Gut 2001;49:467–473 467 PAPERS Gut: first published as 10.1136/gut.49.4.467 on 1 October 2001. Downloaded from Post-immunisation gastritis and Helicobacter infection in the mouse: a long term study P Sutton, S J Danon, M Walker, L J Thompson, J Wilson, T Kosaka, A Lee Abstract Helicobacter pylori is a Gram negative bacterium Background and aims—Helicobacter py- which infects solely the gastric mucosa. It has a lori is a major cause of peptic ulcers and known association with peptic ulcers, gastric gastric cancer. Vaccine development is cancer, and mucosal associated lymphoid progressing but there is concern that tissue (MALT) lymphoma.1–3 The current immunisation may exacerbate Helico- eradication regimen for infection is antimicro- bacter induced gastritis: prophylactic im- bial therapy and concurrent acid suppression. munisation followed by challenge with H Unfortunately, the development of antibiotic felis or H pylori can induce a more severe resistant strains of H pylori is already raising concerns about the long term use of these gastritis in mice than seen with infection 4 alone. The aim of this study was to investi- combination treatments. For this reason, much attention has focused on the production gate the relationship between immunity of an eVective vaccine. Using mouse models of to Helicobacter infection and post- infection with H pylori or the closely related H immunisation gastritis. felis, it has been shown that immunisation with Methods—(1) C57BL/6 mice were prophy- whole cell sonicate or single antigens protects lactically immunised before challenge mice against later bacterial challenge5–10 or even with either H felis or H pylori. Histopath- when given therapeutically11 12 if administered ology and colonisation were assessed one with a mucosal adjuvant. month post-challenge. (2) C57BL/6 mice A consistent observation has been that were prophylactically immunised against prophylactic immunisation is often accompa- http://gut.bmj.com/ H felis infection and gastritis assessed up nied by an increase in the severity of gastritis in to 18 months post-challenge. the challenged animals. This phenomenon has Results—Prophylactic immunisation in- been termed post-immunisation gastritis and is duced a reduction in bacterial colonisa- an important issue which needs to be ad- tion following H felis challenge which was dressed if a human vaccine is to become a real- associated with increased severity of ac- ity. It has mainly been seen following challenge tive gastritis with neutrophil infiltration with H felis13 but has also been reported with H and atrophy. However, immunised mice pylori.14 on September 27, 2021 by guest. Protected copyright. challenged with H pylori SS1 had little Post-immunisation gastritis has only been evidence of pathology. Long term follow studied in short term experiments and its long School of Microbiology up showed that post-immunisation gastri- term eVects are unknown. Similarly, no pub- and Immunology, lished studies have followed immunised and University of New tis was evident at three months. However, South Wales, Sydney, from six months onwards, although challenged mice to assess the life long eVect of NSW 2052, Australia immunised/challenged mice still devel- vaccination on bacterial colonisation. This P Sutton oped gastritis, there was no significant study reports on two immunisation studies in S J Danon diVerence between inflammation in these C57BL/6 mice. The first is a short term study L J Thompson which compares post-immunisation gastritis J Wilson mice and infected controls. Post- immunisation gastritis was not associated induced by challenge of immunised mice with T Kosaka H felis and the Sydney strain of H pylori. The A Lee with the serum antibody response. Immu- second study examines the course of gastritis in nisation prevented the formation of sec- immunised mice for up to 18 months after Department of ondary lymphoid aggregates in the gastric Histopathology, bacterial challenge with H felis. Imperial College tissue. School of Medicine at Conclusion—The H felis mouse model of Materials and methods St Mary’s, Norfolk post-immunisation gastritis is the most ANIMALS Place, London extreme example of this type of pathology. W2 1PG, UK C57BL/6 age matched female mice were M Walker We have shown in this model that post- obtained from the Animal Resources Centre, immunisation gastritis is a transient event Perth, Australia. All protocols involving animal Correspondence to: which does not produce long term exacer- P Sutton, CSL Ltd, Parkville, bation of pathology. VIC 3052 Australia. Abbreviations used in this paper: BHI, brain heart [email protected] (Gut 2001;49:467–473) infusion; PBS, phosphate buVered saline; CT, cholera toxin; GAIS, gland active inflammatory score; BSA, Accepted for