Papillary mucinous adenoma arising in AMH of the gall bladder 967 enomyomatosis. Am J Gastroenterol 1988;83:670-4. clinically important. As AMH can be easily 7 Nakafuji H, Koike Y, Wakabayashi M, Furihata R, Maru- recognised on ultrasonography,'8 any unusual yama Y, Ogata H. Three cases of early stage carcinoma of the gallbladder. GastroenterolJrpn 1981;16:134-40. modifications of the sonographic structure or 8 Paraf F, Molas G, Potet F. Diverticulose intramurale et development of a mass necessitate rigorous cancer de la vesicle biliaire. Gastroenterol Clin Biol 1987- 11:825-7. follow up. Early recognition of degenerative 9 Cartun RW, Pedersey CA. An immunocytochemical tech- change is vital as early gall bladder cancer nique offering increased sensitivity and lower cost with streptavidin-horseradish peroxidase conjugate. J Histo- has a distinctly better prognosis than advanced technol 1980;12:273-7. disease.'7 10 Von Matting H, Muller W, Liebe G, Schulz HG. Gutartiges papillares kystadenom der gallenblase mit inkompletter temporaremcholestase und begleitpankreatitis. Zentralbl 1 Christensen AH, Ishak KG. Benign tumors and pseudo- Chir 1982;107:236-9. tumors ofthe gallbladder. Report of 180 cases. Arch Pathol 11 Weedon D (ed). Adenomyomatosis. In: Pathology of the gall 1970;90:423-32. bladder. New York: Masson Publishing Inc, 1984:185-94. 2 Aldridge MC, Gruffaz F, Castaing D, Bismuth H. Ad- 12 JutrasJA. Hyperplastic cholecystoses. Am_JRoentgenol 1960; enomyomatosis of the gallbladder. A premalignant lesion? 83:795-827. Surgery 1991;109:107-10. 13 Meguid MM, Aun F, Bradford ML. Adenomyomatosis of 3 Arianoff AA, Vielle G, Dewulf E. Le cancer de la vesicule. the gallbladder. Am 7 Surg 1984;147:260-2. A propos de 49 cas cholecystectomises. Acta Gastroentel 14 Ram MD, Midha D. Adenomyomatosis of the gallbladder. Belg 1973;36:310-31. Surgery 1975;78:224-9. 4 Ootani T, Shirai Y, Tsukada K, Muto T. Relationship 15 Fotopoulos JP, Crampton AR. Adenomyomatosis of the between gallbladder carcinoma and the segmental type gallbladder. Med Clin North Am 1964;48:9-36. of adenomyomatosis of the gallbladder. Cancer 1992;69: 16 Shepard VD, Walters W, Dockerty MB. Benign neoplasms 2647-52. of the gallbladder. Arch Surg 1942;45: 1-18. 5 Kawarada Y, Sanda M, Mizumoto R, Yatani R. Early car- 17 Aldridge MC, Bismuth H. Gallbladder cancer: the polyp- cinoma of the gallbladder, noninvasive carcinoma ori- cancer sequence. BrJ Surg 1990;77:363-4. ginating in the Rokitanshy-Aschoff sinus: a case report. 18 Rice J, Sauerbarei EE, Semogas P, Cooperberg PL, Am J Gastroenterol 1986;81:61-6. Burhenne HJ. Sonographic appearance of adeno- 6 Katoh T, Nakai T, Hayashi S, Satale T. Noninvasive car- myomatosis of the gallbladder. J Clin Ultrasound 1981;9: cinoma of the gallbladder arising in localized type ad- 336-7. 7 Clin Pathol 1995;48:967-969 Induction of interleukin-8 secretion from gastric epithelial cells by a cagA negative isogenic mutant of Helicobacter pylori J E Crabtree, Z Xiang, I J D Lindley, D S Tompkins, R Rappuoli, A Covacci Abstract closely associated with the expression of The ability of Helicobacter pylori strains CagA. to induce interleukin-8 (IL-8) gene ex- (J7 Clin Pathol 1995;48:967-969) pression and protein secretion from gast- ric epithelial cell lines in vitro is variable. Division of Medicine, Keywords: Interleukin-8, Helicobacterpylori, CagA, epi- St James's University This cellular response is associated with thelial cells, gastritis. Hospital, bacterial expression of the CagA protein Leeds LS9 7TF present in type I Hpylori strains. To deter- J E Crabtree mine the role of CagA in this host cell The CagA surface protein of Helicobacter pylon Public Health response, an isogenic cagA negative mut- is highly immunogenic and is expressed in Laboratory, ant, N6.XA3, was constructed. The cagA about 60 to 70% of Hpylori strains.' Mucosal Leeds LS15 7TR mutant and the IgA antibody recognition of this protein has D S Tompkins negative isogenic wild-type parental cagA positive strain, N6, were co- been linked with peptic ulcer disease and the Sandoz Research cultured with AGS, ST-42 and KATO-3 activity ofgastritis4 and systemic IgG responses Institute, gastric epithelial cell lines and secreted to CagA are also elevated in ulceration.'5 Vienna, Austria Strains of Hpylori which have the gene coding I J D Lindley interleukin-8 assayed by enzyme linked inmmunosorbent assay. In all three cell for CagA and express this immunogenic protein Immunobiological lines there was no significant difference in usually coexpress the vacuolating cytotoxin Research Institute with this Siena (IRIS), the IL-8 secretion induced by the cagA (VacA).' Strains genotype/phenotype Siena, Italy negative isogenic mutant, N6.XA3, and the have recently been classified as type I bacteria.' Z Xiang wild-type parent strain, N6. These studies Type II strains lack the cagA