Acta medica 3/01

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ACTA MEDICA (HRADEC KRÁLOVÉ). 2001, Vol. 44, No. 3. CONTENTS. REVIEW ...... the battlefield (e.g. Iran-Iraq war) as well as in a civilian sec- tor as a threat ...
ACTA MEDICA (HRADEC KRÁLOVÉ) 2001, Vol. 44, No. 3 CONTENTS REVIEW ARTICLE Petr Nachtigal, Andrea Gojová, Vladimír Semecký The role of epithelial and vascular-endothelial cadherin in the differentiation and maintance of tissue integrity ...............83 René Vobořil Pneumatosis cystoides intestinalis - a review .....................................................................................................89

ORIGINAL ARTICLES Jiří Kassa, Marie Koupilová, Josef Vachek Long term effects of low-level sarin inhalation exposure on the spatial memory of rats in a T-maze...............................93 Cem Kockar , Mustafa Öztürk, Nüket Bavbek Helicobacter pylori eradication with beta carotene, ascorbic acid and allicin ...........................................................97 Pavel Kohout Small bowel permeability in diagnosis of celiac disease and monitoring of compliance of a gluten-free diet (Gut permeability in celiac disease) ....................................................................................101 Dušan Šimkovič, Karel Šmejkal, Milan Široký, Pavel Hladík, Ivo Pospíšil Importance of the anorectal manometry in chronic anal fissure...........................................................................105 Pavel Žáček, Jan Dominik, Jan Harrer, Vladimír Lonský, Jiří Manďák, Pavel Kuneš, Miroslav Solař Morbidity and mortality in patients 70 years of age and over undergoing isolated coronary artery bypass surgery..........109

CASE REPORT Abdurrahman Kadayifci, Yalcin Kepekci, Yavuz Coskun, Ying Huang Wolfram syndrome in a family with variable expression .....................................................................................115

REVIEW ARTICLE

THE ROLE OF EPITHELIAL AND VASCULAR-ENDOTHELIAL CADHERIN IN THE DIFFERENTIATION AND MAINTANCE OF TISSUE INTEGRITY Petr Nachtigal, Andrea Gojová, Vladimír Semecký Charles University in Prague, Faculty of Pharmacy in Hradec Králové: Department of Biological and Medical Sciences

Summary: The present review has focused on the cell adhesion molecules from the cadherin superfamily, in particular on E- and VE-cadherin. In general, cadherins are a large group of cell adhesion molecules located at intercellular junctions called adherent junctions. They play an important role in embryogenesis and morphogenesis in animals and humans due to their adhesive and cell-signalling functions. Disturbances of the expression or function of cadherins and their associated proteins called catenins are crucial for the initiation and development of many pathological states. E-cadherin is an epithelium-specific cadherin that is required for the development and maintenance of the normal function of all epithelial cells in tissues. The loss or down-regulation of E-cadherin is a key event in the process of tumour invasion and metastasis. The assessment of E-cadherin immunoreactivity may be a useful prognostic marker in some cancers, complementary to the established prognostic factors. VE-cadherin is an endothelium-specific cadherin, which plays a relevant role in vascular homeostasis. It has been demonstrated that VE-cadherin is required for normal vasculogenesis, angiogenesis, and for the maintenance of vascular integrity. Disruption of VE-cadherin-catenin complexes by some inflammatory agents such as thrombin, by inflammatory cells, or shear stress is accompanied by an increase in vascular permeability in vivo and in vitro. Key words: Cadherin family; E-cadherin; VE-cadherin; Cancerogenesis; Endothelial permeability

Introduction

Structural properties of the cadherin superfamily

Cell adhesion molecules are substances of a protein character that are necessary for normal embryogenesis, morphogenesis, tissue formation and reparation, but they are also involved in many pathophysiological processes such as inflammation, angiogenesis, thrombosis, tumour invasion and metastasis (19). At present, there are four main classes of cytoadhesion molecules: the immunoglobulin group of adhesion molecules, cadherins, selectins, and integrins (22). In the present review the authors concentrate on cell adhesion molecules from the cadherin family. The cadherins are a family of transmembrane glycoproteins that mediate adhesion through a Ca2+ dependent mechanism. Cadherins are usually localized at intercellular junctions called adherens junctions (41). The cadherins form a superfamily with at least six subfamilies, which can be distinguished on the basis of the protein domain composition, genomic structure, and phylogenetic analysis of the protein sequences. These subfamilies comprise classical or type-I cadherins (E-, N-, R-, P-cadherin), atypical or type-II cadherins (Cadherin-6, -7, -8, -10), desmosomal cadherins (desmocollins, desmogleins), protocadherins (Protocadherin-1, -2, OL-protocadherin, CNR protocadherins), and Flamingo cadherins (25).

In this review the present authors have aimed at the structure of classical cadherins. The cadherins consist of an extracellular adhesive domain, transmembrane segment, and cytoplasmic domain (5). The extracellular domain is responsible for cell-to-cell adhesion between cadherins. It is composed of four or five repeated Ca2+ binding subdomains of about 110 amino acids (39). The structure of this domain is determined by the binding of calcium ions, and the presence of Ca2+ ions is also necessary for the adhesion with cadherin molecules on neighbouring cells. Other classes of adhesion molecules, e.g., members of the Ig superfamily, do not depend on Ca2+ for their adhesive function (29). The adhesive interface is located at the most aminoterminal repeat. Structural data suggest that the extracellular domains form lateral dimers on the cell surface which interact with dimers from the opposing cells to create a zipper-like structure (34). Highly conserved cytoplasmic domains interact with cytosolic proteins from “armadillo family“ called catenins. It has been demonstrated that there are three main catenins in cadherin-catenin complexes that provide the linkage of cadherin to the actin cytoskeleton (18). β-Catenin and plakoglobin (also called γ-catenin), interact with α-catenin that provides connection with the

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actin cytoskeleton. Other catenins can also be associated with the cytoplasmic domain of cadherins, including different isoforms of the so-called p120ctn. It has been found that this catenin interacts with a membrane-proximal region of cadherin that has been shown to be responsible for lateral clustering. This suggest that p120ctn can regulate the strength of cadherin-mediated adhesion (43). In general, the interaction of cytoplasmic domain with the actin cytoskeleton via catenins significantly increases the strength of the intercellular junctions (45).

Function of the cadherins There are two main functional features of cadherins. They are responsible for cell-to-cell adhesion and they have a very important cell signalling function. Cadherins mediate homotypic interactions by binding to their homologues on an adjacent cell. However, it has been found that some members of this family can provide heterotypic interactions, but this is not the predominant adhesion mechanism (27). There are several ways of how cadherin could be involved in cell-signalling events. First, through their homotypic binding they can approach the opposing membranes of neighbouring cells in close proximity and enable interactions of ligands and receptors of these opposing cells and stimulate juxtacrine signalling. Second, because cadherins are able to control the polarity of cells they can affect sig-

nalling via their influence on the distribution of membrane proteins, including transmembrane receptors. Finally, cadherins may behave as ligands or receptors and hence they have direct cell signalling activity (18). All these signalling functions of cadherins are associated with the activation of some regulatory cascade comprising the action of tyrosin kinases and tyrosin phosphatases. Cytoplasmic domains of cadherin, β-catenin and plakoglobin are common targets of these regulatory proteins (38). Adhesive and signalling properties of cadherins can not be separated. Any changes in the expression or function of cadherins might lead to initiation or progression of pathological processes. Within many members of the cadherin superfamily, the present authors have focused on two extensively studied ones, E-cadherin and VE-cadherin.

