Gastroenterology - Microsoft · A prospective study Grazioli B, Matera G, Laratta C, Schipani G, Guarnieri G, Spiniello E, Imeneo M, Amorosi A, Focà A, Luzza F 1945 Clinical characteristics

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Volume 12 Number 12 March 28, 2006

World J Gastroenterol 2006 March 28; 12(12): 1821-1984

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1821 Gastrointestinal manifestations in myotonic muscular dystrophy Bellini M, Biagi S, Stasi C, Costa F, Mumolo MG, Ricchiuti A, Marchi S

1829 Current concept on the pathogenesis of inflammatory bowel disease-crosstalk between genetic and microbial factors: Pathogenic bacteria and altered bacterial sensing or changes in mucosal integrity take “toll”?Lakatos PL, Fischer S, Lakatos L, Gal I, Papp J

1842 Correlation of Epstein-Barr virus and its encoded proteins with Helicobacter pylori and expression of c-met and c-myc in gastric carcinomaLuo B, Wang Y, Wang XF, Gao Y, Huang BH, Zhao P

1849 Anemia and long-term outcome in adjuvant and neoadjuvant radiochemotherapy of stage II and III rectal adenocarcinoma: The Freiburg experience (1989-2002) Weissenberger C, Geissler M, Otto F, Barke A, Henne K, von Plehn G, Rein A, Müller C, Bartelt S, Henke M

1859 Glycine-extended gastrin activates two independent tyrosine-kinases in upstreamof p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cellsFerrand A, Kowalski-Chauvel A, Pannequin J, Bertrand C, Fourmy D, Dufresne M, Seva C

1865 Expression of cytokeratins in Helicobacter pylori -associated chronic gastritis of adult patients infected with cagA + strains: An immunohistochemical studyTodorovic V, Sokic-Milutinovic A, Drndarevic N, Micev M, Mitrovic O, Nikolic I, Wex T, Milosavljevic T, Malfertheiner P

1874 Infl uence of gastric inhibitory polypeptide on pentagastrin-stimulated gastric acid secretion in patients with type 2 diabetes and healthy controlsMeier JJ, Nauck MA, Kask B, Holst JJ, Deacon CF, Schmidt WE, Gallwitz B

1881 Reconstruction of liver organoid using a bioreactor Saito M, Matsuura T, Masaki T, Maehashi H, Shimizu K, Hataba Y, Iwahori T, Suzuki T, Braet F

1889 Interleukin-2 gene-encoded stromal cells inhibit the growth of metastatic cholangiocarcinomasKim MH, Lee SS, Lee SK, Lee SG, Suh CW, Gong GY, Park JS, Kim YH, Kim SH

1895 S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanismdel pilar Cabrales-Romero M, Márquez-Rosado L, Fattel-Fazenda S, Trejo-Solís C, Arce-Popoca E, Alemán-Lazarini L, Villa-Treviño S

1905 Oral administration of S -nitroso-N-acetylcysteine prevents the onset of non alcoholic fatty liver disease in rats

de Oliveira CPMS, Simplicio FI, de Lima VMR, Yuahasi K, Lopasso FP, Alves VAF,

Abdalla DSP, Carrilho FJ, Laurindo FRM, de Oliveira MG 1912 Effect of WeiJia on carbon tetrachloride induced chronic liver injury

Cheung PY, Zhang Q, Zhang YO, Bai GR, Lin MCM, Chan B, Fong CC, Shi L, Shi YF, Chun J, Kung HF, Yang M

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ContentsWorld Journal of Gastroenterology


1918 Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fi brotic rat liver

Zhang LJ, Zheng WD, Shi MN, Wang XZ

1924 Molecular markers (PECAM-1 , ICAM-3 , HLA-DR) determine prognosis in primary non-Hodgkin’s gastric lymphoma patientsDarom A, Gomatos IP, Leandros E, Chatzigianni E, Panousopoulos D, Konstadoulakis MM, Androulakis G

1933 Ileocecal masses in patients with amebic liver abscess: Etiology and management Misra SP, Misra V, Dwivedi M

1937 Up-regulation of NAD (P) H quinone oxidoreductase 1 during human liver injuryAleksunes LM, Goedken M, Manautou JE

1941 Giardia lamblia infection in patients with irritable bowel syndrome and dyspepsia: A prospective studyGrazioli B, Matera G, Laratta C, Schipani G, Guarnieri G, Spiniello E, Imeneo M, Amorosi A, Focà A, Luzza F

1945 Clinical characteristics of a group of adults with nodular lymphoid hyperplasia: A single center experienceRubio-Tapia A, Hernández-Calleros J, Trinidad-Hernández S, Uscanga L

1949 Hyperlactatemia in patients with non-acetaminophen-related acute liver failureTaurá P, Martinez-Palli G, Martinez-Ocon J, Beltran J, Sanchez-Etayo G, Balust J, Anglada T, Mas A, Garcia-Valdecasas JC

1954 Clinical features of hepatopulmonary syndrome in cirrhotic patientsMohammad Alizadeh AH, Fatemi SR, Mirzaee V, Khoshbaten M, Talebipour B, Sharifian A, Khoram Z, Haj-sheikh-oleslami F, Gholamreza-shirazi M, Zali MR

1957 Seroprevalence of Helicobacter pylori in dyspeptic patients and its relationship with HIV infection, ABO blood groups and life style in a university hospital, Northwest EthiopiaMoges F, Kassu A, Mengistu G, Adugna S, Andualem B, Nishikawa T, Ota F

1962 Effect of electroacupuncture on gastric mucosal intestinal trefoil factor gene expression of stress-induced gastric mucosal injury in ratsLi XP, Yan J, Yi SX, Chang XR, Lin YP, Yang ZB, Huang A, Hu R

1966 Large Brunner’s gland adenoma: Case report and literature review Rocco A, Borriello P, Compare D, De Colibus P, Pica L, Iacono A, Nardone G

1969 Metastasis of hepatocellular carcinoma to the small bowel manifested by intussusception

Kim HS, Shin JW, Kim GY, Kim YM, Cha HJ, Jeong YK, Jeong ID, Bang SJ, Kim DH, Park NH

1972 Patients with hepatocellular carcinoma related to prior acute arsenic intoxication and occult HBV: Epidemiological, clinical and therapeutic results after 14 years of follow-up Casanovas-Taltavull T, Ribes J, Berrozpe A, Jordan S, Casanova A, Sancho C, Valls C, Bosch FX

1975 Mucinous cystadenoma of the appendix associated with adenocarcinoma of the sigmoid colon and hepatocellular carcinoma of the liver: Report of a caseDjuranovic SP, Spuran MM, Kovacevic NV, Ugljesic MB, Kecmanovic DM, Micev MT

1978 Cytomegalovirus gastritis after rituximab treatment in a non-Hodgkın’s lymphoma patientUnluturk U, Aksoy S, Yonem O, Bayraktar Y, Tekuzman G

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World Journal of Gastroenterology ( World J Gastroenterol , WJG ), a leading international journal in gastroenterology and hepatology, has an established reputation for publishing fi rst class research on esophageal cancer, gastric cancer, liver cancer, viral hepatitis, colorectal cancer, and Helicobacter pylori infection, providing a forum for both clinicians and scientists, and has been indexed and abstracted in Index Medicus, MEDLINE, PubMed, Chemical Abstracts, EMBASE, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. WJG is a weekly journal published by The WJG Press. The publication date is on 7th, 14th, 21st, and 28th every month. The WJG is supported by The National Natural Science Foundation of China, No. 30224801 and No.30424812, which was founded with a name of China National Journal of New Gastroenterology on October 1,1995, and renamed as WJG on January 25, 1998.

World Journal of GastroenterologyVolume 12 Number 12 March 28, 2006

1980 Acknowledgments to Reviewers of World Journal of Gastroenterology

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HONORARY EDITORS-IN-CHIEFKe-Ji Chen, BeijingLi-Fang Chou, TaipeiDai-Ming Fan, Xi'anZhi-Qiang Huang, BeijingShinn-Jang Hwang, TaipeiMin-Liang Kuo, TaipeiNicholas F LaRusso, RochesterJie-Shou Li, NanjingGeng-Tao Liu, BeijingLein-Ray Mo, TainanFa-Zu Qiu, WuhanEamonn M Quigley, CorkDavid S Rampton, LondonRudi Schmid, CaliforniaNicholas J Talley, RochesterGuido NJ Tytgat, AmsterdamJaw-Ching Wu, TaipeiMeng-Chao Wu, ShanghaiMing-Shiang Wu, TaipeiJia-Yu Xu, ShanghaiHui Zhuang, Beijing

PRESIDENT AND EDITOR-IN-CHIEFLian-Sheng Ma, Beijing

EDITOR-IN-CHIEFBo-Rong Pan, Xi’an

ASSOCIATE EDITORS-IN-CHIEFGianfranco D Alpini, TempleBruno Annibale, RomaJordi Bruix, BarcelonaRoger William Chapman, OxfordAlexander L Gerbes, MunichShou-Dong Lee, TaipeiWalter Edwin Longo, New HavenYou-Yong Lu, BeijingMasao Omata, TokyoHarry H-X Xia, Hong Kong

SCIENCE EDITORSDirector: Jing Wang Deputy Director: Jian-Zhong Zhang

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APPENDIX

Massimo Bellini, Sonia Biagi, Cristina Stasi, Francesco Costa, Maria Gloria Mumolo, Angelo Ricchiuti, Santino Marchi, Gastroenterology Unit, Department of Internal Medicine, University of Pisa, ItalyCorrespondence to: Dr Massimo Bellini, Gastroenterology Unit, Department of Internal Medicine, Via Roma, 67 56100 Pisa, Italy. [email protected]: +39-50-993485 Fax: +39-50-993050Received: 2005-08-09 Accepted: 2005-09-12

AbstractMyotonic dystrophy (MD) is characterized by myotonic phenomena and progressive muscular weakness. Involvement of the gastrointestinal tract is frequent and may occur at any level. The clinical manifestations have previously been attributed to motility disorders caused by smooth muscle damage, but histologic evidence of alterations has been scarce and conflicting. A neural factor has also been hypothesized. In the upper digestive tract, dysphagia, heartburn, regurgitation and dyspepsia are the most common complaints, while in the lower tract, abdominal pain, bloating and changes in bowel habits are often reported. Digestive symptoms may be the first sign of dystrophic disease and may precede the musculo-skeletal features. The impairment of gastrointestinal function may be sometimes so gradual that the patients adapt to it with little awareness of symptoms. In such cases routine endoscopic and ultrasonographic evaluations are not sufficient and targeted techniques (electrogastrography, manometry, e lectromyography, funct iona l u l t rasonography, scintigraphy, etc .) are needed. There is a low correlation between the degree of skeletal muscle involvement and the presence and severity of gastrointestinal disturbances whereas a positive correlation with the duration of the skeletal muscle disease has been reported.

The d r ug s r e c ommended f o r t r e a t i n g t h e gastrointestinal complaints such as prokinetic, anti-dyspeptic drugs and laxatives, are mainly aimed at correcting the motility disorders.

Gastrointestinal involvement in MD remains a complex and intriguing condition since many important problems are still unsolved. Further studies concentrating on genetic aspects, early diagnostic techniques and the development of new therapeutic strategies are needed to improve our management of the gastrointestinal manifestations of MD.

© 2006 The WJG Press. All rights reserved.

Key words: Myotonic dystrophy; Digestive disorders; Gastrointestinal symptoms

Bellini M, Biagi S, Stasi C, Costa F, Mumolo MG, Ricchiuti A, Marchi S. Gastrointestinal manifestations in myotonic muscular dystrophy. World J Gastroenterol 2006; 12(12): 1821-1828

http://www.wjgnet.com/1007-9327/12/1821.asp

INTRODUCTIONMyotonic dystrophy (MD) is an autosomal dominant genetic disorder[1]. It is caused by an unstable trinucleotide repeat expansion containing cytosine-thymidine-guanosine (CTG)n, located in the 3’ untranslated region of chromosome 19q13.3. The CTG trinucleotide is repeated in the normal population from 5 to 36 times[2,3], but has been found to be expanded up to 2 000 times in MD patients[4,5]. This amplification is correlated to the severity of the disease. Patients can be divided into four groups based on the number of CTG repeats. Group E1 (50-100 CTG repeats) may have either the minor or the classical form of MD, group E2 (500-1 000 CTG repeats) have the classical form of MD, group E3 (1 000-1500 CTG repeats) have the classical form of MD with onset during childhood, and group E4 (more than 1 500 CTG repeats) are affected with the congenital form of MD[6].

Although MD is primarily characterised by myotonic phenomena and progressive muscular weakness, multi-system involvement is often present, taking the form of cardiac conduction abnormalities, cognitive deficits, cataracts and diabetes, as well as endocrine, sexual and reproductive disturbances [1]. Involvement of the gastrointestinal (GI) tract is frequent, and may occur at any level from the pharynx to the anal sphincter. Dysphagia, heartburn, emesis, regurgitation, coughing while eating and dyspepsia are the most common complaints involving the upper digestive tract, while abdominal pain and bloating, changes in bowel habits (diarrhoea or constipation) and dyschezia are common signs of impairment of the lower digestive tract [7-9]. These clinical manifestations have generally been attributed to motility disorders caused by striated and more rarely, smooth muscle damage[10],

Gastrointestinal manifestations in myotonic muscular dystrophy

Massimo Bellini, Sonia Biagi, Cristina Stasi, Francesco Costa, Maria Gloria Mumolo, Angelo Ricchiuti, Santino Marchi

PO Box 2345, Beijing 100023, China World J Gastroenterol 2006 March 28; 12(12): 1821-1828www.wjgnet.com World Journal of Gastroenterology ISSN [email protected] © 2006 The WJG Press. All rights reserved.

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although recent studies suggest that neurological alterations may also play a role[11,12].

There appears to be little correlation between the degree of skeletal muscle involvement and the presence and severity of the gastrointestinal disturbance, but some authors have reported a positive correlation with the duration of the skeletal muscle disease[13, 14]. In some patients the impairment of gastrointestinal function is so gradual that they adapt to it with little or no awareness of any disturbance and only a thorough anamnesis may elicit the recall of possible symptoms[12]. In such patients, routine endoscopic and ultrasonographic evaluations are of little value and only highly sensitive, targeted techniques such as electrogastrography, manometry, electromyography, functional ultrasonography, compartmental scintigraphy, etc. may help to reveal the true extent of the involvement.

In this review, the available data on the pathophysiologic mechanisms of GI involvement in myotonic dystrophy, their impact on clinical manifestations of the disease, and the tests that can be used to detect them, are discussed in turn for each segment of the gastrointestinal tract.

PHARYNX AND ESOPHAGUSBecause most complaints by MD patients focus on the pharynx and esophagus, which can be quite easily investigated, these are the manifestations that have been most thoroughly studied thus far. MD patients often report dysphagia, sometimes associated with coughing while eating, chest pain, regurgitation and heartburn. Different studies have reported a prevalence of dysphagia ranging from 25% to 80%[9, 12, 15, 16].This is usually considered by both patients and doctors to be the most serious symptom of MD, probably because it is associated with a high incidence of recurrent pulmonary infection[17].

Manometry is useful to detect motility disturbances, which show in the form of asymmetric contractions of the pharynx and weak contractions of the upper esophageal sphincter (UES)[14]. Studies have reported a reduced basal UES pressure in MD patients compared to controls[11] , although the prevalence and duration of UES relaxation during swallowing are similar between the two groups and more significantly, no difference in the residual pressure during relaxation has been found[11, 12,1 8] .

In the esophageal body significant decreases in the peristaltic amplitude and/or simultaneous waves have often been reported[12, 18]. A study of 14 MD patients showed a marked decrease in the median amplitude of the contractions at every level of the esophagus. A higher percentage of non-transmitted and dropped contraction sequences after swallowing has been observed in patients compared to controls, 43% exhibited complete atony of the esophageal body with a scleroderma-like pattern and no detectable esophageal contractions. In this series, although manometry showed impaired esophageal motor function in 71% of the patients, 50% of these were quite asymptomatic[18].

Involvement of the lower esophageal sphincter (LES) is a somewhat more complex matter. Modolell et al.[12] failed to detect a significant difference in basal LES pressure between patients and controls, only reporting

some cases of incomplete LES relaxation[12]. In contrast, other studies have found a significantly reduced resting tone, competence, and area vector volume of the LES in MD patients than in controls[9, 18]. These data suggest that some alteration in the smooth muscle may take place, leading to a higher risk of developing gastroesophageal reflux disease (GERD)[18].

Esophageal spasm and segmental dilatation can be seen on x-rays, while radiological and scintigraphic techniques are useful for detecting both a lack of tone and a reduced, ineffective or absent peristalsis[12, 15, 19].

Most authors hold that pharyngo-esophageal myotonia, or problems with post-contraction relaxation, should no longer be considered as a typical feature of esophageal involvement[12, 18]. A myotonic response to pharyngeal contractions has been reported by Siegel et al [20], but we now know that this is due to limitations in x-ray technology at the time when the study was conducted.

It has been hypothesized that distal esophageal impairment may be myogenic in origin and abnormalities in both the striated and smooth muscle fibers may play a role in MD. However, the histologic data published to date have failed to demonstrate any alteration in the esophageal smooth muscle fibers[19]. Marked atrophy of the esophageal striated muscle, but only small changes in the smooth muscle were observed in a patient reported by Jéquier et al[21]. In another case, using electron microscopy to study the esophagus, Ludatscher et al[22] detected mild degenerative changes with disoriented filaments of the smooth muscle.

Smooth muscle damage could also explain the reduced LES tonic resting pressure and the elevated frequency of heartburn observed in MD patients. Unfortunately, most of the reported cases did not include an upper digestive tract endoscopic examination or a 24-h pH-metry to determine the presence and degree of GERD[18] .

Recently some authors have suggested that a combined myopathic - neuropathic etiology could provide a more satisfactory explanation for the pharyngo-esophageal symptoms of MD, but at present this remains speculative [12]. One proposed hypothesis is that decrease in the normal amplitude of the peristaltic waves could reflect the inability of the muscle to contract under normal neural control. On the other hand, uncoordinated motor activity in the presence of a normal amplitude contraction could indicate a lack or defect in neural control[12]. This hypothesis is not supported by the study of Eckardt et al[19], who failed to detect any neuropathic alterations in MD patients.

However, there are data to suggest that there is no association between the degree of muscular disability and motor abnormalities of the pharynx and esophagus, the presence and severity of dysphagia, or other esophageal symptoms[12]. For example, manometric findings are not significantly different between symptomatic and asymptomatic patients or in patients with different degrees of striated muscular involvement[9, 12, 18, 19]. In particular, in a study of 18 MD patients Modolell et al[12] showed that while severe pharyngo-esophageal abnormalities are present in all patients, symptoms are spontaneously reported by only 30% of them. This figure rose but only to 55%, when the patients were specifically questioned on

1822 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol March 28, 2006 Volume 12 Number 12

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this point.These conflicting reports may be explained by the

fact that the tongue and pharyngeal muscles can contract sufficiently to push a bolus through the UES into the esophagus, after which the force of gravity is enough to complete the swallowing process. Moreover, because of the slow evolution of the disease, MD patients may develop compensatory mechanisms and thus gradually adapt to the impairment of their esophageal function[18].

STOMACH AND DUODENUMMD patients frequently complain of dyspeptic symptoms such as early satiety, nausea, vomiting, and epigastric pain[9]. Radiographic, manometric, ultrasonographic and scintigraphic evidence of decreased peristaltic activity causing the delayed emptying of liquids and solids, visceral dilatation, gastric bezoars and even gastroparesis have been reported[10, 13, 23-25] .

Kuiper et al [23] have described the case of an MD patient who developed symptoms suggestive of gastric retention. Radiological and endoscopic examination revealed polypoid masses, cytologic washing showed them to be food concretions, probably a consequence of abnormal gastric motility[23]. More recently, scintigraphic studies by Ronnblom et al[25] indicate a delayed emptying of the stomach in MD patients with dyspeptic symptoms, demonstrated by a prolonged lag phase, a slower emptying rate, and a prolonged T/2.

Bellini et al [13] used ultrasound to evaluate gastric emptying abnormalities and their relationship with the severity and duration of the disease in 11 MD patients without dyspeptic symptoms. They found that MD patients and healthy subjects exhibit comparable fasting and maximal antral areas. The findings that (i) the basal and maximal post-prandial values for the gastric antral area between MD patients and controls were quite similar, and (ii) the time required to reach the maximal post-prandial values did not differ in the two groups, are in agreement with the fact that the patients studied were not dyspeptic[13]. Analogous results have been reported for a group of non-dyspeptic patients affected by systemic sclerosis[26].

Indeed, increased antral dimensions have been reported in patients with functional dyspepsia[27,28], suggesting that such abnormalities may play a role in the pathogenesis of the dyspeptic symptoms in MD. In Bellini’s study[13], the final emptying time of the MD patients was longer than that of healthy volunteers and was clearly correlated with the disease duration, but was independent of both the CTG expansion size and the patients’ disability class.

It is not surprising that patients without dyspeptic symptoms may have delayed gastric emptying. Indeed the association between delayed gastric emptying and upper GI complaints is still under debate[29]. Other abnormalities in gastric motor function, such as impaired accommodation of the proximal stomach[30, 31], abnormal distribution of the gastric contents[31], gastric dysrythmias[32, 33], and a lack of antro-pyloro-duodenal coordination[34], could play a role in the pathogenesis of dyspeptic symptoms in these patients.

Bellini et al[13] have confirmed that a direct relationship

between disease severity and the impairment of GI motor functioning remains to be demonstrated, since there is no significant difference in gastric emptying between patients with different degrees of skeletal muscle involvement. These results appear to agree with previous studies, demonstrating that skeletal muscular damage and gastric motor disturbances can progress independently[14]. On the other hand, the direct correlation between final emptying time and disease duration suggests that the impairment of gastric emptying may evolve over time.

The delayed gastr ic emptying in MD could be explained by the muscular disease itself. Unfortunately no definitive conclusions can be drawn on this point because the histological evidence remains limited. Gastric smooth muscle is only rarely affected[7, 8] and as we have already seen in the esophagus, when damage occurs it appears to be similar although earlier and more severe in nature to that occurring in skeletal muscle with severe fatty infiltration[35] . However, a sufficiently large study focusing on the gastric smooth muscle in MD patients is still lacking. It would be important to determine whether the two types of muscle tissue may be damaged to different degrees in the same subjects.

Recent reports suggest that, as in the pharynx and esophagus, muscle involvement alone is not sufficient to explain the altered digestive function in the stomach and duodenum of MD patients. Impaired nervous conduction as well as altered GI hormone secretions could be involved in the motor abnormalities[25]. For example, changes in the electrical impulses controlling duodenal activity could explain the chronic intestinal pseudo-obstruction occasionally reported in MD. Likewise a reduction in the electrical control of gastric activity could be a possible cause of delayed gastric emptying[25].

An electrogastrographic study carried out by Ronnblom et al[36] on 10 patients showed that gastric electric cycling activity manifested as bradygastria, tachygastria and a less stable frequency of the gastric signal are reduced compared with control subjects. The presence of an abnormal EGG in MD patients supports the hypothesis that the disease is a systemic disorder, affecting not only the muscles but also other tissues. Ronnblom et al[36] then studied the gut hormone profile in the same group of patients and found that the post-prandial secretion of motilin and glucagone-like peptide-1 (GLP-1) is decreased in MD patients. Since motilin triggers the antral phase III activity of the migrating motor complex and accelerates gastric emptying, reduced motilin secretion could cause delayed gastric emptying. In contrast, low post-prandial GLP-1 secretion is probably a result of delayed gastric emptying, i.e. a consequence rather than a cause of the disease. The patients with the most markedly delayed gastric emptying showed abnormal gastric electric activity and an abnormal post-prandial gut hormone profile. Both factors may contribute to altering gastric functioning. These findings indicate a partial dysregulation of the GI endocrine system in MD patients rather than a disorder of the entire digestive endocrine system. This conclusion is supported by an immunohistochemistry study carried out earlier by the same authors on the different types of duodenal endocrine cells in MD patients affected with diarrhoea[37].

Bellini M et al . Myotonic dystrophy and digestive disorders 1823

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They reported that the duodenal endocrine cell area containing the cells that produce serotonin, gastrin/CCK, secretin, GIP and somatostatin, is significantly increased. There are various possible explanations for this finding. The increased cell area may be a primary phenomenon or a consequence (via a feed-back mechanism) of the synthesis of defective and biologically inactive peptides or the result of a reduced receptor sensitivity of the effector cells and a malfunctioning effector organ.

If the increase in the number of endocrine cells is a primary disorder, it could play a role in the gastrointestinal manifestations of MD. However, even if it is merely a secondary phenomenon, a possible link can still be hypothesized, because the intestinal peptides may act on many different organs. Further studies are needed to shed light on this intriguing issue. At present, no correlation has been found between the increase in the duodenal cell area and the severity of the disease[37].

Different drugs, particularly prokinetics, have been proposed to treat the dyspeptic symptoms and motor disturbances in MD patients. In their study of 16 patients with delayed gastric emptying of solids and liquids, Horowitz et al [14] showed that oral administration of 10 mg of metoclopramide 3 times per day can improve the gastric emptying of both a solid and a liquid meal, but has no effect on esophageal emptying. In another study the same authors tested the efficacy of cisapride (10 mg, 4 times per day) and reported that it improves both gastric emptying and digestive symptoms such as nausea, vomiting, early satiety, abdominal distension and pain[38]. Metoclopramide also seems to be effective in improving gastric emptying in MD patients with gastroparesis[24]. Its effect may be due to the local release of acetylcholine and/or the hypersensitivity of the smooth muscle cells to this transmitter, and/or a reduction in the inhibitory effect of dopamine on gastric motility[14, 24, 39].

Ronnblom et al[25] administered erythromycin (100 mg, 2 times per day, before lunch and dinner) to dyspeptic MD patients over a period of 4 wk. The rationale for the use of erythromycin, which has been proved effective in accelerating gastric emptying in diabetic gastroparesis, lies in its agonistic action on the motilin receptors[10]. No effect on gastric emptying and no immediate improvement in symptoms were observed, although by the end of the 4-wk period a slight improvement in nausea and early satiety and a marked improvement in diarrhoea were noted by some patients. The authors reported that overall the treatment is effective possibly due to the positive effects of the drug on other GI symptoms, such as bacterial overgrowth in the ileum, which causes diarrhea by inhibiting the absorption of bile acid[25].

SMALL AND LARGE INTESTINEDiarrhea, sometimes accompanied with malabsorption, steatorrhea, and crampy abdominal pain, are frequent complaints in MD patients[9]. Paralytic ileus has also been reported[40, 41]. The pain may be located in any part of the abdomen, with no specific characteristics or eliciting factors. Episodic diarrhoea is the single most common complaint (present in up to 33% of cases). It

is often disabling and may have a marked impact on the patient’s social life, especially when combined with anal incontinence[9].

Diarrhea and possibly malabsorption, have been attributed to reduced peristaltic activity leading to bacterial overgrowth [10]. Anaerobic bacteria seem to be the principal contaminating agents. Their ability to deconjugate bile acids, thereby causing defective uptake in the terminal ileum, could be the main cause of diarrhea[25,

42]. Norfloxacine, either alone or in combination with cholestyramine, can alleviate this symptom in some patients. Other possible mechanisms of diarrhea, such as the reduced secretion of pancreatic amylase, have been hypothesized but not yet been demonstrated, and further evidence is needed to shed light on this issue[42].

Radiological studies have demonstrated reduced or absent peristaltic and/or segmentary activity with delayed intestinal transit[43-45]. Megacolon, sigmoid volvulus and segmental narrowing due to myotonic contractions have also been reported[43, 46-52].

Megacolon with the accompanying risk of ileus, volvulus and rupture, is a significant complication and must be kept in mind during the management of MD patients who are usually at higher risk of complications during anesthesia and surgical operations than normal subjects. The pathophysiological mechanisms underlying megacolon are not entirely clear, but probably smooth muscle damage is not the only factor involved. An electron microscopy study of an MD patient with megacolon showed that the myenteric plexus is degenerated with a paucity of neurons and fragmented axons. Glial cells are increased in number with vacuoles containing electron-dense material. Swelling of the mitochondria, distension of the endoplasmic reticulum and the accumulation of free ribosomes in the cytoplasm are also observed. The neurons are described as being “fewer, and the argyrophilic ones smaller, with less prominent processes and poor staining quality.” The number of nerve fibers exhibiting reactivity to substance P and enkephalin in the muscolaris externa is decreased, while normal smooth muscle cells are reported[52].

In concurrence with pregnancy or episodes of gastroenteritis, MD patients have been reported to suffer from recurrent intestinal pseudo-obstruction presenting with nausea, vomiting, abdominal cramps, distension and sometimes, constipation[53, 54]. However, such complications may occur during any stage of the disease and may even precede significant skeletal muscle weakness by 15 years. Recently, an MD patient with pseudo-obstruction and partial malrotation of the intestine has been reported[55], although it should be noted that this association has never been observed before and could represent an isolated case[56].

Manometric studies of the small intestine (jejunal manometry) in MD patients have shown low amplitude contractions of the migrating motor complex during phases 2 and 3, with high frequency activity during phase 2 and retrograde propagation and interruption of the contractions during phase 3. Low amplitude post-prandial contractions and myotonic phenomena have also been reported. A disturbance of the mechanoreceptors that link the circular muscle layer with the enteric nerves and


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provide the stimulus for the propagation of migrating motor complex phase 3 activity has been hypothesized to explain these phenomena[57].

Very few manometric studies of the colon have been conducted and the results are conflicting. Some report an absence of the normal rhythmic pressure variation in the descending colon[43], while others have failed to detect any motility abnormalities in the sigmoid colon[58]. In any case, intestinal motility disturbances are detectable in both symptomatic and asymptomatic subjects, raising doubts as to the actual role they play in the pathogenesis of MD. The absence of a correlation between disease severity and the presence of symptoms has also been confirmed in the intestinal tract[57].

Only a small number of histologic studies of the muscular layer of the small intestine are now available. They describe the cells as being swollen, fragmented, partially destroyed, or decreased in size, and replaced by fats, quite similar to the abnormalities reported for the skeletal muscles[35, 59]. Biopsies of the mucosa usually show normal histologic patterns[37, 43, 60, 61], although a case of a patient with intestinal villous atrophy and collagenous sprue has been reported by Woods et al[62]. The authors’ suggestion that this type of malabsorption may be frequent in MD patients has not yet been confirmed.

RECTOANAL REGIONAlthough constipation often associated with dyschezia is common, since some patients represent a serious problem, the most burdensome and disabling problem affecting MD patients may be fecal incontinence[9, 63, 64]. Up to 66% of MD patients suffer occasional fecal incontinence, while more than 10% report fecal incontinence one or more times a week. The frequency of urinary incontinence seems to have no difference between MD patients and control subjects[9, 65].

Although the procedure i s s imple and low in invasiveness, manometric studies of the rectoanal region are not often performed in MD patients. Some studies report a decrease in both the resting pressure (based on the tonic activity of the internal anal sphincter) and the squeezing pressure (exerted by the phasic activity of the external anal sphincter)[65-67]. Others have failed to detect any significant modification in the resting pressure and only a slight, statistically insignificant decrease in the squeezing pressure[11].

Lecointe e t a l [11] found that the ampli tude and duration of the recto anal inhibitory reflex (RAIR) in response to rectal distension are markedly decreased in MD patients. This could be explained, however, by a prolonged myotonic contraction of the striated external anal sphincter muscles[67], which could obscure the manometric signal indicating the normal internal anal sphincter relaxation response to rectal distension. Due to this myotonic phenomenon, the duration of the rectoanal contractile reflex (RACR) is longer in patients than in controls[11]. According to Hamel Roy et al[68], the amplitude of this myotonic response is decreased by pudendal nerve blockade, suggesting the possibility of at least a partial neural reflex response. Pudendal nerve terminal motor

latencies are normal in these patients, confirming the absence of a neurogenic lesion[67, 69]. Finally, it was reported that the maximum tolerable volume at rectal distension is significantly lower in MD patients than in controls[11].

Anorectal motility disturbances are as frequent as pharyngo-esophageal disturbances, the degree of involvement of the upper and distal portions of the gastrointestinal tract may be closely correlated both quantitatively and qualitatively[11]. It has been shown that anorectal motility and esophageal motility are altered in a similar number of patients[11].

Although the prevalence of altered motility is high, symptoms such as anal incontinence, diarrhoea and/or abdominal pain have been found in a small number of patients, suggesting that there is a close relationship between smooth and striated muscle motor function in MD but only in the gastrointestinal tract, where smooth and striated muscle abnormalit ies may be interdependent[11]. Unfortunately, there are no histologic data to back up this intriguing hypothesis.

Eckardt et al[66] studied the external anal sphincter using electromyography and have found myopathic potentials with myotonia. Herbaut et al.[65] reported a decreased duration and amplitude of the motor units in the external anal sphincter and the puborectalis muscle of MD patients with fecal incontinence, suggesting that there may be a myopathic component to the pathology. In 25% of these patients, polyphasic high-amplitude motor units were also present, indicating the probable presence of a neuropathic lesion. Taken together, the data suggest that both the striated external anal sphincter and the smooth internal anal sphincter may be altered in MD, even if previous histologic examinations have reported pathological findings only in the striated muscle[66]. More recently, Abercrombie et al[67] used electron microscopy to study the anal sphincter in two siblings with MD and found that the external anal sphincter is atrophic in both patients, with marked fibrosis and a high variation in the diameter of the fibres as well as the striated muscle almost entirely substituted by smooth muscle cells deriving from the internal sphincter. These cells are all different in size and electron density separated by large amounts of fibrous connective tissue. Some show features suggestive of dedifferentiation into myofibroblasts. The puborectalis biopsies revealed an analogous decrease in the amount of normal striated fibres with pronounced fibrosis. Some of the fibres were hypertrophic with increased central nucleation and many showed sarcoplasmic masses. Moreover, type 1 fibres were markedly prevalent (99%). In the ano-rectal region, no close correlation between motor abnormalities and overt symptoms has been found, although defecatory behaviour is often severely altered in these patients.

Constipation in MD patients is usually treated with prokinetics, laxatives and enemas [15, 70], while medical treatment with procainamide (300 mg twice a day) has been proposed for fecal incontinence[71]. Surgery to treat fecal incontinence has been attempted, but post-anal repair only temporarily improves the defecatory function[67]. Treating and curing defecatory problems in MD patients remain a difficult challenge. Rehabilitation involving a combination


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of volumetric rehabilitation, electroanalytical stimulation, kinesitherapy and biofeedback, can be effective in those patients not suffering from severe damage to the pelvic floor muscle[72,73].

CONCLUSIONSSince MD is a relatively rare disease, most of the papers published are hampered by the bias arising from the low number of patients studied. Many report just one or two cases, and very few involve series of more than 10 patients. Gastrointestinal involvement is frequently observed in MD patients and digestive complaints may be the first sign of the disease. According to Ronnblom et al[9], 25% of patients consider their gastrointestinal problems as the most disabling consequence of MD, 28% have digestive symptoms that may appear up to ten years before the typical musculoskeletal features. During this period the impairment of the digestive functions may be so gradual that patients unconsciously adapt to them by compensatory mechanisms, thus masking the symptoms[18,

74]. They may even develop a higher pain threshold (visceral hypoalgesia) similar to that seen in diabetic gastroparesis[75]. For these reasons, patients often do not undergo a thorough examination of the digestive tract, while at the same time they may be subjected to a broad battery of other examinations because of multi-system involvement. Thus, a careful assessment of the digestive tract may be performed only when the symptoms have become severe, although reliable, non-invasive or minimally invasive techniques such as ultrasonography, scintigraphy, breath test and cutaneous electrogastrography are available[13].

Little correlation has been found between the degree of skeletal muscle damage and gastrointestinal disturbances[12,

14], while there does appear to be a relationship between the severity of GI involvement and the duration of the disease[13]. Nevertheless, the pathophysiologic mechanisms of digestive motor disorders certainly suggest that there is damage to the striated muscles in the upper and lower portions of the gastrointestinal tract. Histologic evidence of smooth muscle alterations is scarce and conflicting[12,

14], although some authors have hypothesized that smooth muscle damage may occur earlier and is more severe than striated muscle changes[10].

Moreover, some authors suggest that a common mechanism may be the generat ion of the motor abnormalities in both the smooth and striated muscles. For example, Lecointe et al[11] studied the esophagus and the rectoanal region of MD patients and found that alteration in the smooth muscle is closely related with that in striated muscle, at least in the digestive tract.

A neural factor could also be involved in the digestive symptoms of MD patients. Neural dysfunctions, such as an alteration in the non-adrenergic, non-cholinergic neuronal control of the GI tract, have been suggested to explain the symptoms and instrumental findings that may be present even in the absence of definite histologic damage. Nitric oxide (NO) can mediate non-adrenergic, non-cholinergic nerve-induced relaxation and hyperpolarization of the smooth muscle in the digestive tract[76]. NO is produced from L-arginine via NO synthase,

a key enzyme that is expressed in the myenteric plexus, motor neurons and myenteric fetal interneurons[77-80]. Thus, the NO molecule seems to be common in both the striated and smooth muscles, the inhibition of NO synthase could alter the motor parameters in a manner consistent with the observations of Lecointe et al[11]. This “neuronal hypothesis” can explain the absence of histologic abnormalities in the smooth muscle of MD patients. In addition, it could explain the degeneration and decreased number of argyrophilic neurons in the colonic myenteric plexus of an MD patient affected with megacolon[52].

Gastrointestinal involvement in MD remains a complex and interesting condition. Given the limited data available, there are very few certainties and many important questions to be solved [81]. The relationship between myotonic dystrophy, gastrointestinal motility and clinical symptoms needs to be investigated in greater depth. Studies should begin early in their disease course, using non-invasive diagnostic techniques and concentrating on the genetic and histologic aspects of each case. Such efforts can improve our understanding of the pathophysiology of the digestive system involvement in MD and speed up the development of new therapeutic strategies to manage this difficult condition.

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S- Editor Guo SY L- Editor Wang XL E- Editor Bai SH


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Current concept on the pathogenesis of inflammatory bowel disease-crosstalk between genetic and microbial factors: Pathogenic bacteria and altered bacterial sensing or changes in mucosal integrity take “toll” ?