gene and express R Rappuoli nor the VacA A Covacci show that CagA is not the inducer of IL-8 neither the CagA protein protein.' secretion from gastric epithelial cells. As While the VacA protein is thought to be an Correspondence to: strains studied important mediator ofgastric mucosal damage, Dr J E Crabtree. all wild-type CagA positive to the bacterial factor(s) this protein does not elicit gastric inflammatory Accepted for publication date induce IL-8, 30 March 1995 inducing this inflammatory response is cell infiltration in animal models.6 968 Crabtree, Xiang, Lindley, Tompkins, Rappuoli, Covacci Our previous studies have shown that gastric field of 2-0 Kv, 25 microFD, 200 Ohms. Bac- mucosal production of the neutrophil chemo- teria were recovered and plated onto non-se- tactic and activating peptide interleukin-8 lective plates for two days. Bacteria were then (IL-8) is increased in patients with H pylori isolated and plated onto selective media for infection and epithelial polymorph infiltration three days." Kanamycin resistant H pylori col- (active gastritis).7 In vivo an important source onies were screened for allelic exchange by of IL-8 is the gastric epithelium.8 We have Southern hybridisation and polymerase chain recently demonstrated that type I strains ex- reaction. The expression of the CagA protein pressing the cytotoxin and CagA protein dir- was assessed by western blot assay using a ectly induce IL-8 mRNA expression and IL-8 rabbit anti-CagA specific antiserum. The iso- protein secretion in gastric epithelial cell lines.9 genic cagA negative mutant N6.XA3 showed Using natural phenotypic variant strains of no expression of CagA in western blot assays. H pylori with disparate CagA and VacA ex- pression,3 we have also shown that the up- regulation of epithelial IL-8 is associated with BACTERIAL STIMULATION OF IL-8 SECRETION IN the CagA phenotype and not with expression GASTRIC EPITHELIAL CELL LINES of VacA.10 The isogenic cagA negative mutant N6.XA3, To determine whether the CagA protein is the wild-type parental strain N6 and the type the bacterial mediator inducing gastric epi- strain NCTC 11637 (CagA positive control) thelial IL-8, we have constructed an isogenic were grown on blood agar base Number 2 cagA negative mutant strain of H pylori and (Oxoid, Basingstoke, UK) including 7% fresh examined the ability of the mutant strain and horse blood. Bacteria were harvested on day parental strain to induce IL-8 secretion. 4 and after centrifugation, resuspended at 2-5 x 107/ml in RPMI 1640 (ICN-Flow Laboratories, High Wycombe, UK) containing Methods 10% fetal calfserum (FCS) (Sera Lab, Crawley, Surrey, and used CONSTRUCTION OF ISOGENIC cagA NEGATIVE UK) immediately. MUTANT N6.XA3 Experiments were undertaken with AGS, The cagA gene, cloned in the Bluescript vector ST42 and KATO-3 gastric epithelial cell lines SK+ (pA), was disrupted by the insertion described in previous studies.9"0 Cells were of a kanamycin resistance gene at nucleotide routinely maintained in RPMI 1640 sup- position 2370 (pA::Km). DNA from plasmid plemented with 10% FCS and 40,g/ml gen- pA::Km was transferred into the recipient cagAl tamicin. Confluent monolayers of AGS and CagA positive N6 strain of H pylori (kindly ST42 (approximately 5 x 105/ml) in 24 well provided by Dr A Labigne) by electroporation. plates (ICN-Flow Laboratories) and sus- Briefly, Hpylori, incubated in a microaerophilic pensions of non-adherent KATO-3 (5 x 105/ atmosphere, were collected, resuspended and ml) were cultured for 24 hours in quadruplicate washed three times in WEB solution (10% with N6.XA3, the wild-type parental strain glycerol in deionised water) at 4°C. Bacteria N6 and the type strain NCTC 11637 at a were centrifuged and resuspended at a final concentration of 2-5 x 107/ml as previously concentration of 250 OD/ml. Aliquots of 25 gl described.9"' Cultures were undertaken in gen- were frozen in liquid nitrogen and stored at tamicin free media. After 24 hours, super- - 80°C. For electroporation, 25 ,l of frozen natants were aspirated and stored at - 70°C bacteria were mixed with 1 ,ul of DNA (10 jig/ until assayed in duplicate for IL-8 by enzyme ,ul in deionised water) in pre-cooled 0-1 cm linked immunosorbent assay (ELISA). The cuvettes (BioRad) and exposed to an electric ELISA was carried out as previously described79"0 and uses a mouse monoclonal antibody to IL-8 (Sandoz, Vienna, Austria) and a phosphatase conjugated goat anti-IL-8 polyclonal antibody (Sandoz). Results The 24 hour secretion of IL-8 from AGS and ST42 cells after stimulation with H pyloni is shown in the figure. The cagA positive N6 wild-type strain and the cagA negative isogenic mutant N6.XA3 both induced IL-8 secretion relative to cell only control cultures. IL-8 se- cretion with the isogenic cagA negative mutant strain was 78% (AGS) and 84% (ST42) that of the wild-type parent strain.