Epithelial cadherin (E-cadherin, LCAM, ovomorulin) E-cadherin is an epithelium-specific cadherin. This cadherin is a member of the classical cadherin subfamily. Normal expression and function of E-cadherin is required for proper embryogenesis and morphogenesis of various tissues. Variations in E-cadherin expression have been noted during specific events in embryonic morphogenesis (30). Mutation of the E-cadherin gene leads to early embryonic lethality which is proceeded by a loss of cell-to-cell adhe-

Fig. 1: Structure of cadherin-catenin-complex. Representation of cadherin-catenin complex depicting cadherin ectodomain, transmembrane region (TM) and carboxy-terminal cytodomain (CYTO) and catenins that link cadherin to the cytoskeleton. Ectodamain of classical cadherins consists of five repeated domains (C1-C5) with adhesive interface located at C1 domain. Carboxy-terminal cytodomain has binding sites for β-catenin and γ-catenin (plakoglobin) and membrane proximal binding site for p120. β-catenin and γ-catenin are associated with α-catenin that provide linkage to actin cytoskeleton. 84

sion at the morula stage (5). E-cadherin is necessary during normal neural development (35). It has been demonstrated to occur locally and persistently in the murine central and peripheral nervous system during neural development (36). Expression of E-cadherin (together with P- and N-cadherin) has been observed in murine primordial germ cells. E-cadherin is concentrated at the sites of cell-to-cell contacts of primordial germ cells (PGCs), suggesting an active role in PGCs-PGCs interaction and recognition (6). In the past few years, the alteration in expression and function of E-cadherin has been correlated with cancer development (5). Loss or reduction of E-cadherin expression is in relation with enhanced aggressiveness and dedifferentiation of many carcinomas which has been reviewed by Beavon (4). Asgeirsson et al. have shown that decreased E-cadherin expression is frequent in breast cancer and that a loss of E-cadherin expression is associated with a loss of heterozygosity in the infiltrating lobular breast carcinomas but not in the infiltrating ductal carcinomas. Furthermore, the loss of expression of E-cadherin is an important prognostic marker, especially for disease recurrence in nodenegative breast cancer patients and may even be more informative than tumour size or oestrogen receptor expression (3). Ghadimi et al. have shown that reduced expression of E-cadherin and even α-catenin is observed in primary colorectal carcinoma. Moreover, they were able to demonstrate a significant correlation between the histopathological grading of the tumours and an increased loss of E-cadherin and α-catenin expression. Defective expression was significantly more frequent in less differentiated carcinomas (G3-4) with a pronounced loss of epithelial morphology than in better differentiated tumours (G1/G2) (17). Garcia del Muro et al. have suggested that losses of E-cadherin and β-catenin as confirmed by immunohistochemistry are important prognostic markers in patients with bladder carcinoma. This leakage of E-cadherin expression was associated with high grade and invasive stage of bladder carcinoma. Further, a loss of E-cadherin expression was a significant prognostic indicator of decreased survival, independent of known prognostic factors such as grade, stage, or p53 status (23). There are many other studies which have described a relation between decreased E-cadherin and/or catenin expression and its correlation with dedifferentiation, infiltrative tumour growth, distant metastasis, and poor survival for patients with gastric carcinoma (37), pancreatic carcinoma (40,16), prostate cancer (12). Despite the fact that E-cadherin is extensively studied in relation to cancerogenesis, Bobryshev et al. have elucidated expression of E-cadherin in human atherosclerotic lesions. It has been described that E-cadherin is expressed by macrophage origin intimal cells transforming themselves into foam cells, but there were no expression of E-cadherin in non-atherosclerotic intima. They suggested that E-cadherin might be important for foam cell aggregation. If E-cadherin is involved in foam cell aggregation, it might be also invol-

ved in the development of the lipid core which is an important step of progression of atherosclerosis (9).

Vascular endothelial cadherin (VE-cadherin, cadherin 5 or 7B4) VE-cadherin is endothelium-specific cadherin and it is located strictly at intercellular junctions (zonulla adherens) of essentially all types of vessels both in vitro and in vivo (14,15). VE-cadherin has been first identified by Lampugnani et al. by adopting an indirect approach of developing mouse mAbs to human endothelial cells (20). In term of the structure, VE-cadherin is composed of an extracellular domain, a transmembrane segment, and a cytoplasmic domain that form complexes with catenins and mediate the association of VE-cadherin with the actin cytoskeleton. However, compared with classical cadherins, the VE-cadherin amino acid sequence shows considerable differences (only 23% identity when compared with classical cadherins such as E-, N-, and placental (P)-cadherins) (11,24). VE-cadherin is required for normal vasculogenesis and angiogenesis and for the maintenance of vascular integrity and permeability in adults (13). VE-cadherin is expressed in the embryo at very early stages of vascular development in mesodermal cells of the yolk sac mesenchyme. At later embryonic stages, VE-cadherin expression is restricted to the peripheral layer of blood islands, which gives rise to endothelial cells (10). The role of VE cadherin permeability control is consistent with the observation that the VE-cadherin-catenin complex is the target of the action of permeability-increasing agents. Rabiet et al. have shown that thrombin, which is known to induce profound alterations of endothelial cell monolayer permeability in vitro and in vivo, caused endothelial cell retraction accompanied by a redistribution of VE-cadherin and catenin from adherence junctions. This disassembly of adherence junctions was accompanied by an increase in vascular permeability (28). The proinflammatory cytokines tumour necrosis factorα (TNF-α) and interferon-γ (INF-γ) act synergistically in vitro and in vivo to activate the endothelium, resulting in cellular responses such as altered morphology, loss of barrier function, and adhesion molecule upregulation and/or redistribution (31,32). Wond et al. have described that the tumour necrosis factor-α (TNF-α) and interferon-γ (INF-γ), in combination, affect the barrier function of the vascular endothelial lining by direct stimulation of the endothelium that results in the disruption of VE-cadherin mediated cellto-cell adhesion, which is succeeded by an increase in the permeability of mesenteric venules (44). Andriopoulous et al. have studied the effect of histamine, another mediator of inflammatory reaction, on adherens junction organization in cultured endothelial cells. They have reported that histamine induces tyrosine phosphorylation of VE-cadherin and catenins, which results in an increase in endothelial permeability. The effect of histamine was specific for VE-cadherin, there was no phosphorylation in N-cadherin, another

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major endothelial cadherin (2). In addition to agonists described above, endothelial permeability is affected by a group of inflammatory cells, namely polymorphonuclear leukocytes (PMNs) (33). Several groups have shown that activated polymorphonuclear leukocytes (PMNs) dramatically alter molecular composition and organization of VE-cadherin-catenin complexes in endothelial cells. This disruption of VE-cadherin-catenin complexes leads to disassembly of adherens junctions which is followed by an increase in endothelial permeability (42,1). These inflammatory agents and cells are not the only ones that might exert effects on VE-cadherin-catenin complexes. The structure and physiology of endothelial cells are influenced by shear stresses of blood flow. The most obvious structural responses of endothelium to shear stress are changes in the cell shape and orientation; in areas of low or inconsistent shear stress, in vivo or in vitro endothelial cells form a cuboidal, cobblestone shape, whereas they elongate and align in the direction of flow when shear stress is moderate or high (21). Noria et al. have examined transient and steady-state effects of shear stress on the cadherin-catenin complex at endothelial adherens junctions. They have reported that initiation of shear stress on endothelium causes partial disassembly of adherens junctions followed by a reassembly that reflected shear-induced reorganization of actin distribution. After adaptation to shear stress, adherens junction proteins were localized in adhesion plaques (adherens plaques) that were distinct from the linear belt-like distribution that predominates in static cultures. Thus, adherens junctions in the endothelium exposed to physiological levels of shear stress are structurally distinct from such junctions in static endothelial cell cultures or in other epithelial monolayers (26). As mentioned above, the normal expression and function of VE-cadherin is necessary for the maintenance of normal endothelial permeability; however, it is important in vasculogenesis in the embryo as well as in adults. Bobryshev et al. have examined the expression of VE-cadherin in atherosclerotic lesions. They have demonstrated that VE-cadherin is expressed in early sprouts of neocapillaries and it suggests that VE-cadherin is involved in the ingrowth of medial capillaries into the intima (8). This neovascularization is important for local immune-inflammatory reactions in atherosclerotic plaques (7).