Peter Laszlo Lakatos, Simon Fischer, Laszlo Lakatos, Istvan Gal, Janos Papp

Peter Laszlo Lakatos, Simon Fischer, Janos Papp, 1st Depart-ment of Medicine, Semmelweis University, Budapest, Hungary Laszlo Lakatos, 1st Department of Medicine, Csolnoky F. County Hospital, Veszprem, Hungary Istvan Gal, 2nd Department of Medicine, Kenezi Gy. County Hos-pital, Debrecen, HungaryCorrespondence to: Peter Laszlo Lakatos, MD, PhD, 1st De-partment of Medicine, Semmelweis University, Koranyi str. 2/A, H-1083 Hungary. [email protected]: +36-1-2100278-1500 Fax: +36-1-3130250Received: 2005-10-06 Accepted: 2005-11-10

AbstractThe pathogenesis of inflammatory bowel disease (IBD) is only partially understood. Various environmental and host (e.g. genetic-, epithelial-, immune and non-immune) factors are involved. It is a multifactorial polygenic disease with probable genetic heterogeneity. Some genes are associated with IBD itself, while others increase the risk of ulcerative colitis (UC) or Crohn’s disease (CD) or are associated with disease location and/or behaviour. This review addresses recent advances in the genetics of IBD. The article discusses the current information on the crosstalk between microbial and genetic factors (e.g. NOD2/CARD15, SLC22A46A5 and DLG5). The genetic data acquired in recent years help in understanding the pathogenesis of IBD and can identify a number of potential targets for therapeutic intervention. In the future, genetics may help more accurately diagnose and predict disease course in IBD.


Key words: Inflammatory bowel disease; Ulcerative colitis; Crohn’s disease; Pathogenesis; Microbial factors; Genetics; NOD2/CARD15; SLC22A4/A5; DLG5

Lakatos PL, Fischer S, Lakatos L, Gal I, Papp J. Current concept on the pathogenesis of inflammatory bowel disease-crosstalk between genetic and microbial factors: Pathogenic bacteria and altered bacterial sensing or changes in mucosal integrity take “toll” ? World J Gastroenterol 2006; 12(12):1829-1841


INTRODUCTIONThere has been a sharp increase in the incidence of inflammatory bowel disease (IBD) in the late 1900s in Western countries as well as in some Eastern parts of Europe and North America[1-3]. Both Crohn’s disease (CD) and ulcerative colitis (UC) stem possibly from a common mechanism with an exact etiology that remains obscure[4,5]. Crohn’s disease manifests itself as a chronic granulomatous inflammation of the gastrointestinal tract capable of affecting its entire length with the presence of “skip” lesions[6,7]. It preferentially affects the terminal ileum, as was originally described by Crohn et al [8]. Ulcerative colitis, on the contrary, presents as a continuous inflammatory lesion affecting the rectum and colon, lacking granulomatous characteristics. IBD are multifactorial, polygenic diseases with probable genetic heterogeneity. According to this hypothesis, different genetic backgrounds may explain the various clinical patterns of the disease[4,5]. In addition to genetic predisposition, various environmental and host factors (e.g. genetic-, epithelial-, immune and non-immune) play a major role in the pathogenesis of IBD. Extensive heterogeneity is observed in terms of presentation, extraintestinal manifestations and location in CD, while behavior and response to treatment are heterogeneous in both CD and UC[9]. Furthermore, it is now undisputed that enteric bacterial flora play a key role in the pathogenesis of IBD, both in UC and in CD. The exact mechanism by which the intestinal mucosa loses tolerance to its bacterial neighbors remains elusive. The role of host genetic regulation of the innate immune response in the pathogenesis of CD has been brought to sharp focus by the identification of the NOD2 (CARD15) mutations. The genetic aspect of research is quite focused on nu-merous chromosomal loci. The environmental contribu-tors are diverse and an “infectious origin” of inflamma-tory bowel disease has not been confirmed. This review describes the various environmental, immunologic and genetic components leading to the manifestation of in-flammatory bowel disease.

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REVIEW

MICRObIal faCTORs: aRE PaThOgENIC baCTERIa aND/OR alTERED PERCEPTION Of NORMal flORa ThE kEys IN ThE bREakDOwN Of TOlERaNCE?The normal intestine encounters a high concentration of foreign antigens, bacteria and food. In the stomach and proximal small intestine, secretion of acid and bile, phasic ‘housekeeping’ motility patterns hinder colonization. How-ever, the number of bacteria dramatically increases in the distal small intestine to an estimated 1010 - 1012 bacterial cells/g content in the colon, which contribute to 60% of the faecal mass[10]. More than 400-500 species of bacteria are represented, belonging to 30 genera. Although this antigenic load is separated from the largest complement of lymphocytes in the body (gut associated lymphoid tis-sue, GALT) by only a single layer of polarised intestinal epithelium, most people do not have an immune response to foreign antigens and interaction between the mucosal immune system and the fecal bacterial mass regulates physiologic bowel functions. The mucosal immune system has evolved to balance the need to respond to pathogens while maintaining active tolerance to commensal bacteria and food antigens. In IBD, this tolerance breaks down and inflammation supervenes driven by the intestinal microbial flora. A large part of research has traditionally been devoted to finding a causative biological source of any disease. This has also been the case in IBD, but to date there is no compelling evidence of an etiological role for any single pathogenic microorganism.

PaThOgENIC MICRObEsThe history of IBD is dotted by cyclic reports on the isolation of specific infectious agents responsible for CD or UC. Several microorganisms, such as Mycobacterium paratuberculosis, Listeria monocytogenes, Chlamydia trachomatis, Escherichia coli, Cytomegalovirus, Saccharomyces cerevisiae, have been proposed as having a potential etiologic role. The suggested etiologic role of Mycobacterium paratuber-culosis in CD is also controversial. This bacterium is the causative agent of Johne’s disease, a chronic granuloma-tous ileitis in ruminants, which closely resembles CD. M. paratuberculosis was initially isolated from CD tissues some 20 years ago[11] and follow-up studies have tried to culture M. paratuberculosis for testing specific DNA sequences in intestinal tissues, or measuring serum antibodies against M. paratuberculosis with conflicting or inconclusive results[12-14]. Recently, M. paratuberculosis has been identified by in situ hybridization to the M. paratuberculosis-specific IS900 gene in tissue specimens of Crohn’s disease[15] and in 40% of Crohn’s disease granulomas isolated from surgical speci-mens by laser capture microdissection[16]. Others have localized M. paratuberculosis by PCR to macrophages and myofibroblasts within the lamina propria[17]. However, the possibility of an association between M. paratuberculosis and CD remains inconclusive. Additionally, E. coli bacteria possessing special adhesive properties have been associated with the development of

ulcerative colitis. A fascinating hypothesis was published in the Lancet in 2003[18]: an association between refrigeration and CD. Some so called psychrotrophic bacteria are capable of growing slowly at low temperature (known as “cold chain hypothesis”). Common pathogens are Yersinia enterocolitica, Listeria monocytogenes, and Clostridium botulinum[19]. Several studies have demonstrated members of the Yersinia species in intestinal mucosal samples of Crohn’s disease. The specific pathogens detected are either Y. enterocolitica or Y. pseudotuberculosis, and sometimes even both[20,21]. There are numerous aspects of yersiniosis which resembles the inflammatory reaction seen in Crohn’s disease, making differential diagnoses of these two conditions, including ileitis or ileocolitis, mesenteric adenolymphitis, reactive arthritis and erythema nodosa. Additionally, granulomas may be observed in histological samples[18]. Recent data also demonstrate a role of mucosa-asso-ciated and intramucosal bacteria in the pathogenesis of IBD and colorectal cancer. In the study by Martin et al [22], mucosa-associated or intramucosal E.coli are present in 43% and 29% of CD, and 17% and 9% of controls, re-spectively, supporting a role of mucosa-adherent bacteria in the pathogenesis of Crohn’s disease. Finally, a viral etiology has also been proposed as the cause of IBD, particularly CD. An early measle infection during the perinatal period notably increases the risk of Crohn’s disease[23]. The finding of paramixovirus-like parti-cles in CD endothelial granulomas suggests an association between perinatal measles and predisposition to CD based on some epidemiological and serologic data[24]. However, these preliminary findings are not confirmed by later studies[25]. Importantly, the progressive decline of measle virus infection in the last decades with the concomitant rise of CD during the same period of time speaks against an etiologic role of measles in CD. The hypothesis that measle vaccination rather than measle infection, might be a risk factor for CD, has also been raised, but again results of additional studies fail to confirm this association[26]. In contrast, a role of cytomegalovirus infection is proposed in UC[27].

NON-PaThOgENIC INTEsTINal flORa

In the last decade, the focus of interest in microbial etiol-ogy of IBD has shifted from infectious to commensal agents. Based on fairly solid data, there is a substantial body of evidence that the normal enteric flora plays a key role in the development of IBD[28]. This is even more evident after the discovery of the genetic factors (e.g. NOD2/CARD15, TLR4 and CD14) responsible for al-terations in bacterial perception[29]. The beneficial effect of antibiotics in the treatment of CD, and to a lesser extent UC, has been appreciated for years. Diversion of the fecal stream from inflamed bowel loops has also been known to induce symptomatic improvement in CD patients, while re-emergence of inflammation often occurs upon restora-tion of intestinal continuity[30]. Changes are observed in the normal flora of IBD patients and dysbacteriosis is commonly reported. Based on the clinical picture of IBD, the intestinal flora


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might play a role in the initiation and perpetuation of inflammatory reaction. Fabia et al[31] demonstrated that the concentration of anaerobic bacteria and Lactobacillus are radically decreased in active IBD patients. On the contrary, patients with inactive IBD show no such decrease. Swidsinski et al[32] compared over 300 IBD patients with 40 control individuals and found that abnormalities can be observed in the flora using various laboratory techniques. In addition, a much greater number and concentration of bacteria make up the biofilms covering the epithelium involved in IBD. Furthermore, a direct association has also been reported between bacterial concentration and disease severity. More recently, probiotics (primarily lactic acid bacteria) defined as live microbial feeds that beneficially affects the host by modulating gut microbial balance, have been demonstrated to improve both human IBD and experimental colitis, primarily by preventing relapses, thus adding an important dimension to the role of gut flora in IBD[33,34]. Probably, the most convincing evidence comes from experimental data. In the majority of IBD animal models, intestinal inflammation fails to develop when they are kept in a germfree environment. This critical observation has led to the widely accepted paradigm ‘‘no bacteria, no colitis’’. However, once normal flora is introduced into their environment, the mutant-strain mice quickly develop colitis[35, 36]. It is worth mentioning that bacterial superinfection (most commonly Clostridium difficile, but also Entamoeba histolytica, Campylobacter spp, etc.) is also able to elicit relapse of IBD. In the study of Mylonaki et al[37], 10.5% of all relapses are associated with enteric infections. In another study[38], 20% of all relapses are correlated to C. difficile positivity. The presence of bacterial antigens as the initiating factor in IBD has also been explored. The data support a role of flagellin proteins in activating the innate immune system. Elevated titers of serum anti-flagellin IgG against these proteins could be detected in CD patients[39]. Furthermore, elevated serum IgG2a titers could also be demonstrated in experimental IBD models, similar to results observed in humans. Recently, anti-CBir1 (anti-flagellin) expression has been independently associated with small-bowel, internal-penetrating and fibrostenosing disease features in CD[40]. Finally, the role of bacteria in the pathogenesis of IBD is further supported by the increased intestinal perme-ability in CD patients. Although it is not an environmental factor per se, it has been postulated as an early predisposing factor for the development of this condition. Increased in-testinal permeability is also observed in asymptomatic first-degree relatives of CD patients, possibly encompassing another group of individuals who are at an increased risk of developing this form of IBD[41,42]. Documented obser-vations of increased permeability preceding the onset of symptomatic disease are of further interest[43]. In relating this phenomenon to the UC form, electron microscopy has revealed deficiency of those elements comprising the tight junctions necessary for the wholeness of the intes-tinal epithelium[44]. Recent data reporting an association between genes important in mucosal transport and integ-rity (OCTN and DLG5) and IBD further support this as-

sumption. The phenomenon of decreased bacterial diversity of the intestinal microflora obtained from stool samples in IBD patients has also been suggested, based on culture-dependent microbiological techniques[45]. Recently, a Ger-man group has reported the relative lack of the Bacteroides group (usually accounting for 50%-90% of the anaerobic faecal microflora) by utilizing 16sDNA-based single strand conformation polymorphism (SSCP) fingerprint, cloning experiments and real time PCR. Moreover, the bacterial profiles are stable, at least for the observed period of six weeks. This is in concordance with previous findings, sug-gesting that the mucosa-associated microflora contains only a small number of bacteria of the Bacteroides/Prevotella group[32]. However, in a recent study comparing the fae-cal microflora of healthy controls and CD patients, no differences are noted between patients and healthy con-trols[46]. This lack of significant difference may be partially explained by the differences in mucosal and faecal micro-flora. Why an abnormal response to normal endogenous gut bacteria exists in IBD is not clear, but recent data, espe-cially on the genetic background of CD, suggest an asso-ciation between gut inflammation and abnormal bacterial sensing.

gENETICs aND gENE fUNCTION

Possible genetic loci influencing the presentation of inflammatory bowel disease have already been identified on more than half of all chromosomes, including the X chromosome[47-49]. First, in 1996 Hugot et al[50] reported that the pericentromeric region of chromosome 16 (D16S408) is associated with CD renamed as IBD1[50]. This is confirmed by several studies. To this date, results of numerous studies have revealed a total of nine loci associated with specific linkage requirements, subsequently labeled as IBD1-9[51-55] (Table 1). Furthermore, it is clear that some loci correlate with either UC or CD while others are involved in the pathogenesis of both IBD forms[49].

Table 1 Locations of nine major loci showing linkage to inflammatory bowel disease

IBD locus Chromosome Identified genes Disease IBD1 16q13 NOD2/CARD15 CDIBD2 12q14 Not known

(VDR, STAT6, MMP18, b2-integrin)UC

IBD3 6p Not known(HLA, TNF)

IBD

IBD4 14q11-12 Not known(TCR, LTB4 receptor)

CD

IBD5 5q31-33 SLC22A4/A5 CDIBD6 19p13 Not known

(ICAM1, C3, TBXA2) IBD

IBD7 1p36 Not known(TNF-R family)

IBD

IBD8 16p12 Not known CDIBD9 3p26 Not known

(CCR5, CCR9,hMLH1) IBD

10q23 DLG5 IBD

Lakatos PL et al. Pathogenesis of IBD 1831

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faMIlIal sTUDIEsEvidence of genetic constituents in the induction of inflammatory bowel disease clearly lies within the results of familial studies. Several studies examining the relationship of IBD within families have reported positive findings in 10%-25% of families[56,57]. This finding goes back at least two decades when Farmer and Michener reported 40% concordance for IBD in first-degree relatives. They observed that sibling-sibling involvement occurs in addition to parent-offspring transmission. Solid evidence of genetic predisposition showed that as many as 50% of monozygotic twins are affected by Crohn’s disease whereas ulcerative colitis is seen in approximately 14%[58]. Monozygotic twins are not equally affected but still display greater concordance for IBD than dizygotic twins. The overall risk for IBD is increased by 5-20 times[59], be-ing highest in twins and in siblings. The genetic compo-nent is more prominent in CD than in UC. Finally, genetics certainly play a role in the observed phenotype of IBD, including disease behavior and loca-tion[60]. Genetic anticipation is suggested in early studies with a more severe and earlier presentation of the disease in sub-sequent generation[61], but this has not been replicated in more recent studies[62,63].

gENEs INvOlvED IN ThE RECOgNITION Of baCTERIa – NOD2/CaRD15, NOD1/CaRD4, TlR4 aND CD14NOD2/CARD15 Three independent groups have identified NOD2 CARD15 on chromosome 16 in IBD1 as a candidate gene for CD[29,64,65]. NOD2/CARD15 is an intracellular element responsible for the indirect recognition of bacterial peptidoglycan through the binding of muramyl dipeptide[66,67]. It is a member of the Ced4-APAF1 protein superfamily and is expressed in various cells, including monocytes, dendritic cells, Paneth cells and intestinal epithelial cells[6]. Structur-ally, NOD2/CARD15 is composed of three segments: the first being composed of two CARD units, the central por-tion consisting of nucleotide-binding domain (NBD) and finally, a leucine-rich repeat (LRR) region as is found in TLRs[68]. The most intriguing question that remains to be answered concerns the mechanism whereby mutations in the NOD2/CARD15 gene predispose towards the chronic intestinal inflammation characteristics of CD. At the molecular level, the binding of NOD2/CARD15 to a bacterial motif (muraryl dipeptide-MDP, a component of both Gram negative and positive bacterial cell walls) causes its binding to a second NOD2/CARD15 molecule, thus forming a dimer. Further interaction with other cy-tosolic proteins leads to the ultimate activation of nuclear factor κB (NF-κB), eliciting pro-inflammatory reactions[68]

(Figure 1). Surprisingly, it is still unclear whether NF-κB expression is elevated or depressed in CD due to conflict-ing observations and studies. In vitro experiments demon-strated that the declined activity of this protein indicates a loss-of-function effect[69,70]. Hisamatsu et al[71] showed that

CARD15/NOD2 may function as an antibacterial factor

in CaCo2 intestinal epithelial cells. Cells stably transfected

with a wild-type CARD15/NOD2 gene construct are able to prevent invasion of Salmonella typhimurium. This protec-tive effect is lost in cells transfected with gene constructs of mutant CARD15/NOD2. It has also been demon-strated that NOD2/CARD15 expression in intestinal epithelial cells might be upregulated by the proinflamma-tory cytokine tumor necrosis factor α (TNF-α)[72]. The NF-κB activation is regulated by the LRR region and deletion within this region may theoretically result in its uncon-trolled activation. In concordance with this, increased NF-κB activation is observed in the presence of 3020insC mutation by stimulation of MDP[73], which may explain the findings of higher NF-κB tissue levels in samples from IBD patients[74]. Interestingly, mice lacking NOD2 or pos-sessing mutated NOD2 variants do not spontaneously de-velop Crohn’s disease. These findings suggest that NOD2 mutations create an intestinal environment susceptible to IBD[73,75] rather than playing a direct causative role in dis-ease development expression of α- and β-defensins. Defensins are integral parts of the local intestinal im-mune system, and are secreted by intestinal epithelium as endogenous antimicrobial proteins. The consequential damage could finally lead to an intensified bacterial inva-sion and a chronic inflammation of the intestinal mu-cosa[76]. The possibility exists that NOD2/CARD15 is involved in the regulation of Paneth cell degranulation[77], as NOD2/CARD15 expression is noted in close proxim-ity to the secretory granules of these cells. Kobayashi et al[75] and Wehkamp et al[78] have shown that carriage of NOD2/CARD15 variants may be associated with reduced α-defensin release from Paneth cells in response to bac-terial cell wall components, and the defective defensin release by the Paneth cells could provide the missing link, whereby reduced NOD2/CARD15 activity impairs host defenses against bacteria and underlies persistent intestinal

inflammation. Finally, NOD2/CARD15 is involved in the regulation of TLR2 stimuli by peptidoglycans. Defective

Figure 1 NOD2/TLR4 signaling pathway. NOD2 activation by CARD-CARD dimmer formation results in binding to RICK kinase (RIP-like interacting CLARP kinase). RICK then activates the nuclear factor kB inhibitor (IkB) kinase complex (IKK) via phosphorylation of IKKc. The IKK complex next phosphorylates IkB resulting in nuclear factor kB (NFkB) translocation to the nuclei and transcriptional activation of NFkB responsive genes, such as pro-inflammatory cytokines or defensins. TLR4 induces activation through other molecules (Myd88, IRAK and TRAF6).

←

Caspase9-apoptosis

RICK

MonocyteIntestinal epithelial cell

DNA-gene expression

lipopolysaccharide

Muramyl-dipeptideNOD2

MyD88TRAF6 TLR4

NF-κB /IκB

NF-κB

?

←←

←

←

←

←


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NOD2 function results in a pro-inflammatory cytokine bias after stimulation of mononuclear cells with TLR2 stimuli, which may contribute to the overwhelming inflam-mation in Crohn’s disease[79]. Three major mutations have been identified within NOD2/CARD15: one frame shift (3020insC, SNP13) and two missense mutations (R702W-SNP8 and G908R-SNP12). The presence of NOD2/CARD15 mutations increases the risk for CD by 1.4-4.3-fold in heterozygous patients and 17.6-44.0-fold in homozygous and compound het-erozygous patients. Reports on homozygous individuals who are disease-free are available. One such family has been described by van der Linde et al[80]. It is estimated that any of these three common mutations involving NOD2/CARD15, is present in heterozygous form in 30-50% of CD patients and 7-20% of controls from North-America and Europe[81-85]. However, various geographical differ-ences are noted. Furthermore, other races display a much lower prevalence of this mutation, sometimes lacking it altogether (e.g. African-American[86], Chinese[87] and Japa-nese[88]). The prevalence is also lower in other North Euro-pean countries[89,90]. The association between NOD2/CARD15 and disease phenotype and behavior has also been investigated. The three common mutations are associated with ileal disease and fibrostenosing behavior, but they are relatively less frequent in colonic and fistulous disease[64,66,91,92]. The pres-ence of mutations is not associated with extraintestinal manifestations or the response to infliximab therapy[93]. The mutations are also not associated with UC. In CD, the attributable risk for ileal disease is 40% determined by NOD2/CARD15 and 20% by HLA genes. The numbers are similar for ileo-colonic disease (NOD2/CARD15: 30%, HLA: 40%), while colonic disease is thought to be associated with HLA and other yet unknown genes[91]. It is worth mentioning that NOD2/CARD15 increases the risk for colorectal cancer (CRC). Kurzawski et al[94] have found that the presence of SNP13 mutation increases the risk of developing CRC by 2.23-fold in patients with an age >50 years at diagnosis of CRC. This has not been confirmed in a more recent study by Alhopuro et al[95].

NOD1/CARD4A similar protein to NOD2/CARD15 by its structure and function, is located on chromosome 7p. Its particular location is in the midst of a region with strong IBD correlation, being translated into an intracellular bacterial pathogen-associated molecular pattern[96]. Zouali et al[97] denounced that NOD1/CARD4 plays a role in IBD following an investigation involving 63 IBD patients. The screening process was conducted on the eleven exons constituting the NOD1/CARD4 gene with the goal of identifying polymorphisms. Indeed, several variants were identified, none of which has proved any association with IBD[97]. On the contrary, McGovern et al[96] have located a complex insertion/deletion allele on NOD1, associating it with both an early onset of IBD and extraintestinal manifestations.

Toll-like receptors and CD14 Toll-like receptors (TLRs) expressed in myeloid cells play a

major role both in detecting microbes (lipopolysaccharide) and in initiating innate immune responses. Accordingly, a disturbance in its function predisposes to infections with Gram negative bacterial pathogens, possibly influencing the advancement of IBD[98,99]. In contrast, little is known about the expression, distribution and function of TLRs in epithelial cells per se. TLR4 is also expressed in the Golgi apparatus of intestinal epithelial cells. Thus, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and requires LPS internalization [100]. Recently, it has been suggested that the interaction of LPS with TLR4 / MD2 contributes to the perpetuation of the inflammatory epithelial cell injury via TNFα induced alterations of enterocyte turnover in an autoparacrine/paracrine manner[101]. TLRs may also influence the nature of immune response by skewing T cells toward a Th1 or Th2 profile. Myeloid cells are exquisitely sensitive to TLR ligands and produce significant IL-12p40. They appear to play a key role in the initiation and possibly the Th1/Th2 skewing of inflammatory responses. In this model the inflammation can be normally controlled by myeloid or lymphocyte-derived IL-10 acting through Stat3 in myeloid cells to block further production of IL-12/IL-23 and skewing the responses towards Th1 profile[102]. IBD is characterized by an altered expression pattern of TLRs on the surface of intestinal epithelium and TLR4 expression is up-regulated in patients with CD. In contrast, the expressions of TLR2 and TLR5 are unchanged, while TLR3 that recognizes viral replication is down- regulated[103]. The D299G (Asp299Gly) polymorphism of the TLR4 gene associated with LPS hyporesponsiveness[104]

is associated with CD (OR:2.45-2.80) and UC (OR: 2.05)[98,105]. However, other studies have failed to replicate this association[85,106]. Another TLR which binds to and recognizes bacterial DNA, TLR9, may also play an important role in the pathogenesis of IBD. Rachmilewitz et al[107] reported that the anti-inflammatory effect of probiotics is transmitted through TLR9 in experimental colitis. More recently, an English group has reported a synergy between TLR9 and NOD2 that is lost in the CD patients carrying NOD2 mutation, indicating impairment in innate immunity[108]. Recently, Torok et al[109] reported that the -1237C promoter polymorphism of TLR9 is significantly associated with CD in German patients. Contradictory data are available on the association between the bacterial receptor CD14 and IBD. Klein et al[110] found that the 159 TT genotype is associated with CD but not with UC. In a Japanese study[111], the same genotype is associated with UC (OR: 1.96) but not with CD and also a further study investigating Caucasian patients has failed to demonstrate any association[112].

MUCOsal INTEgRITy aND TRaNsPORT - slC22a4/OCTN1, slC22a5/OCTN2 aND Dlg5SLC22A4/OCTN1, SLC22A5/OCTN2 The association between 5q31 and CD is first noted in genome-wide screens (the genes for Th2 cytokines -e.g. IL-3, 4, 5, 9, 13 and IRF1- maps to this region). The


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risk haplotype is associated only with a moderate risk for CD (OR: 1.4-1.5). The effect of NOD2/CARD15 is additive while the 5q31 haplotype is an independent risk factor[113-115]. A German study reported that the risk haplotype is also associated with UC while the IBD5 haplotype is not associated with the clinical presentation, nor is it correlated to IBD in Japanese[114]. Based on the available data, IBD5 increases mainly the overall risk for IBD, whereas NOD2/CARD15 mutations are primarily responsible for the determination of phenotype. One of the most important findings in the genetics of IBD is the identification of OCTN1/SLC22A4 and OCTN2/SLC22A5 genes coding for integral membrane proteins. The function of these proteins is multispecific in bidirectional transmembrane transport of carnitine and organic cations. The SLC22A4 C1672T and SLC22A5 promoter G207C variants increase the risk for CD by 2-2.5-fold when present as a single copy and by 4-fold in homozygous carriers[55,116]. The elevated risk attributed to OCTN TC haplotype and NOD2/CARD15 mutations, is additive with an odds ratio of 7.3-10.5 in double carriers. A more recent Japanese study on SLC22A4/A5 and DLG5 polymorphisms and CD[117], has shown a weak association, concurring with the Ashkenazi population[118], where the frequency of the allele is lower, indicating racial differences. Variant alleles are associated with functional changes. The SLC22A4 variant encodes exchange of a leucine residue on the OCTN1 transmembrane domain for phenylalanine (L503F), a change which reduces carnitine but augments organic cation transporter activity. The SLC22A5 variant impairs heat shock protein-driven promoter transcriptional activation[116]. Carnitine is an essential mediator of fatty acid oxidation, a role subserved by promoting transport of long-chain fatty acids across the mitochondrial membrane. Inhibition of fatty acid oxidation can evoke clinical and pathologic signs of colitis[119], which may explain why impairments in carnitine transport may confer an increased risk for IBD. Another possibility is that the enhanced cation transporter activity of the OCTN1 503F variant may provoke disease by allowing aberrant uptake of toxic substrates. A possible association between OCTN3 and CD has been reported[120,121]. In contrast, no association has been found between the proton-coupled bivalent metal antiporter located on 2q35 SLC11A1 and IBD[122].

DLG5 The association between DLG5 (Drosophilia Discs Large Homolog 5) at chromosome 10q22-23 is first reported by Stoll et al[123]. DLG5 is a member of the membrane-associated guanylate kinase (MAGUK) gene family which encodes cell scaffolding proteins and is also involved in the maintenance of epithelial integrity and regulation of cell growth[124], a role potentially impaired by expression of the CD-associated DLG5 variants causing increased permeability and disease. The impact on the overall risk of developing IBD is much smaller than NOD2/CARD15. The carriage of the 113A allele increases the risk for CD by 1.3-2.1-fold in German, Italian, Canadian patients and is associated with early onset of IBD in Scottish

children[123,125,126]. Less common variants have also been identified (C4126A). Interaction between DLG5 and NOD2/CARD15 is also detectable. This however, has not been replicated by the same group in English and Scottish CD patients and by the German group from Munich[127,121].

hla, CyTOkINE gENEs aND OThER gENETIC

faCTORs

HLA genes Reports on genetic trait of IBD are available, which investigated the genes associated with the immune response. The main region of interest is that of MHC (main histocompatibility complex) genes located on chromosome 6. The human leukocyte antigen (HLA) class II genes are candidates for a role in the pathogenesis of IBD because their products play a central role in the immune response. More than 100 known genes are located in the area, each is highly variable and several polymorphisms are known. Several studies have addressed the possible association between certain HLA polymorphisms and the risk for IBD. Most of these studies have revealed contradictory results and their findings could not be replicated in different populations. Overall, genes in the HLA region may play a greater role in modifying IBD phenotype rather than in determining overall disease susceptibility[128-130]. The association of UC with HLA genes is stronger than CD[4,130]. In the white Caucasian population, the rare HLA-DRB1*0103 allele is positively associated with UC (OR: 3.42). Others have found that this allele predisposes to extensive disease and is additionally associated with the presence of extraintestinal manifestations[131]. The repeatedly observed association with HLA-DR2 has been confirmed in the cumulative odds ratio of 2.00, while the odds ratio for DRB1*1502 and DR9 is 3.74 and 1.54, respectively. In contrast, the presence of DR4 (OR: 0.54) and DRw6 is preventive for UC[132]. In CD, negative association has been found between DR2 (OR = 0.83) and DR3 (OR = 0.71), while HLA-DR7 and DQ4 seem to be associated with a moderately increased risk (OR = 1.42 and 1.88). The studies on allele DRB3*0301 showed that this allele is positively associated with Crohn’s disease (OR = 2.18) [4,31]. Ahmad et al [91] investigated 340 polymorphisms in HLA genes and found that the 3 common NOD2/CARD15 alleles, the HLA DRB1*0701 (RR: 1.5) and Cw*802 (RR3.0) are associated with increased risk for CD, while the presence of HLA DRB1*1501 (RR: 0.6) is protective. Investigating the genotype-phenotype relations, HLA DRB1*0103 allele in both UC and CD is associated with both extensive and severe disease as judged by the need for colectomy[131,133]. However, the low frequency of this allele even in UC patients suggests that this association is unlikely to be clinically useful in predicting disease course. The same allele is associated with late onset, colonic location (38.5% vs controls 3.2%) of CD [134], while the presence of HLA-DRB1*04 (OR:1.7) and HLA-DRB1*0701 (OR: 1.9) increased the risk for ileal location[135]. MICA*010 (MHC class I chain-related gene A) and HLA-DRB*0103 are associated with perianal


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disease[91]. A number of genetic associations have also been de-scribed in extraintestinal manifestations of IBD. Stud-ies comprising both UC and CD patients showed that migratory pauciarticular large-joint arthritis is associated with HLA-DRB1*0103 and B*27 and B*35 class I al-leles in linkage disequilibrium. In contrast, chronic small-joint symmetric arthritis is associated with HLA-B*44[136]. Uveitis is correlated to HLA-B*27, B*58 and DRB1*0103, while erythema nodosum to B*15 and TNF-1031C[137]. However, due to the relatively small sample size of these studies, the evidence is lacking to draw a firm conclusion.

Cytokine, multidrug resistance and cell adhesion gene polymorphismsInteraction between non-pathogenic, commensal bacteria and epithelial cells, M-cells and dendritic cells of the intestinal mucosa is characterized by a cytokine profile of mainly the Th2/Th3 (suppressor) type (IL-4, IL-5, IL-10, TGF-β), which incapacitates the development of an inadequate, progressive, proinflammatory cycle in healthy individuals[138]. Contrary to this, mucosal immune response against pathogenic species is largely mediated by Th1 cells and a specific cytokine milieu (IL-1, TNF-α, interferon-γ). Both ulcerative colitis and Crohn’s disease are characterized by an increased expression of "general" proinflammatory cytokines (TNF-α, IL-1, IL-6), which suggests an abnormally intense inflammatory response against commensal bacteria. This loss of immune tolerance to non-pathogenic microbes results in pathologic immune reactivity and a self-supporting inflammatory cascade. In healthy individuals, the tight control of the inflammatory reaction between bowel mucosa and bacterial milieu involves antiinflammatory cytokines (IL-10, TGF-β)[139]. In case of cytokine imbalance, the inflammatory reaction intensifies. However, lack of antiinflammatory cytokines plays only a restricted role in the development and maintenance of IBD, which is suggested by their low therapeutic potential in IBD[140]. In contrast, the widely used chimera or humanized anti-TNF antibody represents one of the most fascinating new therapeutic options in severe and/or penetrating CD cases as well as UC[141-143]. Studies are also underway targeting IL-12[144]. Although the above mechanism is pivotal in the induction of local inf lammatory changes from an immunological point of view, lack of reproducibility has challenged many of the other reported genotype-phenotype associations in IBD, such as association between interleukin polymorphisms and IBD. The association between a low-producing allele, allele 2 of the interleukin (IL)-1RA gene and UC (OR: 1.3), extensive colitis, the need for colectomy[145,146] and development of pouchitis (OR: 3.1) in UC after ileal-pouch anastomosis[147] has been reported in Caucasian population. However, another study investigating 529 IBD patients has failed to confirm these results[148]. IL-10 polymorphisms also lack association with UC. However, the 627A allele is more frequent in patients with left sided colitis[149]. In contrast, the frequency of the high IL-10 producer allele (-1082*G) is decreased in IBD[150]. Bone loss and osteoporosis are well recognized in IBD,

but the risk factors have not been clearly identified. Several studies investigated whether genetic markers may predict bone loss and found that variable number of tandem re-peats adjacent to IL-6 or within IL-1RA and genes previ-ously implicated in the paracrine stimulation of osteoclasts are associated with bone resorption[151]. A subsequent study from the same group has failed to identify an association with a putatively functional polymorphism in the IL-6 gene. Nemetz et al[152] reported that the 511*2 allele of IL-1b is associated with increased in vitro production of IL-1b by mononuclear cells in Hungarian CD patients[152]. Furthermore, microsatellite loci of TNF-α are associ-ated with CD[153]. However, only data from a single study suggest that the TNF-308A polymorphism is associated with more intense inflammatory activity and an increased risk of arthritis in CD patients with fistulizing disease[154]. The A6 allele MICA is associated with UC and early onset of disease[155], while the MICA*011 allele increases the risk for UC by almost 2-fold. No association has been found between different polymorphisms of CTLA4, a receptor of activated T cells, which has an inhibitory function in regulating T-cell activation and IBD in different Caucasian and Asian populations[156,157]. Brant et al[158] have described a suggestive linkage on chromosome 7q containing the multidrug resistance (MDR)-1 gene, in association with the appearance of UC and CD. This particular gene is a membrane transport protein with several documented human polymorphisms having effects on intestinal absorption and drug pharma-cokinetics. The significant mutation designated as Ala-893Ser/Thr is originally identified in knockout mice with spontaneous colitis[159,160]. An additional locus of mutation (C3435T) associated with 50% decreased protein secretion corresponding to UC (OR: 1.6-2.0) especially in extensive colitis (OR: 2.64), showed no manifestation in CD[161,162]. Adhesion molecules mediating cell-cell and cell-extracellular matrix interactions, are pivotal mediators of inflammation in IBD. Catenins and catherins are major contributors of integrity of the intestinal mucosa[163]. Dysregulation in E-cadherin-catenin complex formation leads to decomposit ion of the mucosal structure characterized by leaky epithelium, as observed in IBD. Cell surface adhesion molecules conveying leukocyte-endothelial interactions, govern homing of activated inflammatory cells into the bowel mucosa. In order to slow down by rolling along the endothelium, circulating leukocytes in small vessels of the inflamed mucosa interact with adhesion molecules (e.g. CD44) expressed in the endothelium. This is followed by establishing a firm adhesion anchor. Extravasation and migration into the site of inflammation are mediated by integrins (mainly α4β2 and α4β7) and selectins (L-, E- and P-selectin). Vascular endothelium of the inflamed intestine, particularly in CD, is characterized by increased expression of adhesion molecules and integrin ligands, such as E-selectin, ICAM-1, MAdCAM-1, VCAM-1[164,165]. In UC, the soluble E-selectin levels show no preference between active or in-active forms. Also, the sVCAM concentration is far higher in the control group than in the inactive UC patients. Fi-nally, unlike in CD, VEGF levels in UC cases are similar in active, inactive and control subjects[166]. Expression of


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certain adhesion molecules (MAdCAM-1, CCL25) is also increased in tissues not primarily involved in the pathologi-cal process of IBD (e.g. liver), which might account for the extraintestinal manifestations of the disease[167]. Finally, in Japanese CD patients, the ICAM-1 K469 al-lele in IBD6 is associated with CD (OR: 2.6) and UC[168]. This association has also been confirmed in Caucasian population[169]. Targeted therapy against certain adhesion molecules (e.g. a4 and ICAM-1) is currently one of the major focuses of pharmaceutical trials in IBD[170,171]. In conclusion, various factors have been implicated in the pathogenesis of IBD, but its mechanism of action is still obscure. Recent data indicate that altered NOD2/CARD15 (or TLR4)-mediated bacterial sensing of normal commensal flora in the gut and mucosal permeability changes may be the key mechanisms (see Figure 2). At present, most efforts are devoted to a better understanding of the genetic changes underlying IBD and to further clarification of how the altered recognition of pathogenic and/or commensal microbial factors by the mucosal immune system leads to inflammation in IBD subjects but not in the general population (Figure 3).

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CARD-CARDLeucine-rich region

Nucleotide-Binding Domain

SNP12

SNP8 SNP13

←

← ←

Figure 2 Structure of NOD2/CARD15 protein and locations of the three main mutations.

Figure 3 Role of microbial factors and genetics in the pathogenesis of IBD.

Commensal flora and/or Pathogenic microbes

Susceptible host (e.g. altered bacterial sensation and/or altered mucosal integrity in the gut

Development of IBD

←


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S- Editor Guo SY L- Editor Wang XL E- Editor Bi L


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Bing Luo, Yun Wang, Xiao-Feng Wang,Yu Gao, Department of Microbiology, Qingdao University Medical College, Qingdao 266021, Shandong Province, ChinaBao-hua Huang, Department of Oncology, Yantai Yuhuangding Hospital, Yantai 264002, Shandong Province, China Peng Zhao, Department of Pathology, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, ChinaCorrespondence to: Professor Bing Luo, Department of Microbiology, Qingdao University Medical College, 38 Dengzhou Road, Qingdao 266021, Shandong Province, China. [email protected]: +86-532-83812423 Fax: +86-532-83812423Received: 2005-09-23 Accepted: 2005-11-18

AbstractAIM: To investigate the interrelationship of Epstein-Barr virus (EBV) and EBV- encoded proteins with Helicobacter pylori (H pylori ) infection and the expression of c-met and c-myc oncogene proteins in gastric carcinoma, and to explore their role in gastric carcinogenesis.

