight gain than the animals in the control or Ty21a-groups. (Fig. 1) There was no signifi cant difference in weight change between the DSS group (4.75 ± 2.15) and the DSS + Ty21a group (−0.75 ± 2.82; p = 0.25). Stool changes None of the animals got diarrhoea. Blood in stools was found in six animals in both the DSS group and those on DSS + Ty21a, four with occult blood and two with traces of visible blood in each group. Colon length Colon length did not differ signifi cantly between the groups, though there was a tendency to shorter colon in the groups that got DSS (14.13 ± 0.42 cm) or DSS + Ty21a (13.69 ± 0.49 cm; p = 0.35). Macroscopic lesions of the gut Intramucosal bleeding or erosions was observed in the colon of a few animals both in the DSS group (4 animals) and the DSS + Ty21a group (5 animals), 224 Nysœter et al Drug Target Insights 2007:2 while in the other groups, the colons were macro- scopically normal. Granulocyte marker protein Compared with controls, DSS-induced colitis sig- nifi cantly increased faecal GMP by 100 mg/L (95% CI 118-81; p � 0.0001). There was no signifi cant difference of GMP-levels in animals on DSS (105 ± 8) or DSS + Ty21a (108 ± 8; p = 0.5). (Fig. 2). Salmonella antibodies Salmonella antibodies were found in 7 of the 8 animals that got Ty21a and in all of the eight ani- mals that got DSS + Ty21a. The other animals had no salmonella antibodies. There was no correlation between antibody levels and tissue damage. Blood culture There was growth of Gram positive cocci, not salmonella, in one sample from an animal in the control group, no growth in culture from the others. As no rats showed signs of septicaemia, the posi- tive blood culture most likely refl ects contamina- tion upon blood sampling. Histology Histologic fi ndings were normal or close to normal in all animals in the control group and the Ty21a group, while all animals on DSS, with or without the addition of Ty21a developed inflammatory changes in the colon (Fig. 3). The mean value of crypt score was slightly less for the DSS + Ty21a group compared to the DSS group (Fig. 4), but the difference was not statistically signifi cant (95% CI of diff. −0.63 to 1.55; p � 0.05). Analysis of infl am- matory score showed no signifi cant differences between the two groups (95% CI of diff. –0.63 to 0.73; p � 0.05), (Fig. 4). There was a signifi cant correlation between GMP values and histologic fi ndings (Pearson’s r = 0.77; p � 0.0001). Discussion We failed to fi nd a protective effect of salmonella Ty21a in DSS-induced colitis. Both histological signs of colitis and GMP levels in blood indicated similar degree of infl ammation in DSS and DSS + Ty21a treated groups. The two methods support each other as they both depend on the amount of granulocytes infi ltrating the intestinal mucosa. Mucosal damage as measured by crypt score was numerically less pronounced in vaccinated ani- mals, but the difference was not statistically sig- nifi cant. However, neither the initial tolerance study nor the controlled study indicated any adverse effects of the treatment. Although the results should be carefully extrapolated, the fi nding of no deterioration of the colitis, suggests that Control Ty21a DSS DSS + Ty21a-15 -10 -5 0 5 10 15 20 25 ns W ei g h t (g ra m ) p = 0.045 Figure 1. Weight gain from day 14–22 (DSS-period). 225 Effect of Salmonella Ty21a on experimental colitis Drug Target Insights 2007:2 Figure 3.a b c d Figure 2. Salmonella Ty21a does not affect the level of granulocyte marker protein (GMP) in rats. Figure 3. H&E × 20. Histologic normal mucosa in healthy control rat (a) and healthy Ty21a rat (b). Mucosa from DSS-group (c) and DSS- Ty21a-group (d) with severe infl ammatory changes with crypt loss, crypt abscess and infi ltration with infl ammatory cells. 0 50 100 150 Control G M P m g /L Ty21a DSS DSS + Ty21a 226 Nysœter et al Drug Target Insights 2007:2 Figure 4. Histologic fi ndings (crypt score and infl ammatory score) in dextran sulphate induced colitis in rats is not signifi cantly infl uenced by treatment with Salmonella Ty21a. Control Ty21a DSS DSS − Ty21a 0 1 2 3 4 Control Ty21a DSS DSS + Ty21a 0 1 2 3 P > 0.05 P > 0.05 C ry p t sc o re In fl a m m a to ry s co re 227 Effect of Salmonella Ty21a on experimental colitis Drug Target Insights 2007:2 vaccination with Salmonella Ty21a in humans with IBD is safe. This corresponds well with extensive experience in humans. Current guidelines for vac- cination of patients with IBD allow oral adminis- tration of Salmonella Ty21a provided the patient is not signifi cantly malnourished or