Conclusion In this review the authors have described structure and function of cell adhesion molecules from cadherin family. We have focused on two extensively studied E-cadherin and VE-cadherin. Both VE-cadherin and E-cadherin are crucial for proper embryogenesis, morphogenesis but they are involved in many pathological states. Changes in expression and function of E-cadherin seems to be important for development of carcinoma in various tissues. VE-cadherin is specific cadherin which is expressed by endothelial cells and plays important role in vascular homeostasis. It is re-

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quired for vasculogenesis and maintance of vascular permeability. Its expression and function is disturbed during inflammation. In our prospective study we would like to described behaviour of these two cadherins during development and progression of atherosclerosis (on rabbit model af atherosclerosis) because changes in vascular permeability, neovasculogenesis and formation of lipid core are crucial for formation of atherosclerotic plaques. Acknowledgements The authors wish to thank Dr. B. Mánek, CSc. for revising the English text. This work was supported by MŠMT grant 111600002.

References 1. Allport JR, Ding H, Collins T, Gerritsen ME, Luscinskas FW. Endothelial-dependent mechanisms regulate leukocyte transmigration: A process involving the proteasome and disruption of the vascular endothelial–cadherin complex at endothelial cell-to-cell junctions. J Exp Med 1997;186:517–27. 2. Andriopoulou P, Navarro P, Zanetti A, Lampugnani MG, Dejana E. Histamine induces tyrosine phosphorylation of endothelial cell-to-cell adherens junctions. Arterioscler Thromb Vasc Biol 1999;19:2286-97. 3. Asgeirsson KS , Jónasson JG, Tryggvadóttir L et al. Altered expression of E-cadherin in breast cancer: Patterns, mechanisms and clinical significance. Eur J Cancer 2000;36:1098-106. 4. Beavon IRG. The E-cadherin-catenin complex in tumour metastasis: Structure, function and regulation. Eur J Cancer 2000;36:1607-20. 5. Behrens J. Cadherins and catenins: Role in signal transduction and tumor progression. Cancer Metastasis Rev 1999;18:15-30. 6. Bendel-Stenzen MR, Gomperts M, Anderson R, Heasman J, Wylie CH. The role of cadherins during primordial germ cell migration and early gonad formation in the mouse. Mech Dev 2000;91:143-52. 7. Bobryshev YV, Lord RSA. Mapping of vascular dendritic cells in atherosclerotic arteries suggests their involvement in local immune- inflammatory reactions. Cardiovasc Res 1998;37:799–810. 8. Bobryshev YV, Cherian SM, Inder SJ, Lord SA. Neovascular expression of VEcadherin in human atherosclerotic arteries and its relation to intimal inflammation. Cardiovasc Res 1999;43:1003–17. 9. Bobryshev YV, Lord SA, Wanatabe T, Ikezawa T. The cell adhesion molecule Ecadherin is widely expressed in human atherosclerotic lesions. Cardiovasc Res 1998;40:191-205. 10. Breier G, Breviario F, Caveda L et al. Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system. Blood 1996;87:630-41. 11. Breviario FL, Caveda M, Corada I et al. Functional properties of human vascular endothelial cadherin (7B4/cadherin-5) an endothelium-specific cadherin. Arterioscler Thromb Vasc 1995;15:1229–39. 12. Davies G, Jiang WG, Mason MD. E-cadherin and associated molecules in the invasion and progression of prostate cancer. Oncol Rep 1998;5:1567-76. 13. Dejana E, Bazzoni G, Lampugnani MG. Vascular endotelial (VE)-cadherin: Only an intercellular glue? Exp Cell Res 1999;252:13-9. 14. Dejana E, Corada M, Lampugnani MG. Endothelial cell-to cell junctions. FASEB J 1995;9:910-8. 15. Dejana E. Endothelial adherens junctions: Implications in the control of vascular permeability and angiogenesis. J Clin Invest 1996;98:1949–53. 16. El-Hariry I, Jordinson M, Lemoine N, Pignatelli M. Characterization of the Ecadherin-catenin complexes in pancreatic carcinoma cell lines. J Pathol 1999;188:155-62. 17. Ghadimi BM, Behrens J, Hoffmann I, Haensch W, Birchmeier W, Schlag PM. Immunohistological analysis of E-Cadherin, α-, β- and γ-catenin expression in colorectal cancer: Implications for cell adhesion and signaling. Eur J Cancer 1999;35:60-5. 18. Hinck L, Nathke IS, Papkoff J, Nelson WJ. Dynamics of cadherin-catenin complex formation: Novel protein interactions and pathways of complex assembly. J Cell Biol 1994;125:1327–40. 19. Cell adhesion molecules: An overview. Cancer Invest 1998;16:176-82. JosephSilverstein J, Silverstein RL. 20. Lampugnani MG, Resnati M, Raiteri M et al. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J Cell Biol 1992;118:1511-22.

21. Langille BL, Adamson SL. Relationship between blood flow direction and endothelial cell orientation at arterial branch sites in rabbits and mice. Circ Res 1981;48:481-8. 22. Mareckova Z, Heller S, Horky K. Cell adhesion molecules and their role in pathophysiological processes. Vnitr Lek 1999;45:232-7. 23. Muro del GX, Torregrosa A, Muňoz J et al. Prognostic value of the expression of E-cadherin and β-catenin in bladder cancer. Eur J Cancer 2000;36:357-62. 24. Navarro P, Ruco L, Dejana E. Differential Localization of VE- and N-cadherins in human endothelial cells: VE-Cadherin competes with N-cadherin for junctional localization. J Cell Biol 1998;140:1475–84. 25. Nollet F, Kools P, Van Roy F. Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members. J Mol Biol 2000;299:551-72. 26. Noria S, Cowan DB, Gotlieb AI, Langille L. Transient and steady-state effects of shear stress on endothelial cell adherens junctions. Circ Res 1999;85:504-14. 27. Petruzzelli L,Takami M, Humes HD. Structure and function of cell adhesion molecules. Am J Med 1999;106:467-76. 28. Rabiet MJ, Plantier JL, Rival Y, Genoux Y, Lampugnani MG, Dejana E. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler Thromb Vasc Biol 1996;16:488-96. 29. Redies CH. Cadherins in the central nervous system. Prog Neurobiol 2000;61:611-48. 30. Reima I, Lehtonen E, Virtanen I, Flechon JE. The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig. Differentiation 1993;54:35-45. 31. Rival Y, Del Maschio A, Rabiet MJ, Dejana E, Duperray A. Inhibition of platelet endothelial cell adhesion molecule-1 synthesis and leukocyte transmigration in endothelial cells by the combined action of TNF-alpha and IFN-gamma. J Immunol 1996;157:1233-41. 32. Romer LH, McLean NV, Yan HC, Daise M, Sun J, DeLisser HM. IFN-gamma and TNF-alpha induce redistribution of PECAM-1 (CD31) on human endothelial cells. J Immunol 1995;154:6582-92. 33. Romer LH, McLean NV, Yan HC, Daise M, Sun J, DeLisser HM. Interaction of neutrophils and endothelium in isolated coronary venules and arterioles. Am J Physiol 1995;268:490-8. 34. Shapiro L, Fannon AM, Kwong PD et al. Structural basis of cell-cell adhesion by cadherins. Nature 1995;374:327-37. 35. Shimamura K, Hirano S, McMahon, AP, Takeichi M. Wnt-1-dependent regulation of local E-cadherin and alpha N-catenin expression in the embryonic mouse brain. Development 1994;120:2225-34.

36. Shimamura K, Takeichi M. Local and transient expression of E-cadherin involved in mouse embryonic brain morphogenesis. Development 1992;116:1011-9. 37. Shino Y, Watanabe A, Yamada Y et al. Clinicopathologic evaluation of immunohistochemical E-cadherin expression in human gastric carcinomas. Cancer 1995;76:2193-201. 38. Steinberg MS, McNutt PM. Cadherins and their connections: Adhesion junctions have broader functions. Curr Opin Cell Biol 1999;11:554–60. 39. Taga M, Suginami H. Cell adhesion and reproduction. Horm Res 1998;50(suppl 2):2-6. 40. Takao S, Che X, Fukudome T et al. Downregulation of E-cadherin by antisense oligonucleotide enhances basement membrane invasion of pancreatic carcinoma cells. Hum Cell 2000;13:15-21. 41. Telo P, Lostaglio S, Dejana E. Structure of intercellular junctions in the endothelium. Therapie 1997;52:395-8. 42. Tinsley JH, Wu MH, Ma W, Taulman AC, Yuan SY. Activated neutrophils induce hyperpermeability and phosphorylation of adherens junction proteins in coronary venular endothelial cells. J Biol Chem 1999;274:24930-4. 43. Vleminckx K, Kemler R. Cadherins and tissue formation: Integrating adhesion and signaling. Bioessays 1999;21:211-20. 44. Wong RK, Baldwin AL, Heimark RL. Cadherin-5 redistribution at sites of TNF-α and IFN-γ-induced permeability in mesenteric venules. Am J Physiol 1999;276 (Heart Circ Physiol):736-48. 45. Yap AS, Niessen CM, Gumbiner BM. The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strenghtening, and interaction with p120ctn . J Cell Biol 1998;141:779-89.

