Is There an Antigenic Mimicry Between Arteriosclerotic Lesions and H. pylori Antigens? GIOVANNI CAMMAROTA, 1 NATALE FIGURA, 3 ROSSELLA CIANCI, 1 VINCENZO PASCERI, 2 PAOLO FEDELI, 1 GIUSTO CRISTINA LENZI, 3 and GIOVANNI GASBARRINI 1 1 Department of Internal Medicine, 2 Department of Cardiology, Catholic University, Rome, Italy, and 3 Department of Internal Medicine of “Le Scotte” Hospital, Siena, Italy Introduction S everal reports have shown a correlation between the incidence of arteriosclerosis and the presence of at least three types of infectious micro-organisms: Herpesviruses, Chlamydia pneumoniae, and Helico- bacter pylori (1). The main feature shared by all these quite disparate agents is the presence of a chronic infection (often life-long) and of a chronic immune response. But it is unclear how microbes might work in arteries that are distant from their primary infection sites. It has been suggested, for example, that the organisms release toxins into the bloodstream that exacerbate inflammatory reac- tions at blood vessel linings. Helicobacter pylori might influence arteriosclerosis through the gener- ation of a persistent low-grade inflammatory stimu- lus. Indeed, bacterial cytotoxins are able to induce production of several cytokines (including interleu- kin-1, interleukin-6, and tumor necrosis factor), which may activate the vascular endothelium, to change the haemostatic system by increasing ex- pression of procoagulant substances (fibrinogen, plasminogen activator inhibitor (PAI)-1, tissue fac- tor) and down-regulating the fibrinolytic system, and to cause a prolonged endothelial dysfunction (2,3). Another suggestive hypothesis is that Helico- bacter organisms express molecules on their sur- faces, which are responsible of an antigenic mimicry with arteriosclerotic lesions. The purpose of this study was to investigate on the possible presence of an antigenic cross-reactivity between anti-H. pylori antibodies and components of arteriosclerotic plaque. Methods From 9 patients (6 males and 3 females; mean age: 64 years; range 55–73 years), who underwent ca- rotid arteriectomy for advanced (stenotic) arterio- sclerosis, carotid segments were taken. Seven pa- tients had anti-H. pylori antibodies in serum; four of these seven were infected by a virulent strain of H. pylori bearing the cytotoxin-associated gene-A. OCT-embedded surgical samples were snap frozen in liquid nitrogen chilled isopentane slurry and stored at -80 °C. Subsequently, frozen tissue were sectioned on a high-performance cryostat and finely minced with a scalpel. Proteins were extracted with 1% n-Octyl glucoside in phosphate-buffered saline, pH 6.8, added in the ratio of 1:3 w/v. After 20 min of incubation at room temperature with continuous agitation, samples were centrifuged at 12,000  g at 4 °C for 15 min. Supernatants were dialysed vs. phosphate-buffered saline by using membranes with 12 kDa-pore size. Samples were denatured in Lae- mmli’s buffer at 100 °C for 5 min, and proteins were resolved electrophoretically in a 10% polycrylamide gel containing sodium dodecyl sulphate, and trans- ferred onto nitro-cellulose. The free sites blocked with defatted milk in phosphate-buffered saline, pH 7.4, containing 0.1% Triton X (blocking agent, BA). Then, nitro-cellulose was incubated with a hyperim- mune serum against H. pylori, diluted 1:2000 in BA. Antibodies were raised in rabbits to a whole-cell suspension of the H. pylori-type strain CCUG 17874, to a recombinant fragment of cytotoxin-associated gene-A and purified vacuolating cytotoxin proteins, urease, and heat-shock protein (4) (sera were a gift of Dr. R. Rappuoli, Biocine, Siena, Italy). After over-night incubation, nitro-cellulose was washed three times with BA and an antirabbit IgG labeled with peroxidase (Kappel) was added at the dilution of 1:3000 in BA, and incubated for 90 min at room temperature. After further washings with BA and with Tris buffer 0.05 mol/L, pH 6.8, the reaction was visualised by adding the substrate (H 2 O 2 in a solu- Correspondence: Dr. G. Cammarota, Istituto di Medi- cina Interna e Geriatria, Policlinico “A. Gemelli,” Univer- sita ` Cattolica del Sacro Cuore Largo A. Gemelli, 8-00168 Rome, Italy. E-mail: gcammarota@libero.it Received October 28, 1999; revised June 10, 2000; accepted June 16, 2000. Clinical Biochemistry, Vol. 33, No. 5, 419 – 421, 2000 Copyright © 2000 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/00/$–see front matter PII 0009-9120(00)00148-X CLINICAL BIOCHEMISTRY, VOLUME 33, JULY 2000 419