© 2002 Blackwell Science Ltd, Helicobacter , 7, 281–286 281 Volume 7 • Number 5 • 2002 HELICOBACTER Blackwell Science, Ltd Relationship Between Gastric Ulcer and Helicobacter pylori VacA Detected in Gastric Juice Using Bead-ELISA Method Daisuke Shirasaka, * Nobuo Aoyama, † Masanori Sakashita, * Kohei Kuroda, * Shuji Maekawa, * Casmir Marwa Wambura, * Masaki Miyamoto, * Takao Tamura, * Kinnosuke Yahiro, ‡ Akihiro Wada, ‡ Hisao Kurazono, § Toshiya Hirayama ‡ and Masato Kasuga * * Division of Diabetes, Digestive, and Kidney Disease, Department of Clinical Molecular Medicine, † Department of Endoscopy, Kobe University School of Medicine, Kobe; ‡ Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki; § Department of Medical Technology, School of Health Sciences, Okayama University, Okayama, Japan ABSTRACT Background. VacA is an important pathogenetic factor produced by Helicobacter pylori. VacA has often been detected in supernatants of liquid cultures or lysates of whole bacterial cells. However, no studies have ever tried to assay VacA produced in the human stomach. We applied a very sensitive and simple method, bead-ELISA, to detect VacA in gastric juice. Materials and Methods. Forty-eight H. pylori -positive patients (16 nonulcer dyspepsia, 16 gastric ulcer, and 16 duodenal ulcer) and four H. pylori-negative nonulcer dyspepsia patients had endoscopy performed and gastric juice were aspirated. Polystyrene beads coated with the anti-body to VacA, were used in this bead-ELISA method. The nucleotide sequences of vacA in the signal and middle regions were investigated. Results. Of the 48 samples that were positive for H. pylori, 21 [43.8%] were found to be VacA positive in gastric juice. The average and maximum concentra- tions of detected VacA in gastric juice were 143.2 ± 216.5 and 840 pg/ml, respectively. The average density of VacA from gastric ulcer patients (227.5 ± 276.7 pg/ml) was higher than that found in nonulcer dyspepsia (51.8 ± 39.8 pg/ml) and duodenal ulcer (49.2 ± 21.5 pg/ml) patients. There was no relation- ship between VacA in gastric juice and vacA genotype. Conclusions. VacA in gastric juice could be directly detected by bead-ELISA. In this study, the diversity of disease outcome was associated with not the quality but the quantity of VacA. Therefore, not only the quality but also the quantity of VacA is important etiological factors in the pathogenesis of mucosal damage. Keywords. Helicobacter pylori, VacA, bead-ELISA. H elicobacter pylori is the major causative agent of chronic active gastritis, and infection with this organism is an important etiological factor in the pathogenesis of peptic ulcer and gas- tric cancer. An important virulence determinant of H. pylori is the vacuolating cytotoxin (VacA) [1,2], which induces cytoplasmic vacuolation in a variety of mammalian cell lines [3] and pro- duces epithelial cell damage and mucosal ulcera- tion when administered intragastrically to mice [4]. Although the gene that encodes the cyto- toxin, designated vacA, is present in nearly all strains [2,4 – 6], only about 50% of H. pylori strains can produce detectable amounts of this cytotoxin [1]. Cytotoxic strains have been detected more frequently among patients with peptic ulcer than those with chronic gastritis [7,8]. VacA is secreted into the extracellular space and binds to multiple eukaryotic cell-surface receptors in vitro study [9 –11]. The cellular effects induced by VacA include alteration of endo-lysosomal function [12], anion selective channel formation [13], apoptosis [14,15], and epithelial monolayer permeabilisation [16]. VacA has been reported to target several different cell components, including mitochondria [17], the cytoskelton [18], and epithielial cell-cell junc- tions [16]. However, the role of VacA in vivo is still uncertain. VacA has often been detected in supernatants of H. pylori liquid culture [1] or lysates of whole bacterial cells [19]. No studies have ever tried to assay VacA produced in vivo, and it is not clear how much VacA exists in the stomach. We have Reprint requests to: Nobuo Aoyama, M.D., Department of Endoscopy, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650–0017, Japan.