Submitted April 2001. Accepted November 2001. Mgr. Petr Nachtigal, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical sciences, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic. e-mail: [email protected]

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REVIEW ARTICLE

PNEUMATOSIS CYSTOIDES INTESTINALIS - A REVIEW René Vobořil Charles University in Prague, Faculty of Medicine in Hradec Králové: Department of Surgery

Summary: Pneumatosis cystoides intestinalis is a rare disease characterized by presence of multilocular cysts in the gastrointestinal wall. Idiopatic and secondary forms of the disease can be distinguished. There are presented several theories explaining pneumatogenesis in this article. The specific and non-specific symptoms are described. Attention is drawn to the pneumoperitoneum without signs of peritoneal irritation, what is a typical complication of this disease. The suspition of pneumatosis cystoides intestinalis may be based on plain abdominal X-ray, and is usually confirmed by computer tomography or magnetic resonance imaging. The therapy can be conservative or surgical. In conclusion, although pneumatosis cystoides intestinalis is a rare disease, it may represent a problem in differential diagnosis of abdominal pain. Key words: Pneumatosis cystoides intestinalis; Pneumoperitoneum; Cysts

Pneumatosis cystoides intestinalis (PCI) is not too a frequent disease characterized by multilocular gas cysts localized in the wall of the alimentary tract. The rupture of the cysts leads to pneumoperitoneum in the absence of the signs of peritoneal irritation, which is considered to be pathognomic for this disease. Purpose of this paper is to draw attention to pneumatosis cystoides intestinalis – a rare entity, but an important one for clinical practice, futher to show recent view on this problem and notice the supposed etiology, clinical presentation and treatment.

is thought to dissipate via mediastinum, retroperitoneum and mesenterium into the gut wall (37). The second mechanism is represented by increase of intraluminal bowel pressure, which in connection with damage of the mucosa leads to intramural penetration of gas. This can explain the fact that PCI is often present in patients who have gastrointestinal disease – peptic ulcer disease, Crohn’s disease (24,48) or necrotizing enterocolitis (11,27,32). Necrotizing enterocolitis with PCI has been induced experimentally by arterial and lymphatic ligation (46). Some others (41) explain PCI as a consequence of reparation after bowel ischemia.

Etiology and pathogenesis

Bacterial theory

According to Kreiss et al. (33) PCI can be clasified either the idiopathic with unknown etiology (15%) or the secondary one (85%), in which the mechanism of cysts origin has been explained. Several theories elucidating the pathogenesis of this disorder have been proposed.

Bacterial theory explains the pathogenesis of PCI by bacterial infection. This infection either damages the intestinal wall with subsequent intramural penetration of gas, or produces gas, which then penetrates into the gut wall. Gas can also enter the lymphatic vessels and cause their dilatation. This theory is supported by experiments in rats, where PCI was induced by Clostridium perfringens (52). The microorganisms playing role in origin of PCI are Clostridium difficile, cytomegalovirus (44) or Clostridium perfringens (7).

Introduction

Mechanical theory The mechanical theory explains the pathogenesis of pneumatosis by physical factors. Two pathogenetic mechanisms have been proposed: air leakage from lung interstitium to mediastinum, retroperitoneum, mesenterium and intestinal wall, and leakage of intraluminal gas through gaps in intestinal mucosa. The first mechanism has been proposed in patients with chronic obstructive lung disease or with other illness of the respiratory system (48). A rise in intraalveolar pressure leads to alveolar rupture and leakage of air into lung interstitium. Air from lung interstitium 88

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Immunopathological inflammatory reaction Based on observation by Holl et al. (22) and their demonstration of histiocytes and foreign-body giant cells present in the afflicted part of the bowel, immunopathological inflammatory reaction has been proposed as a cause of PCI. The presence of monocytes and similar mononuclear cells has been confirmed by Gagliardi et al. (19). 89

PCI after bone marrow transplantation PCI has been also described after bone marrow transplantation (6). The effect of long term steroid use, infection, immunosuppression, graft-versus-host disease are thought to cause disorder in these cases (36).

PCI in connective tissue disease The increase of PCI incidence in patients with a connective tissue disease has been observed (2,10,21,30,34). PCI in these patients is probably caused by damage of the gut wall primary by this illness or secondary owing to the ischemia after failure of vessels supply.

Failure of activity hydrogen metabolizing bacteria PCI is characterized by high level of breath hydrogen, patients with PCI excrete more hydrogen than others. Clinical features of PCI may be in consequence of abnormal hydrogen metabolism. In normal subjects hydrogen is consumed by methanogenic and sulfatereducing bacteria. The activity of these bacteria is missing in patients with PCI. This leads to the intraluminal gas accumulation, to an increase of intraluminal pressure and thus to intramural gas penetration (12,13). The mechanism just described can explain cysts origin. According to Levitt et al. (35) the hydrogen hyperproduction is only the initial reason for cysts origin. Their futher persistence is caused by nitrogen and oxygen, which diffuse from blood (35).

Clinical presentation Presence and character of symptoms The character of symptoms is dependent on the localization of PCI and on presence or non-presence of basic disease. Symptoms, which can appear, are either non-specific or specific ones. Abdominal distension (29,34), diarrhoea (10,19,29,39), abdominal pain (10,39), constipation (19,39), mucus discharge (19,39), hematemesis (34), rectal bleeding (19,39), meteorism (14) and weight loss (10) belong to non-specific symptoms. Among the specific symptoms there belong cysts, which can be source of origin of invagination (1) or volvulus (5) and can cause interception of motility and the mechanical obstruction (26). It is especially necessary to draw attention to cysts ruptures, which lead to pneumatosis specific complication – pneumoperitoneum without alarming signs of peritoneal irritation (16,23,26,28,31). Pneumoretroperitoneum develops by cyst rupture in retroperitoneal part of bowel. The mentions of pneumoretroperitoneum are repeated in the literature (28,31,37). During these complications patients usually complain of non-specific abdominal disorder. Pneumoperitoneum diagnosed on abdomen X-ray examination is followed by laparotomy when perforation of alimentary tract in common location is not found (3). Clinical course

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of PCI may be benign, however also alarming and requiring surgical intervention (8). Localization of damage Occurence of PCI was described in right colon (21), further in transversum (6) or left colon (18,19). Moote et al. (40) conclude that sigma has predisposition to the occurence of the illness. The cysts in sigmoid localization cause sigmoid colon redundancy by affecting sigmoid mesentery. Rectum is usually spared (49). There is also described the incidence of PCI in other parts of alimentary tract - small intestine (35,42), stomach (4,6). Small intestine infliction can be connected with malabsorption (39) or with coeliac disease (45), the gastric infliction is unusual (15). Age and sex Beyond infancy the PCI is rare (44). In Bertram’s et al. (7) opinion the period with the most frequent incidence of PCI is age between 30 and 50 years, a clear sexual predominance doesn’t exist.

Diagnostics When PCI suspected the first examination is plain radiography of abdomen (26,51). There are seen the cysts in the bowel wall or free air under diaphragm in the case of their rupture and pneumoperitoneum appearance. The next diagnostic method can be represented by barium enema examination (7,40). Further it is possible to complete other investigations, which are more sensitive but also more expensive. Sonography that is able to diagnose the cysts is according to some authors (20,51) suitable method, too. Computer tomography remains the most successful technique for initial diagnosis and subsequent follow up (17,51). Its disadvantage is high radiation stress and financial severity. It is also possible to use magnetic resonance in PCI diagnostics (43). Furthermore, but less frequently, PCI can be diagnosed by other ways, as diagnostic laparoscopy (38), endoscopical methods (15,18,34,51) and H2 test, which enables to detect higher breath level of hydrogen by patients with PCI (12).