METHODS: One hundred and eighty-five gastric carcinoma tissues were detected by polymerase chain reaction (PCR)-Southern blot for EBV genome and in situ hybridization (ISH) for EBV-encoded small RNA 1 (EBER1). Gastric carcinoma with positive EBER1 signals was confirmed EBV-associated gastric carcinoma (EBVaGC). The status of H pylori infection in 185 gastric carcinomas was assessed by rapid urease test and PCR. The samples with positive PCR and urease test were defined as H pylori infection. The expression of c-met and c-myc oncogene proteins in tissues of EBVaGC and matched EBV-negative gastric carcinoma (EBVnGC) were examined by immunohistochemistry. RT-PCR and Southern hybridization were used to detect the expression of nuclear antigens (EBNAs) 1 and 2, latent membrane protein (LMP) 1, early genes BARF1 and BHRF1 in EBVaGC cases.

RESULTS: The positive rate of H pylori and EBV in 185 gastric carcinomas was 59.45% (110/185) and 7.03% (13/185) respectively. No difference was found in sex, age, pathological differentiation, clinical stages and lymph node metastasis between H pylori -positive and H pylori -negative gastric carcinomas. However, the positive rate of H pylori infection in the antrum gastric

carcinomas was higher than that of cardia and body gastric carcinomas. In our series, age, pathological differentiation, clinical stages, lymph node metastasis and location of cancer were not different between EBVnGC and EBVaGC, while the positive rate of EBV in male patients was significantly higher than that of female patients. The positivity of H pylori in EBV-associated and EBV-negative gastric carcinomas was 46.15% (6/13) and 81.40%(104/172) respectively. There was no significant correlation between EBV and H pylori infection. The c-met overexpression was significantly higher in the EBVaGC group than in the EBVnGC group. However, c-met and c-myc expression did not show significant difference between the two groups. Transcripts of EBNA1 were detected in all 13 EBVaGCs, while both EBNA2 and LMP1 mRNA were not detected. Six of the 13 cases exhibited BARF1 transcripts and 2 exhibited BHRF1 transcripts.

CONCLUSION: The positivity of H pylori in EBVnGCs is higher than that of EBVaGCs, but no significant correlation is found between EBV infection and H pylori infection. H pylori -positive gastric carcinoma is predominant in antrum location, while EBVaGC has a tendency of predominance in cardia/body location. EBV infection is associated with c-met abnormal expression but not with c-myc protein in EBVaGC. c-met overexpression is not induced by LMP1. BARF1 and BHRF1 may play important roles in the tumorigenesis of EBVaGC through different pathways.


Key words: Epstein-Barr virus; Helicobacter pylori ; Gastric carcinoma; c-met; c-myc

Luo B, Wang Y, Wang XF, Gao Y, Huang BH, Zhao P. Correla-tion of Epstein-Barr virus and its encoded proteins with Heli-cobacter pylori and expression of c-met and c-myc in gastric carcinoma. World J Gastroenterol 2006; 12(12): 1842-1848


INTRODUCTIONHelicobacter pylori (H pylori) infection is one of the important environmental risks for gastric carcinoma. In 1994, the

GASTRIC CANCER

Correlation of Epstein-Barr virus and its encoded proteins with Helicobacter pylori and expression of c-met and c-myc in gastric carcinoma

Bing Luo, Yun Wang, Xiao-Feng Wang, Yu Gao, Bao-Hua Huang, Peng Zhao


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Working Group Meeting of the International Agency for Research on Cancer (IARC) concluded that H pylori is a definite carcinogen to gastric carcinoma(GC). Chronic gastritis caused by H pylori infection may progress to intestinal metaplasia and even GC[1,2]. The correlation between Epstein-Barr virus (EBV) infection and gastric carcinoma is well known. EBV infection is found in 2%-16% of ordinary gastric adenocarcinoma cases and 80%-100% of gastric lymphoepithelioma-like carcinoma cases[2-5]. However, the pathogenic role of EBV in gastric carcinogenesis remains to be elucidated. Recent studies have shown that the expressions of EBV encoded genes in gastric carcinoma are different from those in Burkitt’s lymphoma and nasopharyngeal carcinoma (NPC), suggesting that the oncogenic mechanism of EBV in gastric carcinoma may be unique[6-8]. The development of gastric carcinoma is a multistep event proceeding from normal to preneoplastic lesions to highly malignant tumors, accompanied by participation of multiple factors and multiple genes. To shed further light on gastric carcinogenesis, we determined the clinicopathologic characteristics and EBV status in 185 patients with gastric carcinoma and correlated with the status of H pylori infection, genetic alterations in proto-oncogenes c-myc and c-met.

MATERIALS AND METHODSSpecimens and cases One hundred and eighty-five surgically resected specimens of gastric carcinoma were collected from the Affiliated Hospital of Qingdao University Medical College, Qingdao Municipal Hospital and Yantai Yuhuangding Hospital. Tumor tissues from each surgical specimen were separately dissected. Partial tissue was used to detect H pylori by urease test. DNA was extracted by the standard proteinase K-sodium dodecyl sulfate (SDS) method and purified with phenol-chloroform. Total RNA was extracted with TRIzol reagent (Gibco BRL, Gaithersburg MD, USA) according to the manufacturer’s instructions. The tissue sections were used for histopathological diagnosis, in situ hybridization (ISH) and immunohistochemical analysis.

Detection of EBV infectionThe cases positive for EBV DNA by PCR-Southern blot assay were further confirmed by ISH for EBER1 as previously described[6]. The cases with EBER1 positive signals were classified as EBVaGC group.

Detection of H pylori infectionThe resected tissues were used to detect H pylori infection by urease test kit. Simultaneously, PCR was used to detect 16 sRNA of H pylori. The specific primers were designed as previously described[9]. The sequence of sense primer is 5΄-CTGGAGAGACTAAGCCCTCC-3΄, and that of antisense primer is 5΄-ATTACTGACGCTGATTGTGC-3΄. The PCR products were 109 bp. Three microliter DNA was added into a solution containing 200 μmol/L dNTPs, 0.5 μmol/L each primer, 1.5 mmol/L MgCl2 and 1 U Taq DNA polymerase (Promega, USA) in a total volume of 25

Luo B et al . EBV, H pylori , c-met and c-myc in gastric carcinoma 1843

μL. PCR was carried out under the following conditions: first denaturation at 94℃ for 5 min; then denaturation for 30 s at 94℃, annealling for 30 s at 55℃, extension for 45 s at 72 ℃ in 35 amplification cycles; and finally extension for 5 min at 72℃. The amplified products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining and ultraviolet illumination. DNA from the culture of H pylori was used as positive control, and that from human leukocyte as negative control.

ImmunohistochemistryParaffin-embedded sections of tissues from EBVaGC c a s e s a n d 4 5 c a s e s o f E B V n G C w i t h s i m i l a r clinicopathological data were immunostained by the standard streptavidin-biotin-peroxidase method. Anti-human mouse polyclonal antibodies against c-met and anti-human mouse monoclonal antibodies 9E10 against c-myc (Santa Cruz Biotechnology Inc) were used as primary antibodies. Phosphate buffered saline (PBS), instead of the primary antibody, was used for negative control sections. The sections of breast carcinoma tissue with highly expressed c-met and c-myc served as the positive controls. The percentage of positively stained tumor cells in each tumor section was evaluated by counting at least 1 000 cells in 10 randomly selected high-power fields. Brown staining for c-myc was located in nuclei, staining for c-met protein was located in both membrane and cytoplasm. The section was considered as expressing the protein if cellular staining ≥5%, following the methods described previously [10,11]. c-met positivity was divided into three grades: 5%-30%, 30%-50% and >50%. Positive cells >30% (++) was regarded as overexpression.

RT-PCR and Southern hybridization analysis for EBV genes expressionAccording to the methods previously described[12], RT-PCR and Southern hybridization were used to detect the expression of nuclear antigens (EBNAs) 1 and 2, latent membrane protein (LMP) 1, early genes BARF1 and BHRF1 in EBVaGC cases. cDNAs from EBV-immortalized lymphoblastoid cell lines (LCL) were used as positive controls, and those from EBV-negative Ramos cells as negative controls. The integrity of RNA was checked by parallel amplification of endogenous control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.

Statistical analysisQualitative data were analyzed by c2 test or the Fisher’s exact test (two-tail). Quantitative data were expressed as mean±SD and compared between the 2 groups by Student’s t-test or t’-test. P <0.05 was considered statistically significant. Software SAS 6.12 was employed to process the data.

RESULTSDetection of H pylori infection H pylor i infect ion in 185 gastr ic carcinomas was assessed by rapid urease test and PCR. The samples

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with positive PCR and urease test were defined as H pylori- positive. No difference was found in sex, age, pathological differentiation, clinical stages or lymph node metastasis between H pylori-positive and H pylori-negative gastric carcinomas. However, the location of tumor was significantly different between the two groups (c2=13.817，P = 0.003) . The positive rate of H pylori infection in the antrum gastric carcinomas was higher than that of cardia and body gastric carcinomas (q = 4.528, P = 0.005；q = 3.681，P = 0.010) (Table 1).

Detection of EBV infectionThe positive rate of EBV in 185 gastic carcinomas was 7.03% (13/185). Age, pathological differentiation, clinical stages, lymph node metastasis and location of cancer were not different between EBV-negative gastric carcinomas (EBVnGC) and EBVaGC (P = 0.669, 0.141, 0.259, 0.818, 0.064, respectively), while sex was significantly different between the two groups (c2=3.940，P = 0.047) (Table 2).

Relationship of EBV and H pylori The positivity of H pylori in EBV-positive and EBV-negative gastric carcinomas was 46.15% (6/13) and

81.40%(104/172) respectively. There was no significant cor re la t ion between EBV and H py lo r i infect ion (c2=1.027，P = 0.317，r = -0.075) (Table 3).

Immunohistochemistry of c-met and c-mycF o r t y - f i v e c a s e s o f E B V n G C w i t h s i m i l a r clinicopathological data were chosen as the control group. No statistical difference was found in age, sex, tumor location, histological subtype, stage, or lymphnode metastasis between the two groups [12]. Immunostaining results of c-met and c-myc are shown in Figure 1. The c-myc and c-met expression was 61.5% (8/13) and 76 .9% (10/13), and c-met overexpress ion was 69.2% (9/13) in EBVaGC group, while they were 55.6 (25/45), 64.4% (29/45) and 37.8% (17/45) respectively in EBVnGC group. The difference in c-met overexpression between EBVaGC and EBVnGC was s ignif icant . However, the difference in c-myc and c-met expression between the two groups was not significant (Table 4).

Expression of EBV-associated genes in EBVaGC We investigated the expression of EBV-associated g e n e s i n 1 3 E B Va G C c a s e s b y R T- P C R a n d

Table 1 H pylori status in relation to clinicopathologic characteristics

n H pylori (+) H pylori (-) P

Sex Male 134 78 56 0.574 (χ2=0.315) Female 51 32 19 Age (yr) 30- 11 6 5 40- 29 15 14 50- 41 23 18 0.779 (χ2=1.764) 60- 59 38 21 ≥70 45 28 17Histological subtype LDAC1 125 75 50 MDAC2 49 29 20 0.986 WDAC3 7 4 3 Signet ring carcinoma 4 2 2Tumor stage Ⅰ 27 16 11 Ⅱ 98 57 41 0.975 (χ2=0.216) Ⅲ 41 25 16 Ⅳ 19 12 7Lymph node metastasis Present 130 79 51 0.577 (χ2=0.311) Absent 55 31 24 Tumor location Cardia5 23 8 15 Body6 47 23 24 Antrum 95 68 27 0.003 (χ2=13.817) Multiple 14 8 6 Remanent carcinoma4 6 3 3

1LDAC: Lowly-differentiated adenocarcinoma; 2MDAC: Moderately-differentiated adenocarcinoma; WDAC: Well-differentiated adenocarcinoma; 46 cases of remanent gastric carcinoma were not statistically analyzed; 5Compared with antrum, q = 4.528, P = 0.005; 6Compared with antrum, q = 3.681, P = 0.010.

Table 2 EBV status in relat ion to cl inicopathologic characteristics

n EBV (+) EBV (-) P

Sex Male 134 13 121 0.047 (c2=3.940) Female 51 0 51 Age (yr) 30- 11 0 11 40- 29 3 26 50- 41 2 39 0.669 60- 59 6 53 ≥70 45 2 43Histological subtype LDAC1 125 11 114 MDAC2 49 1 48 0.141 WDAC3 7 0 7 Signet ring carcinoma 4 1 3Tumor stage Ⅰ 27 1 26 Ⅱ 98 5 93 0.259 Ⅲ 41 4 37 Ⅳ 19 3 16Lymph node metastasis Present 130 10 120 0.818 (c2=0.053) Absent 55 3 52 Tumor location Cardia5 23 1 22 Body6 47 7 40 Antrum 95 3 92 0.064 Multiple 14 1 13 remanent carcinoma4 6 1 5

1LDAC: Lowly-differentiated adenocarcinoma; 2MDAC: Moderately-differentiated adenocarcinoma; WDAC: Well-differentiated adenocarcinoma; 46 cases of remanent gastric carcinoma were not statistically analyzed.

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Southern hybr id izat ion ana lys i s (F igure 2) . The transcripts of EBNA1 were detected in all 13 cases, while neither EBNA2 nor LMP1 mRNA was detected. Six of the 13 cases exhibited BARF1 transcripts and 2 exhibited BHRF1 transcripts. GAPDH mRNA was amplified to check pertinent RNA extraction. The result showed that the RNA was in integrity.

DISCUSSION H pylori is believed to be a carcinogen of gastric carcinoma. Recently, it was found that EBV is also linked with the development of partial gastric carcinomas. In this study, we simultaneously detected the status of EBV and H pylori infection in gastric carcinomas. No statistical relationship was found between H pylori infection rate and sex, age, pathological differentiation, clinical stages and lymph node metastasis. However, the positive rate of H pylori infection in the antrum gastric carcinomas was higher than that of cardia and body gastric carcinomas. These results are consistent with those of previous studies[1,13]. In our series, age, pathological differentiation, clinical stages, lymph node metastasis and location of cancer were not different between EBVnGC and EBVaGC, while the positive

rate of EBV in male patients was significantly higher than that of female patients. Eleven cases of EBVaGC were low differentiated adenocarcinoma, and 8 cases of EBVaGC were body or cardia cancer. Although no statistical difference was found, it revealed a tendency that included predominance of cardia/body location and low differentiated adenocarcinoma. It remains controversial whether there is a significant clinicopathologic difference between EBVaGC and EBVnGC. Several reports showed that EBVaGC was characterized by male predominance, preferential location in proximal stomach, and a high prevalence of low differentiated adenocarcinoma[14,15]. Wu et al[2] found EBV-positive lymphoepithelioma-like carcinoma (LELC) showed less node metastasis and higher survival rate, and tended to be at cardia/body location in contrast to EBV-negative gastric carcinomas, while no significant clinicopathologic difference was found between EBV-positive ordinary gastric carcinoma and EBV-negative gastric carcinoma.

In the present study, no significant correlation between EBV and H pylori infection was found in gastric carcinomas. Currently, only a few studies have investigated the effect and interaction of EBV and H pylori infection in gastric carcinomas, but no conclusive results have been

Table 3 Relationship between EBV and H pylori in gastric carcinoma

EBV(+) EBV( -) Total H pylori (+) 6 104 110 H pylori (- ) 7 68 75 Total 13 172 185

χ2 =1.02, P = 0.317, r = -0.075

Table 4 c-myc and c-met expression in EBVaGC and EBVnGC

n c-myc expression c-met expression c-met overexpression + - + - + - EBVaGC 13 8 5 10 3 9 4 EBVnGC 45 25 20 29 16 17 28 c2 0.147 0.259 4.035P value 0.701 0.611 0.045

Figure 1 Immunohistochem‑istry of c‑met and c‑myc. A: Positive expression of c‑met; B: Negative expression of c‑met; C: Positive expression o f c ‑ m y c ; D : N e g a t i v e e x p r e s s i o n o f c ‑ m y c . (Original magnification ×400).


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A B

C D

reported[2,16-18]. Levine et al[16] documented that specific IgG antibody levels against H pylori in 39 EBV-negative gastric carcinomas were significantly higher than those of 5 EBV-positive gastric carcinomas. It was thus inferred that an inverse relationship between specific IgG titer against H pylori and EBV status existed. In the present study, the positivity of H pylori in EBVnGCs was higher than that of EBVaGCs, but no reverse correlation is found between EBV infection and H pylori infection. Wu et al[2] found EBV was detected in 11 (100%) gastric LELC and 19 (13.7%) of 139 non-LELC. Compared with the EBV-negative gastric carcinomas (68.4%) and EBV-positive non-LELC (68.3%), EBV-positive gastric LELC had a significant lower positive rate of H pylori IgG (36.4%). This finding implicates that there is a tendency for gastric LELC, with the high frequency of EBV and predominant proximal location, to have less association with H pylori infection than the other two groups, and also suggests that EBV may play a role in H pylori-seronegative gastric carcinomas, especially those located at the proximal stomach. Same results were found in other two reports[19,20]. Our result showed that in ordinary non-LELC gastric carcinomas, H pylori-positive gastric carcinoma is predominant in antrum location, while EBVaGC has a tendency of predominance in cardia/body location．Several studies found equal H pylori infection and distribution of intestinal metaplasia

and atrophic gastritis among EBV-positive and -negative gastric carcinomas, indicating that EBV and H pylori may play roles together in the pathogenesis of gastric carcinomas. Chronic atrophic gastritis and subsequent intestinal metaplasia caused by H pylori infection enhances the susceptibility of EBV to gastric mucosal epithelia, and then EBV facilitated the carcinogenesis of gastric carcinoma[17,18].

Many studies have been focused on the relationship between EBV and the oncogenes or tumor suppressor genes in the carcinogenic mechanism of EBV. c-met protein is a receptor for hepatocyte growth factor (HGF), also known as scatter factor (SF), a tyrosine kinase encoded by the proto-oncogene c-met. HGF/SF-met signaling has been shown to affect a wide range of biological activities in mammalian cells, including cellular proliferation, migration, invasion, morphogenesis, and angiogenesis[21,22]. The oncogene c-myc also has numerous biological activities, such as transformation, immortalization, blockage of cell differentiation and induction of apoptosis[23]. Amplification and abnormal expression of oncogenes of the c-myc and /or c-met are involved in the development of tumor. In this study, EBVaGC had a higher rate of c-met overexpression than EBVnGC, however the difference in c-myc and c-met expression between the two groups was not significant, indicating that EBV infection induces c-met over-expression. The results are inconsistent with previous studies [11,24-30]. The study about the amplification and expression of c-met oncogene and EBV infection in tumor tissues has been little reported，majoring in lymphoproliferative disorder such as Hodgkin’s disease (HD). Kij ima e t a l [24] found the amplif icat ion and overexpression of c-met in EBV-positive and -negative gastric carcinomas did not differ significantly. Teofili et al[25] also reported c-met expression was independent of the presence of EBV in HD patients. However, Weimar et al[26] found a strong correlation between the expression of the c-met proto-oncogene and EBV infection in patients with HD. Six out of eight EBV-positive samples from HD patients expressed c-met protein, while none of the 10 EBV-negative samples from HD expressed c-met. Furthermore, circulating B lymphocytes,c-met-negative, express c-met after infection by EBV. In the same way, no consistent results were found in the study of c-myc expression in EBV-associated tumor. Several studies showed that EBV facilitates the development of tumor by inducing c-myc and inhibiting p53 expression[27-29]. However, Park et al[30] found that significant increase of c-myc gene copy numbers was only found in 12 out of 31 non-Hodgkin’s malignant lymphomas (MLs) (38.7%), in which 6 cases were EBV positive and 6 cases were EBV negative, indicating c-myc gene amplification did not correlate with EBV infection. Ishii et al[11] found c-myc expression in early stage of EBVaGC was higher than that of EBVnGC, while the c-myc expression in advanced stage of EBVaGC was lower than that of EBVnGC. It was inferred that EBV might cause the host cell to induce c-myc expression and inhibit p53 overexpression in the initial development of the cancers (early stage), but then influence c-myc expression negatively in advanced

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Figure 2 Detection of EBV‑associated gene expression by RT‑PCR and Southern hybridization in EBVaGC. M: DIG‑labeled DNA molecular weight marker VIII (Roche); lane 1: EBV‑positive LCL (positive control); lane 2: EBV‑negative Ramos cells (negative control); lanes 3‑13: EBV‑positive gastric carcinoma samples.


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stage cancers, making them less likely to have a natural regression via apoptosis. In the present study, most of the samples were advanced stage gastric carcinomas. Apparently, it could not infer whether EBV induces c-myc expression in early stage EBVaGC, but it was certain that EBV does not inhibit c-myc expression in advanced stage EBVaGC.

Some in vitro and in vivo studies have shown that EBV-encoded genes, such as LMP1, EBNA, BHRF1 and BARF1, can interact with oncogenes and tumor suppressor genes in the carcinogenesis of tumor[28,31,32]. LMP1 are the essential genes for cell transformation. LMP1 can induce c-met expression through the activation of Ets-1 transcription factor in vitro, which may contribute in part to the highly metastatic potential of NPC[31]. The expression of c-myc can be induced by LMP1 and EBNA2 in vitro and in vivo[28, 29, 32, 33]. Our study and other studies failed to detect LMP1 and EBNA2 mRNA in EBVaGC[6-8], suggesting that LMP1 and EBNA2 may not be essential for EBVaGC formation and may not be related with c-met and c-myc expression in EBVaGC. EBV early gene BHRF1 shows partial sequence homologous to the human bcl-2 proto-oncogene, which is involved in inhibiting cell apoptosis. BHRF1 protein can inhibit apoptosis of B lymphocytes and epithelial cells and promote cell growth and transformation[34,35]. BARF1 shares homology with the cellular proto-oncogene c-fms and is able to immortalize epithelial cells and fibroblast cells and B lymphocyte in vitro[8,36,37]. Furthermore, it can activate the expression of bcl-2[38]. We demonstrated that 6 of 13 EBVaGC cases exhibited BARF1 mRNA and 2 exhibited BHRF1 mRNA. Zur Hausen et al[8] also detected 9 BARF1-positive cases and 2 BHRF1-positive cases in 10 EBV-related gastric adenocarcinomas. Because EBVaGC lacks the expression of LMP1[6-8], BARF1 and BHRF1 might provide an alternative way for the pathogenesis of EBVaGC independent of LMP1. In vitro studies showed that BARF1 can activate the expression of c-myc in BARF1 transformed cells, and EBNA1 and c-myc cooperate in lymphomagenesis[38,39]. In our study no correlation was found between c-met and c-myc expression with the expression of BARF1 or BHRF1 in 13 EBVaGCs. So further study is needed to elucidate the relationship of EBV infection and EBV-encoded proteins with expression of c-met and c-myc.

In summary, this study shows that H pylori infection is closely linked to the distal location gastric carcinoma, but EBVaGC is predominant proximal location. No correla-tion exists between EBV infection and H pylori infection in the development of gastric carcinomas. EBV infection is associated with c-met abnormal expression but not with c-myc protein in EBVaGC. c-met overexpression is not induced by LMP1. BARF1 and BHRF1 may play important roles in tumorigenesis of EBVaGC through different pathways.

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S-Editor Wang J L-Editor Zhang JZ E-EditorBai SH


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Weissenberger Christian, Department of Radiotherapy, University Hospital of Freiburg, 79106 Freiburg, GermanyGeissler Michael, Department Oncology, Gastroenterology and Internal Medicine, Städtische Kliniken Esslingen, 73730 Esslingen a. N., GermanyOtto Florian, Department of Internal Medicine I, Division of Oncology, University Hospital of Freiburg, 79106 Freiburg, GermanyBarke Annette, Henne Karl, von Plehn Georg, Rein Alex, Müller Christine, Bartelt Susanne, Henke Michael, Department of Radiotherapy, University Hospital of Freiburg, 79106 Freiburg, GermanyCorrespondence to: Dr. Christian Weissenberger, Department of Radiotherapy, University Hospital of Freiburg, 79106 Freiburg, Germany. [email protected]: +49-761-2709401 Fax: +49-761-2709582Received: 2005-09-12 Accepted: 2005-10-26

AbstractAIM: To evaluate the long-term outcome of standard 5-FU based adjuvant or neoadjuvant radiochemotherapy and to identify the predictive factors, especially anemia before and after radiotherapy as well as hemoglobin increase or decrease during radiotherapy.

METHODS: Two hundred and eighty-six patients with Union International Contre Cancer (UICC) stage II and III rectal adenocarcinomas, who underwent resection by conventional surgical techniques (low anterior or abdominoperineal resection), received either postoperative (n = 233) or preoperative (n = 53) radiochemotherapy from January 1989 until July 2002. Overall survival (OAS), cancer-specific survival (CSS), disease-free survival (DFS), local-relapse-free (LRS) and distant-relapse-free survival (DRS) were evaluated using Kaplan-Meier, Log-rank test and Cox’s proportional hazards as statistical methods. Multivariate analysis was used to identify prognostic factors. Median follow-up time was 8 years.

RESULTS: Anemia before radiochemotherapy was an independent prognostic factor for improved DFS (risk ratio 0.76, P = 0.04) as well as stage, grading, R status (free radial margins), type of surgery, carcinoembryonic antigen (CEA) levels, and gender. The univariate analysis revealed that anemia was associated with impaired LRS

(better local control) but with improved DFS. In contrast, hemoglobin decrease during radiotherapy was an independent risk factor for DFS (risk ratio 1.97, P = 0.04). During radiotherapy, only 30.8% of R0-resected patients suffered from hemoglobin decrease compared to 55.6% if R1/2 resection was performed (P = 0.04). The 5-year OAS, CSS, DFS, LRS and DRS were 47.0%, 60.0%, 41.4%, 67.2%, and 84.3%, respectively. Significant differences between preoperative and postoperative radiochemotherapy were not found.

CONCLUSION : Anemia before radiochemotherapy and hemoglobin decrease during radiotherapy have no predictive value for the outcome of rectal cancer. Stage, grading, R status (free radial margins), type of surgery, CEA levels, and gender have predictive value for the outcome of rectal cancer.


Key words: Rectal cancer; Adjuvant radiotherapy; Adjuvant radiochemotherapy; Anemia; Prognostic factor

Weissenberger C, Geissler M, Otto F, Barke A, Henne K,

von Plehn G, Rein A, Müller C, Bartelt S, Henke M. Anemia

and long-term outcome in adjuvant and neoadjuvant radio-chemotherapy of stage II and III rectal adenocarcinoma: The Freiburg experience (1989-2002). World J Gastroenterol 2006; 12(12): 1849-1858


IntroductIonAn improved therapeutic strategy for stage II and III rectal adenocarcinomas is urgently needed because up to 30% of patients still develop recurrent disease after curative surgical resection[1]. Several studies are ongoing aiming at evaluation of new multimodality treatment strategies[2]. However, pelvic radiotherapy per se is nowadays undisputed and accepted as standard therapy in all trials concerning locally advanced rectal cancer. The current standard treatment is the result of two independent, multi-institutional, prospective randomized trials more than 12 years ago by the Gastrointestinal Tumor Study Group

Anemia and long-term outcome in adjuvant and neoadjuvant radiochemotherapy of stage II and III rectal adenocarcinoma: The Freiburg experience (1989-2002)

Christian Weissenberger, Michael Geissler, Florian Otto, Annette Barke, Karl Henne, Georg von Plehn, Alex Rein, Christine Müller, Susanne Bartelt, Michael Henke


COLORECTAL CANCER

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(GITSG 7175) in 1985[3] and the North Central Cancer Treatment Group (NCCTG 79-47-51) in 1991[4]. Both studies demonstrated that combined radiotherapy and chemotherapy following surgical resection of stage II and III rectal cancer can improve the overall survival. These results have prompted the National Institute of Health to publish the NIH Consensus recommending postoperative radiochemotherapy for stage II and III tumors.

Anemia has been shown to have impact on the rate of local control or distant metastasis of other tumor entities[5, 6]. To identify such prognostic factors and to evaluate the outcome of preoperative or postoperative radiochemotherapy, we conducted a retrospective study including 286 patients with stage II and III rectal carcinoma treated with radiochemotherapy in the Department of Radiotherapy at the University Hospital Freiburg from 1989 until 2002.

materIals and methodsPatient cohortThe retrospective study enrolled patients with stage II or III rectal cancer who were treated with pre- or postoperative adjuvant radiochemotherapy between January 1989 and July 2002. Pretreatment evaluation included complete blood test , chemistr y prof i le, determination of carcinoembryonic antigen (CEA), chest radiography, liver ultrasonography and computer tomography (CT) of abdomen and pelvis[7]. Tumor location was divided in to the lower third (less than 7 cm from the anal verge), the middle third (less than 12 cm) and the upper third (12 or more than 12 cm) according to Phang et al[8]. However, there are other definitions[9], and even the anatomic length of the rectum is unclear[10]. If patients suffered from other diseases leading to a Karnofski lower than 80, these patients were counted as having “other serious disease(s)”. Concerning survival a complete set of data were available. But due to the missing data about hemoglobin during radiotherapy, the effect of anemia (haemoglobin <120 g/L in women or <130 g/L in men) was analyzed using a subgroup of 192 patients with complete patient documentation.

Multi-modal therapyAccording to the surgical reports from the different hospitals, all patients were treated with standard surgical technique. Total mesorectal excision (TME) was performed in all patients with abdominal-perineal resection (APR) but less consequently and not quality-controlled in patients with low anterior resection (LAR). If proximal and distal surgical margins were microscopically free of tumor, the patients were defined as “radically resected” (R0). According to the patient documentation analyzed, the circumferential margins were not systematically assessed. The scheduled radiotherapy delivered 45 - 56 Gy in 25 - 31 sessions using 6 / 18 MeV linear accelerator. The treatment included two parallel opposing right and left portals (using wedges with 40 or 50 % absorption) and a dorsal field. These three (or four, if external iliac lymph nodes were included) field box arrangements were used, representing

the generally approved radiotherapeutic scheme during the retrospective study[11]. The upper margin was fixed 1.5 cm cranial of the promontorium whereas the lower margin was chosen depending on the exact tumor localization. The lower margin including the perineal scar (if the tumor was located less than 8 cm from the anal verge or if abdominoperineal resection was performed), was marked by the tuber ischiadicum (between 8 and 12 cm above the anal verge), or covered by the lower border of the obturator foramen (12 cm above the anal verge). The width of posteroanterior portals covered the pelvic inlet with 2 cm margin. Radiotherapy was administered with cycles 1 and 2 instead of cycles 3 and 4 as recommended by the NIH[12], renewed by the 1996 Patterns of Care Rectal Cancer Committee[13]. In the NCCTG study, cycles 1 and 2 of 5-FU plus semustine were given, followed by pelvic radiotherapy plus chemotherapy.

Depending on their R status, patients were treated with combined modality therapy either according to the NIH recommendations[12] or to the protocol[14] of the Arbeitsgemeinschaft Radioonkologie (ARO) of the German Cancer Society. Patients with no evidence of microscopical residuals of the disease (R0 resection) were treated according to the NIH protocol[12]. Following R1 or R2 resection, patients were treated according to the ARO protocol. When the NIH protocol was used, the concurrent bolus 5-FU was given at a daily dose of 500 mg/m² for 3 d during cycles 1-3, the following three cycles were given for 5 d. When the ARO protocol was used, patients received 350 mg/m² 5-FU iv continuously during 24 h for 14 d. Additionally, bolus of 200 mg/m² leucovorin and 4 mg/m² mitomycin C, was given daily for 1 h. In both protocols radiotherapy and chemotherapy were started simultaneously.

Statistical analysisThe data were analyzed using SAS. The statistical methods included Student’s t-test, Chi-square test, and Kruskal-Wallis-test. Survival was analyzed using univariate and multivariate methods (step down analysis). Kaplan-Meier curves[15] were used to estimate the distribution of overall survival (OAS), cancer-specific survival (CSS) and disease-free survival (DFS). For analysis, the rates of treatment failure were adjusted, local-relapse-free survival (LRS) and distant-relapse-free survival (DRS) were determined as life table analysis referring to freedom of locoregional relapse and freedom of distant recurrence (metastasis). LRS was defined as the rate of local control. Log-rank test (Cox-Mantel) was used to compare the survival distributions between different patient subgroups[16]. Multivariate analysis and proportional hazard models[17] were used to determine the prognostic factors with significant impact on survival, including hemoglobin effect, grading, staging, adjuvant therapy, surgical method and tumor marker.

resultsTwo hundred and eighty-six patients (186 men and 100 women) fulfilled the inclusion criteria and were enrolled in the study (Table 1). Their age ranged from 30 to 84


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years (median 62 years). The distribution of stages was as follows: 92 patients (32.2%) were assigned to stage II (lymph node negative) and 194 (67.8%) to stage III (lymph node positive). One hundred and forty-six patients (51.0%) were treated with low anterior resection (LAR), 140 patients (49.0 %) with abdominoperineal resection (APR). According to the surgical reports from different hospitals, all patients were treated with standard surgical technique. Total mesorectal excision (TME) was performed in all patients with APR but less consequently and not quality-controlled in patients after LAR. Two hundred and thirty four patients (81.8 %) were defined as R0 (Table 1). Sixteen patients were known to have oncological diseases

in their histories. Other severe diseases referred to cardial (n = 62), pulmonary (n = 5), hepatic or gastric (n = 18), psychiatric diseases (n = 8), or diabetes (n = 6).

Survival and life table analysisThe calculated overall 5-year survival (OAS) of all stage II and III patients was 47% (Figure 1 and Figure 2), the 10-year survival was 36.3%. The cancer-specific survival (CSS) and disease-free survival (DFS) decreased from 60.0% and 41.4% respectively to 52.0% and 34.9 % between 5 and 10 years (Table 3). Local-relapse-free survival (LRS) was 67.2% (5 years) and 65.7% (10 years), distant-relapse-free survival (DRS) was 84.3% (5 and 10

Table 1 Patient characteristics

Adjuvant Neoadjuvant All radiochemotherapy radiochemotherapy n (%) n (%) n (%)

Gender Female 148 (63.5) 38 (71.7) 186 (65.0) Male 85 (36.5) 15 (28.3) 100 (35.0)Age ≤Median age of 62 yr 134 (57.5) 32 (60.4) 166 (58.0) > Median age of 62 yr 99 (42.5) 21 (39.6) 120 (42.0)Tumor location Upper third 35 (15.1) 0 (0.0) 35 (12.2) Middle third 100 (42.9) 23 (43.4) 123 (43.0) Lower third 87 (37.3) 25 (47.2) 112 (39.2) Not known 11 (4.7) 5 (9.4) 16 (5.6)Surgical Resection Anterior (LAR) 133 (56.8) 13 (24.6) 146 (51.0) Abdominoperineal (APR) 100 (43.2) 40 (75.4) 140 (49.0)R status R0 197 (84.5) 37 (69.8) 234 (81.8) R1 17 (7.3) 5 (9.4) 22 (7.7) R2 4 (1.8) 2 (3.8) 6 (2.1) not known 15 (6.4) 9 (17.0) 24 (8.4)Stage II 75 (32.2) 17 (32.1) 92 (32.2) III 158 (67.8) 36 (67.9) 194 (67.8)N stage N0 74 (31.8) 17 (32.1) 91 (31.8) N1 81 (34.8) 27 (50.9) 108 (37.8) N2 78 (33.4) 9 (17.0) 87 (30.4)Grading G I 11 (4.7) 9 (17.0) 20 (7.0) G II 161 (69.1) 29 (54.7) 190 (66.5) G III 44 (18.9) 9 (17.0) 53 (18.5) Not known 17 (7.3) 6 (11.3) 23 (8.0)CEA < 3 ng/mL 102 (43.8) 16 (30.2) 118 (41.3) ≥ 3 ng/mL 102 (43.8) 16 (30.2) 118 (41.3) Not known 29 (12.4) 21 (39.6) 50 (17.4)RTOG RTOG 0 96 (41.2) 30 (56.6) 126 (44.1) RTOG I 75 (32.2) 14 (26.4) 89 (31.1) RTOG II 46 (19.7) 9 (17.0) 55 (19.2) RTOG III 13 (5.6) 0 (0.0) 13 (4.6) RTOG IV 3 (1.3) 0 (0.0) 3 (1.0) RTOG V 0 (0.0) 0 (0.0) 0 (0.0)

Weissenberger C et al. Anemia and advanced rectal cancer 1851

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years). The mean overall survival time was 4.4 years and the mean disease-free survival time was 2.73 years. The mean follow-up time was 8 years.

Survival and patient characteristicsComplete data about hemoglobin were available from 192 patients. When this subgroup (Table 2) was analyzed, anemia was found in 52.1% of the patients. Of the patients who received adjuvant radiotherapy, 64.6% showed increased hemoglobin during radiotherapy compared to 35.7% of the patients who were treated neoadjuvantly. Patients showed more frequently anemia if they were older than the median age or if R0 resection was performed. Anemia after radiotherapy was less frequently seen if postoperative radiotherapy was given instead of preoperative radiotherapy (37.8% vs 71.4%, respectively) or if low anterior resection (LAR) instead of abdominoperineal resection (APR) was performed (36.8% vs 51.4%, respectively), while 43.8% of the patients with stage III tumors showed increased hemoglobin during radiotherapy compared to 30.0% of the patients with stage II tumors (Table 7). Furthermore, patients less frequently suffered from increased hemoglobin if they received postoperative radiochemotherapy or their tumors underwent R0-resection. Decrease of hemoglobin was

found to be an independent risk factor for DFS with a risk ratio of 1.97 (CI 1.02 - 3.43). Conversely anemia before radiotherapy had a risk ratio of 0.76 (CI 0.57 - 0.99) (Table 6).