publication Keywords: Helicobacter; immunisation; bovine serum albumin; MALT, mucosal associated 12 February 2001 post-immunisation gastritis lymphoid tissue. www.gutjnl.com 468 Sutton, Danon, Walker, et al Table 1 Time frames used for the studies SAMPLING At termination of the experiments, mice were Bacterial Collection Immunisation challenge (time (months) anaesthetised by intraperitoneal injection of Gut: first published as 10.1136/gut.49.4.467 on 1 October 2001. Downloaded from (week) (week) post-challenge) 50 mg/kg of ketamine and xylazine (Parnell Study11234 8 1 Laboratories, NSW, Australia). Blood was col- Study21234 8 3,6,12,18 lected from the aortic arch and sera stored at −20°C. Mice were sacrificed by cervical dislo- experimentation were approved by the Animal cation and the stomachs removed. Half of each Care and Ethics Committee at the University stomach was fixed in 10% formal buVered of New South Wales (ACE 96/113). saline and processed for histopathology. For H pylori infected mice, half of the stomachs were EXPERIMENTAL PROCEDURE collected into a preweighed bijou containing The time frames used for these studies are 2 ml of BHI broth for quantitative culture. shown in table 1. HELICOBACTER: ANTIGEN PREPARATION AND ASSESSMENT OF COLONISATION AND GASTRIC BACTERIAL CHALLENGE PATHOLOGY H felis CS1 (ATCC 49179)15 was grown on H felis does not form colonies in culture and Campylobacter selective agar consisting of 5% therefore colonisation was assessed histologi- (v/v) sterile horse blood in blood agar base No 2 cally. Sections of stomach (4 µm) were cut and (Oxoid Ltd, Basingstoke, UK) and Skirrow’s stained by the modified Steiner silver method. supplement (10 µg/ml vancomycin (Sigma The degree of colonisation in H felis infected Chemical Co., St Louis, Missouri, USA), mice was assessed by semiquantitative analysis 5 µg/ml trimethoprim lactate (Sigma), 2500 IU/l of bacteria. The most dense region of bacterial polymyxin B (Sigma), and 5 µg/ml amphotericin colonisation was noted and graded from 0 to 4 B (ER Squibb & Sons, Princetown, New Jersey, where 0=no bacteria, 1=1–2 bacteria/crypt, USA)). Plates were incubated in an anaerobic jar 2=3–10 bacteria/crypt, 3=11–20 bacteria/ with a microaerophilic gas generating kit (code crypt, and 4=>20 bacteria/crypt. No BR 56; Oxoid) for two days at 37°C. H pylori For assessment of colonisation by H pylori, strain SS116 was grown for two days at 37°C with half stomachs were weighed and homogenised with an Ultra Turrax homogeniser (John Mor- 10% CO2 and 95% humidity in brain heart infusion (BHI) broth culture (Oxoid) with 5% ris Scientific Ltd). One in 10 serial dilutions (v/v) horse serum and Skirrow’s supplement. were prepared in BHI broth and 200 µl aliquots For antigen preparation, cells were harvested spread over GSSA selective agar plates—a in phosphate buVered saline (PBS) and blood agar base with 5% horse blood supple- sonicated with a Branson Sonifier fitted with a mented with vancomycin (10 mg/ml), poly- microtip (Branson Ultrasonics Corporation, myxin B (0.33 mg/ml), bacitracin (20 mg/ml), http://gut.bmj.com/ Danbury, Connecticut, USA). The protein nalidixic acid (1.07 mg/ml), and amphotericin content of the sonicate was determined using B (5 mg/ml). After five days of incubation the Bio-Rad DC protein assay (Bio-Rad, under humidified microaerophilic conditions, Regents Park, Australia) and then stored at colonies were counted. Colony forming units −70°C until use. per gram of stomach tissue were calculated by For challenge, Helicobacter were harvested in multiplying the number of colonies counted by BHI broth (Oxoid) and the final concentration the dilution factor and dividing by the weight of was adjusted to approximately 108 bacteria/ml. the stomach tissue. on September 27, 2021 by guest. Protected copyright. Mice were inoculated intragastrically with a For assessment of gastric histopathology, single dose of 0.1 ml of bacterial suspension blinded sections stained with haematoxylin and (107 bacteria). eosin were examined by light microscopy. Antral and body mucosa were graded sepa- IMMUNISATION rately; active inflammation was assessed by the Mice were immunised with four weekly presence of neutrophils and chronic inflamma- orogastric doses of 1 mg of either H felis or H tion by the presence of lymphocytes. The scor- pylori whole cell sonicate plus 10 µg of cholera ing system was graded as: 1=mild multifocal; toxin (CT) as adjuvant (Sigma) in PBS. Age 2=mild widespread or moderate multifocal; and sex matched control animals were dosed 3=mild widespread and moderate multifocal or with PBS alone. severe multifocal; 4=moderate widespread; Table 2 Corpal gastritis in immunised and challenged C57BL/6 mice. Post-immunisation gastritis in short term immunised/challenged