Therapy In asymptomatic patients with PCI no special therapy is recommended (9,18). If a basic disease is present, then it is necessary to treat it and secondary cysts regression is usually observed. PCI therapy could be conservative or surgical one. Conservative therapy Conservative therapy can be causal or symptomatical. The causal therapy includes ways suppressing supposed etiological mechanisms. Inhibition principles of these mechanisms consist either in restriction of intestinal gas producting microflora – administration of antibiotics, es-

pecially metronidazol (7,29,31,47), or in inhibition of process leading to the hydrogen hyperproduction - hyperbaric oxygen inhalation (7,31,42). In another way of treatment there is possible to include a diet low in flatulence-producing carbohydrates (14), parenteral nutrition (29,31), endoscopical puncture and cysts sclerotherapy (25), therapy with long-acting somatostatin analogue (30). Symptomatical therapy suppresses single symptoms (19) – as pain, constipation, diarrhoea. Surgical therapy Patients with pneumoperitoneum without signs of peritoneal irritation when diagnosis of PCI is known are not necessary to operate (23,44), it is sufficient enough to observe them (23). Surgery is indicated only in fulminant cases (3). The most frequent surgical solution is gut resection (7) or limited colectomy (50).

Conclusion To conclude: pneumatosis cystoides intestinalis is a rare entity with uncomplicated recognition by modern diagnostic methods. The practical importance of this paper is to inform about this problem and thus enable to avoid the laparotomy in patients suffering from PCI with pneumoperitoneum without signs of peritoneal irritation.

References 1. Ahrar K, Watkins GE, Gardiner G. Colocolic intussusception caused by pneumatosis cystoides coli. Abdom Imaging 1997;22(4):392-4. 2. Alcocer-Gouyonnet F, Chan-Nunez C, Hernandez J, Guzman J, GamboaDominguez A. Acute abdomen and lupus enteritis: thrombocytopenia and pneumatosis intestinalis as indicators for surgery. Am Surg 2000;66(2):193-5. 3. Andresen SJ, Gronneberg KG, Oppedal T. Pneumatosis cystoides intestinalis. Tidsskr Nor Laegeforen 1999;119(12):1756-7. 4. Antela CC, Antela LA, Masa VL et al. Intestinal and gastric cystoid pneumatosis associated with duodenal stenosis. Rev Esp Enferm Dig 1990; 77(5):361-4. 5. Azimuddin K, Bourne R. Pneumatosis cystoides intestinalis in a case of sigmoid volvulus. Br J Hosp Med 1997;57(9):468-9. 6. Bates FT, Gurney JW, Goodman LR, Santamaria JJ, Hansen RM, Ash RC. Pneumatosis intestinalis in bone-marrow transplantation patients: diagnosis on routine chest radiographs. Am J Roentgenol 1989;152(5):991-4. 7. Bertram P, Treutner KH, Winkeltau G, Booss HJ, Staatz G, Schumpelick V. Pneumatosis cystoides intestini. Langenbecks Arch Chir 1993;378(4):249-54. 8. Boerner RM, Fried DB, Warshauer DM, Isaacs K. Pneumatosis intestinalis. Two case reports and a retrospective review of the literature from 1985 to 1995. Dig Dis Sci 1996; 41(11):2272-85. 9. Brewaeys P, Ysebaert D, Hubens G, Vaneerdeweg W, Eyskens E. Pneumatosis cystoides intestinalis. Conservative approach in non surgical pneumoperitoneum: a case report and literature review. Acta Chir Belg 1995;95(4 Suppl):195-8. 10. Cabrera GE, Scopelitis E, Cuellar ML, Silveira LH, Mena H, Espinoza LR. Pneumatosis cystoides intestinalis in systemic lupus erythematosus with intestinal vasculitis: treatment with high dose prednisone. Clin Rheumatol 1994;13(2):312-6. 11. Chabot VH, Slovis TL, Cullen M. Recurrent pneumatosis intestinalis in young infants. Pediatr Radiol 1992;22(2):120-2. 12. Christl SU, Gibson GR, Murgatroyd PR, Scheppach W, Cummings JH. Impaired hydrogen metabolism in pneumatosis cystoides intestinalis. Gastroenterology 1993;104(2):392-7. 13. Christl SU, Scheppach W, Kasper H. Hydrogen metabolism in the large intestine-physiology and clinical implications. Z Gastroenterol 1995;33(7):408-13. 14. Christl SU, Scheppach W, Kasper H. Pneumatosis cystoides intestinalis. Dtsch Med Wochenschr 1996;121(7):195-9. 15. Cordum NR, Dixon A, Campbell DR. Gastroduodenal pneumatosis: endoscopic and histological findings. Am J Gastroenterol 1997;92(4):692-5.

16. Daly BD, Guthrie JA, Couse NF.Pneumoperitoneum without peritonitis. Postgrad Med J 1991;67(793):999-1003. 17. Dorenberg E. Diagnostic imaging in intestinal pneumatosis. Tidsskr Nor Laegeforen 1999;119(22):3266-8. 18. Estermann F, Denis B, Gaucher P, Regent D, Sondag D. Pneumatosis cystoides of the colon: knowing how to recognize it. Apropos of 8 cases. Ann Gastroenterol Hepatol Paris 1994;30(4):151-5. 19. Gagliardi G, Thompson IW, Hershman MJ, Forbes A, Hawley PR, Talbot IC. Pneumatosis coli: a proposed pathogenesis based on study of 25 cases and review of the literature. Int J Colorectal Dis 1996;11(3):111-8. 20. Goske MJ, Goldblum JR, Applegate KE, Mitchell CS, Bardo D. The ”circle sign”: a new sonographic sign of pneumatosis intestinalis - clinical, pathologic and experimental findings. Pediatr Radiol 1999;29(7):530-5. 21. Hiraishi T, Tokuda M, Mitsunaka H, Dobashi H, Takahara J. Asymptomatic pneumatosis cystoides intestinalis in a patient with systemic lupus erythematosus. Ryumachi 1999;39(3):580-5. 22. Holl K, Nolte H, Zornig C, Schroder S. Pneumatosis intestinalis – histology, immunocytochemistry and new theory of morphogenesis. Pathologe 1993;14(4):199-204. 23. Hoover EL, Cole GD, Mitchell LS, Adams CZ Jr, Hassett J. Avoiding laparotomy in nonsurgical pneumoperitoneum. Am J Surg 1992;164(2):99-103. 24. Iitsuka T, Kobayashi M, Izumi Y, Koyama A. Pneumatosis cystoides intestinalis following steroid treatment in a nephrotic syndrome patient: report of a case. Nippon Jinzo Gakkai Shi 1993;35(3):293-7. 25. Johansson K, Lindstrom E. Treatment of obstructive pneumatosis coli with endoscopic sclerotherapy: report of a case. Dis Colon Rectum 1991;34(1):94-6. 26. Keene JG. Pneumatosis cystoides intestinalis and intramural intestinal gas. J Emerg Med 1989;7(6):645-650. 27. Keller KM, Schmidt H, Wirth S, Queisser-Luft A, Schumacher R. Differences in the clinical and radiologic patterns of rotavirus and non-rotavirus necrotizing enterocolitis. Pediatr Infect Dis J 1991;10(10):734-8. 28. Kirchner J, Lorenz M, Heyd R, Gute P, Jacobi V. Pneumoperitoneum and pneumoretroperitoneum without perforation. Zentralbl Chir 1996;121(10):861-5. 29. Kirchner J, Seipelt G, Heyd R, Dietrich CF, Jacobi V. Disseminated pneumoperitoneum during the therapy of lymphoma with methotrexate and cytosine arabinoside. Dtsch Med Wochenschr 1996;121(42):1288-91. 30. Kobayashi T, Kobayashi M, Naka M, Nakajima K, Momose A, Toi M. Response to octreotide of intestinal pseudoobstruction and pneumatosis cystoides intestinalis associated with progressive systemic sclerosis. Intern Med 1993;32(7):6079. 31. Kopp AF, Gronewaller E, Laniado M. Pneumatosis cystoides intestinalis with pneumoperitoneum and pneumoretroperitoneum following chemotherapy. Abdom Imaging 1997;22(4):395-7. 32. Kosloske AM, Musemeche CA, Ball WS Jr, Ablin DS, Bhattacharyya N. Necrotizing enterocolitis: value of radiographic findings to predict outcome. Am J Roentgenol 1988;151(4):771-4. 33. Kreiss C, Forohar F, Smithline AE, Brandt LJ. Pneumatosis intestinalis complicating C. difficile pseudomembranous colitis. Am J Gastroenterol 1999;94(9):2560-1. 34. Lanspa SJ, Liu MW, Jenkins HJ Jr. Giant bulla in pneumatosis cystoides intestinalis. J Clin Gatroenterol 1988;10(4):437-40. 35. Levitt MD, Olsson S. Pneumatosis cystoides intestinalis and high breath H2 excretion: insights into the role of H2 in this condition. Gastroenterology 1995;108(5):1560-5. 36. Lipton J, Patterson B, Mustard R et al. Pneumatosis intestinalis with free air mimicking intestinal perforation in a bone marrow transplant patient. Bone Marrow Transplant 1994;14(2):323-6. 37. Mack P. Pneumoretroperitoneum associated with pneumatosis cystoides intestinalis. Ann Acad Med Singapore 1988;17(4):600-2. 38. Mehta SN, Friedman G, Fried GM, Mayrand S. Pneumatosis cystoides intestinalis: laparoscopic features. Am J Gastroenterol 1996;91(12):2610-2. 39. Micklefield GH, Kuntz HD, May B. Pneumatosis cystoides intestinalis: case reports and review of the literature. Mater Med Pol 1990;22(2):70-2. 40. Moote DJ, Fried LA, LeBrun GP, Fraser DB. Pneumatosis coli: is there a relationship with sigmoid colon redundancy? Gastrointest Radiol 1989;14(1):79-82. 41. Morali GA, Braverman DZ, Zimran A, Gottschalk S, Dollberg M. Pneumatosis cystoides intestinalis of the splenic flexure. Harefuah 1990;119(12):428-30. 42. Paw HG, Reed PN. Pneumatosis cystoides intestinalis confined to the small intestine treated with hyperbaric oxygen. Undersea Hyperb Med 1996;23(2):115-7. 43. Rabushka LS, Kuhlman JE. Pneumatosis intestinalis. Appearance on MR examination. Clin Imaging 1994;18(4):258-61. 44. Reynolds HL Jr, Gauderer MW, Hrabovsky EE, Shurin SB. Pneumatosis cystoides intestinalis in children beyond the first year of life: manifestations and management. J Pediatr Surg 1991;26(12):1376-80. 45. Sackier JM, Smith EJ, Wood CB. Cystic pneumatosis in coeliac disease. Gut 1988; 29(6):852-5.