Tumor grading was found to be an independent prognostic factor for CSS and DFS (Table 3). The overall 5-year survival rate of patients with stage III tumors was about 10% compared to patients with stage II tumors (50.6% vs 40.7%; P = 0.023) (Table 3). Patients with stage II tumors showed a 5-year overall survival of 57 % (CSS: 65.8%; DFS: 53.3%) whereas patients with stage III tumors showed a 5-year overall survival of 42.6% (CSS: 57.2 %; DFS: 36.3%). Univariate analysis showed that the N stage was found to be significant for OAS and CSS (P = 0.06). The patients’ gender was found to be an independent factor for LRS. The 5-year LRS for female was significantly worse than that for males (56.8% vs 73.1%). No dependency between tumor location and outcome could be seen (Table 5). Tumor adherence to adjacent structures predicted survival (OAS P < 0.001, CSS P = 0.004, DFS P < 0.001, DRS P = 0.019) regardless of the surgical method, but was not significant for LRS (P = 0.13).

Survival and treatment characteristicsThe overall survival did not vary depending on the radiotherapy treatment type (either pre- or post-operative radiotherapy) (Table 4). Neither gross nor microscopic evidence of disease could be achieved in 37 of 53 (69.8%) preoperatively irradiated patients, and in 197 of 233 (84.5%) of postoperatively irradiated patients. Sphincter-saving surgery could be performed in 24.6% of patients receiving preoperative radiotherapy and in 56.8% of the patients receiving postoperative radiochemotherapy.

Multivariate analysis showed that R status (R0 vs R1/2) was an independent prognostic factor for disease-free survival (risk ratio 3.45, CI 1.56 -7.96) whereas surgical method (low anterior or abdominoperineal resection) was an independent prognostic factor for local control, determined as LRS (risk ratio 1.3, CI 1.01-1.60) (Table 4). Chi-square test showed no significant relationship between surgical method and staging (P = 0.93) or R status (P = 0.07) but a significant relationship between R status

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Cancer-specific survival

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Figure 1 Kaplan-Meier curves of OAS, CSS and DSF.

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Figure 2 Log-rank test of staging (5-year disease-free survival, P = 0.015).

Table 2 Anemia and hemoglobin values during radiotherapy (subgroup analysis of 192 patients)

Adjuvant Neoadjuvant All radiochemotherapy radiochemotherapy n (%) n (%) n (%)

Anemia before RT No 78 (47.6) 14 (50.0) 92 (47.9) Yes 86 (52.4) 14(50.0) 100 (52.1)Anemia after RT No 102 (62.2) 8 (28.6) 110 (57.3) Yes 62 (37.8) 20 (71.4) 82 (42.7)Hemoglobin during RT Hb increase 106 (64.6) 10 (35.7) 116 (60.4) Hb decrease 58 (35.4) 18 (64.3) 76 (39.6)


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Table 3 Survival rates and results of uni- and multivariate analysis (patient-related variables)

OAS (%) CSS (%) DFS (%) LRS (%) DRS (%)

All stages II & III 5 yr / 10 yr: 47.0 / 36.0 60.0 / 52.0 41.4 /34.9 67.2 / 65.7 84.3 / 84.3at risk (5 yr / 10 yr): 78 / 34 81 / 36 69 / 34 72 / 46 74 / 34median survival: 1602 996

Gender Male 5 yr / 10 yr: 49.2 / 36.3 64.9 / 56.8 24.7 / 12.9 73.1 / 73.1 85.1 / 85.1 Female 5 yr / 10 yr: 43.2 / 37.0 51.7 / 44.3 21.0 / 8.0 56.8 / 53.0 83.2 / 83.2

uni- / multivariate: P = 0.93 / NS 0.16 / NS 0.17 / NS 0.01 / 0.018 0.66 / NSmultivariate risk ratio: male: 0.75

(CI 0.59 – 0.95)Age < 62 yr 5 yr / 10 yr: 52.2 / 42.0 62.0 / 55.8 31.1 / 16.0 65.3 / 63.4 85.9 / 85.9 > 62 yr 5 yr / 10 yr: 41.0 / 27.0 57.1 / 42.6 13.6 / 5.1 70.4 / 70.4 80.2 / 80.2*Median age uni- / multivariate: P = 0.02 / NS 0.22 / 0.005 < 0.01 / NS 0.47 / NS 0.59 / NS

multivariate risk ratio: < 61 yr: 0.48(CI 0.28 – 0.80)

Staging II 5 yr / 10 yr: 57.0 / 49.9 65.8 / 62.5 53.3 / 45.5 75.1 / 75.1 85.5 / 85.5 III 5 yr / 10 yr: 42.6 / 30.2 57.2 / 47.4 36.3 / 28.7 63.3 / 61.0 83.8 / 83.8

uni- / multivariate: P = 0.02 / NS 0.09 / 0.009 0.02 / NS 0.06 / (0.07) 0.7 / NSmultivariate risk ratio: stage II: 0.45 stage II: 0.79

(CI 0.21 – 0.84) (CI 0.59 – 1.03)Grading II 5 yr / 10 yr: 50.6 / 39.7 62.0 / 55.1 49.3 / 41.7 73.3 / 70.7 86.8 / 86.8 III 5 yr / 10 yr: 40.7 / 36.6 55.3 / 49.8 23.3 / 23.3 58.8 / 58.8 64.4 / 64.4

uni- / multivariate: P = 0.023 / (0.06) 0.07 / NS < 0.01 / NS 0.04 / NS < 0.01 / 0.04multivariate risk ratio: G III: 1.68 G III: 2.87

(CI 0.97 – 2.78) (CI 1.08 – 7.01)

Risk ratio with confidence interval (CI) is given if multivariate analysis (step down analysis) revealed a significant result. Five- and 10- year survival rates are given as OAS, CSS, DFS, LRS and DRS.

Table 4 Survival rates and results of uni- and multivariate analysis (treatment-related variables)


Adjuvant therapy Postop RCT 5 yr / 10 yr: 47.6 / 35.6 59.0 / 48.4 40.2 / 34.0% 66.1 / 64.1 82.2 / 82.2 Preop RCT 5 yr / 10 yr: 43.9 / 38.0 62.6 / 62.6 45.4 / 39.3 69.8 / 69.8 92.8 / 92.8

uni- / multivariate: P = 0.82 / NS 0.56 / NS 0.61 / NS 0.96 / NS 0.12 / NSProtocol NIH 5 yr / 10 yr: 60.7 / 45.2 72.5 / 59.4 49.8 / 44.2 84.1 / 84.1 73.2 / 73.2 ARO 5 yr / 10 yr: 26.9 / 21.5 45.6 / 36.5 24.5 / 16.3 67.6 / 67.6 64.0 / 64.0

uni- / multivariate: P = < 0.001 / NS 0.003 / 0.004 0.002 / NS 0.024 / NS 0.57 / NSmultivariate risk ratio: ARO: 2.66

(CI 1.37 – 5.21)Surgery risk ratio: LAR 5 yr / 10 yr: 36.4 / 25.9 49.5 / 41.1 27.8 / 22.7 64.7 / 64.7 81.3 / 81.3 APR 5 yr / 10 yr: 35.2 / 26.0 46.7 / 39.9 27.6 / 22.9 52.4 / 51.1 86.4 / 86.4

uni- / multivariate: P = 0.4 / NS 0.51 / NS 0.27 / NS 0.015 / 0.039 0.6 / NSmultivariate risk ratio: LAR: 1.3

(CI 1.01 – 1.60)R status R0 5 yr / 10 yr: 53.2 / 40.3 64.2 / 55.1 45.6 / 39.5 66.2 / 64.5 91.2 / 91.2 R1 or R2 5 yr / 10 yr: 14.2 / 14.2 20.7 / 20.7 17.3 / 17.3 62.9 / 62.9 51.2 / 51.2 5

uni- / multivariate: P = < 0.001 / NS 0.011 / (0.09) < 0.001 / 0.026 0.95 / NS < 0.001 / NSmultivariate risk ratio: R1/R2: 1.65 R1/R2: 3.45

(CI 0.91 – 3.10) (1.56 – 7.96)

Risk ratio with confidence interval (CI) is given if multivariate analysis (step down analysis) revealed a significant result. Five- and 10- year survival rates are given as OAS, CSS, DFS, LRS and DRS.


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and grading (P = 0.01). The chemotherapy protocol was proved to be an independent prognostic factor (risk ratio 2.66, CI 1.37-5.21), showing a higher 5-year cancer-specific survival rate for patients treated with NIH protocol (72.5%) compared to ARO protocol (45.6%). Elevated CEA

(> 3 ng/mL) levels were proved to be an independent prognostic factor for OAS and DFS (Table 5). The risk ratios were 1.37 (CI 1.09- 1.73) for OAS and 3.21 (CI 1.61 - 6.82) for DFS, respectively. According to our patient documentation, 126 patients (44%) had no documented

Table 5 Results of uni- and multivariate analysis (other variables)

OAS CSS DFS LRS DRS

N stage uni- / multivariate: P = 0.009 / NS 0.05 / NS 0.06 / NS 0.09 / NS 0.15 / NSCEA uni- / multivariate: P = < 0.001 / 0.007 < 0.001 / NS < 0.001 / 0.001 < 0.001 / NS 0.55 / 0.055 multivariate risk ratio: CEA increase: 1.37 CEA increase: 3.21 CEA decrease: 0.91

(CI 1.09 – 1.73) (CI 1.61 – 6.82) (CI 0.76 – 1.00)CA 19-9 uni- / multivariate: P = < 0.001 / NS 0.033 / NS 0.22 / NS 0.79 / NS 0.62 / NSTumor location uni- / multivariate: P = 0.34 / NS 0.43 / NS 0.4 / NS 0.83 / NS 0.37 / NSAdherence to adjacent structures uni- / multivariate: P = < 0.001 / NS 0.004 / NS < 0.001 / NS 0.13 / NS 0.019 / NSBMI before RT uni- / multivariate: P = 0.47 / NS 0.55 / NS 0.83 / NS 0.83 / NS 0.15 / NSSmoking uni- / multivariate: P = 0.79 / NS 0.49 / NS 0.71 / NS 0.22 / NS 0.31 / NSHkt before vs after RT uni- / multivariate: P = 0.047 / NS 0.35 / NS 0.31 / NS 0.87 / NS 0.99 / NSLDH before vs after RT uni- / multivariate: P = 0.32 / NS 0.69 / NS 0.77 / NS 0.37 / NS 0.21 / NS

Risk ratio with confidence interval (CI) is given if multivariate analysis (step down analysis) revealed a significant result. Five- and 10- year survival rates are given as OAS, CSS, DFS, LRS and DRS

Table 6 Anemia and hemoglobin increase versus decrease during radiotherapy (subgroup analysis of 199 patients with documented hemoglobin values before and after radiotherapy)


Anemia before RCT No 5 yr / 10 yr: 44.6 / 31.4 66.4 / 66.4 41.5 / 33.7 76.6 / 76.6 74.9 / 74.9 Yes 5 yr / 10 yr: 49.5 / 47.2 55.4 / 45.6 48.5 / 39.2 61.5 / 59.6 88.1 / 88.1

uni- / multivariate: P = 0.39 / NS 0.14 / NS 0.83 / 0.04 0.04 / NS 0.01 / NSmultivariate risk ratio: anemia present: 0.76

(CI 0.57 – 0.99)Anemia after RCT No 5 yr / 10 yr: 47.5 / 34.0 57.4 / 48.4 42.5 / 34.7 87.6 / 87.6 62.7 / 60.7 Yes 5 yr / 10 yr: 47.0 / 41.5 64.7 / 59.3 39.6 / 35.9 80.0 / 80.0 73.0 / 73.0

uni- / multivariate: P = 0.76 / NS 0.56 / NS 0.83 / NS 0.12 / NS 0.06 / NSmultivariate risk ratio:

Hemoglobin (Hb) increase or decrease during RT

Hb increase 5 yr / 10 yr: 67.7 / 59.3 58.8 / 51.4 45.2 / 45.2 83.8 / 83.8 62.8 / 62.8 Hb decrease 5 yr / 10 yr: 35.3 / 35.3 38.7 / 33.9 36.2 / 36.2 73.9 / 73.9 75.9 / 75.9

uni- / multivariate: P = < 0.01 / NS < 0.01 / NS 0.08 / 0.04 0.38 / NS 0.79 / NSmultivariate risk ratio: Hb decrease: 1.97

(CI 1.02 – 3.43)

Survival rates and results of the uni- and multivariate analysis as well as risk ratio with confidence interval (CI) are given if multivariate analysis (step down analysis) revealed a significant result. Five- and 10-year survival rates are given as OAS, CSS, DFS, LRS and DRS


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side effects (Table 1), while 89 (31.1%), 55 (19.2%), 13 (4.5%), and 3 (0.7%) patients had RTOG grade I, II, III or IV side effects, respectively. Diarrhoea, dysuria and skin reaction were the leading problems whereas other side effects were rarely reported.

dIscussIonConcerning the outcome of rectal cancer, patients with younger age, lower stage, tumor grading GII or better, no elevated tumor markers, and a higher distance from the anal verge may fare better[8, 18, 19]. There is also some evidence that R0 resection, no adherence to adjacent structures, and lower anterior resection (LAR), performed with quality-controlled total mesorectal excision (TME), are beneficial factors for the local control whereas positive lymph nodes are risk factors mainly for distant metastases. The impact of radiotherapy is still debated. Moreover, valid data from other tumor entities suggest that anemia may have predictive value for local relapse or distant recurrence[5, 6]. At the Department of Radiotherapy, University Hospital Freiburg from 1989 to 2002, 286 patients with stage II or III cancer were treated and qualified for our retrospective study analyzing patient- or treatment-related variables and their impact on treatment outcome. This study, however, has some limitations mainly due to lack of data about the quality of surgery and incomplete patient documentation.

Therefore, the extent and quality of total mesorectal excision could not be assessed. The impact of anemia on 192 patients with complete data about hemoglobin during radiochemotherapy was analyzed.

The overall 5-year survival rate (47.0%) and disease-free survival rate (41.1%) were lower than those of other large randomized trials[3, 4]. It is known that the outcome of therapeutic interventions obtained in studies usually exceed “the reality” of population-based reports of cancer treatment[21]. The rate of distant recurrence (16.7%) was slightly lower in our patient cohort than that reported in literature (Table 4), which may be due to the modified sequence of radiochemotherapy. This scheme of early radiochemotherapy can achieve significantly improved rates of local recurrence and distant metastasis[22], suggesting that early radiotherapy as performed in our study is more effective concerning local control and consequently more effective concerning distant outcome.

The Mayo study and other studies are influenced by a local relapse rate of about 30 %[3, 4]. These studies could reflect the situation before the “era” of trained and quality-controlled total mesorectal excision. Similarly, we found that a local recurrence rate of 32.8% (local-relapse-free survival of 67.2%) is consistent with current literature[23]. After conventional and non-standardized surgery, local recurrence was 15%-55%[24-26]. However, it was reported that the 5-year local control rate was higher than 90 %, even without chemotherapy or any adjuvant treatment[27-29].

Tumor adherence to the adjacent structures can predict local recurrence and survival[30-32]. Consistent with these data, the Freiburg patient cohort including 286 cases showed that all survival rates (OAS, CSS, DFS) were associated with tumor adherence. This may be due to the effect of sample size or support the contention that out-spread of the gross tumor should be estimated as an important indicator for systemic tumor progression leading to worse outcome. According to this, it has been recently shown that even among N2 patients (4 or more positive lymph nodes), T stage influences overall 5-year survival[22]. Furthermore, T and N classifications can predict survival, but only N stage is correlated with local recurrence[30]. On the contrary, in our retrospective study N stage was significant for overall survival (P = 0.009) and cancer-specific survival (P =0.05), whereas a trend for local recurrence (P =0.09) and disease-free survival (P = 0.06) could be seen.

When an average radiation dose of 50 Gy is used for preoperative or postoperative radiochemotherapy, patients classified as N0 have better results than N positive ones[33]. Similarly, analysis of eligible patients has confirmed the effect of N stage[19]. Notably our data suggest that N positive patients are even more substantially at risk for local failure rather than for distant metastasis. Apparently, spread-out to lymph nodes indicates a more aggressive nature of local tumor growth. Concerning treatment intervention, these results support the hypothesis that sterilization of tumor cells in lymph nodes could be achieved in many patients, preventing them from distant metastasis. As a consequence, this contradicts to any efforts to minimize radiation fields. Only if adequately included in the radiation field, the tumor cells in regional

Table 7 Anemia and patient characteristics.

Hb decrease during RT n (%) P value

Age ≤ 621yr 42 (42.9) 0.31 > 621yr 32 (35.6)Stage II 18 (30.0) 0.07 III 56 (43.8)Other serious diagnoses No 38 (35.2) 0.17 Yes 36 (45.0)BMI ≤ 24.22 44 (50.0) 0.03 > 24.22 18 (26.5)Radiochemotherapy Postoperative 56 (35.0) 0.03 Preoperative 18 (64.3)Surgical Resection LAR 42 (38.2) 0.96 APR 28 (37.8)R status R 0 24 (30.8) 0.04 R 1/2 10 (55.6)Side effects RTOG 0 8 (80.0) 0.27 RTOG I / II 62 (38.8) RTOG III / IV 4 (22.2)

Results of chi-square test. 1Median age, 2median BMI (body mass index).


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lymph nodes can be killed.In the present study, when anemia and hemoglobin

were analyzed, conflicting results were obtained from the multivariate analysis. When hemoglobin values decreased during radiotherapy it meant to be a risk factor for patients, possibly indicating bad performance status, prolonged convalescence, side effects of therapy or progressive disease (Table 7). But anemia had a risk ratio of 0.76 for DFS (Table 6), suggesting that rectal cancer patients with anemia before radiotherapy have better results than patients without anemia. Considering the adjuvant setting at a time interval of 3 - 5 wk between surgery and radiochemotherapy, anemia can be regarded as a consequence of a more radical surgical intervention. This hypothesis is supported by the analysis of 192 patients with complete data about hemoglobin during radiochemotherapy: 60.0% of patients after R0-resection had anemia compared to 55.6% of patients after R1/2-resection. After radiotherapy only 42.5% patients after R0-resection still had anemia compared to 44.4% patients after R1/2-resection. Only 30.8% patients after R0-resection suffered from hemoglobin decrease during radiotherapy compared to 55.6 % patients after the R1/2-resection, suggesting that R status is an independent prognostic factor for disease-free survival. According to our data, anemia before radiochemotherapy or hemoglobin decrease during radiotherapy does not imply any predictive value for the outcome of rectal cancer. It should be considered as a readout of age, stage, type of surgical resection, or performance status. Taken together, if there is any hemoglobin effect on the outcome of rectal cancer, it is completely biased by the extent of surgical intervention, which is one of the most important predictors for the outcome of rectal cancer.

Multivariate analysis of our data revealed that tumor grading was the most important prognostic factor for CSS and DFS. In a retrospective study of 214 patients with primary rectal carcinoma, Luna-Perez et al[33] have identified well-differentiated cancer as a prognostic factor for achieving local control. Martijn et al[31] also found that tumor grading has a prognostic value, which is consistent with our study. We found that there was no significant difference in survival rate regarding tumors in the lower, middle or upper third. Thus, the hypothesis that tumor location influences response rate or survival is not supported by our data. Phang et al[8] reported that survival is affected by tumor distance from the anus. Lower distance significantly can worsen survival and distant recurrence rate but not the rate of local recurrence. Finally, an independent detrimental influence on local recurrence has been proved by a retrospective study of 197 patients, using conventional resection technique[32].

By analyzing the influence of the resection type, we revealed a better control rate of local relapse (34%) if the patients were treated by low anterior resection (LAR) compared to abdominoperineal resection (APR). Out data are consistent with a study of Stocchi et al[30] showing a better rate for LAR (28%) but not with a study of Kuru et al[32] who identified anterior resection as a negative independent prognostic factor for local control. However, Stocchi et al[30] stated that the overall rate of

local recurrence (16%) is unexpectedly low in their study, considering the high-risk patient cohort and the data from literature. Unlike this, our data (32.8 % local recurrence in five years) could obviously reflect the “surgical reality today”[34] or, more accurately, the surgical results in the era (Table 6) before comprehensive surgical quality control of TME[36].

In the Freiburg cohort male gender was found to be an independent prognostic factor for local recurrences. No difference was found in OAS, CSS, DFS and DRS. It was reported that DFS and local recurrence do not differ significantly in gender, but differ significantly in OAS with a risk ratio 1.2 (P = 0.03) for men. Furthermore, increased grade III or IV toxicity in females has been described. As in our study, no interaction between sex and age was seen. Therefore sex as an indicator for higher age can be ruled out. Studies evaluating psycho-oncological aspects have identified gender-related factors which influence cancer treatment and outcome[38]. However, if sex difference has any significant impact on outcome, this effect seems to be inconsistent and may be influenced by local differences in lifestyle or environmental factors.

Among the patients-related factors, age is known to be crucial, because administration of adjuvant chemotherapy to patients aged over 70 years remains a difficult choice for the clinicians with respect to the expected benefit versus toxicity. Sargant et al[39] and Popescu et al [40] showed that old patients (with good performance status and renal and hepatic function) share the same benefit from adjuvant chemotherapy as younger patients without increasing toxicity. CEA and CA 19-9 levels are reliable tumor markers for rectal cancer[18, 41, 42]. In our study, the prognostic value of CEA could be shown by uni- and multivariate analysis. Additionally, the increase of CEA during radiotherapy indicated a decreased survival rate. Our results are consistent with that of Behbehani et al[43], who demonstrated that patients with preoperatively elevated CEA levels have a 2-year DFS of 23% whereas patients without CEA elevation have a 2-year DFS of 71%.

Our retrospective review of the Freiburg patient cohort did not detect any difference between preoperative and postoperative radiochemotherapy. Cancer-specific survival was 62.6% and 59.0%, respectively. Our data correspond to Rinkus et al[34] who reviewed 292 patients showing no significant difference in survival between preoperative versus postoperative radiotherapy (combined with chemotherapy in 66% and 48% of the cases, respectively).

It was reported that preoperative radiation can significantly decrease local failure rate. The 5-year survival curve does not differ significantly between both strategies. Until now the German trial (Protocol CAO/ARO/AIO 94) is the only prospective study with patients randomized to conventional preoperative and postoperative radiochemotherapy[20]. This study showed that preoperative radiochemotherapy could improve local control and reduce toxicity but overall survival was not different between pre- and postoperative radiochemotherapy. However, our retrospective, non-randomized data might be biased as preoperative radiochemotherapy was mainly given if the tumor was inoperable. Therefore these patients were in more


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advanced tumor stages. Supposing an advantage of preoperative radiochemotherapy, as shown by Sauer et al[20], this may explain why preoperative and postoperative radiochemotherapy seem to be equivalent according to our data.

In conclusion, new therapeutic strategies are urgently needed, preferably based on a better understanding of beneficial or disadvantageous factors determining the outcome of rectal cancer. Tumor stage, grading, R status (free radial margins), type of surgery, CEA levels, and gender are predictive factor for the outcome of rectal cancer. However, anemia before radiochemotherapy or hemoglobin decrease during radiotherapy does not imply any predictive value for the outcome of rectal cancer. It should be considered as an indicator for higher age, stage, type of surgical resection, or performance status. Any hemoglobin effect of radiotherapy on outcome of rectal cancer is completely biased by the extent of surgical intervention.

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19 GundersonLL, Sargent DJ, Tepper JE, O‘Connell MJ, Allmer C, Smalley SR, Martenson JA, Haller DG, Mayer RJ, Rich TA, Ajani JA, Macdonald JS, Goldberg RM. Impact of T and N sub-stage on survival and disease relapse in adjuvant rectal cancer: a pooled analysis. Int J Radiat Oncol Biol Phys 2002; 54: 386-396

20 SauerR, Becker H, Hohenberger W, Rödel C, Wittekind C, Fi-etkau R, Martus P, Tschmelitsch J, Hager E, Hess CF, Karstens JH, Liersch T, Schmidberger H, Raab R. Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med 2004; 351: 1731-1740

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22 LeeJH, Lee JH, Ahn JH, Bahng H, Kim TW, Kang YK, Lee KH, Kim JC, Yu CS, Kim JH, Ahn SD, Kim WK, Kim SH, Lee JS. Randomized trial of postoperative adjuvant therapy in stage II and III rectal cancer to define the optimal sequence of chemo-therapy and radiotherapy: a preliminary report. J Clin Oncol 2002; 20: 1751-1758

23 Arnaud JP, Nordlinger B, Bosset JF, Boes GH, Sahmoud T, Schlag PM, Pene F. Radical surgery and postoperative radio-therapy as combined treatment in rectal cancer. Final results of a phase III study of the European Organization for Research and Treatment of Cancer. Br J Surg 1997; 84: 352-357

24 McCall JL, Cox MR, Wattchow DA. Analysis of local recur-rence rates after surgery alone for rectal cancer. Int J Colorectal Dis 1995; 10: 126-132

25 PhillipsRK, Hittinger R, Blesovsky L, Fry JS, Fielding LP. Local recurrence following ‘curative’ surgery for large bowel cancer: I. The overall picture. Br J Surg 1984; 71: 12-16

26 HarnsbergerJR, Vernava VM 3rd, Longo WE. Radical abdom-inopelvic lymphadenectomy: historic perspective and current role in the surgical management of rectal cancer. Dis Colon Rectum 1994; 37: 73-87

27 EnkerWE, Merchant N, Cohen AM, Lanouette NM, Swallow C, Guillem J, Paty P, Minsky B, Weyrauch K, Quan SH. Safety and efficacy of low anterior resection for rectal cancer: 681 consecutive cases from a specialty service. Ann Surg 1999; 230:544-52; discussion 552-554

28 MacFarlane JK, Ryall RD, Heald RJ. Mesorectal excision for rectal cancer. Lancet 1993; 341: 457-460

29 Tang R, Wang JY, Chen JS, Chang-Chien CR, Lin SE, Leung S, Fan HA. Postoperative adjuvant radiotherapy in Astler-Coller stages B2 and C rectal cancer. Dis Colon Rectum 1992; 35: 1057-1065

30 Stocchi L, Nelson H, Sargent DJ, O‘Connell MJ, Tepper JE, Krook JE, Beart R Jr. Impact of surgical and pathologic vari-ables in rectal cancer: a United States community and coopera-tive group report. J Clin Oncol 2001; 19: 3895-3902

31 Martijn H, de Neve W, Lybeert ML, Crommelin MA, Ribot JG. Adjuvant postoperative radiotherapy for adenocarcinoma of the rectum and rectosigmoid. A retrospective analysis of locoregional control, survival, and prognostic factors on 178 patients. Am J Clin Oncol 1995; 18: 277-281

32 KuruB, Camlibel M, Dinç S, Erdem E, Alagöl H. Prognostic factors affecting local recurrence and survival for operable rec-tal cancers. J Exp Clin Cancer Res 2002; 21: 329-335

33 Luna-PérezP, Rodríguez-Ramírez S, Vega J, Sandoval E, La-bastida S. Morbidity and mortality following abdominoperi-neal resection for low rectal adenocarcinoma. Rev Invest Clin 2001; 53: 388-395

34 RinkusKM, Russell GB, Levine EA. Prognostic significance of nodal disease following preoperative radiation for rectal ad-enocarcinoma. Am Surg 2002; 68: 482-487


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35 LinkKH, Staib L, Schatz M, Suhr P, Röttinger E, Beger HG. Adjuvant radiochemotherapy--what is the patients benefit? Langenbecks Arch Surg 1998; 383: 416-426

36 Kapiteijn E, van de Velde CJ. Developments and quality as-surance in rectal cancer surgery. Eur J Cancer 2002; 38: 919-936

37 Tepper JE, O‘Connell M, Niedzwiecki D, Hollis DR, Benson AB 3rd, Cummings B, Gunderson LL, Macdonald JS, Marten-son JA, Mayer RJ. Adjuvant therapy in rectal cancer: analysis of stage, sex, and local control--final report of intergroup 0114. J Clin Oncol 2002; 20: 1744-1750

38 MoynihanC. Men, women, gender and cancer. Eur J Cancer Care (Engl ) 2002; 11: 166-172

39 SargentDJ, Goldberg RM, Jacobson SD, Macdonald JS, Labi-anca R, Haller DG, Shepherd LE, Seitz JF, Francini G. A pooled analysis of adjuvant chemotherapy for resected colon cancer in elderly patients. N Engl J Med 2001; 345: 1091-1097

40 Popescu RA, Norman A, Ross PJ, Parikh B, Cunningham D. Adjuvant or palliative chemotherapy for colorectal cancer in

patients 70 years or older. J Clin Oncol 1999; 17: 2412-2418 41 Reiter W, Stieber P, Reuter C, Nagel D, Lau-Werner U, Pahl

H, Fateh-Moghadam A. Preoperative serum levels of CEA and CA 19-9 and their prognostic significance in colorectal carci-noma. Anticancer Res 1997; 17: 2935-2938

42 NelsonRL. The usefulness of carcinoembryonic antigen in postoperative colorectal cancer patients. Dis Colon Rectum 1997; 40: 866-867

43 BehbehaniAI, Al-Sayer H, Farghaly M, Kanawati N, Mathew A, al-Bader A, Van Dalen A. Prognostic significance of CEA and CA 19-9 in colorectal cancer in Kuwait. Int J Biol Markers 2000; 15: 51-55

44 FrykholmGJ, Glimelius B, Påhlman L. Preoperative or post-operative irradiation in adenocarcinoma of the rectum: final treatment results of a randomized trial and an evaluation of late secondary effects. Dis Colon Rectum 1993; 36: 564-572

45 PåhlmanL, Glimelius B Pre-operative and post-operative ra-diotherapy and rectal cancer. World J Surg 1992; 16: 858-865

S-Editor Wang J L-Editor Wang XL E-EditorBai SH


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Key words: Colon cancer; Src; JAK2; Phosphatidylinositol 3-kinase; Glycine-extended gastrin

Ferrand A, Kowalski-Chauvel A, Pannequin J, Bertrand C, Fourmy D, Dufresne M, Seva C. Glycine-extended gastrin activates two independent tyrosine-kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cells. World J Gastroenterol 2006; 12(12): 1859-1864


INTRODUCTIONHyperproliferation of the colonic mucosa is associated with an increased risk of tumour development, and cor-responds to an early stage in the adenoma-carcinoma sequence. It is now well established that glycine-extended gastrin (G-gly), the precursor of the mature amidated gas-trin, plays an important role in colonic mucosa hyperpro-liferation. Proliferative effects of G-gly were first described in a pancreatic tumour cell line, AR4-2J[1]. Afterwards, numerous studies have confirmed its mitogenic effects, es-pecially on colonic mucosa, and in vitro studies have shown that G-gly is a growth factor for non transformed cell lines from colon origin or human colon cancer cells[2-5]. Trophic effects of G-gly have also been confirmed in vivo and MTI/G-gly transgenic mice overexpressing G-gly, as well as gastrin-deficient mice perfused with this peptide, dis-play hyperproliferation of the colonic mucosa[6]. Further-more, perfusion of G-gly into rats results in proliferation of colonic mucosal cells forming aberrant crypt foci and increases the sensitivity to azoxymethane, a colon carcino-gen[7].

The p85/p110 PI3K is a lipid kinase composed of two constitutively associated subunits: p85, the regulatory subunit; and p110, the catalytic subunit. Upon stimula-tion, PI3K phosphorylates the D3 position of phosphoi-nositides leading to second messengers, namely phosphati-dylinositol 3, 4 biphosphate and phosphatidylinositol 3, 4, 5 triphosphate[8]. The PI3K pathway is involved in the regulation of many cellular processes including prolifera-

Glycine-extended gastrin activates two independent tyrosine-kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cells

Audrey Ferrand, Aline Kowalski-Chauvel, Julie Pannequin, Claudine Bertrand, Daniel Fourmy, Marlene Dufresne, Catherine Seva

Audrey Ferrand, Aline Kowalski-Chauvel, Claudine Bertrand, Daniel Fourmy, Marlene Dufresne, Catherine Seva, INSERM U.531, Groupe de Recherche de Biologie et Pathologie Digestives, IFR31, Institut Louis Bugnard, BP 84225, 31432 toulouse Cedex 4, FranceJulie Pannequin, University of Melbourne Department of Surgery, Austin Campus, ARMC, Heidelberg, Victoria 3084, AustraliaSupported by INSERM, the "Association pour la Recherche Contre le Cancer" Grants # 3664, # 4430, and the "Ligue Contre le Cancer"Correspondence to: Catherine Seva, IFR31, Institut Louis Bugnard, BP 84225, Unité INSERM 531, Biologie et Pathologie Digestives, 31432 toulouse Cedex 4, France. [email protected] Telephone: +33-5-61322408 Fax: +33-5-61322403Received: 2005-09-13 Accepted: 2005-10-26

AbstractAIM: To investigate whether Src, JAK2 and phosphati-dylinositol 3-kinase (PI3K) pathways are involved in the proliferation of human colonic tumour cells induced by glycine-extended gastrin (G-gly), the precursor of the mature amidated gastrin and to elucidate the molecular interaction between these three kinases in response to this peptide.

METHODS: Using the human colonic tumour cell line HCT116 as a model, we first measured the activation of PI3K, p60-Src and JAK2 in response to G-gly by in vitro kinase assays. Then we investigated the involvement of these kinases in G-gly-induced cell proliferation by MTT test.

RESULTS: G-gly stimulation induced p60-Src, JAK2 and PI3K activation in HCT116. The different pathways were involved in proliferation of human colon cancer cells induced by G-gly. Furthermore, we found that both Src and JAK2 were necessary to PI3K regulation by this pep-tide. However, we did not find any cross-talk between the two tyrosine kinases.

CONCLUSION: Our results suggest that the p60-Src/PI3K and JAK2/PI3K pathways act independently to me-diate G-gly proliferative effect on human colonic tumour cells.


COLORECTAL CANCER

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tion and survival. During the last years, many studies have shown the implication of the PI3K in colon carcinogen-esis. In particular, the PI3K has been found to play an important role in colon cancer development and progres-sion by promoting cell growth and allowing cells to escape apoptosis[9]. Activation of this pathway is also involved in the progression of human colon adenocarcinoma[10].

Different groups including ours, have demonstrated the involvement of the phosphatidylinositol 3-kinase in G-gly functions. We have reported the activation of lipid kinase in response to this peptide in a pancreatic tumoral cell line, AR4-2J[11]. Afterwards, Hollande et al[12] have described the involvement of the phosphatidylinositol 3-kinase in adhesion and migration of gastric epithelial cells regulated by G-gly. More recently, we described the overexpression of the regulatory subunit p85 as well as an overactivation of the downstream effector Akt, in the hyperprolifera-tive colonic mucosal epithelium of MTI/G-Gly mice[13]. Moreover, in the same study, we have demonstrated the involvement of the PI3K pathway as well as Src and JAK2 pathways in the proliferation of isolated normal murine colonic epithelial cells in response to G-gly.

Our aim here was to investigate whether these three kinases are involved in G-gly-induced proliferation of hu-man colonic tumour cells and to elucidate the molecular interaction between Src, JAK2 and PI3K in response to the peptide. In HCT116, a human colon tumour cell line, we first measured the activation of these kinases by G-gly as well as their involvement in G-gly-induced cells prolifer-ation. The results indicate that both p60-Src and JAK2 are necessary to PI3K regulation by the peptide. However, we did not observe any cross-talk between the two tyrosine ki-nases, suggesting that the p60-Src/PI3K and JAK2/PI3K pathways act independently.

MATERIALS AND METHODSMaterialsPolyclonal anti-JAK2 antibody was purchased from Up-state Biotechnology Inc. Monoclonal anti-p60-Src anti-body was obtained from Oncogene Science. Rabbit poly-clonal antibody specific to p85, the regulatory subunit of the phosphatidylinositol 3-kinase, was kindly provided by Drs. Y. Le Marchand-Brustel and J. F. Tanti (Nice, France). DFO, LY 294002, PP2 and AG490 were from Calbiochem. [γ-32P] ATP (7000 Ci/mmol) was from ICN. Phosphati-dylinositol was purchased from Sigma.

Cell cultureHCT116, human colonic tumour cells, were grown in DMEM containing 4.5g/L glucose-glutamax, supplement-ed with 10% fetal calf serum at 37 ℃ in a 50mL/L CO2 atmosphere.

Proliferation assayApproximately 75 000 cells/well were plated into 96-well plates. Forty-eight hours after plating, cells were treated for 48 h with G-gly (10 pmol/L) with or without inhibitors (10µmol/L). MTT colorimetric assay (MTT, Sigma) was used to measure proliferation as previously described[13].

Immunoprecipitation and Western-blot analysisHCT116 cells were serum-starved for 18 h before peptide addition. After stimulation, the cells were lysed, and the soluble fractions containing identical levels of proteins were immunoprecipitated and analyzed by Western-blot with the indicated antibodies as described previously[14]. Band intensity was analyzed using the image analyzer Bio-com (France).

Src kinase assaySrc kinase activities were determined on anti-p60-Src-im-munoprecipitates as previously described[15]. Briefly, kinase assay was carried out at 37 ℃ for 10 min in kinase buffer (12.5 mmol/L MnCl2, 1.25 mmol/L DTT, 25 mmol/L Hepes, pH 7.4) containing 3.75 µmol/L ATP, 10 µCi/point ATP[γ-32P]. Proteins were separated by SDS–PAGE and the gel was autoradiographied. The gels were then treated with 1N KOH at 50 ℃ for 1h and autoradiographied. Band intensity was analyzed using the image analyzer Bio-com (France).

JAK2 kinase assayJAK2 kinase activities were determined on anti-JAK2-im-munoprecipitates as previously described[14]. Briefly, kinase assay was carried out at 37 ℃ for 10 min in kinase buffer (10 mmol/L MnCl2, 5 mmol/L MgCl2, 0.1 mmol/L Vn2+, 10 mmol/L Tris, pH 7.4) containing 3.75 µmol/L ATP, 10 µCi/point ATP[γ-32P]. Proteins were separated by SDS–PAGE and the gel was autoradiographed. Band intensity was analyzed using the image analyzer Biocom (France).

Phosphatidylinositol 3-kinase assayCell lysates were immunoprecipitated with the indicated antibodies. PI3K assay was performed on immunoprecipi-tates as described previously[16]. Briefly, phosphatidylinositol used as an exogenous substrate, was prepared in 5 mmol/L HEPES and then sonicated for 15 min at 4 ℃. The sam-ples were incubated for 15 min at room temperature with phosphatidylinositol (0.2 mg/mL) and 50 µmol/L ATP[γ-32P] (10 µCi/point). The reaction was stopped by adding HCl 4N and lipids were extracted using agitation in chloro-forme/methanol (1/1) solution for 45 s. After centrifuga-tion, the upper phase containing the lipids was analyzed. The phospholipids were separated by thin layer chromatog-raphy and analyzed by autoradiography. Spot intensity was analyzed using the image analyzer Biocom (France). When indicated, the cells were pretreated for 1h with 1µmol/L desferrioxamine (DFO).