immune com- promised (Sands, Cuffari, Katz, Kugathasan, Onken, Vitek and Orenstein, 2004). Any informa- tion as to whether the vaccine infl uences the IBD itself is not available. For human use salmonella Ty21a is taken in the encapsulated form to avoid destruction of the microbes by pepsin and acid in the stomach. Our rats got unprotected vaccine by gavage through a metallic sonde, assuming that a suffi cient propor- tion of the high dose of live microbes (half that given to an adult person) would reach the gut. The three fi rst doses of vaccine were given on day 1, 3 and 5 as is the recommended schedule for human use. The following two weeks interval enabled the animals to face the DSS-challenge from day 15 with an immune system triggered by Ty21a. All the vaccinated rats, except one, produced salmo- nella antibodies, thus proving the vaccine’s contact with the immune system. To achieve a possible probiotic effect, a higher vaccine dose could be necessary. But instead we chose to give a fourth dose the day after DSS-treatment was started (day 16), in order to have live salmonella present in the gut during the colitis period. The choice of timing for vaccination as well as duration of the colitis period may be essential for the effect (Di Giacinto et al. 2005; Jun et al. 2005). To have a better parallel to a clinical situation, we might have induced a chronic colitis fi rst, and then introduced the vaccine to study its therapeutic effect. All ani- mals receiving DSS got colitis, particularly in the distal part of the colon, but the infl ammation was remarkably weak compared with what we observed in a recent study performed by us (Erichsen et al. 2005; Milde and Murison, 2002). The relevance of this fact, is not known. A vaccine containing live bacilli could theo- retically have the advantages of both probiotic and vaccine components. Accordingly we gave the vaccine both two weeks before and during the induction of colitis. As assumed, the adaptive immune system had started production of salmo- nella antibodies after two weeks. Others have found that in humans, oral vaccination with live Salmonella Ty21a stimulated, during the fi rst few weeks, large amounts of IFN-γ but no IL-10 or TGF-β (Lundin, Johansson, and Svennerholm, 2002), which theoretically could be an unfavour- able effect of Salmonella Ty21a in IBD. However, the activity of such pro-infl ammatory Th1 cyto- kines may be subdued by subsequent induction of CD4+CD25+ regulatory T lymphocytes producing both IL-10 and TGFβ with a healing effect on colitis (Holmen et al. 2006; Uhlig et al. 2006). In case of the latter, a study with a longer observation period would be of interest. A live vaccine against enterotoxigenic Eschericia coli (ETEC) suppressed the proinfl ammatory response (Jun, Gilmore, Cal- lis, Rynda, Haddad and Pascual, 2005) and reduced the production of TNF-α, IL-1 and IL-6, while it increased the production of IL-4, IL-10, and IL-13. Interestingly, toxins associated with another tropic diarrhoeal disease, cholera, has an anti-infl amma- tory effect in experimental TNBS-colitis. Oral administration of recombinant cholera toxin sub- unit B has been shown to inhibit murine TNBS- induced experimental colitis (Boirivant, Fuss, Ferroni, De Pascale and Strober, 2001) and pro- mote a Th2 response with Tr1 cells, enhanced IL-10 production and inhibition of IL-12 and TNF- α secretion (Lavelle et al. 2004). Even vaccines unrelated to intestinal disease may alleviate exper- imental colitis. Thus, a three-component Bordetella pertussis vaccine attenuated colitis in Gαi2- defi cient mice (Ohman, 2005). Contrary to the adaptive immunity, the innate immune system would respond immediately (Fell- ermann et al. 2003). The innate immune system is supposed to play a key role in the protection against IBD through production of defensins and other protective mechanisms (Wehkamp et al. 2003; Wehkamp, 2005; Wehkamp, Schauber and Stange, 2007). However, an important pathogenetic trait of Salmonella is to down-regulate the production of defensins, enabling bacterial invasion of the gut mucosa (Salzman et al. 2003). Experiments on pigs have shown that Salmonella enterica serovar typhimurium infection might up-regulate catheli- cidine genes and stimulate the production of another family of antimicrobial peptides prominent in the host defence mechanisms of several mam- malian species (Wu et al. 2000). If immunisation with the non-pathogenous salmonella Ty21a has the same effect, it would contribute to protection of the host. 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