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46. Sibbons P, Spitz L, van Velzen D. The role of lymphatics in the pathogenesis of pneumatosis in experimental bowel ischemia. J Pediatr Surg 1992;27(3):339-43. 47. Tak PP, Van Duinen CM, Bun P et al. Pneumatosis cystoides intestinalis in intestinal pseudoobstruction. Resolution after therapy with metronidazole. Dig Dis Sci 1992;37(6):949-54. 48. Tompolska KA, Wulff C. Air cysts in the small intestine. Ugeskr Laeger 1993;155(35):2713-4. 49. Trittmacher S, Felsenberg D, Bachmann G. Diagnosis of pneumatosis intestinalis coli in CT. Radiologe 1992;32(10):523-4. 50. Woodward A, Lai L, Burgess B, Beynon J, Carr ND. A case of pneumatosis coli managed by restorative proctectomy and ileal pouch - anal anastomosis. Int J Colorectal Dis 1995;10(3):181-2. 51. Xavier JL, Boscagi G, Claudel N et al. Pneumatosis cystoides intestinalis. Apropos of a case. Ann Radiol Paris 1991;34(6-7):401-6. 52. Yale CE, Balish E. The natural course of Clostridium perfringens – induced pneumatosis cystoides intestinalis. J Med 1992;23(34):279-288.

Submitted September 2001. Accepted November 2001. MUDr. René Vobořil, Rusek 106, 500 03 Hradec Králové, Czech Republic. e-mail: [email protected]

ORIGINAL ARTICLE

LONG TERM EFFECTS OF LOW-LEVEL SARIN INHALATION EXPOSURE ON THE SPATIAL MEMORY OF RATS IN A T-MAZE Jiří Kassa, Marie Koupilová, Josef Vachek Purkyně Military Medical Academy in Hradec Králové: Department of Toxicology

Summary: 1. To study the influence of low-level sarin exposure on cognitive functions, male albino Wistar rats were exposed to three various low concentrations of sarin (LEVEL 1–3) for 60 minutes in the inhalation chamber. Testing of cognitive functions was carried out using the T-maze evaluating learning and spatial memory. The behavior of sarin-exposed rats in the T-maze was tested several times within five weeks following sarin inhalation exposure to look for any cognitive impairments. The alteration of cognition was evaluated by using a method studying memory elicitation in response to appetitive motivation in a multiple T-maze. 2. Statistically significant, short-term deficiency in the T-maze performance was observed in rats exposed to symptomatic (LEVEL 3) as well as clinically asymptomatic concentration (LEVEL 2) of sarin. The repeated exposure of rats to clinically asymptomatic dose of sarin (LEVEL 2R) did not change the effect of lowlevel sarin exposure on spatial memory compared to the single exposure to the same dose of sarin. 3. Thus, sarin is able to influence the cognitive functions (e.g. spatial memory) even at low doses that do not cause clinically manifested intoxication following the inhalation exposure. Nevetheless, the alteration of spatial memory lasts for a short time only, in contrast with the severe sarin poisoning. Key words: Sarin; Low-level inhalation exposure; Spatial memory; T-maze; Rat

Introduction The potential for the exposure to highly toxic organophosphorus compounds (OPs), called nerve agents, exists on the battlefield (e.g. Iran-Iraq war) as well as in a civilian sector as a threat by a terrorist group (e.g. Tokyo subway incident – 12) or as an accident as a part of current demilitarization efforts. OPs elicit their toxic effects by irreversible inhibiting acetylcholinesterase (AChE, EC 3.1.1.7) in the central as well as peripheral nervous system allowing accumulation of acetycholine (ACh), excessive stimulation of postsynaptic cholinergic receptors and consequent signs of neurotoxicity. Signs of acute toxicity with extensive AChE inhibition include autonomic dysfunction (e.g. excessive salivation, lacrimation, urination and defecation), involuntary movements (e.g. tremor, fasciculation), respiratory dysfunction and other signs and symptoms (9, 19). OP-induced cholinergic effects are usually manifested immediately following high-level exposure (9, 19), nevertheless, there are numerous studies in both humans and animals showing that survivors of high-level OP exposure can experience subtle but significant long-term neurological and neuropsychological outcomes that are detectable months or even years following the recovery from acute poisoning (2). The rapid onset of signs and symptoms of poisoning following OP exposure can be explained in terms of 92

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ACh accumulation following AChE inhibition but no mechanism has been identified for the induction of long term effects. In addition, very little is known about possible neurological and neuropsychological effects including the impairments of cognitive functions of single or repeated low-level, asymptomatic exposure to OPs. The purpose of this study is to find out whether a nerve agent sarin might cause adverse effects on cognitive functions following the single or repeated low-level inhalation exposure in rats.