Statistical analysisStatistical analysis was carried out by Student’s t test using GraphPad Prism.

RESULTSGlycine-extended gastrin activated phosphatidylinositol 3-kinaseWe first tested whether the p85/p110 phosphatidylinositol 3-kinase (PI3K) was activated in HCT116 cells after G-gly stimulation. Cells were treated with the peptide for varying


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lengths of time. Lipid kinase assay was performed in vitro on anti-p85 immunoprecipitates using inositol phosphate as an exogenous substrate. Lipids were then separated by thin layer chromatography. Increase in phosphatidylinosi-tol phosphorylation was detected within 3-5 min after treatment with G-gly. This stimulation similar to our pre-vious observation in pancreatic tumour cells[11], indicated that PI3K activation was also an early event of G-gly signalling in colon cancer cells. The ferric ion chelator, desferrioxamine (DFO) could inhibit both the binding of G-gly to its receptor and biological activity[17]. To test the specificity of the activation by G-gly, we performed PI3K assays after pretreatment of the cells with DF0 for 1h. DFO could completely inhibit PI3K activation in response to the peptide (Figure 1).

Glycine-extended gastrin activated tyrosine kinases p60-Src and JAK2To investigate whether glycine-extended gastrin could reg-ulate p60-Src activation, serum-starved HCT116 cells were treated with G-gly for various times and lysed. Tyrosine kinase assays were performed in anti-p60-Src immunopre-cipitates using enolase as an exogenous substrate. Our re-sults indicated that p60-src was very rapidly and transiently

activated in HCT116 cells in response to G-gly. The activa-tion of p60-Src was detected 1 min after peptide addition. The stimulation was maximal at 3 min and decreased after 5 min (Figure 2A).

To test whether JAK2 was activated by G-gly, lysates from cells stimulated for various times were immunopre-cipitated with anti-JAK2 antibodies and in vitro kinase as-says were performed on immunoprecipitates as described in “Methods”. We observed a rapid and transient increase in JAK2 autophosphorylation in response to G-gly stimu-lation. The maximal activation obtained within 5 min after peptide addition decreased toward the basal level at 15 min (Figure 2B).

Involvement of JAK2, Src and phosphatidylinositol 3-ki-nase pathways in glycine-extended gastrin-induced prolif-erationIn order to identify the role of PI3K, Src and JAK2 path-ways in the proliferation of human colonic tumour cells induced by G-gly, we measured HCT116 proliferation in the presence or absence of specific inhibitors for each pathway, LY290042, PP2 and AG490 respectively. Stimula-tion of HCT116 cells for 48h by G-gly induced a signifi-cant increase of cell proliferation (mean ± SE; 1.38 ± 0.07; P<0.05; n = 5) (Figure 3). Treatment of the cells with PP2, AG 490 or LY 290042 completely inhibited G-gly-induced HCT116 proliferation while inhibitors alone did not af-fect basal cell proliferation (data not shown). The result indicated that these three pathways could mediate G-gly proliferative effects not only on normal colonic epithelial cells but also on human colonic tumoral cells.

Involvement of both p60-Src and JAK2 upstream of PI3K activation in response to G-glyIn order to determine whether p60-Src was involved in PI3K activation by G-gly, we first tested whether PI3K activity was detected in association with p60-Src. After peptide treatment for various times, PI3K assays were car-ried out on anti-p60-Src immunoprecipitates. In response to G-gly, we observed an increased PI-kinase activity co-precipitated with maximal p60-Src after 5 min of peptide stimulation (Figure 4A). To test the PI3K specificity, we

IP: anti-p85

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Figure 1 G-gly- induced PI3K (A ) and G-gly (B ) activation. HCT116 cells were stimulated with G-gly (10 pmol/L) for the indicated t ime. PI3K act iv i ty was determined in an immune complex kinase assay as desc r i bed i n Me thods . The speci f ic i ty of G-gly activation was accessed by pretreat ing the cel ls with ferric chelator DFO (1µmol/L). Representative autoradiograms from at least three separate experiments are shown.

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Figure 2 G-gly-induced p60-Src (A) and JAK2 (B) activation. HCT116 cells were stimulated with G-gly (10 pmol/L) for the indicated time. p60-Src and JAK2 activities we re de te rm ined i n an immune complex k i n a s e a s s a y a s described in Methods. Immunoprec ip i ta ted p r o t e i n s w e r e a l s o analysed by Western-b lo t us ing the an t i -p60-Src and anti-JAK2 antibody respectively. R e p r e s e n t a t i v e autoradiogram from at least three separate experiments is shown.150

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Figure 3 Role of PI3K, Src or JAK2 pathways in human colonic tumour cell proliferation induced by G-gly. Serum-starved HCT116 cells were treated with G-gly for 48h in the presence or absence of the indicated inhibitor (10 µmol/L). The proliferation was determined as described in Methods. Each point represents data from triplicate and is representative of 5 independent experiments. Data are presented as means ± SE.

Ferrand A et al. Kinases in human colon cancer cell growth 1861

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added the PI3K inhibitor, LY 294002, in the in vitro li-pid kinase assay. Under these conditions, G-gly induced-PI-kinase activity was completely blocked. These results demonstrated that glycine-extended gastrin was associated with p60-Src and PI3K. In order to confirm that an Src family member could act upstream of the PI3K pathway activated by G-gly, we pretreated the cells for 1h with PP2 before G-gly stimulation. PI3K activation was inhibited in PP2 pretreated cells, indicating the role of Src in PI3K activation (Figure 4B).

We then examined the role of JAK2 in PI3K activation by G-gly. After peptide treatment for the time indicated, the cells were lysed and PI3K assays were performed on anti-JAK2 immunoprecipitates. Increased PI-kinase activity was associated with JAK2, detectable at 1 min and maxi-

mal after 5 min of peptide stimulation (Figure 5A). The inhibition of PI-kinase activity observed when LY 294002 was added in the in vitro lipid kinase assay indicated that glycine-extended gastrin was associated with JAK2 and PI3K. To confirm the involvement of JAK2 upstream of the G-gly-induced PI3K pathway, the cells were pretreated for 1h with AG 490 prior to G-gly addition. The PI3K activation in response to G-gly was inhibited in the pres-ence of JAK2 inhibitor, indicating the role of this tyrosine kinase upstream in PI3K activation (Figure 5B).

No cross-talk between p60-Src and JAK2 pathways in response to G-glyTo investigate the possible association between p60-Src and JAK2, solubilized proteins were immunoprecipitated with an anti-JAK2 antibody and the precipitates were analyzed by immunoblot using an anti-p60-Src antibody. No p60-Src protein associated with JAK2 was detected in control cells or in cells stimulated with G-gly, whereas comparable amounts of JAK2 proteins were detected in immunoprecipitates (data not shown).

Independently of any association, we studied whether Src-family kinases were involved in JAK2 activation by G-gly. We tested the effect of PP2 on JAK2 activity. Cells were pretreated for 1h with PP2 5 min before stimulation with G-gly. JAK2 kinase assay was performed on JAK2 im-munoprecipitates. PP2 did not inhibit G-gly-induced JAK2 activation, indicating that Src kinases were not involved in upstream of JAK2 activation by G-gly (Figure 6A). Similarly, using AG490, we tested a possible role of JAK2 upstream in p60-Src activation. The presence of AG490 in the incubation medium did not significantly change p60-Src activation by G-gly (Figure 6B). Indeed, in this human colon tumoral cell line, activation of the two tyrosine-ki-nases, p60-Src and JAK2, in response to G-gly was entirely independent.

DISCUSSIONThe p85/p110 phosphatidylinositol 3-kinase (PI3K) is a

PIP

Time: 0 1 5 15 5(min) LY 294002

IP: Src

PIP

Time: 0 3 5 15 5(min) LY 294002

IP: JAK2

PIP

IP: p85

G-gly - + - + PP2

PIP

G-gly - + - + AG490

IP: p85

A

B

A

B

Figure 4 Involvement of p60-Src (A) and PI3K (B) activation in response to G-gly. HCT116 cells pretreated with or without 10 µmol/L of LY290042 and anti-p85 antibody were stimulated (+) or not (-) with glycine-extended gastrin (10 pmol/L) for the indicated time. After immunoprecipitation of cell lysates with anti-p60-Src and anti-p85 antibody, precipitates were assayed for PIK activity using inositol phosphate as an exogenous substrate. Lipids were separated by thin layer chromatography and autoradiographied. The result shown is representative from five and three separate experiments.

Figure 5 Involvement of PI3K (A) and JAK2 (B) activation in response to G-gly. HCT116 cells pretreated with or without 10µmol/L of LY290042 and were 10µmol/L of AG 40, stimulated (+) or not (-) with glycine-extended gastrin G-gly (10 pmol/L) for the indicated time. After immunoprecipitation of cell lysates with anti-JAK2 and anti-p85 antibody, precipitates were assayed for PIK activity using inositol phosphate as an exogenous substrate. Lipids were separated by thin layer chromatography and autoradiographied. The result shown is representative from five separate experiments.

P-JAK2

G-gly - + - + PP2

IP: JAK2

IB: JAK2

Enolase

IB: Src

G-gly - + - + AG490

IP: Src

A

B

Figure 6 No involvement of p60-Src (A) and JAK2 (B) activation in response to G-gly. Cells pretreated with or without PP2 and AG 490 (10 µmol/L), were stimulated (+) or not (-) by G-gly (10 pmol/L) for 5 min. JAK2 and p60-Src kinase activities were determined by immune complex kinase assay using anti-JAK2 and anti-p60-Src antibodies as described in Materials and Methods. Proteins were separated by SDS-PAGE and JAK2 autophosphorylation was detected by autoradiography. A representative autoradiogram from at least three separate experiments is shown.


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lipid kinase which plays an important role in human colon cancer. Its specific inhibitor, LY 294002, has been shown to inhibit cell growth and to induce apoptosis. Treatment of human colon cancer cell lines in vitro with LY 294002 or transplanted in vivo abolishes tumour cell growth and leads to apoptosis[9]. In addition, Philp et al[18] have identified somatic mutations in the gene of the p85 subunit leading to a constitutive active form of PI3K. These mutations are present in primary human colon tumours and cancer cells, suggesting that PI3K is involved in human colonic tumori-genesis

Non-amidated gastrin precursors, including G-gly, are produced by colorectal cancers and exert growth factor ef-fects on these tissues[19, 20]. They are expressed in 80% - 90% of colorectal tumours and polyps in human beings[21-24]. Identification of signalling pathways involved in colonic mucosa hyperproliferation is important for the understand-ing of tumour processes. In a previous study we have iden-tified that Src, JAK2 and PI3K pathways are overactivated in the colonic epithelium of mice overexpressing G-gly and involved in G-gly-induced proliferation of normal colonic epithelial cells isolated from control mice[13].

In the current study, we used the human colon tumour cell line, HCT116, to investigate whether these three ki-nases interact to regulate the growth of human tumoral colon cells. The results indicate that both p60-Src and JAK2 are necessary to PI3K regulation by the peptide. Theses results are in accordance with previous reports showing that JAK2 are involved upstream of the PI3K pathway of other cellular models. Pretreatment of human neutrophils with the JAK2 inhibitor, AG 490 could abol-ish the stimulation of the p85/p110 PI3K in response of the granulocyte-macrophage colony-stimulating factor. In addition, Attoub et al[25] have shown that leptin can induce a transient elevation of the PI3K lipid products in JAK2 immunoprecipitates prepared from MDCK cells. Similarly, Src family kinases are involved upstream of the PI3K/AKT pathway[26, 27]. Previous studies suggest that the IRS proteins could serve as scaffolding intermediates between tyrosine kinases and PI3K[28]. This mechanism is likely involved in PI3K activation by G-gly. In HCT116 cells we observed a basal association between JAK2 and IRS1 and the tyrosine phosphorylation of IRS-1 in response to G-gly (data not shown). In addition, we have previously re-ported that tyrosine phosphorylation of IRS-1 in pancre-atic tumour cells, leading to the rapid recruitment of the regulatory subunit of PI3K, might represent a common mechanism for PI3K activation by different factors includ-ing insulin and G-gly[11]. Obviously, we cannot exclude other hypotheses such as interaction between the SH2 domain of p85 and phospho-tyrosine of p60-Src or JAK2. Different studies have previously described the involve-ment of Src family kinases upstream of JAK2 activation. For example, cell transformation by the oncogene v-Src[29] or the overexpression of certain members of the Src family-kinases as Lck[30] can lead to a constitutive activation of JAK2. However, we did not find any cross-talk between the two tyrosine kinases, suggesting that the p60-Src/PI3K and JAK2/PI3K pathways act independently. The involve-ment of two different mechanisms upstream of the lipid kinase might be a way to amplify the PI3K signal. Howev-

er, in our study, the level of PI3K activity associated with JAK2 or p60-Src was not different from that observed in anti-p85 immunoprecipitates. As for many other signalling molecules, it seems that the cellular localization of PI3K is important for the function of the enzyme. Therefore the recruitment of PI3K to different cell compartments by the two independent tyrosine-kinases allows the enzyme to play a complementary role. The finding that p60-Src and JAK2 do not associate in response to G-gly stimulation, demonstrates that the p60-Src/PI3K and JAK2/PI3K pathways act independently and indicates that they might be involved in different cell compartments. The analysis of the cellular localization of phosphatidylinositol 3-kinase, might be crucial to understand the role of this signalling molecule in different biological effects of G-gly.

Our previous studies indicate that gastrin precursors contribute to the initiation phases of colon carcinogenesis by upregulating the signalling pathways involved in cellular proliferation and cell survival. The results of this study demonstrate that G-gly is also able to regulate proliferation of tumoral colonic cells and support the idea that gastrin precursors are not only involved in the initiation steps of the colon tumour process but also involved in the later stages of tumour progression after genetic alterations, by conferring a growth advantage to the cells via the constitu-tive activation of numerous signalling pathways.

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Shulkes A, Baldwin GS. Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line. Gastroenterology 1997; 113: 1576-1588

3 Singh P, Owlia A, Espeijo R, Dai B. Novel gastrin receptors mediate mitogenic effects of gastrin and processing intermedi-ates of gastrin on Swiss 3T3 fibroblasts. Absence of detectable cholecystokinin (CCK)-A and CCK-B receptors. J Biol Chem 1995; 270: 8429-8438

4 Iwase K, Evers BM, Hellmich MR, Guo YS, Higashide S, Kim HJ, Townsend CM Jr. Regulation of growth of human gastric cancer by gastrin and glycine-extended progastrin. Gastroen-terology 1997; 113: 782-790

5 Stepan VM, Sawada M, Todisco A, Dickinson CJ. Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells. Mol Med 1999; 5: 147-159

6 Koh TJ, Dockray GJ, Varro A, Cahill RJ, Dangler CA, Fox JG, Wang TC. Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation. J Clin Invest 1999; 103: 1119-1126

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8 Duronio V, Scheid MP, Ettinger S. Downstream signalling events regulated by phosphatidylinositol 3-kinase activity. Cell Signal 1998; 10: 233-239

9 Semba S, Itoh N, Ito M, Harada M, Yamakawa M. The in vitro and in vivo effects of 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a specific inhibitor of phosphatidylinositol 3’-kinase, in human colon cancer cells. Clin Cancer Res 2002; 8: 1957-1963

10 Khaleghpour K, Li Y, Banville D, Yu Z, Shen SH. Involvement of the PI 3-kinase signaling pathway in progression of colon adenocarcinoma. Carcinogenesis 2004; 25: 241-248

11 Kowalski-Chauvel A, Pradayrol L, Vaysse N, Seva C. Tyro-sine phosphorylation of insulin receptor substrate-1 and acti-

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12 Hollande F, Choquet A, Blanc EM, Lee DJ, Bali JP, Baldwin GS. Involvement of phosphatidylinositol 3-kinase and mi-togen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells. J Biol Chem 2001; 276: 40402-40410

13 Ferrand A, Bertrand C, Portolan G, Cui G, Carlson J, Praday-rol L, Fourmy D, Dufresne M, Wang TC, Seva C. Signaling pathways associated with colonic mucosa hyperproliferation in mice overexpressing gastrin precursors. Cancer Res 2005; 65: 2770-2777

14 Ferrand A, Kowalski-Chauvel A, Bertrand C, Pradayrol L, Fourmy D, Dufresne M, Seva C. Involvement of JAK2 up-stream of the PI 3-kinase in cell-cell adhesion regulation by gastrin. Exp Cell Res 2004; 301: 128-138

15 Daulhac L, Kowalski-Chauvel A, Pradayrol L, Vaysse N, Seva C. Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors. J Biol Chem 1999; 274: 20657-20663

16 Kowalski-Chauvel A, Pradayrol L, Vaysse N, Seva C. Gastrin stimulates tyrosine phosphorylation of insulin receptor sub-strate 1 and its association with Grb2 and the phosphatidyli-nositol 3-kinase. J Biol Chem 1996; 271: 26356-26361

17 Pannequin J, Barnham KJ, Hollande F, Shulkes A, Norton RS, Baldwin GS. Ferric ions are essential for the biological activity of the hormone glycine-extended gastrin. J Biol Chem 2002; 277: 48602-48609

18 Philp AJ, Campbell IG, Leet C, Vincan E, Rockman SP, White-head RH, Thomas RJ, Phillips WA. The phosphatidylinositol 3’-kinase p85alpha gene is an oncogene in human ovarian and colon tumors. Cancer Res 2001; 61: 7426-7429

19 Aly A, Shulkes A, Baldwin GS. Gastrins, cholecystokinins and gastrointestinal cancer. Biochim Biophys Acta 2004; 1704: 1-10

20 Ferrand A, Wang TC. Gastrin and cancer: a review. Cancer Lett 2006; 238: 15-29

21 Ciccotosto GD, McLeish A, Hardy KJ, Shulkes A. Expression, processing, and secretion of gastrin in patients with colorectal carcinoma. Gastroenterology 1995; 109: 1142-1153

22 Nemeth J, Taylor B, Pauwels S, Varro A, Dockray GJ. Identifi-cation of progastrin derived peptides in colorectal carcinoma extracts. Gut 1993; 34: 90-95

23 Kochman ML, DelValle J, Dickinson CJ, Boland CR. Post-translational processing of gastrin in neoplastic human colonic tissues. Biochem Biophys Res Commun 1992; 189: 1165-1169

24 Smith AM, Watson SA. Gastrin and gastrin receptor activa-tion: an early event in the adenoma-carcinoma sequence. Gut 2000; 47: 820-824

25 Attoub S, Noe V, Pirola L, Bruyneel E, Chastre E, Mareel M, Wymann MP, Gespach C. Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-ki-nase-, rho-, and rac-dependent signaling pathways. FASEB J 2000; 14: 2329-2338

26 Frame MC. Src in cancer: deregulation and consequences for cell behaviour. Biochim Biophys Acta 2002; 1602: 114-130

27 Penuel E, Martin GS. Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways. Mol Biol Cell 1999; 10: 1693-1703

28 Yamauchi T, Kaburagi Y, Ueki K, Tsuji Y, Stark GR, Kerr IM, Tsushima T, Akanuma Y, Komuro I, Tobe K, Yazaki Y, Kad-owaki T. Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly PI3-kinase activation via JAK2 kinase. J Biol Chem 1998; 273: 15719-15726

29 Murakami Y, Nakano S, Niho Y, Hamasaki N, Izuhara K. Con-stitutive activation of Jak-2 and Tyk-2 in a v-Src-transformed human gallbladder adenocarcinoma cell line. J Cell Physiol 1998; 175: 220-228

30 Yu CL, Jove R, Burakoff SJ. Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase. J Immunol 1997; 159: 5206-5210

S- Editor Wang J L- Editor Wang XL E- Editor Liu WF


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Vera Todorovic, Neda Drndarevic, Olivera Mitrovic, Institute for Medical Research, Department for Immunohistochemistry and Electron Microscopy, Belgrade, Serbia and MontenegroAleksandra Sokic-Milutinovic, Tomica Milosavljevic, Clinic for Gastroenterology and Hepatology, Institute for Digestive Diseases, Clinical Center of Serbia, Belgrade, Serbia and Montenegro Marjan Micev, Pathology Department, Institute for Digestive Diseases, Clinical Center of Serbia, Belgrade, Serbia and Montenegro Ivan Nikolic, Institute of Histology and Embryology, Faculty of Medicine, University of Nis, Serbia and MontenegroThomas Wex, Peter Malfertheiner, Clinic for Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University, Magdeburg, GermanySupported by a grant from Serbian Ministry for Science and Environmental Protection, No. 1752Correspondence to: Aleksandra Sokic Milutinovic, MD, PhD, Clinical Center of Serbia, Clinic for Gastroenterology and Hepatology, Koste Todorovica 6, 11000 Belgrade, Serbia and Montenegro. [email protected]:+ 381- 11- 3617777 - 37-34Fax: + 381-11-361-5432Received: 2005-07-08 Accepted: 2005-08-26

AbstractAIM: To invest igate the expression of di fferent cytokeratins (CKs) in gastric epithelium of adult patients with chronic gastritis infected with Helicobacter pylori (H pylori ) cagA+ strains.

METHODS: The expression of CK 7, 8, 18, 19 and 20 was studied immunohistochemically in antral gastric biopsies of 84 patients. All the CKs were immunostained in cagA+H pylori gastritis (57 cases), non-H pylori gastritis (17 cases) and normal gastric mucosa (10 cases).

RESULTS: In cagA + H pylor i gastr i t is, CK8 was expressed comparably to the normal antral mucosa from surface epithelium to deep glands. Distribution of CK18 and CK 19 was unchanged, i.e. transmucosal, but intensity of the expression was different in foveolar region in comparison to normal gastr ic mucosa. Cytokeratin 18 immunoreactivity was significantly higher in the foveolar epithelium of H pylori -positive gastritis compared to both H pylori -negative gastritis and controls. On the contrary, decrease in CK19 immunoreactivity occurred in foveolar epithelium of H pylori -positive

gastritis. In both normal and inflamed antral mucosa without H pylori infection, CK20 was expressed strongly/moderately and homogenously in surface epithelium and upper foveolar region, but in H pylori -induced gastritis significant decrease of expression in foveolar region was noted. Generally, in both normal antral mucosa and H pylori -negative gastritis, expression of CK7 was not observed, while in about half cagA+ H pylori -infected patients, moderate focal CK7 immunoreactivity of the neck and coiled gland areas was registered, especially in areas with more severe inflammatory infiltrate.

CONCLUSION: Alterations in expression of CK 7, 18, 19 and 20 together with normal expression of CK8 occur in antral mucosa of H pylori -associated chronic gastritis in adult patients infected with cagA+ strains. Alterations in different cytokeratins expression might contribute to weakening of epithelial tight junctions observed in H pylori -infected gastric mucosa.


Key words: cytokeratin 7; cytokeratin 8; cytokeratin18; cytokeratin19; cytokeratin20; Helicobacter pylori ; Gastritis; CagA

Todorovic V, Sokic-Milutinovic A, Drndarevic N, Micev M, Mitrovic O, Nikolic I, Wex T, Milosavljevic T, Malfertheiner P. Expression of cytokeratins in Helicobacter pylori -associa-ted chronic gastritis of adult patients infected with cagA+ strains: An immunohistochemical study. World J Gastroen-terol 2006; 12(12): 1865-1873


INTRODUCTIONCytokeratins (CKs), a family of important cytoskeleton structural proteins, have specific spatial and temporal dynamic locations along the epithelial axis of the gastrointestinal tract (GIT), and their expression is linked to the degree of epithelial differentiation[1-4]. Cytokeratins 7, 8 (intermediate-sized and basic), 18 and 19 (smallest in size and acidic) are exclusively expressed in nearly all simple epithelia, pseudostratified respiratory epithelium and

Expression of cytokeratins in Helicobacter pylori –associated chronic gastritis of adult patients infected with cagA+ strains: An immunohistochemical study

Vera Todorovic, Aleksandra Sokic-Milutinovic, Neda Drndarevic, Marjan Micev, Olivera Mitrovic, Ivan Nikolic, Thomas Wex, Tomica Milosavljevic, Peter Malfertheiner


Helicobacter pylori

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transitional epithelium. CK8 and CK18 pair together and have a similar distribution, while CK19 can be detected in a broad range of epithelial tissues, including many simple epithelia, diverse stratified epithelia, and cultured keratinocytes. CK20 (intermediate sized and acidic) is expressed in gastric foveolar epithelium, intestinal villi and crypt epithelium, cutaneous and oral Merkel cells[4]. Various changes in CK expression and distribution profile have been noted in inflammatory[5], preneoplastic[6-10] and neoplastic[2,11-13] disorders along GIT, including gastric mucosa. Structural changes in the gastric epithelium of adult and pediatric H pylori-infected patients with chronic gastritis have been recently demonstrated using cytokeratin immunohistochemistry[14-19].

Results of previous studies postulated that only a subset of individuals infected with H pylori develop severe gastritis and/or metaplasia, peptic ulcer or gastric cancer [14,20]. Bacterial strain, environmental and host factors can converge in the gastroduodenal milieu and control the final outcome of H pylori infection. However, to the best of our knowledge, relationship between cagA+ H pylori and changes in CK expression in the gastric epithelium has not yet been studied in patients with chronic gastritis. Since we have previously demonstrated high seroprevalence of antibodies to cagA in H pylori-infected patients in Serbia and Montenegro[21], this study aimed to identify and describe immunohistochemical pattern of antral CK expression in H pylori –associated chronic gastritis of adult patients infected with cagA+ strains.

MATERIALS AND METHODSSubjectsWe conducted an outpatient-based prospective study at the Clinic for Gastroenterology and Hepatology (Clinical Center of Serbia, University of Belgrade). All patients gave informed consent for participation in the study and the study protocol was approved by the local Ethics Committee. Adult patients with dyspeptic symptoms and histological signs of gastritis entered the study. Dyspepsia was defined as upper abdominal or retrosternal pain, discomfort, nausea, vomiting or other symptoms referable to the upper abdominal tract lasting for at least one month[22]. Exclusion criteria were in concordance with the recommendations from European H pylori Study Group[20].

Ninety-one pat ients entered the s tudy. After histological examination, we excluded 17 patients (15 with and 2 without H pylori infection) due to the presence of intestinal metaplasia (IM) in the antral mucosa. Since specific IM- related changes in CK expression were reported, we aimed to identify changes related exclusively to the presence of H pylori infection. Therefore, data from 74 patients (57 H pylori-positive and 17 H pylori-negative) with chronic gastritis were analyzed. Control group (CG) consisted of 10 asymptomatic healthy volunteers with no histological changes in the gastric mucosa (mean age 32±11 years, 3 males, 7 females). In the H pylori-positive group mean age was 44 ± 13 years (26 males, 31 females), while in the non-infected group mean age was 47 ± 17 years (5 males, 12 females). Esophagogastroduodenoscopy with biopsies from antral and corpus mucosa was performed

in all patients and blood was taken for serology and immunoblot.

H pylori infection was diagnosed by rapid urease test (RUT), histology, immunohistochemistry and serology. A patient was defined as H pylori positive if histology and at least one of the other applied diagnostic methods were positive.

Routine endoscopy and H pylori statusEach patient underwent upper endoscopy and testing for the presence of H pylori by RUT and histology. Six biopsies were taken, 3 from the antrum and 3 from the corpus (2 for histology and 1 for RUT). Biopsies selected for histological examination were stained with hematoxylin-eosin, alcian blue pH 2.5/periodic acid Schiff (AB/PAS) and high iron diamine/alcian blue pH2.5 (HID/PAS). Both Giemsa and immunohistochemical stainings (polyclonal antibody to H pylori, dilution 1:10, DAKO A/S, Glostrup, Denmark) were applied for the detection of H pylori.

H pylori serology and immunoblot assayBlood samples were taken from al l pat ients after endoscopic examination and sera were separated by centrifugation and stored at -20 oC until analyzed. The presence of anti-cagA antibodies was detected using the Helicobacter pylori Vira blot test kit (Viramed Biotech AG, Lich, Germany). The concentration of anti-H pylori IgG antibodies was analyzed using Pyloriset EIA-G III (Orion Diagnostica, Finland)[23]. Both tests were performed according to the manufacturer’s instructions.

ImmunohistochemistryImmunohistochemical reactions were performed on consecutive sections of one selected antral biopsy of each patient to detect cytokeratins 7, 8, 18, 19 and 20. Only well-oriented biopsies allowing assessment of full mucosal thickness, were selected for immunohistochemical study. The sections were stained with monoclonal antibodies to CK7 (dilution 1:25, DAKO A/S, Glostrup, Denmark), CK8 (dilution 1:20, DAKO Carpinteria, CA, USA), CK18 (dilution 1:25, DAKO A/S, Glostrup, Denmark), CK19 (dilution 1:50, DAKO A/S, Glostrup, Denmark) and CK20 (dilution 1:25, DAKO A/S, Glostrup, Denmark). Immunohistochemical staining was performed according to the manufacturer’s instructions using streptavidin-biotin/HRP detection system (DAKO LSAB+/HRP kit, Glostrup, Denmark), followed by counterstaining with hematoxylin. For negative controls, no staining was detectable when the pre-immune serum was used instead of primary antibodies. In addition, human fetal esophagus of the 13th gestation week was used as positive control for evaluation of CK7 immunoreactivity.

Histological and immunohistochemical evaluationMucosal biopsies were evaluated by an experienced pathologist blinded to clinical and endoscopic data. In addition, an experienced gastrointestinal pathologist and a cytologist independently evaluated immunohistochemistry. Differences in immunohistochemical evaluation were resolved by re-examination and consensus. Chronic gastritis was diagnosed and graded according to the


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updated Sydney system[24]. Biopsies showing intestinal metaplasia classified according to previous proposals [13] were not included in this study. Mucosal distribution (surface epithelium, foveolar region, glandular necks and deep glands) and intensity of cytoplasm staining (without staining, weak or moderate/strong staining) were registered together with the expression pattern (focal or diffuse) for each analyzed CK, while an additional semi-quantitative assessment of percentages of immunoreactive cells in each mucosal compartment was performed and graded for CK7 in 3 groups (<10%, 10-20%, and >20% immunoreactive cells).

Statistical analysisResults were analyzed using non-parametric tests, Kruskall-Wallis, chi-square or Fisher’s exact test for independent samples. P < 0.05 was considered statistically significant.

RESULTSResults of our study showed that presence of intestinal metaplasia could be detected in 18.6% (17/91) of dyspeptic patients with histological signs of gastritis (15 with and 2 without H pylori infection). These patients were excluded from further analysis, since we aimed to investigate the influence of H pylori infection on normal gastric epithelium. Out of the remaining 74 patients, 57 (77%) were infected with H pylori, while infection was not found in 17. All infected patients harbored cagA+ bacterial strain in the gastric mucosa.

Histology evaluationHistological examination of antral and corpus mucosa in H pylori-infected individuals revealed signs of antral

gastritis in 5 (9%), while pangastritis was diagnosed in the remaining 52 (91%) patients. In the H pylori negative group antral gastritis was diagnosed in 7 (42%) patients and histological signs of pangastritis were found in 10 (58%). Histological changes in the gastric mucosa were graded using Sydney classification both for antral (Table 1) and corpus mucosa (data not shown).

In the antrum of H pylor i - infected individuals, inflammatory infiltrate density and activity of inflammation were higher than in the uninfected group (P < 0.001 for both histological parameters). Presence of atrophic changes was not different between the two groups, while moderate density of H pylori-colonization was most frequent in infected patients.

Immunohistochemical evaluation of CK expression and distributionCytokeratin 8 was identified immunohistochemically in antral mucosa of both H pylori positive and negative patients with gastritis and controls (Table 2). Diffuse immunoreactivity to CK8 was the predominant expression pattern in surface epithelium, foveolae and glandular neck (moderate/strong immunoreactivty was observed in 90% of controls, 70.6% of H pylori negative and 63.1% of H pylori+ gastritis and weak in only 11.8% with and 12.3% without H pylori infection). On the other hand, deeper glandular structures did not express CK8 in about 10-30% of cases. No significant difference was found in any of the examined antral mucosa regions between the three groups (Figure 1).

Normal antral mucosa was in general immunostained transmucosal (from the surface to the gland region) when antibodies to CK18 and CK19 were applied (Tables 3 and 4), while in foveolar region inconsistent CK18 immunoreactivity with the expression rate of only 10% was observed. In the foveolar epithelium of H pylori+ patients more intense diffuse (P < 0.05) and focal (P < 0.01) CK18 immunoreactivity was detected when compared to H pylori-negative gastritis and controls (Figure 2). As opposed to these results, lower antral CK19 patchy immunoreactivity was noted in foveolar epithelium of H pylori+ gastritis compared to H pylori-negative gastritis and controls (P < 0.05) (Figure 3).

Al l examined antra l b iopsies showed posi t ive immunostaining of CK20 (Table 5) restricted to the surface and upper foveolar epithelium. In the surface epithelium strong and homogenous CK20 immunoreactivity was predominant (moderate/strong diffuse immunoreactivity was observed in 90% of controls, 70.6% of H pylori negative and 64.9% of H pylori+ gastritis, as opposed to the patchy staining observed in 10%, 29.4% and 35.1% of individuals, respectively). No significant differences were noted between controls and patients with gastritis irrespective of the presence of H pylori infection. In the antral mucosa of the majority of H pylori negative patients with gastritis and controls CK20 was expressed strongly/moderately in all foveolar cells while in H pylori positive gastritis patients a significantly higher percentage of patients had focal expression of CK20 (Figure 4). Further analysis revealed different CK20 expressions in upper foveolar region related to the presence of gastritis but not

Table 1 Antral histology according to Sydney classification of gastritis in patients with and without Helicobacter pylori infection

Antral mucosa H pylori+ H pylori - P(Sydney classification) (n=57) (n=17)

Inflammatory infiltrate0 0 1 0.0001 19 142 31 23 7 0Activity of inflammation0 2 9 0.0001 39 62 15 23 1 0Atrophy0 45 13 NS1 12 4H pylori-colonization density 0 0 171 19 02 25 03 13 0

H pylori+: Helicobacter pylori positive patients; H pylori -: Helicobacter pylori negative patients; NS: P > 0.05.

Todorovic V et al. Cytokeratins in cagA+ H pylori gastritis 1867

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H pylori infection. Namely, patchy staining pattern was detected in 44% of H pylori+ and 29% of H pylori negative gastritis patients and only 10% of controls (P < 0.05).

The main findings of CK7 immunoreactivity are listed in Table 6. In both controls and H pylori-negative gastritis patients almost no immunoreactivity of CK7 was found in the antral mucosa, with the exception of some

inconsistent and faint CK7 immunoreactivity observed in single cells of glandular necks and deep glands in about 2/3 of cases (Figure 5). However, moderate focal CK7 immunoreactivity in the same area was more frequently observed in H pylori-positive gastritis compared to both H pylori-negative gastritis and control group (P < 0.01). Namely, about half in H pylori-infected patients (28/55,

Table 2 Expression of CK8 in antral mucosa of patients with gastritis (H pylori positive and negative) compared to the control group

Cytokeratin 8 Antral mucosa epitheliumexpression H pylori+G H pylori -G CG P n % n % n % Surface epithelium, foveolae and glandular necksDiffuse immunoreactivity 43 75.4 14 82.4 9 90 NSFocal immunoreactivity 14 24.6 3 17.6 1 10 NSWithout staining 0 0 0 0 0 0 NSn (%) 57 100 17 100 10 100

Deep glands Diffuse immunoreactivity 24 42.1 11 64.7 5 50 NSFocal immunoreactivity 15 26.3 2 11.8 4 40 NS Without staining 18 31.6 4 23.5 1 10 NSn (%) 57 100 17 100 10 100

CG: control group; H pylori+G: H pylori positive patients with gastritis; H pylori-G -: H pylori negative patients with gastritis; NS: P > 0.05

Figure 1 CK8 immunoreactivity in antral mucosa of patients with cagA+ H pylori chronic gastritis (A), non-H pylori chronic gastritis (B) and control subjects with normal gastric mucosa (C) with differences of CK8 immunoreactivity in antral mucosa. Original magnification: x10 (A,C); x20 (B).

A CB

A B

Figure 2 CK18 immunoreactivity in antral mucosa of patients with cagA+ H pylori chronic gastritis (A) and control subjects without gastritis (B). Original magnification: x10. Cytokeratin 18 immunoreactivity was significantly higher in the foveolar epithelium of H pylori-positive gastritis compared with H pylori-negative gastritis.

A B

Figure 3 CK19 immunoreactivity in antral mucosa of patients with cagA+ H pylori chronic gastritis (A) and non-H pylori chronic gastritis (B). Decrease in CK19 immunoreactivity was found in foveolar epithelium of antral mucosa of H pylori-positive gastritis compared to non-H pylori chronic gastritis. Original magnification: x20 (A, B)


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50.1%) moderate focal immunoreactivity of CK7 in more than 10% of cells in neck and coiled gland areas was registered, especially in individuals with more severe inflammatory infiltrate (Figure 6).

DISCUSSIONH pylori-induced proinflammatory cytokine expression and inflammation affects various gastric cell populations controlling gastric acid secretion[25], including somatostatin-producing D cells[26], gastrin-producing G cells[27], and

parietal cells[28]. In addition, in the course of H pylori-induced gastritis, acute foveolitis of the pit proliferative zone with characteristic morphological change of epithelial cells, which is often described as the malgun (clear) cell change[29] occurs. It is postulated that malgun cell change in the course of H pylori gastritis reflects epithelial genomic damage and repair processes. In addition, various structural changes of mucosal epithelial cells in H pylori gastritis have been reported in both adult[14- 16, 19] and pediatric patients[18].