Material and methods Male albino Wistar rats weighing 180-200 g were purchased from VÚFB Konárovice (Czech Republic). They were kept in an air-conditioned room and allowed access to standard food and tap water ad libitum. The rats were divided into groups of ten. Handling of the experimental animals was done under supervision of the Ethics Committee of the Medical Faculty of Charles University and the Purkyně Military Medical Academy in Hradec Králové (Czech Republic). The rats were exposed to various low concentrations of sarin (obtained from Military Technical Institute, Zemianské Kostolany, Slovak Republic) for 60 minutes in the inhalation chamber. Three low concentrations of sarin were chosen: 93

– clinically nad laboratory asymptomatic concentration (0.8 µg/L) – LEVEL 1 – clinically asymptomatic concentration with a significant inhibition of erythrocyte AChE by 30% (1.25 µg/L) – LEVEL 2. This concentration was used for a single (LEVEL 2) or repeated (three times during one week) exposure (LEVEL 2R) – non-convulsive symptomatic concentration (2.5 µg/L) – LEVEL 3 Cognitive functioning was tested using a T-maze, consisting of five segments, a starting and a goal compartment to evaluate learning, spatial memory and spatial orientation (6,7). The rats were trained, with the food reward, to run through the maze in less than 10 seconds without entering the side arm. The time necessary to reach the goal box was recorded. Before inhalation exposure to sarin, the rats were trained to reach the goal box as soon as possible by moving to the correct segment in the T-maze. It usually took 4-6 weeks of training to reach the criterion which was 80% or more correct behavior. The exposure started the day after the animals had reached this criterion. The spatial memory was tested 1 hour, 2 hours, 1 day and 1 week following the sarin inhalation exposure and then, once a week till the end of the fifth week following the exposure. The time of reaching the goal box by sarin-exposed rats was compared to the values obtained from the same rats immediately before sa-

rin exposure and from control rats exposed to pure air instead of sarin. Analysis of variance (ANOVA) with Bonfferoni’s corrections for multiple comparisons was used for the determination of significant differences between experimental and control values (1). The differences were considered significant when P < 0.05.

Results While the rats exposed to LEVEL 1 of sarin did not show any significant changes in the rapidity of spatial discrimination in T-maze following their exposure in comparison with the control rats exposed to the pure air, the significant impairment of spatial memory of rats exposed to other low concentrations of sarin (LEVEL 2 and 3) was observed. The results of the influence of various sarin concentrations on the T-maze performance of rats following single inhalation exposure are shown in Figure 1. While a spatial memory of rats exposed to LEVEL 1 of sarin was not significantly influenced, rats exposed to LEVEL 2 and 3 of sarin showed a significant decrease in T-maze performance for a short time (till the first day following the exposure). Their latency time in the choice of the correct segment and reaching the goal box of the T-maze was extended. In addition, the effects of low-level sarin inhalation

exposure was dose-dependent. When the rats were exposed to LEVEL 3 of sarin, their time of passage through the maze was more lengthened at 1 and 2 hours following the inhalation exposure compared to the rats exposed to LEVEL 2 of sarin (Fig. 1). The results of T-maze performance of rats repeatedly exposed to LEVEL 2 of sarin are given in Figure 2. The repeated exposure of rats to clinically asymptomatic concentration of sarin (LEVEL 2) did not change the effect of low-level sarin exposure on spatial memory in comparison with the single exposure to the same dose of sarin (Fig. 2).

persist for a relatively long time following the termination of toxicant exposure. The results from several studies have demonstrated the presence of OP-induced learning impairments several days after the behavioral signs of OP toxicity have subsided (3,4,10). The chronic exposure to OP compounds can also result in specific long-term cognitive deficits even when signs and symptoms of excessive cholinergic activity are not present (14,15). Recently, the ability of a nerve agent sarin to cause subtle long-term neurobehavioral and neurophysiological effects in rats exposed to its low level without a significant inhibition of AChE activity and a clinically manifested alteration of cholinergic nervous system has been described (5). Our data clearly demonstrate that sarin is also able to induce dose-dependent alteration of cognitive functions in the case of the inhalation exposure of rats to its low concentrations. The adverse effect of low-level sarin inhalation exposure was manifested in the time determining rate of orientation (latency time). Therefore, the significant, clinically manifested AChE inhibition in the central nervous system leading to the neuronal degeneration of some brain regions including hippocampus, associated with the spatial learning and memory, is not necessary for the clinically manifested cognitive impairments. These findings correspond with earlier published data about neurological and neurophysiological outcomes detectable months or even years following recovery from acute OP poisoning (17,20). In addition, a current study attempts to show a temporal rela-

Discussion The exposure to high doses of OPs has been demonstrated to result in severe brain neuropathology that involves not only neuronal degeneration and necrosis of various brain regions (8,11,13) but also persistent severe alteration in behavior and cognitive functions, especially impairment of learning and memory (4,10,16). The most significant injury caused by OP poisoning is neuronal degeneration of the hippocampus that is associated with the spatial learning and memory. Therefore, impairment of cognitive functions, especially incapacitation of learning and memory, belongs to the most frequent central signs of acute OP poisoning (9,10). In addition, the adverse effects of OP compounds on cognitive functions, such as learning and memory, may

14,00 Controls Level 1 Level 2 Level 3

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Fig. 2: The alteration of the T-maze performance in rats repeatedly exposed (A – the first exposure, B – the second exposure, C – the third exposure) to LEVEL 2 of sarin. Statistical significance - see Figure 1. 95

tionship between OP-induced impairment in performance of a spatial memory task and the protracted decrease in the expression of cholinergic receptors in specific brain regions caused by asymptomatic exposure to an OP compound (18). Although these findings are difficult to extrapolate directly to human low-level exposures to OPs, they indicate that short cognitive impairments without clinically manifested disturbance of central cholinergic nervous system could occur in humans following the inhalation exposure to clinically asymptomatic concentrations of sarin. Aknowledgements The authors thank to Mrs. H. Antlová and Mrs. E. Vodáková for their skilful technical assistance and to Mgr. V. Bláha for the statistical evaluation. This study was supported by the grant of Ministry of Defence, No. 03021100006.

References 1. Afifi AA, Azen SP. Statistical analysis and computer oriented approach. 2nd ed. New York: Academic Press, 1979:442-5. 2. Brown MA, Kelley AB. Review of health consequences from high-, intermediateand low-level exposure to organophosphorus nerve agents. J Appl Toxicol 1998;18:393-408. 3. Buccafusco JJ, Heithold DL, Chon SH. Long-term behavioral and learning abnormalities produced by the irreversible cholinesterase inhibitor soman: effect of a standard pretreatment regimen and clonidine. Toxicol Lett 1990;52:319-29. 4. Bushnell PJ, Padilla SS, Ward T, Pope CN, Olszyk VB. Behavioral and neurochemical changes in rats dosed repeatedly with diisopropylfluorophosphate. J Pharmacol Exp Ther 1991;256:741-50. 5. Kassa J, Pecka M, Tichý M, Bajgar J, Koupilová M, Herink J, Kročová Z. Toxic effects of sarin in rats at three months following single or repeated low-level inhalation exposure. Pharmacol Toxicol 2001;88:209-12 . 6. Koupilová M, Herink J. Effects of mescaline and its derivative N-(3,4,5- trimethoxyethyl)-aziridine on the spatial orientation of rats in T-maze. Physiol Bohem 1989;38:497-502. 7. Koupilová M, Herink J. An attempt to antagonize DSP-4 induced impairment of the performance of rats in a T-maze. Homeostasis 1995;36:41-2.

8. Lemercier G, Carpentier P, Sentenac-Roumanou H, Morelis P. Histological and histochemical changes in the central nervous system of the rat poisoned by an irreversible anticholinesterase organophosphorus compound. Acta Neuropathol 1983;61:123-9. 9. Marrs TC. Organophosphate poisoning. Pharmacol Ther 1993;58:51-66. 10. McDonald BE, Costa LG, Murphy SD. Spatial memory impairment and central muscarinic receptor loss following prolonged treatment with organophosphates. Toxicol Lett 1988;40:47-56. 11. McLeod CG, Singer AW, Harrington DG. Acute neuropathology in soman poisoned rats. Neurotoxicology 1982;297:681-3. 12. Ohtomi S, Takase M, Kunagoi F. Sarin poisoning in Japan. A clinical experience in Japan Self Defense Force (JSDF) Central Hospital Int Rev Arm For Med Ser 1996;69:97-102. 13. Petras JM. Soman neurotoxicity. Fundam Appl Toxicol 1981;1:242-9. 14. Prendergast MA, Terry AV, Buccafusco JJ. Chronic, low-level exposure to diisopropylfluorophosphate causes protracted impairment of spatial navigation learning. Psychopharmacology 1997;130:276-84. 15. Prendergast MA, Terry AV, Buccafusco JJ. Effects of chronic, low-level organophosphate exposure on delayed recall, discrimination and spatial learning in monkeys and rats. Neurotoxicol Teratol 1998;20:115-22. 16. Rafaelle K, Olton D, Annau Z. Repeated exposure to diisopropylfluorophosphate (DFP) produces increased sensitivity to cholinergic antagonists in discrimination retention and reversal. Psychopharmacology (Berlin) 1990;100:267-74. 17. Savage EP, Keefe TJ, Mounce LM, Heaton RK, Lewis JA, Burcar PJ. Chronic neurological sequelae of acute organophosphate pesticide poisoning. Arch Environ Health 1988;43:38-45. 18. Stone JD, Terry AVJr, Pauly JR, Prendergast MA, Buccafusco JJ. Protractive effects of chronic treatment with an acutely subtoxic regimen of diiopropylfluorophosphate on the expression of cholinergic receptor densities in rats. Brain Res 2000;882:9-18. 19. Taylor P. Anticholinesterase agents. In: Hardman JG, Limbird LE (eds): The Pharmacological Basis of Therapeutics, 9th ed. New York: McGraw Hill, 1996:161-76. 20. Yokoyama K, Araki S, Murata K et al. Chronic neurobehavioral and central and autonomic nervous system effects in Tokyo subway sarin poisoning. J Physiol (Paris) 1998; 92:317-23

Submitted May 2001. Accepted November 2001. Doc. MUDr. Jiří Kassa, CSc., Purkyně Military Medical Academy, P.O. Box 35/T, 500 01 Hradec Králové, Czech Republic. e-mail: [email protected]

ORIGINAL ARTICLE

HELICOBACTER PYLORI ERADICATION WITH BETA CAROTENE, ASCORBIC ACID AND ALLICIN Cem Kockar , Mustafa Öztürk, Nüket Bavbek Fatih University Medical School, Ankara, Turkey: Department of Gastroenterology Summary: In this study, in vivo effectiveness of ascorbic acid (AA), beta carotene (BC) and allicin in HP eradication were evaluated. 210 patients who are HP positive in biopsy were involved in this study. The patients randomised to seven treatment groups (each group consisting of 30 patients). The first group was given standard eradication treatment (lansaprasol 30 mg bid, clarithromycin 500 mg bid, amoxicillin 1 g bid for 14 days). Second group received AA 1000 mg/day in addition to the standard treatment. Third group received only AA 1000 mg/day for 14 days. Fourth group was treated with standard regiment plus 120 mg/day BC. Fifth group was given only BC 120 mg/day for 14 days. Sixth group was given standard regiment and allicin 4200 µg/day. Seventh group received only Allicin 1200 µg/day for 14 days. The eradication was achieved in 20 (66.6 %) in group I, 15 (50 %) in group II, 3 (10 %) in group III, 15 (50 %) in group IV, 0 (0 %) in group V, 27 (90 %) in group VI and 7 (23.3 %) in group VII. Allicin seemed to be potentially effective agent for HP eradication but ascorbic acid, beta caroten was found to be ineffective. Key words: Helicobacter Pylori; Allicin; Ascorbic Acid; Beta Carotene

Introduction Helicobacter pylori (HP) is one of the most common infectious agents and associated with numerous gastrointestinal system disorders (12). It is a known risk factor for gastric cancer (18). Eradication of HP not only provides symptomatic relief in most patients but also prevents subsequent complications (9). Effectiveness of several double or triple antibiotic combinations with concurrent proton-pump inhibitors were studied and success rates reaches 80-95% in some regiments (11,16). Cost effectiveness of drug therapy is an important issue particularly in developing countries where HP prevalence is extremely high (3). HP induces oxidant stress on gastric mucosa. HP colonisation was shown to reduce gastric antioxidant (e.g. ascorbic acid) levels (22). On the other hand in vitro studies ascorbic acid (AA) was proved to be bactericidal for HP (23). In this study, in vivo effectiveness of AA, and two other antioxidants - beta carotene (BC) and Allicin – in HP eradication were evaluated.

Material and Methods Study group Two hundred and ten patients (113 female, 97 male; mean age ± SD, 38 ± 10 years) who are HP positive in biopsy were involved in this study. The patients randomised to seven treatment groups (each group consisting of 30 pati96

ACTA MEDICA (Hradec Králové) 2001;44(3):97-100

ents). Informed consent was taken in each patient. The first group was given standard eradication treatment (lansaprazole 30 mg bid, claritromisin 500 mg bid, amoxicilline 1 g bid for 14 days). Second group received AA 1000 mg/day in addition to the standard treatment. Third group received only AA 1000 mg/day for 14 days. Fourth group was treated with standard regiment plus 120 mg/day BC. Fifth group was given only BC 120 mg/day for 14 days. Sixth group was given standard regiment and Allicin 1200 µg/day (Cirkulin dragé, 0.3 mg, Munir Sahin). Seventh group received only Allicin 4200 µg/day for 14 days. Endoscopic examination of the patients was conducted before and four weeks after the treatments by the same blinded endoscopist. Four biopsies from antrum and two biopsies from corpus were taken endoscopically in each patients. Biopsy specimens were painted with Giemsa for the detection of HP. The same expert who was blinded to the clinical data of the patients evaluated histologic specimens. For semiquantitative grading of presence of HP, the following criteria were used: Grade 0: no bacteria was detected; grade I: sporadic bacteria observed; grade II: many bacteria seen in most microscopic fields at high power magnification (x400); grade III: clusters of microorganisms were found in the superficial mucus layer in all fields examined and grade IV: dense infiltration of bacteria in all fields. Average points of HP colonisation grade in each group were calculated for statistical purposes. Histopathologic changes were evaluated as chronic atrophic gastritis, chronic superficial gastritis, chronic ero97

sive gastritis and acute erosive gastritis. The eradication was described as histologicaly negative examination (grade 0). Statistical analysis Student’s t test was used for comparison of the age between the groups. Chi square test were used for comparison of results of treatment groups. Spierman test was used for correlation of HP positivity and symptoms. P < 0.05 was considered to indicate statistical significance.

Results The patient characteristics and distribution of risk factors for gastritis were shown in table 1. There were no statistically significant differences between groups in age, alcohol intake, smoking, non steroidal antiinflammatory drug use and HP positivity. At the end of the treatment, eradication was achieved in 20 patients (66.6 %) in group I, 15 patients (50 %) in

Tab. 1: Patients characteristic. Group/ Characteristic Age (year) Sex (F/M) Smoking (+/-) Alcohol (+/-) NSAID (+/-)

Group I N=30 (%) 38.9±10 18/12 7/23 -/30 7/23

Group II N=30 (%) 40±10 16/14 7/23 5/25 8/22

Group III N=30 (%) 39±10 18/12 9/21 5/25 6/24

Group IV N=30 (%) 41±10 14/16 8/22 4/26 4/26

Group V N=30 (%) 38±10 16/14 8/22 5/25 3/27

Group VI N=30 (%) 37±10 15/15 8/22 6/24 4/26

Group VII N=30 (%) 40±13 16/14 5/25 2/28 4/26

F: Female M: Male (+/-): used or not used NSAID: Non Steroidal Anti Inflammatory Drugs. There was no statistically difference between the groups, P>0.05.

Tab. 2: At the end of the treatment eradication rates. Group/ Characteristic Eradication (+) Eradication (-)

Group I N=30 (%) 20 (66.7) 10 (33.3)

Group II N=30 (%) 15 (50) 15 (50)

Group III N=30 (%) 3 (10) 27 (90)

Group IV N=30 (%) 15 (50) 15 (50)

Group V N=30 (%) - (0) 30 (100)

Group VI N=30 (%) 27 (90) 3 (10)

Group VII N=30 (%) 7 (24.4) 23 (76.6)

P: value for eradication rates: Group I and II, IV >0.05, Group I and III,