Strain-specific H pylori gene, cagA, has been recognized as a marker of strains that confer increased risk for peptic


Cytokeratin 18 Antral mucosa epitheliumexpression H pylori+G H pylori -G CG P n % n % n % Surface epithelium Diffuse immunoreactivity 32 56.1 14 82.3 5 50.0 NSFocal immunoreactivity 24 42.1 2 11.8 3 30.0 NSWithout staining 1 1.7 1 5.9 2 20.0 0.043n (%) 57 100 17 100 10 100

Foveolar epitheliumDiffuse immunoreactivity 31 54.4 8 47.1 1 10 0.035Moderate/Strong 30 52.7 8 47.1 1 10 0.045Weak 1 1.7 0 0 0 0 NSFocal immunoreactivity 20 35.1 1 5.8 0 0 0.008Moderate/Strong 5 8.8 0 0 0 0 NSWeak 15 26.3 1 5.8 0 0 0.045Without staining 6 10.5 8 47.1 9 90 <0.001n (%) 57 100 17 100 10 100

Glandular necksDiffuse immunoreactivity 36 63.2 13 76.5 6 60 NS

Focal immunoreactivity 18 31.6 2 11.8 2 20 NSWithout staining 3 5.2 1 5.9 2 20 NSn (%) 57 100 17 100 10 100

Deep glandsDiffuse immunoreactivity 15 26.3 8 47.0 4 40 NSModerate/Strong 15 26.3 5 29.4 3 30 NSWeak 0 0 3 17.6 1 10 0.008Focal immunoreactivity 18 31.6 3 17.7 4 40 NSWithout staining 24 42.1 6 10.3 2 20 NSn (%) 57 100 17 100 10 100


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B CA

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Figure 4 CK20 immunoreactivity in antral mucosa of patients with cagA+ H pylori chronic gastritis (A), non-H pylori chronic gastritis (B) and control subjects with normal gastric mucosa (C). In the cagA+ H pylori chronic gastritis, CK20 expression was decreased in upper foveolar epithelium in comparison to non-H pylori chronic gastritis and control subjects with normal gastric mucosa. Numerous CK20 strong immunoreactive endocrine cells could be observed ( ) in the glandular epithelium. Original magnification: x20 (A-C)

CG:control group; H pylori+G -:Helicobacter pylori positive patients with gastritis; H pylori-G: Helicobacter pylori negative patients with gastritis; NS: P > 0.05

B

D

ulcer disease and gastric cancer[25]. Since the seroprevalence of cagA+ H pylori strains was high in chronic gastritis patients of Serbia and Montenegro[21] and there is no evidence that bacterial strain is related to alterations in cytokeratin expression in gastric epithelium, we aimed in the current study to evaluate the changes in distribution and expression pattern of different CKs in the course of cagA+ H pylori -associated chronic gastritis in adult

patients.In normal antral gastric mucosa, results of our

study revealed broad panmucosal (from the surface to the gland) expressions of CKs8,18 and 19, while CK20 immunoreactivity was strong and homogenous in the tip


Cytokeratin 19 Antral mucosa epitheliumexpression H pylori+G H pylori -G CG P n % n % n % Surface epitheliumExpression in all cells 34 60.6 12 70.6 5 55.6 NSPatchy staining 21 37.4 5 29.4 4 44.4Without staining 1 2.0 0 0 0 0n (%) 56 100 17 100 9 100

Foveolar epitheliumExpression in all cells 39 69.7 9 53.0 5 55.6 NSPatchy staining 7 12.5 5 29.4 4 44.4 0.027Moderate/Strong 2 3.6 2 11.8 2 22.2Weak 5 8.9 3 17.6 2 22.2Without staining 10 17.8 3 17.6 0 0 NSn (%) 56 100 17 100 9 100

Glandular neckExpression in all cells 37 58.1 9 53 6 66.7 NSPatchy staining 10 17.8 6 25.2 3 33.3Without staining 9 16.1 2 11.8 0 0n (%) 56 100 17 100 9 100

Deep glandExpression in all cells 22 39.2 6 35.2 3 33.3 NSPatchy staining 10 17.8 5 29.5 2 22.2Without staining 24 43.0 6 35.3 4 44.5n (%) 56 100 17 100 9 100

CG: control group; H pylori+G: Helicobacter pylori positive patients with gastritis; H pylori-G: Helicobacter pylori negative patients with gastritis; NS: P > 0.05.


Cytokeratin 20 Antral mucosa epitheliumexpression H pylori+G H pylori -G CG P n % n % n % Surface epitheliumExpression in all cells 37 64.9 12 70.6 9 90Patchy staining 20 35.1 5 29.4 1 10 NSWithout staining 0 0 0 0 0 0n (%) 57 100 17 100 10 100

Upper foveolar regionExpression in all cells 32 56.1 12 70.6 9 90 NSPatchy staining 25 43.9 5 29.4 1 10Moderate/Strong 20 35.1 4 23.5 1 10Weak 5 8.8 1 5.9 0 0 0.044Without staining 0 0 0 0 0 0n (%) 57 100 17 100 10 100

CG:control group; H pylori+G: Helicobacter pylori positive patients with gastritis; H pylori-G: Helicobacter pylori negative patients with gastritis; NS: P > 0.05.

Figure 5 CK7 immunoreactivity in antral mucosa of patients with cagA+ H pylori chronic gastritis (A), non-H pylori chronic gastritis (B), control subjects with normal gastric mucosa (C), and control specimen of human fetal esophagus (D). Moderate focal CK7 positivity ( ) in the pits and the glands of antral mucosa in patients with cagA+ H pylori chronic gastritis could be observed. CK7 positive cells covering a single gland ( ) of antral mucosa could be found in patients with non-H pylori chronic gastritis. Normal antral mucosa with a few single cells or cell clusters ( ) were strongly decorated with antibody to CK7. Strong CK7 immunoreactivity was displayed in the stratified columnar epithelium ( ) of human fetal esophagus of the 13th gestation week. Original magnification: x10 (A-D).

A

C


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and upper portion cells of foveolae. As opposed to other examined cytokeratins, CK7 was mostly undetectable. Similar results were obtained for gastritis patients without H pylori infection and overall these findings are in line with previous reports[1,2,14,15,17].

Several immunohistochemical studies in the past few years demonstrated that altered gastric cytokeratin expression is closely related with H pylori infection in adult patients[1,2,14-17,19]. However only two studies by Schwerer et al[15] and Louwers et al[17] have investigated multiple CKs simultaneously. To the best of our knowledge, all other studies are focused on particular CK. None of these studies however have provided data concerning bacterial strain.

According to previous observations, CK7 is present in fetal but largely absent in normal adult and is transiently de novo expressed in metaplastic and neoplastic epithelial cells[2,11]. Our results suggest that CK7, largely absent in normal adult antral mucosa, is expressed in H pylori chronic gastritis patients, which is in line with reports in adults describing slight[17] and markedly increased CK7 expression in H pylori-associated chronic gastritis in children[18]. As opposed to these findings, a study by Schwerer and Baczako[14] when investigating CK7 expression in normal foveolar epithelium found that H pylori can induce gastritis and intestinal metaplasia with CK7 immunoreactivity detected only in intestinal metaplasia.

Study by Kirchner et al[2] using animal experimental model of gastritis-cancer sequence in Mongolian gerbils revealed signs of mild gastritis 2 mo after H pylori infection together with CK7 expression in epithelial cells of basal glands followed by loss of specific differentiation and changes to duct-like appearance, while after 6, 12 and 24 mo moderate to severe gastritis with loss of differentiated gastric glands and switch to CK7 positive duct-like structures in large mucosal segments was observed. These results may provide evidence that non-neoplastic stomach with non-atrophic H pylori gastritis constantly exhibits low score of CK7 positive cells in antrum and corpus, thus supporting our findings. Furthermore, there is evidence that CK7 expression in metaplastic and neoplastic stomach is related to dedifferentiated epithelial cells that can phenotypically be linked to fetal cells at the beginning of gastric pit development[2]. The dedifferentiated cells exhibit low proliferation and beta-catenin accumulation similar to stem cells. Therefore, observations of Kirchner et al[2] imply that metaplasia, gastric intraepithelial neoplasia, early gastric cancer and dedifferentiated epithelial cells defined by CK7 expression are related with each other in H pylori induced gastritis. Based on the above stated findings, we speculate that CK7 de novo expression in gastric mucosa of patients infected with cagA+ H pylori strains represents the proliferative/regenerative cells rather than pure dedifferentiated cells because CK7 positive flat duct-like


Cytokeratin 7 Antral mucosa epitheliumexpression H pylori+G H pylori -G CG P n % n % n % Surface and foveolar epitheliumWeak focal immunoreactivity 0 0 0 0 0 0 NSModerate focal immunoreactivity 5 9.1 0 0 0 0 NS<10% cells 0 0 0 0 0 010-20% cells 3 5.5 0 0 0 0>20% cells 2 3.6 0 0 0 0Without staining 50 90.9 16 100 10 100 NSn (%) 55 100 16 100 10 100

Glandular necks and deep glands Weak focal immunoreactivity 6 10.9 1 6.3 3 30.0<10% cells 3 5.5 1 6.3 3 30.0 0.0110-20% cells 2 3.6 0 0 0 0 NS>20% cells 1 1.8 0 0 0 0 NS

Moderate focal immunoreactivity 38 69.1 10 62.5 7 70.0<10% cells 10 18.3 10 62.5 7 70.0 0.00210-20% cells 14 25.4 0 0 0 0 0.002>20% cells 14 25.4 0 0 0 0 0.002Without staining 11 20.0 5 31.2 0 0n (%) 55 100 16 100 10 100

CG: control group; H pylori+G: Helicobacter pylori positive patients with gastritis; H pylori-G: Helicobacter pylori negative patients with gastritis; NS: P > 0.05.

A B

C D

Figure 6 Typical expression pattern of CK18 (A), CK19 (B), CK20 (C) and CK7 (D) in antral mucosa of patients with cagA+ H pylori chronic gastritis. Strong diffuse cytokeratin 18, moderate focal CK19 and CK20 (C) immunoreactivities were demonstrated in upper foveolar epithelium ( ) of the antral mucosa. Moderate focal CK7 positivity ( ) was revealed in the pits and the glands of antral mucosa. CK7 immunoreactivity was prominent (*) in some cell clusters of the foveolar epithelium (insert). Original magnification: x10 (A-C), insert x20.


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structures have not been identified.Our results did not reveal any significant difference in

CK8 expression between patients with cagA+ H pylori-induced gastritis and normal mucosa. These findings are supported by results of other authors[15,17], but not by study of Baek et al[19] that described under-expressed CK8 in the gastric mucosa of H pylori infected-individuals as a result of oxidative stress-induced cytoskeleton damage.

Higher expression of CK 18 in foveolar epithelium together with a decrease in CK19 expression was noted in patients with cagA+ H pylori-induced gastritis. These findings differ from previous studies, since two independent studies have revealed unchanged CK18 and CK19 expression in gastric mucosa of H pylori-infected patients[15,17]. Normal stomach expresses less CK19 in the upper mucosal compartment in comparison to other mucosal compartments[1]. CK19 expression is thought to be inversely related to cell proliferation, strong CK19 expression implying weak proliferation and vice versa[1]. Intestinal metaplasia of the stomach however exhibits more intense CK19-immunoreactivity than gastric cancer tissue[1]. If CK19 immunoreactivity is negatively correlated with cell proliferation and differentiation in fetal, normal and pathologically transformed adult gastric mucosa, our results may suggest good differentiation and enhanced proliferation of upper foveolar cells in patients with cagA+ H pylori-induced gastritis.

Botta et al[3] studied CK20 immunoreactivity in fetal and neonatal human gut, including stomach and demonstrated that CK20 expression is progressive increased during gestation, suggesting that the degree of CK20 positivity is related to the epithelial maturation stage in gastric mucosa. CK20 expression in adults is restricted to the surface foveolar epithelium and is not detectable in gastric pit or glandular region. Previous investigations revealed that CK20 expression is significantly lower in foveolar epithelium of H pylori-induced chronic gastritis[15-17] supporting our results, while available studies suggest that this is a reversible change and CK20 expression is normalized within 6 mo after eradication of H pylori infection[15].

Taken together, all these findings imply alterations in epithelial cell maturation in the course of H pylori-induced chronic gastritis.

Our results suggest that bacterial strain is of importance in inducing alterations of CK expression. It is well known that cagA present in 50-60% of all strains is a part of H pylori genome termed cag pathogenicity island (cagPAI) and that proteins encoded by cagPAI are responsible for both NFkB and MAPK activation in gastric epithelial cells. It has also been demonstrated that infection with cagA+ strains is more likely to result in peptic ulceration, atrophic gastritis and gastric carcinoma[25]. Therefore, presence of cagPAI in H pylori genome might play a role in signal transduction leading to H pylori-induced host gene expression, thus regulating inflammation, proliferation and carcinogenesis. In addition, bacterial strain-related differences in host gene expression have been reported, implying that protein expression profile including CKs, depends on bacterial strains and is related to the presence of cagA+ H pylori strains. Amieva et al.[31]

reported that cagA appears to target H pylori host cell intercellular junctions and to disrupt junction-mediated functions. Since predominant genotype of H pylori in Serbia and Montenegro has been reported to be the cagA+ genotype[21], it is very important to further investigate the presence and reversibility of different epithelial alterations induced by different H pylori strains.

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3 Botta MC, Ambu R, Liguori C, Van Eyken P, Pisanu A, Cabras A, Hofler H, Werner M, Faa G. [CK20 expression in the gastro-intestinal tract of the embryo and fetus]. Pathologica 2001; 93: 640-644

4 Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathology 2002; 40: 403-439

5 Stosiek P, Kasper M. [Neo-expression of cytokeratin 7 in chronic atrophic gastritis with pernicious anemia]. Pathologe 1990; 11: 14-17

6 Couvelard A, Cauvin JM, Goldfain D, Rotenberg A, Robasz-kiewicz M, Fléjou JF. Cytokeratin immunoreactivity of intesti-nal metaplasia at normal oesophagogastric junction indicates its aetiology. Gut 2001; 49: 761-766

7 DeMeester SR, Wickramasinghe KS, Lord RV, Friedman A, Balaji NS, Chandrasoma PT, Hagen JA, Peters JH, DeMeester TR. Cytokeratin and DAS-1 immunostaining reveal similari-ties among cardiac mucosa, CIM, and Barrett’s esophagus. Am J Gastroenterol 2002; 97: 2514-2523

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14 Schwerer MJ, Baczako K. Expression of cytokeratins typi-cal for ductal and squamous differentiation in the human stomach: an immunohistochemical study of normal foveolar epithelium, Helicobacter pylori gastritis and intestinal meta-plasia. Histopathology 1996; 29: 131-137

15 Schwerer MJ, Kraft K, Baczako K. Structural changes in the gastric foveolar epithelium in Helicobacter pylori-positive gas-tritis revealed by keratin immunohistochemistry. Hum Pathol 1997; 28: 1260-1267

16 Sales MG, Nasciutti LE, Lorena DE, Muzzi M, Porto LC. Dif-ferential expression of laminin isoform (alpha2), integrins (alpha3beta1 and alpha6beta4) and cytokeratin 20 in H. pylori


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toskeletal and kinetic epithelial differences between NSAID gastropathy and Helicobacter pylori gastritis: an immunohisto-chemical determination. Histopathology 2001; 39: 133-140

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19 Baek HY, Lim JW, Kim H, Kim JM, Kim JS, Jung HC, Kim KH. Oxidative-stress-related proteome changes in Helicobacter pylo-ri-infected human gastric mucosa. Biochem J 2004; 379: 291-299

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21 Sokic Milutinovic A, Wex T, Todorovic V, Milosavljevic T, Malfertheiner P. Anti-CagA and anti-VacA antibodies in Heli-cobacter pylori-infected patients with and without peptic ulcer disease in Serbia and Montenegro. Scand J Gastroenterol 2004; 39: 222-226

22 Colin-Jones DG. Acid-related disorders: what are they? Scand J Gastroenterol Suppl 1988; 155: 8-11

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24 Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. Interna-tional Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol 1996; 20: 1161-1181

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26 Milutinovic AS, Todorovic V, Milosavljevic T, Micev M, Spuran M, Drndarevic N. Somatostatin and D cells in patients with gastritis in the course of Helicobacter pylori eradication: a six-month, follow-up study. Eur J Gastroenterol Hepatol 2003; 15: 755-766

27 Sokic-Milutinovic A, Todorovic V, Milosavljevic T, Micev M, Drndarevic N, Mitrovic O. Gastrin and antral G cells in course of Helicobacter pylori eradication: six months follow up study. World J Gastroenterol 2005; 11: 4140-4147

28 Beales IL, Calam J. Interleukin 1 beta and tumour necrosis fac-tor alpha inhibit acid secretion in cultured rabbit parietal cells by multiple pathways. Gut 1998; 42: 227-234

29 Jang J, Lee S, Jung Y, Song K, Fukumoto M, Gould VE, Lee I. Malgun (clear) cell change in Helicobacter pylori gastritis re-flects epithelial genomic damage and repair. Am J Pathol 2003; 162: 1203-1211

30 Stosiek P, Bräutigam E, Kasper M. Expression of cytokeratin 7 in human glandular epithelium of fetal stomach. Acta Histo-chem 1991; 91: 21-23

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Juris J Meier, Bartholomaeus Kask, Wolfgang E Schmidt, Baptist Gallwitz, Department of Medicine I, St. Josef-Hospital, Ruhr-University Bochum, Germany Michael A Nauck, Diabeteszentrum Bad Lauterberg, Germany Jens J Holst, Carolyn F Deacon, Department of Medical Physiology, The Panum Institute, University of Copenhagen, DenmarkBaptist Gallwitz, Department of Medicine IV, Eberhard-Karls-University, Tübingen, GermanySupported by the Wilhelm-Sander-Stiftung (No. 2002.025.1 to JJM), Deutsche Forschungsgemeinschaft (grants Me 2096/2-1, Na 203/6-1 and Ga 386/8-1) and the Deutsche Diabetes Gesellschaft (to JJM)Correspondence to: Dr. Juris J Meier, Department of Medicine I, St. Josef-Hospital, Ruhr-University of Bochum Gudrunstr. 56 44791 Bochum, Germany. [email protected]: +49-234-509-2712/-1 Fax: +49-234-509-2309Received: 2005-06-09 Accepted: 2006-08-26

AbstractAIM: Gastric inhibitory polypeptide is secreted from intestinal K-cells in response to nutrient ingestion and acts as an incretin hormone in human physiology. While animal experiments suggested a role for GIP as an inhibitor of gastric secretion, the GIP effects on gastric acid output in humans are still controversial.

METHODS: Pentagastrin was administered at an infusion rate of 1 µg . kg-1 . h-1 over 300 min in 8 patients with type 2 diabetes (2 female, 6 male, 54 ± 10 years, BMI 30.5 ± 2.2 kg/m2; no history of autonomic neuropathy) and 8 healthy subjects (2/6, 46 ± 6 years., 28.9 ± 5.3 kg/m2). A hyperglycaemic clamp (140 mg/dl) was performed over 240 min. Placebo, GIP at a physiological dose (1 pmol . kg-1 . min-1), and GIP at a pharmacological dose (4 pmol . kg-1 . min-1) were administered over 60 min each. Boluses of placebo, 20 pmol GIP/kg, and 80 pmol GIP/kg were injected intravenously at the beginning of each infusion period, respectively. Gastric volume, acid and chloride output were analysed in 15-min intervals. Capillary and venous blood samples were drawn for the determination of glucose and total GIP. Statistics were carried out by repeated-measures ANOVA and one-way ANOVA.

RESULTS: Plasma glucose concentrations during the hyperglycaemic clamp experiments were not different

between patients with type 2 diabetes and controls. Steady-state GIP plasma levels were 61 ± 8 and 79 ± 12 pmol/l during the low-dose and 327 ± 35 and 327 ± 17 pmol/l during the high-dose infusion of GIP, in healthy control subjects and in patients with type 2 diabetes, respectively (P = 0.23 and p 0.99). Pentagastrin markedly increased gastric acid and chloride secretion (P < 0.001). There were no significant differences in the rates of gastric acid or chloride output between the experimental periods with placebo or any dose of GIP. The temporal patterns of gastric acid and chloride secretion were similar in patients with type 2 diabetes and healthy controls (P = 0.86 and P = 0.61, respectively).

CONCLUSION: Pentagastrin-stimulated gastric acid secretion is similar in patients with type 2 diabetes and healthy controls. GIP administration does not influence gastric acid secretion at physiological or pharmacological plasma levels. Therefore, GIP appears to act as an incretin rather than as an enterogastrone in human physiology.


Key words: Gastric inhibitory polypeptide; Gastric acid secretion; Type 2 diabetes; Hyperglycemic clamp; Pentagastrin-stimulated acid secretion

Meier JJ, Nauck MA, Kask B, Holst JJ, Deacon CF, Schmidt WE, Gallwitz B. Influence of gastric inhibitory polypeptide on pentagastrin-stimulated gastric acid secretion in patients with type 2 diabetes and healthy controls. World J Gastro-enterol 2006; 12(12): 1874-1880


INTRODUCTIONGastric inhibitory polypeptide (GIP) is secreted from in-testinal K-cells in response to nutrient ingestion and acts to augment insulin secretion from pancreatic beta-cells[1-4]. Together with glucagon-like peptide 1 (GLP-1) it confers approximately 60 % of total postprandial insulin secretion, thus acting as an incretin hormone[5, 6]. Since the insulino-tropic effect of GIP appears to be dependent on the pre-vailing plasma glucose concentrations[7], the term “glucose-

BASIC RESEARCH

Influence of gastric inhibitory polypeptide on pentagastrin-stimulated gastric acid secretion in patients with type 2 diabetes and healthy controls

Juris J Meier, Michael A Nauck, Bartholomaeus Kask, Jens J Holst, Carolyn F Deacon, Wolfgang E Schmidt, Baptist Gallwitz


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dependent insulinotropic polypeptide” was coined later to denominate the peptide in accordance with its major physiological role[8]. In addition, GIP administration aug-ments glucagon secretion under certain circumstances[7, 9]. While there is little doubt regarding GIP effects on en-docrine pancreatic secretion, the case is different for its role in the regulation of gastrointestinal functions. In fact, original studies in canine gastric preparations pointed to an inhibition of gastric acid secretion by GIP[10, 11] and subsequent in vivo experiments demonstrated an increase in gastric acid output following antibody blockade of endog-enous GIP in dogs, suggesting a physiological importance of GIP in the stomach[12]. These inhibitory effects of GIP on gastric acid secretion were ascribed to the local antral release of somatostain[13].

In humans, a significant inhibition of gastric acid out-put was only reported during the administration of highly supraphysiological doses of GIP[14, 15], whereas no effects were observed at physiological GIP plasma levels[15, 16].

While GIP is the major mediator of the incretin ef-fect in healthy subjects[1, 5], the incretin activity of GIP is almost absent in patients with type 2 diabetes[17-19]. It is yet unclear, whether this is due to a general impairment in beta-cell function in type 2 diabetes or whether the reduced insulinotropic effect of GIP reflects a specific defect, for example of the GIP receptor, in patients with type 2 diabe-tes[20, 21]. In the latter case, one would expect a loss of GIP action not only with respect to insulin secretion, but also regarding other physiological actions like the inhibition of gastric acid secretion. Therefore, in the present study, the effects of GIP on pentagastrin-stimulated gastric acid se-cretion at both physiological and supraphysiological plasma levels were investigated in patients with type 2 diabetes and healthy controls. The effects of GIP on insulin secretion have been reported in a separate communication[22].

MATERIALS AND METHODSStudy protocolThe study protocol was approved by the ethics committee of the medical faculty of the Ruhr-University, Bochum on 01-24-2002. Written informed consent was obtained from all participants.

Study designAt a screening visit a clinical examination was performed and laboratory parameters were screened. If the subjects met the inclusion criteria (see below), they were recruited for the following procedure: Pentagastrin-stimulated gastric acid secretion was assessed over 300 min. A hyperglycemic clamp aiming at a steady capillary plasma glucose concen-tration of 140 mg/dL (7.8 mmol/L) was performed over 240 min. Placebo (1 % human serum albumin in 0.9 % NaCl), GIP at a low infusion rate (1.0 pmol/(kg . min)), and GIP at a high infusion rate (4.0 pmol/(kg . min)) were administered consecutively, each over 60 min. Boluses of placebo, GIP at a low dose (20 pmol/kg) and GIP at a high dose (80 pmol/kg) were administered intravenously at the beginning of each infusion period (at 0, 60, and 120 min, respectively).

In order to exclude a time-dependent order effect on

Meier JJ et al . GIP and gastric acid secretion 1875

gastric secretion, five of the control subjects were studied on an additional occasion with the administration of pla-cebo only (1 % human serum albumin in 0.9 %NaCl from 0 to 180 min) instead of GIP. Boluses of placebo were administered at 0, 60, and 120 min.

Subjects/patients Eight patients with type 2 diabetes and eight healthy con-trol subjects without a family history of diabetes were studied. Patient/subject characteristics are presented in Table 1.

Subjects with anemia (hemoglobin < 11 g/dl), elevation in liver enzymes more than double the respective upper normal limits, or with elevated creatinine concentrations (> 1.3 mg/dl) were excluded. Among the patients with type 2 diabetes, 2 were treated with metformin, whereas the other 6 were on a dietary regimen. All antidiabetic treatment was withdrawn at least 48 hours prior to the experiments. None of the participants had a history of gastrointestinal disease or was taking any kind of medication with a known influence on gastric secretion. In addition, none of the patients had a history or any clinical signs of autonomic or sensory neuropathy. No abnormalities in the responsiveness of both the patellar and Achilles tendon reflexes, as assessed using a reflex hammer, were found. The results of a vibration perception threshold performed at both medial malleoli were within the normal limits

PeptidesSynthetic GIP was purchased from PolyPeptide Laborato-ries GmbH, Wolfenbüttel, Germany, and processed for infusion as described[18]. Pen-tagastrin was purchased from Cambridge Laboratories, UK, Lot-No. 1TT.

Experimental proceduresThe tests were performed in the morning after an over-night fast. Two forearm veins were punctured with a tef-lon cannula, and kept patent using 0.9 % NaCl (for blood sampling and for glucose and peptide administrations, respectively). Both ear lobes were made hyperemic using Finalgon (Nonivamid 4 mg/g, Nicoboxil 25 mg/g). A

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Table 1 Characteristics of the participants (mean±SE)

Statistics: ANOVA or c2 test. 1Normal range: < 6.5

Parameter Healthy Patients with Significance(unit) controls type 2 diabetes (P -value)Sex (female/male) 2/6 2/6 1.0 Age (years) 46 ± 6 54 ± 10 0.07Body mass index (kg/m2) 28.9 ± 5.3 30.5 ± 2.2 0.43Waist-to-hip ratio (cm/cm) 0.97 ± 0.08 1.00 ± 0.05 0.40Blood pressure Systolic (mmHg) 118 ± 13 138 ± 21 0.044 Diastolic (mmHg) 79 ± 10 92 ± 10 0.026HbA1c (%)1 6.0 ± 0.5 8.3 ± 2.2 0.013Diabetes duration (years) - 9 ± 4 -Total-cholesterol (mg/dL) 223 ± 52 210 ± 35 0.57HDL-cholesterol (mg/dL) 49 ± 22 34 ± 14 0.15LDL-cholesterol (mg/dL) 145 ± 37 164 ± 31 0.32Triglycerides (mg/dL) 161 ± 110 167 ± 92 0.90Creatinine (mg/dL) 0.86 ± 0.13 1.05 ± 0.29 0.12

nasogastric tube (Total length: 144 cm, diameter 6 mm, Sherwood Medical, Gosport, UK) was positioned for removing gastric acid by intermittent suction at 15 min intervals (HICO Gastrovac, Hirtz & Co., Germany). Basal gastric acid output was assessed over 15 min following a 30 min equilibration period. From -90 to 210 min, penta-gastrin was administered intravenously at an infusion rate of 1 µg . kg body weight-1 . h-1. All participants remained in a semi-recumbent position, turned to their left side throughout the experiments.

A hyperglycemic clamp test aiming at a steady capil-lary plasma glucose concentration of 7.8 mmol/l (140 mg/dl) was started by injecting 40 % glucose as a bolus at -30 min and maintained by infusing glucose (20 % in wa-ter, weight/vol.) until 210 min, as appropriate, based on

glucose determinations performed every 5 min and until 210 min. At 0 min, an intravenous infusion of placebo (1 % human serum albumin in 0.9 % NaCl) was started and maintained until 60 min. At 60 min, an intravenous infu-sion of GIP was started with an infusion rate of 1.0 pmol .

kg-1 . min-1. At 120 min, the GIP infusion rate was increased to 4.0 pmol . kg-1 . min-1 and maintained until 180 min. At 0 min an intravenous bolus of placebo was administered. GIP was injected as a bolus at 60 min (20 pmol/kg) and at 120 min (80 pmol/kg). Venous blood samples were drawn frequently, as seen in Figure 1.

Blood specimenVenous blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol; 20 000 KIU/ml, 200 µl per 10 ml blood; Bayer AG, Leverkusen, Germany) and kept on ice. After centrifugation at 4 ℃, plasma for hormone analyses was kept frozen at -28 ℃. Capillary blood samples collected from the ear lobe (approximately 100 µl) were stored in NaF (Microvette CB 300; Sarstedt, Nümbrecht, Germany) for the immediate measurement of glucose.

Laboratory determinationsGlucose was measured with a Glucose Analyser 2 (Beckman Instruments, Munich, Germany). GIP immunoreactivity was determined as described[18] using a C-terminal assay involving antiserum R65, which reacts fully with intact GIP (1-42) and the truncated metabolite (3-42), but not with so-called 8-Ku GIP, of which the chemical nature and molecular relation to GIP is uncertain. The assay has a detection limit of less than 2 pmol/l and an intra-assay variation of approximately 6 %. Human GIP (Peninsula Laboratories, Europe, Ltd.) was used as standard, and ra-diolabeled GIP was obtained from Amersham Pharmacia Biotech Ltd. (Aylesbury, UK).

Gastric volume output was measured in 15-min frac-tions to the nearest 0.5 ml. Acidity was determined by titration to pH 7.0 using phenol red as an indicator dye. Chloride concentration was measured by a Corning EEL 920 chloride meter (CIBA Corning Diagnostics, Fernwald, Germany).

Statistical analysisResults are reported as mean ± SEM. Statistical calculations were carried out using repeated-measures analysis of vari-ance (RM-ANOVA), using Statistica Version 5.0 (Statsoft Europe, Hamburg, Germany). This analysis provides p-values for differences between groups (A), differences over time (B), and for the interaction of groups with time (AB). If a significant interaction of treatment and time was documented (P < 0.05), values at single time points were compared by one-way ANOVA. A P-value < 0.05 was taken to indicate significant differences.

RESULTSFasting plasma glucose concentrations were significantly higher in patients with type 2 diabetes compared to control subjects (P < 0.001; Figure 1). During the hyperglycemic clamp period, similar glucose levels were obtained in both groups. As expected, glucose infusion rates required to


Pentagastrin i.v.

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Figure 1 Plasma concentrations of glucose (A), glucose infusion rates (B) and plasma concentrations of total GIP (C) during hyperglycemic clamp experiments with the intravenous infusion of pentagastrin (1 µg . kg-1 . h-1; -90 to 210 min), placebo (0 - 60 min), GIP at a low dose (1 pmol . kg-1 . min-1; 60-120 min), and GIP at a high dose (4 pmol . kg-1 . min-1; 120 -180 min) in eight patients with type 2 diabetes (open circles) and eight healthy controls (filled diamonds). Arrows indicate bolus administrations of placebo, 20 pmol GIP/kg and 80 pmol GIP/kg, respectively. Data are presented as means ± SEM; P-values were calculated using repeated measures ANOVA and one-way ANOVA and denote A: differences between the groups, B: differences over time and AB: differences due to the interaction of group and time. aP < 0.05 patients with type 2 diabetes at individual time points.

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maintain the hyperglycaemic clamp conditions were much higher in the controls than in the patients with type 2 dia-betes (P < 0.0001).

GIP plasma levels increased to steady-state levels of 61 ± 8 and 79 ± 12 pmol/l during the low-dose and 327 ± 35 and 327 ± 17 pmol/l during the high-dose infu-sion of GIP, in healthy control subjects and in patients with type 2 diabetes, respectively (P = 0.23 and P = 0.99).

Basal gastric acid output was 1.9 ± 0.4 mmol/15 min and 1.6 ± 0.4 mmol/15 min (in healthy controls and in patients with type 2 diabetes, respectively; P = 0.56). The secretion of both gastric acid and chloride was mark-

edly increased during the administration of pentagastrin (P < 0.001; Figures 2 and 3). In contrast, intravenous glucose administration had no effects on gastric acid or chloride output (Figure 2 and 3; Table 2). There were no differences in the rates of gastric acid or chloride output between the experimental periods with the administration of placebo, the low GIP dose, and the high GIP dose (Figure 3; Table 2). The lack of GIP effect on gastric secretion was confirmed in the five control experiments with the administration of placebo only and with GIP (P = 0.87 for acid output and P = 0.59 for chloride output, respectively; details not shown). The temporal pattern of

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Figure 2 Gastric volume (A), as well as gastric acid (B) and chloride (C) concen-trations determined in 15-min intervals in eight patients with type 2 diabetes (open circles) and eight healthy controls (filled diamonds) studied during hyperglycemic clamp experiments with the intravenous infusion of pentagastrin (1 µg . kg-1 . h-1; -90 to 210 min), placebo (0 - 60 min), GIP at a low dose (1 pmol . kg-1 . min-1; 60-120 min), and GIP at a high dose (4 pmol . kg-1 . min-1; 120 -180 min). Arrows indicate bolus administrations of placebo, 20 pmol GIP/kg and 80 pmol GIP/kg, respectively. Data are presented as means ± SEM; P -values were calculated us-ing repeated measures ANOVA and one-way ANOVA and denote A: differences between the groups, B: differences over time and AB: differences due to the inter-action of group and time.

A B

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Figure 3 Gastric acid (A), and chloride (B) secretion rates per 15 min in eight patients with type 2 diabetes (open circles) and eight healthy controls (filled diamonds) studied during hyperglycemic clamp experiments with the intravenous infusion of pentagastrin (1 µg . kg-1 . h-1; -90 to 210 min), placebo (0 - 60 min), GIP at a low dose (1 pmol . kg-1 . min-1; 60-120 min), and GIP at a high dose (4 pmol . kg-1 . min-1; 120 -180 min). Arrows indicate bolus administrations of placebo, 20 pmol GIP/kg and 80 pmol GIP/kg, respectively. Data are presented as means ± SEM; p-values were calculated using repeated measures ANOVA and one-way ANOVA and denote A: differences between the groups, B: differences over time and AB: differences due to the interaction of group and time.

t /mint /min

t /min t /min


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gastric acid and chloride secretion was similar in patients with type 2 diabetes and healthy controls (P = 0.86 and P = 0.61, respectively; Figure 3).

DISCUSSIONThe present experiments were undertaken to investigate the influence of GIP on pentagastrin-stimulated gas-tric acid secretion in patients with type 2 diabetes and in healthy controls. No effects of GIP on gastric acid output were observed at both physiological and pharmacological plasma concentrations.

A role for GIP in the regulation of gastric acid secre-tion was suggested from initial experiments in dogs[10-13]. Moreover the reduced rates gastric of acid secretion ob-served after jejuno-ileal shunt operation, when GIP plasma levels are increased, lent support to the concept of GIP as a negative regulator of gastric secretion[23]. In humans however, inhibitory effects of GIP on gastric acid secre-tion were only described, when supraphysiological GIP doses were used[14, 15], whereas GIP plasma levels that re-sembled those typically reached following meal ingestion failed to inhibit gastric acid secretion[15, 16]. In the present experiments, no effect of GIP on gastric acid output was observed even at supraphysiological plasma concentra-tions. The differences between the present and some of the previous studies[14, 15] may be explained by the use of different GIP preparations. In fact, both studies demon-strating inhibitory effects of GIP on gastric acid secretion in humans employed porcine GIP[14, 15], which differs from human GIP in two amino acid positions[24]. Moreover, some earlier peptide preparations contained impurities with cholecystokinin (CCK)-33 and -39, thereby limiting the interpretation of those studies[25]. Therefore, all aspects considered, the role of GIP in the regulation of gastric acid secretion in humans appears negligible.

The present data give rise to reconsider the role of GIP in human physiology. In fact, the peptide was initially considered to act as an enterogastrone[10, 11]. This term was proposed to describe (hormonal) factors from the duodenum that are secreted in response to nutrient inges-tion and inhibit upper GI-functions at their typical plasma concentrations[26]. Such effects have been demonstrated for GLP-1 as well as for peptide-YY (PYY)[27-30]. In contrast, GIP does not appear to have any effects on gastric empty-ing[31] or gastric acid secretion in humans, and therefore does not fulfil the criteria for an enterogastrone. Rather, GIP acts as an incretin hormone, as demonstrated by

numerous previous experiments[1, 5, 32]. Of note, a potent augmentation of insulin secretion by GIP was observed in the present experiments as well[22], thereby affirming the activity of the GIP preparations used.

The lack of GIP effect on gastric acid output rep-resents another interesting difference in the biological actions of GIP and the other incretin hormone GLP-1, which is known as a potent inhibitor of gastric secretion[27,

33]. In fact, even though both hormones are secreted almost simultaneously[5, 34] and act in concert to augment glucose-stimulated insulin secretion[5, 32], they do exhibit a number of characteristic differences: Thus, GLP-1 suppresses pan-creatic glucagon release[35, 36], whereas GIP has no effect or, at normoglycemic fasting conditions, even stimulates glucagon release[7, 9]. In addition, GLP-1 dose-dependently decelerates gastric emptying, thereby reducing the rise in glycemia following meal ingestion[30, 33, 37, 38]. In contrast, GIP administration has no effect on the velocity of gastric emptying[31]. Taken together, GLP-1 apparently possesses both incretin and enterogastrone activity, whereas GIP mainly acts as incretin hormone.

Another purpose of the present experiments was to compare gastric acid secretion in patients of type 2 dia-betes and healthy controls. Since hyperglycemia itself has been shown to inhibit gastric acid output[39], hypergly-caemic clamp conditions were applied to match plasma glucose concentrations in both groups. Under these condi-tions, no differences occurred in the rates of gastric acid or chloride output. It is important to point out that in the present study only patients without a history or clinical signs of neuropathy were included. In fact, a number of previous studies have demonstrated reduced rates of gas-tric acid secretion in diabetic patients with overt autonomic neuropathy[40] [41-43]. In contrast, no abnormalities in gastric secretion were reported in patients with diabetes without signs of neuropathy[41, 43]. Therefore, it appears that, unless neuropathy develops, gastric acid secretion is similar in pa-tients with type 2 diabetes and non-diabetic controls, when plasma glucose levels are matched.

In conclusion, gastric inhibitory polypeptide has no effect on gastric acid secretion in patients with type 2 dia-betes and healthy controls. Therefore, GIP seems to act as an incretin rather than as an enterogastrone in human physiology.

ACKNOWLEDGMENTSThe excellent technical assistance of Birgit Baller and Lone Bagger is greatly acknowledged.

Table 2 Gastric acid and chloride secretion in patients with type 2 diabetes and healthy controls (mean±SE)

Experimental period: Basal Pentagastrin Hyperglycemia Placebo GIP low GIP high SignificanceTime (min): (-90) (-45 to -30) (-15 to 0) (45 to 60) 105 to 120) (165 to 180) (P -value)Healthy controls H+ -secretion [mmol/15 min] 1.9 ± 0.4 6.2 ± 0.9 7.2 ± 0.7 8.1 ± 1.1 7.9 ± 1.0 6.5 ± 0.5 < 0.001 Cl- -secretion [mmol/15 min] 1.9 ± 0.4 7.9 ± 1.0 9.0 ± 0.8 9.9 ± 1.3 9.6 ± 1.2 8.0 ± 0.7 < 0.001Patients with type 2 diabetes H+ -secretion [mmol/15 min] 1.6 ± 0.4 8.3 ± 1.7 8.3 ± 1.7 9.5 ± 1.8 8.6 ± 1.7 8.2 ± 1.6 < 0.01 Cl- -secretion [mmol/15 min] 1.6 ± 0.4 11.1 ± 1.4 11.7 ± 2.1 12.5 ± 2.1 11.8 ± 1.7 11.4 ± 1.5 < 0.001

Statistics: ANOVA


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AE. Gastric acid and pancreatic polypeptide responses to sham feeding are impaired in diabetic subjects with autonomic neuropathy. Diabetes 1985; 34: 1181-1185

42 Feldman M, Corbett DB, Ramsey EJ, Walsh JH, Richardson CT. Abnormal gastric function in longstanding, insulin-depen-dent diabetic patients. Gastroenterology 1979; 77: 12-17

43 Fiorucci S, Bosso R, Scionti L, DiSanto S, Annibale B, Delle Fave G, Morelli A. Neurohumoral control of gallbladder motility in healthy subjects and diabetic patients with or without autonomic neuropathy. Dig Dis Sci 1990; 35: 1089-1097

S- Editor Guo SY L- Editor Wang XL E- Editor Liu WF


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cyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cul-tures and cocultures in the RFB.

RESULTS: By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (VVO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate syn-thetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased.

CONCLUSION: Co-culture in the RFB system can dra-matically change the structure and function of all cell types, including the functional characteristics of hepato-cytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.


Key words: Liver organoid; Organ reconstruction; Bio-artificial liver; Coculture; Liver sinusoidal endothelial cell; Hepatocytes; Fenestrae; Vesiculo vacuolar organelles; Radial flow bioreactor

Saito M, Matsuura T, Masaki T, Maehashi H, Shimizu K, Hataba Y, Iwahori T, Suzuki T, Braet F. Reconstruction of liver organoid using a bioreactor. World J Gastroenterol 2006; 12(12): 1881-1888


INTRODUCTIONLiver regeneration technology has made many advances in recent years. Efforts now are being made toward develop-ment of embryonic stem cells (ES cells), differentiation of hemopoietic stem cells, and development of isolation and culture methods for somatic stem cells originating from different organs. Hemopoietic stem cells, hepatoblasts originating from fetal liver, hepatocytes, and pancreatic duct epithelial cells have been included in the list of candi-

Reconstruction of liver organoid using a bioreactor

Masaya Saito, Tomokazu Matsuura, Takahiro Masaki, Haruka Maehashi, Keiko Shimizu, Yoshiaki Hataba, Tohru Iwahori, Tetsuro Suzuki, Filip Braet

Masaya Saito, Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, JapanTomokazu Matsuura, Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, JapanTakahiro Masaki, Tetsuro Suzuki, Department of Virology II, National Institute of Infectious Disease, Tokyo, JapanHaruka Maehashi, Keiko Shimizu, First Department of Biochemistry, The Jikei University School of Medicine, Tokyo, JapanYoshiaki Hataba, DNA Medical Institute, The Jikei University School of Medicine, Tokyo, JapanTohru Iwahori, Fifth Division of Blood Purification, Department of Surgery, Tokyo Medical University, Tokyo, JapanF i l ip Braet , Austra l ian Key Center for Microscopy & Microanalysis, Electron Microscope Unit, The University of Sydney, NSW 2006, AustraliaSupported by grants-in-aid from the University Start-Up Creation Support System, the Promotion and Mutual Aid Corporation for Private Schools of Japan, and The Japan Health Sciences Foundation (Research on Health Sciences on Drug Innovation, KH71068)Correspondence to: Tomokazu Matsuura, MD, PhD, Department of Laboratory Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan. [email protected]: +81-3-34331111-3210 Fax: +81-3-34350569Received: 2005-09-12 Accepted: 2005-10-26

AbstractAIM: To develop the effective technology for reconstruc-tion of a liver organ in vitro using a bio-artificial liver.

METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional high-density culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 107 FLC-5 cells were incubated in the RFB. After 5 d, 2 x 107 A7 cells were added in a similar manner followed by an-other addition of 107 M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepato-


BASIC RESEARCH

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date cells for liver regeneration[1]. Development of immor-talized cells by introduction of the simian virus 40 (SV40) large T antigen gene or human telomerase reverse tran-scriptase (hTERT) is also under investigation[2]. To date, however, no technique for regenerating and reconstructing parenchymal organs using these cells has been established. Conventional cell culture methods have achieved this goal clinically for skin, cornea, and bone tissue[3,4].

Reconstruction of organs such as the liver requires maintenance of viable cells at a high density and cocul-ture under conditions favorable to several different cell types that constitute a liver. To make a culture system is important in reconstructing a liver organoid. Conventional stationary culture techniques are not well suited to the culture of cells in a layered form, i.e., in a structural and functional organoid a simple air/CO2 incubator does not deliver adequate oxygen supply to layered cells. Further-more, high-density culture cannot be maintained with the limited nutrients available in conventional cultures. For these reasons, construction of a bioreactor that allows 3-dimensional growth in a high-density perfusion culture has been advocated for reconstructing a liver organoid. In our study a radial-flow bioreactor (RFB) developed in Japan was used as a candidate model for high-density perfusion culture. Filled with a porous carrier, this bioreac-tor permits culture at a cell density 10 times higher than that allowed by a hollow-fiber culture system[5, 6]. Another important point is to select a cell source. Clinically, cells using bio-artificial liver are required to be highly functional and supplied quickly in large quantities. Therefore we established a functional human hepatocellular carcinoma cell line (FLC-5), which can express drug-metabolized en-zymes (e.g., human-type carboxyl esterase or cytochrome) and liver-specific proteins such as albumin. In vitro this cell line retains its three-dimensional form, developing distinct microvilli on the surface. These cells can be cultured in se-rum-free ASF104 medium (Ajinomoto, Tokyo). A liver or-ganoid cannot be reconstructed with hepatocytes only. At minimum, coculture of hepatocytes with nonparenchymal cells, such as sinusoidal endothelial cell (SEC) and hepatic stellate cell (HSC) is required. So we established immortal-ized SEC line M1[7] and an immortalized HSC line A7[8] by isolating nonparenchymal cells from an H-2Kb-tsA58-transgenic mouse liver transfected with the SV40 large T antigen gene[9].

Reconstruction of the liver sinusoid is important for activity of the liver organoid as a functional unit. Also, the open pores on the surfaces of SEC in fenestrae have an important functional role in the liver sinusoid. Fenestrae are the most remarkable characteristics of SEC, as first de-scribed by Wisse in 1970[10] using transmission electron mi-croscopy (TEM). Diameters of these pores vary between species, ranging from 100 to 200 nm[11]. These fenestrae facilitate the transport of materials and solutes from the luminal to the abluminal side of the liver parenchymal cells and vice versa[12]. The process and mechanism of formation of these pores remain largely unclarified[13, 14]. The presence of actin filaments at the margin of these pores has been demonstrated by electron microscopic studies[15, 16]. Swin-holide A, a most potent microfilament-disrupting drug available, has been demonstrated to increase the number

of SEC fenestrae[13]. However, when immortalized SEC was treated in a monolayer culture or as a monoculture in the RFB, an increase in number of fenestrae could not be observed when the Swinholide A was introduced. The po-tential for drug-induced increase also has been reported to disappear in long-term cultures[7].

In developing a high functioning organoid using a bio-artificial liver, the function, form and reactivity of phar-macological agent should be near in vivo. In the present study, we reconstructed a functional liver organoid using immortalized cell lines in the RFB.

MATERIALS AND METHODSCell culture and mediumWe used the three cell lines mentioned above, FLC-5, M1, and A7. As reported, culture of M1 cells was possible in serum-free conditions while supplementation of ASF104 medium with 2% fetal bovine serum (FBS) was required for culture of A7 cells. Therefore, in coculture experi-ments, ASF104 medium was enriched with 2% FBS.

Coculture in radial-flow bioreactorAs reported elsewhere, the RFB system is composed of a 15-mL radial-flow chamber (RA-15; ABLE, Tokyo), a mass flow controller (RAD925, ABLE), a reservoir (Figure 1), a computer, and a tissue incubator as described previously[17] (Figure 1). The culture medium was oxygenated within the reservoir, and the pH was adjusted automatically to 7.4. Oxygen pressure in the culture medium was measured both within the reservoir and at the outlet of the bioreac-tor. Relative oxygen consumption was monitored on the basis of the oxygen pressure gradient. During the study the temperature within the reservoir was kept constantly at 37 ℃. Two × 107 FLC-5 cells were inoculated into the res-ervoir. The bioreactor was perfused in a closed circuit for 2 h to aid cells in adhering to the porous carrier cellulose beads (Asahi Kasei, Tokyo). Subsequently the bioreactor was switched to the open-circuit mode, and incubation was continued with addition of fresh culture medium to the reservoir. After 5 d, 2 ×107 A7 cells were added in a simi-lar manner followed by another addition of 107 M1 cells 5 d later. Retinol (10-6mol/L) was added during the first 2 d. After three days of perfusion cellulose beads with the adherent cells were harvested, and cells deposited at the bottom of the bioreactor also were recovered. Beads with attached cells were fixed in 1.2% or 2.0% glutaraldehyde as described below.

Swinholide A experimentsWe cultured the three cell lines as described above. The

Figure 1 The sys tem o f radial-flow bioreactor. A 15-mL radial-flow bioreactor (large arrow), a mass flow controller (arrow head), and a reservoir (small arrow) are connected each other. Culture medium is perfused in the RFB. Medium cond i t ions (PH, oxygen, CO2 and temperature) are controlled by computer.


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last incubation included perfusion with 200 nmol/L swin-holide A (Sigma catalog number S9810; S) for 2 h. The cel-lulose beads along with adherent cells were harvested from the bioreactor. Beads with attached cells were fixed in glu-taraldehyde and prepared for morphologic observation as follows.

Electron microscopyFor scanning electron microscopy (SEM), cultured cells were fixed with 1.2% glutaraldehyde in 0.1 mol/L phos-phate buffer (PB) at pH 7.4 and postfixed with 1% OsO4

in 0.1mol/L PB. The fixed cells were rinsed twice with PBS, subsequently dehydrated in ascending concentrations of ethanol, critical point-dried using carbon dioxide, and coated by vacuum- evaporated carbon and ion-spattered gold. Specimens were observed under JSM-35 scanning electron microscope (JEOL, Tokyo) at an accelerated volt-age of 10 kV.

For transmission electron microscopy (TEM), cultured cells were fixed with 2.0% glutaraldehyde in 0.1mol/L PB for 1 h and postfixed with 1% OsO4 in 0.1mol/L PB for 1 h at 4 ℃. Specimens were dehydrated in ethanol and sub-sequently embedded in a mixture of Epon-Araldite. Thin sections (60 nm) were cut with a diamond knife mounted on an LKB ultratome, and stained with aqueous uranyl acetate. Specimens were examined under a JEOL 1200EX electron microscope.

Amino acid analysis of supernatantsFor analysis of amino acid fractions by high-performance liquid chromatography (HPLC), supernatants were collect-ed from FLC-5 alone and from cocultures of the three cell types in the bioreactor. Supernatants were mixed with 5% sulfosalicylic acid and allowed to stand at 4 ℃ for 15 min. After centrifugation to precipitate protein, supernatants were injected into amino acid analysis columns (L-8500, Hitachi, Tokyo).

Quantitative TaqMan RT-PCRWe measured mRNA expression for the urea cycle en-zymes, carbamoyl phosphate synthetase (CPS1), ornithine carbamoyltransferase (OCT), argininosuccinatesynthetase (ASS), argininosuccinatelyase (ASL), and arginase (ARG), as well as mRNA expression for albumin, hepatocyte nuclear factor (HNF)-1 and HNF-4, by quantitative Taq-Man reverse transcription polymerase chain reaction (RT-PCR). RT-PCR was performed on the ABI PRISM 7700 sequence detection system using random hexamers from TaqMan reverse transcription reagents and the RT reac-tion mix (Applied Biosystems, Rockville, M) to reverse-transcribe RNA. TaqMan universal PCR Master Mix and Assays-on-Demand gene expression probes (Applied Biosystems) were used for PCR. A standard curve for serial dilution of 18S rRNAs was generated similarly. A relative standard curve method (Applied Biosystems) was used to calculate the amplification difference in urea cycle-related enzymes between cocultured and control cells, and elongation factor 1 (EF1), for each primer set and between albumin, HNF-1, HNF-4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Specificity was evaluated using GAPDH mRNA as an internal control (4310884E; Perkin-

Elmer Applied Biosystems). Each amplification was per-formed in triplicate, and averages were obtained.

Based on DNA sequences in GenBank, primers and the TaqMan probe for albumin, HNF-1, and HNF-4 were designed using the primer design software Primer Express TM (Perkin-Elmer Applied Biosystems, Foster City, CA). AmpliTaq DNA polymerase extended the primer and dis-placed the TaqMan probe through its 5’-3’ exonuclease ac-tivity. Probes were labeled with a reporter fluorescent dye either 6-carboxy-fluorescein (FAM) or 2, 7 dimethoxy-4,5-dichloro-6-carboxy-fluorescein (JOE) at the 5’ end and a quencher fluorescent dye [6-carboxytetramethyl-rhodamine (TAMRA)] at the 3’ end.

Primers/probes were as follows: ornithine transcar-bamoylase (OTC) forward primer 5’-CCAGGCAATA-AAAGAGTCAGGATT-3’, reverse primer/ 5’-TTAT-CAAAG TCCCCTGGTTAGAGATACT-3’, probe/ 5’-(FAM)- TTCAAATGCTCCTACACCCTGCCCTG-(TAMRA)-3’; arginosuccinase (ASL) forward primer/ 5’-TGGCCAAGGAGGTCGTCA-3’, reverse primer 5’-TTCCTCGTCGTCCGGAAG-3’, probe 5’-(FAM)-TGTCTTCCAGACCCGGAGACCGAA-(TAMRA)-3’; a lbumin forward pr imer/ 5 ’ -CGATTTTCTTTT-TAGGGCAGTAGC-3 ’ , r everse pr imer/ 5 ’ -TG-GAAACTTCTGCAAACTCAGC-3’, probe/ 5’-(FAM)-CGCCTGAGCCAGAGATTTCCCA-(TAMRA)-3’; HNF-1 forward primer/ 5’-AGCGGGAGGTGGTC-GATAC-3’, reverse primer/ 5’-CATGGGAGTGCCCTT-GTTG-3’, probe/ 5’-(FAM)-TCAACCAGTCCCACCT-GTCCCAACA-(TAMRA)-3’; HNF-4 forward primer/ 5 ’ -GGTGTCCATACGCATCCTTGA-3’ , reverse primer/ 5’-TGGCTTTGAGGTAGGCATACTCA-3’, probe/ 5’-(FAM)-CCTTCCAGGAGCTGCAGATC-GATGAC-(TAMRA)-3’; GAPDH forward primer/ 5’-CTCCCCACACACATGCACTTA-3’, reverse primer/ 5’-CCTAGTCCCAGGGCTTTGATT-3’, probe/ 5’-(VIC)-AAAAGAGCTAGGAAGGACAGGCAACTTGGC- (TAMRA)-3’.

RESULTSStructure of cells cultured in bioreactorIn the bioreactor, cells cultured in high density assumed layered form on the cellulose beads. Lumen-like structure was observed. Endothelial cells were exits with flat shape at the surface of the lumen and the perfusion side (Figure 2). Multiple layers of FLC-5 cells adhered to the cellulose beads, while A7 and M1 cells were predominantly localized to the side where perfusion occurred. Layered cells were seen in a hole of porous cellulose beads. Sinusoid-like lu-men was observed at perfusion side in the cellulose beads

Figure 2 Light microscopic image of coculture in the RFB. High-density and layered cells at tached on the ce l lu lose beads (C ) . S inuso id - l i ke lumen structure (L) could be observed. SEC was observed with flat shape on surface of the lumen and perfusion side (arrow).

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(Figure 3A). TEM showed that cocultured cells assumed layered form from cellulose beads to the perfusion side (Figure 3B). M1 and A7 cells containing vitamin A-laden fat droplets were seen mainly at the perfusion side, while dense layers of FLC-5 cells were observed beneath (Fig-ure 3C). At sites where the three cell lines were in contact with each other bile canaliculus-like structures were pres-ent between neighboring FLC-5 cells. Lumens of these structures contained electron-dense bile components, tight junctions and desmosomes also could be observed (Figure 3D). This side showed growth of endothelial cells with the formation of sinusoid-like vascular structures (Figures 4A and 4B). Tight junctions were seen between endothe-

lial cells (Figure 4C). Fenestrae which are characteristic of SEC in vivo, were absent in monocultures of M1 cells on plastic dishes (Figures 5A and 5B). Because a long time subculture would change the character of M1 cells, pores were represent on the surface of M1 cells co-cultured in the RFB system (Figure 5C). The pores had a diameter of 100 to 200 nm, being similar in morphology and size to those of fenestrae shown by SEC in vivo (Figure 5D).

Morphology of M1 cells incubated with swinholide ACells incubated for 2 h with 200 nmol/L of the actin-dis-rupting agent swinholide A showed the increased number of pores (Figure 6), while some pores were dilated (about

Figure 3 Transmission electron microscopic images of cocultures in the RFB. A: The cells are arrayed on the cellulose beads. Several cell clusters could be seen in a gap of cellulose beads (arrow). Vascular lumen structure surrounding cell clusters could be seen in the beads (small arrow). Culture media flow through inside of lumen structure; B: The cells are arrayed in layers on cellulose beads. Part of a cellulose bead (arrow) is visible at the bottom of the layer. A process of a sinusoidal endothelial cell (arrowhead) is seen at the perfusion side. Scale bar: 5 μm; C: Sinusoidal endothelial cells (EC) can be seen at the perfusion side. Hepatic stellate cells (SC) containing fatty vitamin A droplets are seen overlying the FLC-5 cells (H). FLC-5 cells (H) below EC and SC show bile-canaliculus-like structures (B). Scale bar: 5 μm. D: Bile canaliculus-like structures (B) containing electron dense bile components. Tight junctions (t) and desmosomes (d) are visible, as are fatty vitamin A droplets (L). Scale bar: 2 μm.

Figure 4 Ultrastructure of sinusoidal endothelial cells. A: Scanning electron microscopic image of sinusoidal endothelial cells localized at the perfusion side. They form a thin layer (arrowhead), showing the typical appearance of a sinusoid-like vascular structure. Scale bar: 5 μm; B: Transmission electron microscopic image showing sinusoidal endothelial cell growth at the perfusion side forming a thin layer (arrowhead) overlying the A7 cells (SC). Scale bar: 1 μm; C: Transmission electron microscopic view showing tight junctions (arrow) between sinusoidal endothelial cells (EC). Scale bar: 200 nm.

Figure 5 Scanning electron microscopic image of the surface of sinusoidal endothelial cells. A: Low-magnification scanning electron microscopic images of the surface of sinusoidal endothelial cells cultured on plastic dishes. The sinusoidal endothelial cells formed a thin layer on the plastic dish substrate. Scale bar: 5 μm; B: High-magnification scanning electron microscopy images of the surface of sinusoidal endothelial cells cultured on plastic dishes. Fenestrae could not be detected on the surface of endothelial cells. Only small pits are seen (arrow). Scale bar: 1 μm; C: Low-magnification scanning electron microscopic view of the surface of sinusoidal endothelial cells cocultured in the RFB. Fenestrated pores could be observed (arrow). Scale bar: 5 μm; D: High-magnification scanning electron microscopic view of the surface of sinusoidal endothelial cells cocultured in the RFB. Pores have a diameter of 100 - 200 nm. Scale bar: 1 μm.

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1 mm). Small pores (tens of nanometers in size) that prob-ably resembled coated pits were abundant in the nonfenes-trated areas.

TEM investigation showed that treatment with swin-holide A resulted in fenestrated pores with a diameter between 100 and 200 nm. The pores fused with each other formed labyrinthine structures (Figure 7A). In addition, vacuoles with a diameter of about 200 nm, similar to pre-viously described vesiculo vacuolar organelles (VVO), were noted. These structures typically were seen in areas where relatively regular overlap was seen in FLC-5, A7, and M1. The number of VVO increased when cells were treated with 200 nmol/L swinholide A, which was associated with partial fusion (Figure 7B).

Amino acid fractions from supernatantsAt the end of culture, the supernatant was subjected to amino acid analysis. Urea production was not seen in monocultures of FLC-5 cells in the RFB, while FLC-5 cells cocultured with M1 and A7 cells produced 523 nmol urea /mL in the culture medium, suggesting that the urea cycle was activated in the coculture RFB system (Figure 8). Several amino acids were increased in the medium.

We compared mRNA expression of CPS1, OTC, ASS, ASL, and ARG in FLC-5 monolayer cultures with those of monocultures in the RFB system. In addition, mRNA expression in cocultures in the RFB also was assessed. We

could not detect OTC in any type of culture. Expression of other urea cycle enzymes showed no notable difference between monolayer culture of FLC-5 and monoculture of FLC-5 in the RFB. However, ASS and ARG expressions in co-culture in the RFB were about 7 and 3 times greater than those in FLC-5 monolayer culture (Figure 9).

Albumin synthesis and expression of nuclear factorsWe compared mRNA expression of albumin and HNF-1 and HNF-4 as transcription regulation factors between ex-perimental conditions. Expression of mRNA encoding the three proteins was less in FLC-5 co-cultures in the RFB system than in FLC-5 RFB monocultures or in FLC-5 cells in monolayer culture (Figures 10A-10C). In a previous study, albumin production was enhanced in the RFB using the immortalized cell line[17]. However it was different cell line in this study.

DISCUSSIONIntroduction of a functional human hepatocellular carci-noma cell line (FLC) in our system can allow the cells to be cultured at high density in a layered array and maintain viability for long periods[17,18].

Immortalized cells can be used for artificial liver. The reason is that it can supply cells in large quantities and quickly. Immortalized cells lose several characteristics in

Figure 6 Scanning electron micrographs of the surface of swinholide A - treated SEC cells in the RFB culture system. Large open pores have a fenestra-like appearance (short arrow). Small pores were detectable in the nonfenestrated area (long arrow). Scale bar: 1 μm.

Figure 7 Transmission electron micrographs of sectioned SEC cells after swinholide A - treatment; A: Numerous open pores or fenestrae in the cytoplasm (arrow). Fine cytoskeletal elements showing a close spatial relationship with these pores (arrowhead). Scale bar: 200 nm; B: VVO could be observed in SEC cells (arrows) in response to stress or actin fibers (arrowheads). Scale bar: 200 nm. Inset shows the overall composition of the cells. Scale bar: 1 μm.

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Figure 8 Amino acid and urea analysis in supernatants. Urea was detectable only in coculture, at 523 nmol/ml. ASF 104 designates culture medium. Mean value ± SD.

Figure 9 Comparison of expressions of CPS1, OTC, ASS, ASL, and ARG mRNA in FLC-5 incubated under different conditions as assessed by TaqMan 1-step RT-PCR. The mRNA expression of each enzyme in different conditions is relative to that in monolayer cultures. Mean value ± SD.

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morphology and function. However, three cell lines were studied in our RFB culture system, including their fine structure according to electron microscopy. Layers of FLC-5, A7, and M1 were arranged respectively from the carrier attachment side to the perfusion side. In some ar-eas, liver-like architectures, sinusoid-like lumen structure, bile-canaliculi and functional complex, were observed, comparable to in vivo tissue relationship. The M1 cell line well covered the perfusion side, mimicking vascular struc-tures, indicating that this cell type forms an arrangement similar to that in vivo. Furthermore, M1 cells in monolayer culture did not express fenestrae, probably reflecting a long culture or subculture time[19]. In a previous study, we found that M1 cells also lack fenestrae in monocultures in the RFB[7]. In contrasts, fenestrated pores were seen in M1 cells cocultured according to the present RFB experimental design. Coculture and cell to cell contact have an influence on these morphological changes. Because fine structures in vivo could be observed better than monoculture in the RFB.

The electron microscopic observations in the present study clearly showed that if an appropriate environment for cell growth was provided in a perfusion culture system, the individual cell types could arrange themselves accord-ing to their in vivo characteristics, even in a high-density layered culture.

This study also examined the numerical dynamics of fenestrae. For this we exposed the cocultures to the actin-disrupting drug swinholide A[13]. When the cells were treat-ed with swinholide A, the number of pores with a diam-eter of about 100 to 200 nm increased 2 h after swinholide A treatment. Furthermore, by TEM, cytoplasmic vesicles about 200 nm in diameter could be seen and were much larger than the caveolae in the cytoplasm, and their num-ber increased in the presence of swinholide A. These vac-uolar-like vesicles probably represent the vesiculo vacuolar organelle (VVO) as described by Feng et al[20]. The VVO is an organelle contributing to transport of macromolecules between luminal and abluminal sides of endothelial cells, thus increasing transcellular permeability. Vascular perme-ability factor and vascular endothelial growth factor (VPF/VEGF) can induce formation of VVO[21]. FLC-5 used in this study, could express VPF/VEGF (data not shown).

The presence of vascular factors may partially explain why VVO is noted in cocultures and why fenestrae could be observed in our experiments[22]. VVO is thought to be formed by fusion of caveolae, when multiple VVOs fuse together, a structure extending from the luminal to the ab-luminal sides of endothelial cells is formed. In the present study, fused VVOs also were seen in swinholide A - treated specimens by transmission electron microscopy, suggest-ing that this fusion represents a process culminating in formation of the labyrinthine structures in SEC[23]. The mechanism of pore formation in immortalized SEC and under cocultured perfusion conditions remains unknown from the present study. However, pore formation may result from multiple effects or factors working in concert upon endothelial cells, such as cytoskeletal dynamics rep-resented by actin and/or the influence of a yet unknown factor secreted by other cell types present in the cocultures such as VEGF. The observation that hepatic endothelial cells maintain one of their typical morphological features (i.e. an abundant number of membrane-bound coated-pits, uncoated vesicles/vacuoles and fenestrae) is an indication that the bioreactor mimics a nearby physiological cultiva-tion environment for the various liver cell types. However, the mechanism by which the bioreactor and its culture environment bring about and maintain these membrane-bound vesicles and fenestrae in endothelial cells remain to be elucidated and consequently open up new directions for future experiments.

To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes and albumin synthesis by FLC-5 in monolayer culture compared to these single-type cultures and cocultures in the RFB. Previously, we have demonstrated hepatocyte functions such as albumin synthesis and cytochrome expression are enhanced in the RFB[24, 25]. Urea production is among the most primitive functions of liver cells. We could not detect urea in me-dium from monolayer cultures or monocultured FLC-5 in the RFB. In contrast, FLC-5 cells cocultured in the RFB exhibit ability to produce urea, and mRNA expression for ASS and ARG is enhanced. The medium used in this experiment, ASF 104 contained arginine, so urea produc-tion was observed in cocultures in the RFB although OTC was not expressed. One report showed that urea produc-

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Figure 10 Expression of mRNA for albumin (A), HNF-4 (B) and HNF-1 (C) as transcription regulation factors in each condition. Messenger RNA expression for these three proteins decreased in cocultured FLC-5 in the RFB compared with FLC-5 monocultures in the RFB and FLC-5 cultured in a monolayer. Mean value ± SD. The ratio of mRNA for each protein versus GAPDH is shown. Differences with respect to each condition were statistically significant (bP < 0.01) according to Student’s t-test.

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tion in OTC-deficient mice could be detected under the same condition[26]. Glutamic acid, glutamine and alanine were also increased in supernatant co-cultured in the RFB, indicating that amino acid metabolism becomes active.

It was reported that three-dimensional spherical cul-ture induces albumin synthesis, a particularly important hepatocytic function[27, 28]. However, in the present study, mRNA expression of albumin was decreased under co-culture conditions in the RFB. Nuclear transcriptional factors HNF-4 and HNF-1, which regulate albumin syn-thesis, were decreased under coculture conditions in the RFB. Albumin in supernatant was also decreased during culture (data not shown). The results suggest that the cul-ture environment (cell-to-cell communication, cell polarity, shear stress, and other factors) can control manifestations of intracellular nuclear transcription factors and therefore dramatically influence albumin production by liver cells. Immortalized cells can be used for artificial liver. The rea-son is that it can supply cells in large quantities and quickly. But immortalized cells may change the characteristics of its original cells. In this study, albumin synthesis was decreased. It was not useful for artificial liver. In future study, we have to try other cell sources (ES cell, oval cell, and other immortalized cell lines).

Finally, several points should be noted concerning our culture system. First, controlling the mixture ratio of the three cell types used is very difficult since each type pos-sesses its own potential for active growth. Thus, growth rates vary between cell types and are difficult to control. For examples, A7 cells grew less rapidly and tended to be less than the other two cell types in the coculture system. Second, the hepatic lobule spans about 140 μm in vivo, ex-tending from the portal to the central area, toward which portal blood flows in a radial manner. According to Matsu-moto et al[29], the liver is an organ composed of numerous groups of microscopic three-dimensional units (minimal radial-flow bioreactors) extending from the inflow side (composed of combinations of parabola-shaped inflow fronts) to the central vein[30]. According to this model, the liver microcirculation as observed in vivo could not be re-produced faithfully with a radial-flow bioreactor, since the

distance between inflow and outflow sides in the bioreac-tor is about 1.5 cm (Figure 11). Third, bile canaliculus-like structures are formed between hepatocytes. Since we did not use the bile duct cells in this study, whether different cell types can reconstruct bile ducts remains to be eluci-dated[31]. Finally, although several questions remain, the re-sults of the present study suggest that liver reconstruction is possible in vitro. Such organ reconstruction technology is expected to contribute greatly to the development of so-phisticated artificial livers and other organs for transplanta-tion. Our culture system may be a very important tool to maintain liver organ.

ACKNOWLEDGMENTSThe authors thank Mr. Hideki Saito, Mrs. Emi Kikuchi, and Mrs. Hisako Arai of the DNA Medical Institute at The Jikei University School of Medicine for technical assistance with electron microscopy. The authors also thank the members of the Australian Key Center for Mi-croscopy and Microanalysis of The University of Sydney for their excellent administrative, technical, and practical support.

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4 Tabata Y. Tissue regeneration based on growth factor release. Tissue Eng 2003; 9 Suppl 1: S5-S15

5 Sussman NL, Chong MG, Koussayer T, He DE, Shang TA, Whisennand HH, Kelly JH. Reversal of fulminant hepatic failure using an extracorporeal liver assist device. Hepatology 1992; 16: 60-65

6 Sussman NL, Kelly JH. Improved liver function following treatment with an extracorporeal liver assist device. Artif Or-gans 1993; 17: 27-30

7 Matsuura T, Kawada M, Hasumura S, Nagamori S, Obata T, Yamaguchi M, Hataba Y, Tanaka H, Shimizu H, Unemura Y, Nonaka K, Iwaki T, Kojima S, Aizaki H, Mizutani S, Ikenaga H. High density culture of immortalized liver endothelial cells in the radial-flow bioreactor in the development of an artificial liver. Int J Artif Organs 1998; 21: 229-234

8 Matsuura T, Kawada M, Sujino H, Hasumura S, Nagamori S, Shimizu H. Vitamin A metabolism of immortalized hepatic stellate cell in the bioreactor. In: Wisse E, Knook D L, De Zanger R, Arthur M J P, eds. Cells of the Hepatic Sinusoid 7. Leiden: Kupffer Cell Foundation, 1999: 88-89

9 Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Lars-en L, Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci U S A 1991; 88: 5096-5100

10 Wisse E. An electron microscopic study of the fenestrated en-dothelial lining of rat liver sinusoids. J Ultrastruct Res 1970; 31: 125-150

11 Braet F. How molecular microscopy revealed new insights into the dynamics of hepatic endothelial fenestrae in the past

Figure 11 RFB and intact organ. A: In the RFB system, culture medium flows from outside the column toward the center of the reactor. Medium flows faster at the center than at the periphery. Biases in distribution of oxygen and nutrition at inflow and outflow are minimized. B: In the hepatic lobe, blood flows from the portal vein to central vein. The RFB system is similar to the organization of the hepatic primary lobe[31]. Figure 11B is reproduced from Figure 9 in reference 31.

200 mm

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decade. Liver Int 2004; 24: 532-53912 Fraser R, Dobbs BR, Rogers GW. Lipoproteins and the liver

sieve: the role of the fenestrated sinusoidal endothelium in lipo-protein metabolism, atherosclerosis, and cirrhosis. Hepatology 1995; 21: 863-874

13 Braet F, Spector I, De Zanger R, Wisse E. A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide. Proc Natl Acad Sci U S A 1998; 95: 13635-13640

14 Braet F, Spector I, Shochet N, Crews P, Higa T, Menu E, de Zanger R, Wisse E. The new anti-actin agent dihydrohalichon-dramide reveals fenestrae-forming centers in hepatic endothe-lial cells. BMC Cell Biol 2002; 3: 7

15 Oda M, Tsukada N, Komatsu H, Kaneko K, Nakamura M, Tsuchiya M: Electron microscopic localizations of actin, cal-modulin and calcium in the hepatic sinusoidal endothelium in the rat. In: Kirn A, Knook DL, Wisse E des. Cells of the Hepatic Sinusoid 1. Rijswijk, Kupffer Cell Foundation 1986; 511-512

16 Gatmaitan Z, Varticovski L, Ling L, Mikkelsen R, Steffan AM, Arias IM. Studies on fenestral contraction in rat liver endothe-lial cells in culture. Am J Pathol 1996; 148: 2027-2041

17 Kawada M, Nagamori S, Aizaki H, Fukaya K, Niiya M, Mat-suura T, Sujino H, Hasumura S, Yashida H, Mizutani S, Ike-naga H. Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: ultrafine structure of functionally enhanced hepatocarcinoma cell lines. In Vitro Cell Dev Biol Anim 1998; 34: 109-115

18 Aizaki H, Nagamori S, Matsuda M, Kawakami H, Hashimoto O, Ishiko H, Kawada M, Matsuura T, Hasumura S, Matsuura Y, Suzuki T, Miyamura T. Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor. Virology 2003; 314: 16-25

19 Braet F, de Zanger R, Seynaeve C, Baekeland M, Wisse E. A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells. J Electron Microsc (Tokyo) 2001; 50: 283-290

20 Feng D, Nagy JA, Hipp J, Dvorak HF, Dvorak AM. Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin. J Exp Med 1996; 183: 1981-1986

21 Feng D, Nagy JA, Pyne K, Hammel I, Dvorak HF, Dvorak AM. Pathways of macromolecular extravasation across mi-crovascular endothelium in response to VPF/VEGF and other vasoactive mediators. Microcirculation 1999; 6: 23-44

22 Yokomori H, Oda M, Yoshimura K, Nagai T, Ogi M, Nomura M, Ishii H. Vascular endothelial growth factor increases fenes-tral permeability in hepatic sinusoidal endothelial cells. Liver Int 2003; 23: 467-475

23 Oda M, Yokomori H, Han J Y, Kamegaya Y, Ogi M, Nakamu-ra M. Hepatic sinusoidal endothelial fenestrae are a stationary type of fused and interconnected caveolae. In: Wisse E, Knook D L, De Zanger R, Arthur M J P, eds. Cells of the Hepatic Sinu-soid 8. Leiden: Kupffer Cell Foundation, 2001: 94-98

24 Nagamori S, Hasumura S, Matsuura T, Aizaki H, Kawada M. Developments in bioartificial liver research: concepts, perfor-mance, and applications. J Gastroenterol 2000; 35: 493-503

25 Iwahori T, Matsuura T, Maehashi H, Sugo K, Saito M, Hoso-kawa M, Chiba K, Masaki T, Aizaki H, Ohkawa K, Suzuki T. CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver. Hepatology 2003; 37: 665-673

26 Li MX, Nakajima T, Fukushige T, Kobayashi K, Seiler N, Saheki T. Aberrations of ammonia metabolism in ornithine carbamoyltransferase-deficient spf-ash mice and their preven-tion by treatment with urea cycle intermediate amino acids and an ornithine aminotransferase inactivator. Biochim Biophys Acta 1999; 1455: 1-11

27 Glicklis R, Merchuk JC, Cohen S. Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and he-patocellular functions. Biotechnol Bioeng 2004; 86: 672-680

28 Ma M, Xu J, Purcell WM. Biochemical and functional changes of rat liver spheroids during spheroid formation and main-tenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activi-ties of some enzymes. J Cell Biochem 2003; 90: 1166-1175

29 MacSween R. N. M. Pathology of the Liver, 4th ed. In: Devel-opmental anatomy and normal structure, Churchill Livingstone, 2002: 16-22

30 Matsumoto T, Komori R, Magara T, Ui T, Kawakami M, Hano H. A study on the normal structure of the human liver, with special reference to its angioarchitecture. Jikei Med J 1979; 26: 1-40

31 Ishida Y, Smith S, Wallace L, Sadamoto T, Okamoto M, Auth M, Strazzabosco M, Fabris L, Medina J, Prieto J, Strain A, Neuberger J, Joplin R. Ductular morphogenesis and functional polarization of normal human biliary epithelial cells in three-dimensional culture. J Hepatol 2001; 35: 2-9



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Kim MH, Lee SS, Lee SK, Lee SG, Suh CW, Gong GY, Park JS, Kim YH, Kim SH. Interleukin-2 gene-encoded stromal cells inhibit the growth of metastatic cholangiocarcinomas. World J Gastroenterol 2006; 12(12): 1889-1894


INTRODUCTIONCholangiocarcinoma arising from bile duct epithelium is the second most common primary liver cancer in the world[1]. In the United States, 2000-3000 new cases of cholangiocarcinoma are diagnosed per year[2]. At present, only surgical excision of all detectable tumors can improve 5-year survival[3-5]. However, most patients are not candi-dates for surgery and undergo only endoscopic or percuta-neous biliary drainage procedures, such as plastic or metal stents. Systemic chemotherapy and radiation therapy are not able to enhance the survival of patients with cholangiocarcinoma[6-11].

Introduction of specific genes into the body by infu-sion of vehicle cells that have been modified ex vivo offers a potential therapy for a number of diseases. Vehicle cells most commonly used for gene transfer include hemato-poietic stem cells, peripheral blood lymphocytes and bone marrow stromal cells (BMSCs). Among these, BMSCs have several advantages including ease of culture and gene transduction, as well as specificity to hematopoietic organs such as bone marrow, spleen, and liver. These qualities make BMSCs ideal for gene transfer in hematologic dis-eases[12].

BMSCs have been shown to support hematopoiesis and have the potential to differentiate into cells of multiple lineages, including osteogenic, chondrogenic and adipo-genic cells[13-15]. Human BMSCs can be easily obtained and grown in conventional ex vivo culture. In addition, BMSCs can be infused safely and have been shown to home to the bone marrow, spleen, lung, liver and kidney. Using a mu-rine lymphoma model, we found that immunocell therapy with ad/hIL-2 encoded stromal cells shows promising therapeutic results[16]. Here we further investigated immu-nocell therapy for biliary cancer induced by the KIGB-5 cell line in Syrian golden hamsters, and found that Ad/hIL-2 genetically-modified BMSCs could provide adoptive immunocell therapy for biliary cancer.

Interleukin-2 gene-encoded stromal cells inhibit the growth of metastatic cholangiocarcinomas

Myung-Hwan Kim, Sang Soo Lee, Sung Koo Lee, Seung-Gyu Lee, Chul-Won Suh, Gyung-Yub Gong, Jung-Sun Park, Young-Hoon Kim, Sang-Hee Kim

Myung-Hwan Kim, Sang Soo Lee, Sung Koo Lee, Department of Gastroenterology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, KoreaSeung-Gyu Lee, Department of Surgery, University of Ulsan College of Medicine, Asan Medical Center, Seoul, KoreaChul-Won Suh, Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, KoreaGyung-Yub Gong, Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, KoreaJung-Sun Park, Young-Hoon Kim, Sang-Hee Kim, Asan Institute for Life Sciences, Seoul, KoreaSupported by The Asan Institute for Life Sciences of South Korea, No. 2003-013Co-first-authors: Myung-Hwan KimCorrespondence to: Dr. Sang-Hee Kim, Asan Institute for Life Sciences, 388-1 Poongnapdong, Songpagu, Seoul 138-736, South Korea. [email protected]: +82-2-30104175 Fax: +82-2-30104182Received: 2005-08-23 Accepted: 2005-10-09

AbstractAIM: To demonstrate bone marrow stromal cel ls (BMSCs) can be used as an attractive target for genetic modification in the treatment of malignant diseases.

METHODS: Using a hamster model of biliary cancer, we investigated the therapeutic effects of interleukin-2 (IL-2) gene-modified BMSCs. Syrian golden hamsters were injected via the femoral vein with 5×105 cells of the KIGB-5 biliary cancer cell line (n=20). One week later, the hamsters were injected intraperitoneally with BMSCs containing Ad/hIL-2 and Ad/∆E1, unmodified BMSCs, or RPMI only (control) and observed for 12 wk (n=5 /each group).

RESULTS: All hamsters treated with BMSCs containing Ad/hIL-2 survived with no evidence of the disease during this period. In contrast, hamsters in the other three groups showed disseminated metastases involving the lungs as early as 4 wk.

CONCLUSION: Ad/IL-2 therapy is effective in the treatment of biliary cancer.


Key words: Bone marrow stromal cell; Adenovirus/hIL-2; Biliary cancer


BASIC RESEARCH

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MATERIALS AND METHODSAnimalsFemale Syrian golden hamsters aged 6-8wk, purchased from Harlan (Indianapolis, Indiana, USA) were housed in the specific pathogen-free unit of the Animal Resource Center at the Asan Institute for Life Sciences and Technology.

Cholangiocarcinoma cell lineThe KIGB-5 cell line, a gallbladder carcinoma cell line derived from Syrian golden hamsters, was established and kindly provided by Dr. Tajima (Nagasaki University, Nagasaki, Japan). This cell line was maintained in com-plete RPMI-1640 (GIBCO BRL, Grand Island, NY, USA) supplemented with 100 mL/L fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA), 100 U/mL peni-cillin (Sigma, St. Louis, MO, USA), 0.1 mg/mL streptomy-cin (Sigma), and 10-5 mol/L β-mercaptoethanol (Sigma)[17]. The 293 cell line, derived from human embryonic kidney, was maintained in high glucose DMEM (GIBCO BRL) and 100 mL/L FBS (GIBCO BRL).

BMSC cultureBone marrow cells were obtained by flushing the femurs and tibias of 6-8 wk old Syrian golden hamsters with RPMI-1640 medium (GIBCO BRL) using 2mL syringes with 23-guage needles. After a single-cell suspension was prepared, the cells were plated at a concentration of 5.0 ×105 /mL in 75 cm2 plastic tissue culture flasks, and cul-tured in McCoy’s 5A medium (Sigma) supplemented with 125 mL/L FBS, 125 mL/L horse serum (HS), 200mmol/L L-glutamine, 0.05 mg/mL hydrocortisone, 1 ng/mL recombinant human basic fibroblast growth factor (rh bFGF, Invitrogen Corporation, Groningen, Netherlands), and 0.5ng/mL rh IL-1α (Invitrogen) at 37 ℃ in 50 mL/L CO2 and 950 mL/L air. The cultures were fed weekly by replacing 500 mL/L of the supernatant with fresh culture medium. When the cells became confluent, they were tryp-sinized and harvested for passage. Gene transduction was performed on cells after 5 passages.

Preparation of recombinant adenovirusThree replication-deficient adenoviral vectors were used, namely Ad/Lac-Z containing the β-galactosidase gene, Ad/∆E1 and Ad/hIL-2 harboring the human IL-2 gene. Each of these vectors was constructed from human ad-enovirus serotype 5 by homologous recombination. The expression of the inserted genes was driven by a CMV promoter. The recombinant adenoviruses were propagated into human 293 embryonic kidney cells, and the adenovi-ral titers were determined by a plaque-forming assay with these cells. Recombinant adenoviruses were diluted to a titer of 5.0 × 108 PFU/mL in viral supernatant and stored at -70 ℃.

Stromal cell transductionStromal cells (SCs) were seeded at a density of 1.0 × 105 /mL in 75 cm2 plastic tissue culture flasks and allowed to grow. The medium was removed, the cells were washed twice with PBS, and the modified adenovirus was added at

various multiplicities of infection (MOI). The cells were cultured at 37℃ for 24h in a humidified atmosphere of 50 mL/L CO2.

The transduction efficiency of the recombinant adeno-viruses was determined by an X-gal assay. BMSCs were transduced with Ad/Lac Z for 24h at 0, 10, 20, 50, 100 MOI. After one round of transfection the transduction ef-ficiency was determined by the percentage of blue-stained cells, after subtraction of the percentage of non-specifical-ly stained, uninfected BMSCs[7]. Stained positive cells were 5-10% at 10 MOI, 10-15% at 20 MOI, 40-50% at 50 MOI, 60-65% at 100 MOI (Figure 1).

From the results of the preliminary experiments we decided to use 50 MOI for further experiments.

X-gal assayBMSCs were seeded in 12-well plates and transfected 24 h later with Ad/Lac Z. The cells were cultured for 24 h at 37 ℃ in a humidified atmosphere containing 50 mL/L CO2. The media were removed and the cells were washed with PBS. Fixing solution was added to the transfected cells at room temperature for 5 min and removed. The cells were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside(X-gal) overnight at 37℃ in a humidified atmosphere containing 50 mL/L CO2.

Stromal cell transplantationTo test for homing of gene-transduced stromal cells, 6-8 wk old Syrian gold hamsters were infused intraperitoneally (IP) or injected via the femoral vein with transduced stro-mal cells mixed in 0.1 mL PBS at a dose of 2.5×106 cells per animal. After one week, the hamsters were sacrificed and the bone marrow, spleen, liver, kidneys, and lungs were obtained. Single cells prepared from these organs using collagenase (GIBCO BRL) were cultured for four days and stained with X-gal to test for homing of the transfected cells.

RT-PCRTotal RNA was extracted using Trizol (Life Technologies, Invitrogen) following the manufacturer’s instructions. The extracted RNA was resuspended in diethylpyrocar-bonate-treated water at a concentration of 0.5 μg/μL. Two ug of each total RNA preparation was reverse-tran-scribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Boeringer, Manheim, Germany) in 20 μL reaction mixture containing 50 mmol/L DTT, 1 μg oligo (dT) and 0.125 mmol/L dNTPs, as described by the manufacturer. The cDNAs were stored at -20℃ or directly used for subsequent amplification. Amplification of human IL-2 mRNA was performed using primers: 5’-TTGCATTGCACTAAGTCTTGC-3’ (forward) and 5’-CAATGGTTGCTGTCTCATCAG-3’ (reverse), whereas amplification of GAPDH mRNA was performed using primers: 5’-ACCACAGTCCATGCCATCAC-3’ (forward) and 5’-TCCACCACCCTGTTGCTGTA-3’ (reverse). PCR was performed in reaction mixtures containing 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 200 μmol/L of each dNTP, 1 μmol/L of each primer, 0.5 units of Taq polymerase, and 1×concentration of PCR buffer, in a total volume of 50 μL. The amplification pro-


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tocol consisted of 32 cycles of denaturation at 94℃ for 30 s, annealing at 55℃ (IL-2) or at 65℃ (GAPDH) for 30 s, and extension at 72℃ for 40 s (IL-2) or 80 s (GAPDH), using a Perkin-Elmer 9600 thermal cycler. The PCR prod-ucts were separated on 15 g/L agarose gels stained with ethidium bromide.

hIL-2 ELISA BMSCs were seeded at a concentration of 2.5 × 105

cells/mL and transduced with Ad/hIL-2 for 1, 2, 3, or 5 d .The supernatants were collected each day, filtered and tested for the presence of IL-2 by ELISA (Endogen, Woburn, MA, USA).

Preparation of cholangiocarcinoma-bearing hamster modelKIGB-5 cells were cultured for 6-7 passages, washed three times and resuspended in PBS. Each hamster was infused through the femoral vein with 5.0×105 KIGB-5 cells. After seven days, the tumor-bearing hamsters were divided into four groups and injected intraperitoneally with PBS (n = 5), unmodified BMSCs (n = 5), BMSCs+Ad/hIL-2 (n = 5) or BMSCs+Ad/∆E1 (n = 5). Hamsters were sacrificed 4, 8, and 12 wk after tumor inoculation, and the number of metastatic lesions in each was assessed.

RESULTSEvaluation of BMSC homingFollowing transduction of BMSCs with Ad/LacZ at 50 MOI for 24 h, 2.5×106 cells were transplanted into each hamster. After one week, the hamsters were sacrificed and

various organs including bone marrow, spleen, kidneys, liv-er, and lungs, were obtained. Single cell preparations were made from each of these organs and examined by X-gal assay. We found that the BMSCs made in our laboratory homed primarily into the bone marrow, spleen and liver, with fewer in the lungs and kidneys (Figure 2).

Expression of human IL-2The Ad/hIL-2 construct was designed to express human IL-2 protein in BMSCs. To confirm hIL-2 expression in BMSCs transduced with Ad/hIL-2, we tested these cells by RT-PCR and ELISA. Both showed IL-2 expression in transduced BMSCs (Figure 3).

Gross and microscopic findings of metastatic lesionsFollowing injection of KIGB-5 cells into hamsters, we ob-served gross metastatic lung lesions after 8 wk (Figure 4).

Hamsters subsequently injected with RPMI, unmodi-fied BMSCs, or BMSCs containing Ad/∆E1 had multiple metastatic lung lesions 8 and 12 wk after tumor cell injec-tion, but no metastatic lesions were observed in the liver or other organs. In contrast, no evidence of metastatic tu-mors was observed in hamsters treated with BMSCs con-taining Ad/hIL-2. These findings were further confirmed by microscopic examination (Figure 5).

DISCUSSIONCholangiocarcinoma is an extremely aggressive tumor most frequently detected at an advanced stage. Fewer than 50% of patients with cholangiocarcinoma have a chance of curative surgery. Patients with advanced disease have a

Figure 1 X-gal assay of hamster BMSCs transfected with Ad/ Lac Z at 0 (A), 10 (B), 20 (C), 50 (D), and 100 (E) MOI.

A B C

D E

Kim MH et al. Ad/h IL-2 encoded BMSC on metastatic cholangiocarcinomas 1891

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median survival time of less than 6 mo and respond poor-ly to conventional chemotherapy[18]. Patients who undergo curative surgery have a 5 year-survival rate ranging from 16% to 32% because of early lymph node metastases[19, 20].

There have been no clinical trials using adoptive im-munotherapy (IL-2 cytokine or dendritic cell and tumor lysates) for biliary cancer. We previously showed that BM-SCs can be targeted to bone marrow in mice as well as to other organs, including spleen, liver, kidneys and lungs. Moreover, BMSCs containing Ad/hIL-2, with a low calcu-lated dose of IL-2, are therapeutically effective for malig-nant lymphoma, because animals treated with this recom-binant demonstrate 100% survival with no evidence of the disease, whereas control mice show extensive metasta-ses[16]. These findings encouraged us to investigate whether Ad/hIL-2 would be effective in hamsters bearing biliary

cancer induced by the KIGB-5 cell line. IL-2 is a cytokine with multiple biologic effects, including enhancement of the cytotoxic activity of cytotoxic T lymphocytes (CTLC). Previous clinical trials have shown that IL-2 has anti-tumor modulating effects in melanoma, renal cell carcinoma and hematologic malignancies[22]. In each of these models, the IL-2 dose is extremely high and has moderate to severe clinical side effects whereas the response rate ranges from 20-25%[21, 22].

Adenoviruses can bind efficiently to epithelial cells and efficiently transfer genes into both replicating and non-replicating cells, although the transfection efficiency varies depending on cell type. Therapy with BMSCs containing Ad/hIL-2 has potential advantages for gene delivery in vivo due to their ability to home to specific organs and to se-crete IL-2 into the local microenvironment. This may en-

A B C

D E

Figure 2 X-gal assay of post -transfection in BM (A), spleen (B), kidney (C), lung (D), and liver (E).

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The hIL-2 ELISA assayA B F i g u r e 3 E x p r e s s i o n a n d quantitative analysis of human IL-2. A: RT-PCR of in vi tro cultured BMSC. Lane 1: 1x106 stromal cells; lane 2: 1x106 stromal cells containing 20 MOI Ad/∆E1; lane 3: 1x106 stromal cells containing 20 MOI Ad/hIL-2; B: ELISA assay of IL-2 secretion from in vitro cultured BMSCs (2.5 X 105/mL) of Syrian golden hamster after transduction with Ad/hIL-2 at MOI of 0(◆), 25(■), 50(▲), and 100 (●). Culture supernatants were harvested on different days and tested for hIL-2 secretion levels by ELISA.


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hance cytotoxicity against tumor cells while reducing side effects.

Using BMSCs containing Ad/Laz ((β-galactosidase gene), we observed strong positive X-gal staining in the bone marrow, spleen, liver, lungs and kidneys. The trans-duction efficiency of these BMSCs was 70% at 50 MOI and 90% at 100 MOI. At 50 MOI, BMSCs containing Ad/hIL-2 showed effective anti-tumor effects. These animals showed no evidence of the disease, whereas the control animals developed widespread metastases during the 12

wk observation period. In addition, hamsters treated with BMSCs containing Ad/hIL-2 showed no evidence of side effects until the time of sacrifice. These findings suggest that BMSCs containing Ad/hIL-2 can implant into various organs and that locally-transplanted IL-2 stimulates and ac-tivates T4 and T8 cells leading to an attack on tumor cells via paracrine effects, thus killing tumor cells at metastatic sites.

One limitation of this study is that BMSCs were infused prior to the formation of measurable tumor mass

Figure 4 Gross findings of metastatic lung lesions in Syrian hamsters 8 wk after injection of KIGB-5 tumor cells in RPMI control (A), BMSCs (2.5 x 106 cells/d) (B), BMSCs containing 50 MOI Ad/∆E1 (2.5 x 106

cells/day) (C), and BMSCs containing 50 MOI Ad/hIL-2 (2.5 x 106 cells/d) (D). Hamsters injected with RPMI, BMSCs, or BMSCs containing Ad/∆E1 showed multiple metastatic lung lesions 8 wk after tumor injection, whereas hamsters injected with BMSCs containing Ad/IL-2 showed no evidence of disease.

A B

C D

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F i g u r e 5 M i c r o s c o p i c findings in Syrian hamsters 8 wk after injection of KIGB-5 tumor cells in RPMI control ( A ) , B M S C s ( 2 . 5 x 1 0 6 c e l l s / d ) ( B ) , B M S C s containing 50 MOI Ad/∆E1 (2.5 x 106 cells/d) (C), and BMSCs containing 50 MOI Ad/hIL-2 (2.5 x 106 cells/d) (D). Hamsters injected with RPMI, BMSCs, or BMSCs containing Ad/∆E1 showed multiple metastatic lesions in bo th lungs , whereas hams te rs i n jec ted w i th BMSCs containing Ad/IL-2 showed no ev idence o f disease.

Kim MH et al. Ad/h IL-2 encoded BMSC on metastatic cholangiocarcinomas 1893

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in hamsters, thus not allowing us to determine whether the effect of IL-2 was dependent on tumor burden. However, given that BMSCs containing Ad/hIL-2 could suppress cholangiocarcinoma development in hamsters, this therapy may be effective as an adjuvant treatment after curative resection in minimizing residual disease after debulking surgery. These findings suggest that treatment with BMSCs containing Ad/hIL-2 may be one of the potential modalities for cholangiocarcinoma, especially for eradicating residual metastatic biliary cancer.

ACKNOWLEDGMENTSThe authors thank Dr. Tajima for providing the KIGB-5 cholangiocarcinoma cell line.

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19 Jan YY, Jeng LB, Hwang TL, Wang CS, Chen MF, Chen TJ. Factors influencing survival after hepatectomy for peripheral cholangiocarcinoma. Hepatogastroenterology 1996; 43: 614-619

20 Berdah SV, Delpero JR, Garcia S, Hardwigsen J, Le Treut YP. A western surgical experience of peripheral cholangiocarci-noma. Br J Surg 1996; 83: 1517-1521

21 Ju DW, Wang BM, Cao X. Adenovirus-mediated combined suicide gene and interleukin-2 gene therapy for the treatment of established tumor and induction of antitumor immunity. J Cancer Res Clin Oncol 1998; 124: 683-689

22 Rosenberg SA, Yannelli JR, Yang JC, Topalian SL, Schwartz-entruber DJ, Weber JS, Parkinson DR, Seipp CA, Einhorn JH, White DE. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleu-kin 2. J Natl Cancer Inst 1994; 86: 1159-1166



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S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

María del Pilar Cabrales-Romero, Lucrecia Márquez-Rosado, Samia Fattel-Fazenda, Cristina Trejo-Solís, Evelia

Arce-Popoca, Leticia Alemán-Lazarini, Saúl Villa-Treviño

María del Pilar Cabrales-Romero, Lucrecia Márquez-Rosado, Samia Fattel- Fazenda, Evelia Arce-Popoca, Leticia Alemán-Lazarini, Department of Cell Biology, Centro de Investigación y Estudios Avanzados IPN, Av. IPN No. 2508 México DF, MéxicoCristina Trejo-Solís, Department of Neuroimmunology, Instituto Nacional de Neurología y Neurocirugía MVS, Insurgentes Sur 3877, Col La Fama DF MéxicoSupported by CONACyT, México City, Grant 39525-MCorrespondence to: Dr. Saúl Villa-Treviño, Centro de Investig-ación y Estudios Avanzados IPN, Av. IPN No. 2508 México DF CP 07360, México. [email protected]: +52-55-50613800-3993 Fax: +52-55-50613393Received: 2005-10-09 Accepted: 2005-11-11

AbstractAIM: To determine the role of c-Jun N-terminal kinase (JNK) activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosyl-methionine (AdoMet).

METHODS: P r imary hepatocyte cu l tures were pretreated with 100 µmol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays. JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by Ellman’s method and reactive oxygen species (ROS) generation by cell cytometry.

RESULTS: The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decrease in ethanol-induced apoptosis (P < 0.05). AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity, and prevented cytochrome c release and pro-caspase 3 cleavage.

CONCLUSION: The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease (ALD) treatment.


Key words: Alcoholic liver disease; c-Jun N-terminal kinase; Apoptosis; SP600125; S-Adenosyl methionine; Bid; Reactive oxygen species

del pilar Cabrales-Romero M, Márquez-Rosado L, Fattel-Fazenda S, Trejo-Solís C, Arce-Popoca E, Alemán-Lazarini L, Villa-Treviño S. S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism. World J Gastroenterol 2006; 12(12): 1895-1904


INTRODUCTIONEthanol abuse increases the risk of developing liver damage, such as fatty liver, hepatitis, cirrhosis and the development of viral hepatitis and hepatocarcinomas[1-3].Alcoholic l iver disease (ALD) is a common health problem in Western countries, therefore the diagnostic evaluation and clinical management of ALD are major issues of concern. Clinical management depends on the extent of ALD and, although alcohol abstinence and supportive care are the classical treatments for ALD, the development of new therapies to improve ALD outcome are being examined. Among these new therapeutic agents are small c-jun N–terminal kinase (JNK) inhibitors, such as SP600125 and S-Adenosyl methionine (AdoMet), the principal biological methyl donor and also a glutathione (GSH) precursor in the liver[4-6]. However, an incomplete knowledge of the molecular mechanisms regulated by these agents in ALD development has complicated the validation of these new therapeutic approaches.

BASIC RESEARCH

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The JNK signaling pathway, a member of the mitogen-activated protein kinases family, regulates cell differ-entiation, proliferation, inflammation, cell survival and cell death in response to stress signals[7,8]. Acute ethanol exposure induces JNK activation, promoting hepatocyte apoptosis[9-11], which has been strongly correlated with liver disease and postulated as the “nexus of liver injury and fibrosis”[12]. Small molecule JNK inhibitors, such as SP600125[13], have been proved to exert a protective effect in reperfusion liver injury and are suggested as optional treatment for liver disease[14-16]. Thus, the JNK signaling transduction pathway has emerged as an interesting ele-ment in liver disease development, opening the possibility that its modulation may represent an important approach for ALD treatment. Besides specific small molecule in-hibitors, JNK activity may be modulated by other thera-peutic agents, such as antioxidants[17-19]. Indeed, in diverse aspects of cellular function, JNK is implicated as a redox stress cell sensor[20]. Alcohol ingestion promotes oxida-tive stress[21-24] and causes changes in intrahepatic GSH[25,

26]. This events may be related to JNK activation induced by ethanol. AdoMet, which has been shown to inhibit he-patocyte apoptosis[27], is increasingly used for liver disease treatment, although its protective mechanisms still remain unclear. A possible hepatoprotective mechanism has been attributed to AdoMet effects on the regulation of cytosol and mitochondrial GSH levels[28-30]. The up-regulation of GSH levels may have a down-regulatory effect on JNK signaling, since GSH acts as a key cell signaling modula-tor[31,32].

Although JNK has been identified as a pro-apoptotic signaling pathway involved in ALD development, the mechanism by which JNK induces programmed cell death remains far less clear[9,14,16], limiting the identification and validation of JNK-activity modulator agents as a potential ALD treatment. In this study, we determined the effect of the small JNK inhibitor SP600125 and AdoMet on the apoptotic pathway activated by ethanol and a possible modulating effect of AdoMet on JNK activity as part of its hepatoprotective mechanism. We found that JNK ac-tivation played a critical role in ethanol-induced apoptosis by means of its effect on Bid fragmentation, a key pro-apoptotic target of JNK. Even though AdoMet increased GSH concentration and protected against ethanol-induced apoptosis, we did not find an effect of AdoMet on JNK activity. These results strongly suggest that JNK activation induced by ethanol is independent of ROS generation, and that the AdoMet protective effects lie mainly at mitochon-drial level.

MATERIALS AND METHODSMaterials Anti-JNK-P and DeadEndTM Colorimetric TUNEL System were obtained from Promega (Madison, WI). Antibodies against JNK and c-jun-P were purchased from Cell Signal-ing. c-jun, Bid, caspase 3 and cytochome c antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin monoclonal antibody was kindly provided by Dr. Manuel Hernández (CINVESTAV, IPN México). JNK inhibitor SP600125 and caspase 8 inhibitor Z-IETD-

fluoromethyl ketone were purchased from Calbiochem (San Diego, CA). Collagenase, ethanol, dimethyl sulfoxide (DMSO), AdoMet in p-toluene sulfonate salt form, 2’, 7’-dichloro-dihydrofluorescin diacetate (DCFH-DA) and all other reagents were purchased from Sigma Chemical Com-pany (St. Louis,MO). Electrophoresis reagents and Protein assay kit were from Bio-Rad (Richmond, Calif.)

Animals Male Fisher 344 rats with an average weight of 200 g were obtained from the Lab Animal Facility UPEAL, CIN-VESTAV. Animal care was performed according to the guidelines established by the Institutional Animal Care Committee

Hepatocyte isolation and treatmentsRat hepatocytes were isolated from male Fisher rats (weigh-ing 180-200 g) by collagenase perfusion as previously de-scribed[33]. Viable hepatocytes were separated by Percoll gradient centrifugation at 1 500 r/min for 2 min at 4 °C. Subsequently, cell viability was examined by trypan blue exclusion, and was over 90%. Hepatocytes were plated in Dulbecco’s modified Eagle’s medium (Gibco-BRL) supple-mented with 100 mL/L bovine serum (Gibco-BRL). After 2 h incubation, the medium was changed to William’s E medium containing 100 mL/L bovine serum, 1.5 U/mL insulin, 20 nmol/L dexamethasone, 100 µg/mL strep to-mycin and 100 U/mL penicillin. Cells were cultured over-night at 37 °C in a humidified atmosphere containing 50 mL/L CO2. The day after plating, cells were washed with Hank’s balanced salt solution (HBSS), and incubated in William’s E medium (supplemented with 1 mL/L of fe-tal calf serum, 100 µg/mL streptomycin and 100 U/mL penicillin) with either 100 µmol/L SP600125, 25 µmol/L Z-IETD-FMK or 1 mL/L DMSO as a vehicle control for 2 h. Another set of cultures was pre-incubated with 4 mmol/L AdoMet for 1 h. Then 100 mmol/L ethanol was added and the dishes were sealed with parafilm to prevent evaporation. The cells were incubated for different periods of time at 37 °C before collection.

Cell viability assessmentCell viability was determined using the MTT (methyl thia-zole tetrazolium) assay as previously described[34]. Briefly, at indicated times after pretreatments and ethanol exposure, media were removed and MTT solution (0.4 mg/mL in media, filter sterilized) was added and the cells were incu-bated for 2 h at 37 °C. Plates were washed twice with PBS. The resultant formazan product was dissolved by addition of 500 µL DMSO to the plate. Optical density was mea-sured at A595 nm and cell survival was expressed as percent-age of absorbance.

DNA gel electrophoresisAfter different pretreatments and 24 h of ethanol expo-sure, hepatocytes (detached and attached) were harvested and washed in ice-cold PBS by centrifugation at 4°C at 1500 r/min for 5 min in an Eppendorf microcentrifuge. Approximately 8 × 105 cells were resuspended in lysis buffer (200 mmol/L EDTA; 100 mmol/L Tris, pH 8.0;


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8 g/L sodium lauryl sarcosine; 20 µg/mL DNase-free RNasa ) and incubated at 37 ℃ for 2 h, followed by addi-tion of proteinase K (200 µg/mL) and another overnight incubation at 50 °C. DNA was electrophoresed on 18 g/L agarose gel at 35 mv, stained with ethidium bromide and visualized under UV light[35].

TUNEL assayThe TUNEL assay was performed using a DeadEnd colo-rimetric apoptosis detection system kit. Rat hepatocytes were cultured on Falcon Chamber slides pre-coated with poly-L-lysine. After different pretreatments and 24 h etha-nol exposure, slides were fixed by immersion in 40 g/L paraformaldehyde in PBS for 25 min at room temperature. Then, the TUNEL assay was performed as previously de-scribed[36] following the manufacturer’s instructions. One hundred cells were counted in three randomly selected microscopic fields and cell apoptotic rate was expressed as a percentage of the total cells counted.

Isolation of mitochondriaMitochondria were isolated from 4.8 × 106 cells. Cells were washed twice with HBSS, scrapped and centrifuged for 10 min at 1 500 r/min at 4 ℃ in an Eppendorf microcen-trifuge. Cell pellets were resuspended in permeabilizing buffer (210 mmol/L D-manitol, 10 mmol/L HEPES, 0.2 mmol/L EGTA, 50 mmol/L succinate, and addition just before use of 70 mmol/L sacarose, 1.5 g/L bovine serum albumin and 80 µg/mL digitonine, pH 7.2 ) and incubated at 4 ℃ for 20 min. Permeabilized cells were centrifuged at 170 r/min for 10 min at 4 ℃. The supernatant was centri-fuged at 13 000 g for 10 min at 4 ℃, the second superna-tant obtained contained the cytosolic fraction. The pellet resultant from the first centrifugation after the permea-bilization step was incubated with 1 mL/L Triton X-100 in PBS for 20 min at 4 ℃ and centrifuged at 13 000 g for 10 min at 4 ℃, thus the supernatant contained the mito-chondrial fraction. Supernatants were precipitated with 50 g/L sulfosalicylic acid and resuspended in PBS[37]. Pro-tein concentration in the supernatant was determined by the Lowry method.

Immunoblot analysisCellular protein was extracted at 4 ℃ in kinase lysis buffer (20 mmol/L Hepes pH 8.0, 136 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L KCl, 2 mmol/L MgCl2, 50 mmol/L sodium fluoride, 1 mmol/L phenyl-methylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 2 g/L SDS) or Chaps buffer for samples to determine Bid and pro-caspase cleavage (50 mmol/L Tris-HCl, 2 mmol/L EDTA, 1 g/L Chaps, 1 mmol/L phenylmethylsulfonyl fluoride and Complete (BioRad) 25 × ). Cell lysates were centrifuged at 12 000 g for 10 min at 4 ℃. The super-natant protein concentration was determined by Lowry method. Whole cell extracts and cytosolic or mitochondrial fractions were resolved on SDS-PAGE and transferred onto nitrocellullose membrane. Blots were incubated overnight at 4 ℃ with antibodies against JNK, phospho JNK, c-jun, phospho c-jun, Bid, caspase 3 (Bid antibody detected full length Bid and caspase 3 antibody recognized

the precursor of pro-caspase 3 form) and cytochome c. Then, blots were incubated with horseradish peroxidase-conjugated antibody and developed by chemiluminescence.

Intracellular GSH content determinationHepatocytes were incubated under control conditions in William’s E medium only, or were pretreated with 4 mmol/L AdoMet for 1 h and then exposed to ethanol, or only exposed to ethanol. After the indicated times, cells were harvested and washed with HBSS buffer (pH 7.4). Cells (3 ×106 ) were sonicated for 1 min in ice-cold 50 g/L sulfosalicylic acid, incubated for 20 min on ice and centrifuged at 12 000 g at 4 ℃ for 30 min. The resultant thiol extract was assayed by the method of Ellman with previously reported modifications[38,39]. The pellet obtained from the centrifugation was washed twice with 50 g/L sulfosalicylic acid and resuspended in 0.5 mol/L NaOH. Protein concentration was determined by the method of Lowry. The GSH content (including other eventual thiols) was expressed as nmol per mg of protein.

Measurement of intracellular reactive oxygen species (ROS)The level of intracellular ROS was measured by the change in fluorescence resulting from oxidation of DCFDA. After treatment for indicated times, hepatocytes were washed with HBSS and incubated with 5 µmol/L DCFH-DA for 30 min at 37 ℃. Hepatocytes were harvested and resus-pended in HBSS, and then 5 g/L propidium iodide was added to detect dead cells. Intracellular ROS levels were measured on a Benckton Dickinson FACS Calibur. Ten thousand events were recorded for the analysis[40].

Statistical analysisData were analyzed using one-way ANOVA to determine differences between all independent groups. Differences between 2 groups were tested using two-tailed unpaired Student’s t-test. P<0.05 was considered statistically significant.

RESULTSInhibition of ethanol-induced JNK activation by SP600125We first confirmed that 100 mmol/L ethanol exposure promoted JNK activation in our system. Ethanol induced JNK phosphorylation. We detected that the JNK1 basal activity was lower than JNK2, while JNK1 activity increment was higher than that for JNK2. Maximal JNK activation induced by ethanol was detected after 30 min and remained above control levels until 8 h after, as detected by c-jun phosphorylation, one of the most commonly measured parameters of JNK activity (Figure 1A-B). Next, we determined the effect of 100 µmol/L SP600125 on JNK activity. JNK activity significantly decreased in SP600125-pretreated hepatocytes compared to hepatocytes exposed to ethanol only (P < 0.05), as determined by a decrease in c-jun phosphorylation (Figure 2A-B). The Western blot and densitometric analyses shown are representative of three individual experiments.

del Pilar Cabrales-Romero M et al. Hepatoprotective mechanisms of SP600125 and AdoMet 1897

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Inhibition of ethanol-induced apoptosis by SP600125 and AdoMet Once we identified an effect of both ethanol and SP600125 on JNK activity, we determined the effect of SP600125 and AdoMet on the apoptosis process induced by ethanol. Ethanol-induced apoptosis was significantly reduced (P < 0.05) in hepatocyte cultures pretreated with 100 µmol/L SP600125 or 4 mmol/L AdoMet, but not in hepatocytes pretreated with 1 g/L DMSO, the vehicle for SP600125, as shown by the DNA laddering assay (Figure 3A) or TUNEL assay (Figure 3B). A 24-h exposure to 100 mmol/L ethanol produced approximately 47% cell death in non-pretreated hepatocytes as compared to 23% and 25% cell death observed in SP600125- and AdoMet-pretreated cultures, respectively. Cells pretreated with

DMSO but not stimulated with ethanol showed 5% cell death (Figure 1C).

Protection of SP600125 and AdoMet on ethanol hepatocyte injuryIn order to determine whether or not the survival of hepatocytes exposed to 100 mmol/L ethanol was modulated by SP600125 and AdoMet, we determined survival rates after different periods of 100 mmol/L ethanol exposure. Cell metabolic activity, determined by the MTT assay, significantly decreased after 12 h in cells only exposed to ethanol (P < 0.05) compared to the cells pre-incubated with AdoMet, SP600125 or incubated under control conditions. Cell metabolic activity, measured as the percentage of tetrazolium salt reduction, decreased from 75% observed in pretreated hepatocytes to 40% observed in hepatocytes exposed to ethanol only (Figure 4).

AdoMet effect on GSH intracellular levels from ethanol-incubated hepatocytesThe JNK signaling pathway activation may be modulated by antioxidants. AdoMet, a GSH precursor in liver, pre-vented ethanol-induced apoptosis as aforementioned. We tested if, through an antioxidant effect, AdoMet may be related to an inhibitory action of JNK activity as a pos-sible hepatoprotective mechanism. We first determined the effect of 4 mmol/L AdoMet pretreatment on GSH intracellular levels in ethanol-incubated hepatocytes. Pre-incubation with 4 mmol/L AdoMet significantly preventedGSH decrease induced by the exposure of cultured hepa-tocytes to 100 mmol/L ethanol (P < 0.05), and moreover, maintained GSH levels above those found in control hepa-tocyte cultures. A significant increase in GSH concentra-

Figure 1 Inhibition of 100 mmol/L ethanol-induced JNK activation by SP600125.A: Western blot; B: JNK; C: P-c-jun densitometric analysis.

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tion was observed between 8 and 12 h in hepatocytes pre-treated with AdoMet compared to hepatocytes incubated

in control medium (Figure 5) (P < 0.05). Furthermore, ROS generation in hepatocytes pretreated with 4 mmol/L AdoMet and then exposed to ethanol was maintained as in control cells in comparison to hepatocyte cultures ex-posed to ethanol only, in which a significant ROS genera-tion was observed (Figure 6) (P < 0.05).

Effect of AdoMet on JNK activity Once we determined that AdoMet had antioxidant ef-fects, we tested if AdoMet exerted an inhibitory action on JNK activity. We first measured AdoMet effect on c-jun phosphorylation. AdoMet pretreatment slightly decreased JNK activity induced by ethanol treatment by 25.4% with respect to the cultures exposed to ethanol only, contrasting with the effect of the selective JNK inhibitor which pro-duced a c-jun phosphorylation inhibition of approximately 68.8% with respect to ethanol- treated samples. So, the de-crease on c-jun phosphorylation induced by AdoMet was

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Figure 4 Protective effect of SP600125 and AdoMet on ethanol hepatocyte injury. Data represent percentage of MTT reduction in mean±SEM of n = 3. arepresents a significant difference (P < 0.05) in MTT reduction between pretreated and non-pretreated cells.

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not comparable to the decrease produced by SP600125, a selective JNK inhibitor (Figure 7).

Effects of SP600125 and AdoMet on Bid fragmentation, another JNK target We next determined the effects of SP600125 and AdoMet on Bid cleavage. Bid is a proapoptotic member of the Bcl-2 family, which may represent another important JNK target. We observed that 100 mmol/L ethanol hepatocyte exposure promoted Bid cleavage between 4 h and 24 h. SP600125 hepatocyte pretreatment significantly prevented Bid fragmentation compared to cells stimulated with ethanol only between 4 h to 24 h (P < 0.05). Bid has been reported as a caspase 8 target. Indeed, we found that pre-

incubation with 25 µmol/L Z-IETD-fmk, a selective cas-pase 8 inhibitor, significantly decreased Bid fragmentation induced by ethanol (Figure 8) (P < 0.05). These results suggested that Bid cleavage induced by ethanol not only depends on JNK, but also on caspase 8 activity. Neverthe-less, even if AdoMet exerted antioxidant properties, it did not prevent Bid fragmentation induced by ethanol. West-ern blot and densitometric analyses shown are representa-tive of three independent experiments. Decrease in ethanol-induced cytochrome c release and caspase 3 activation by SP600125 and AdoMet Apoptosis induced by 100 mmol/L ethanol involves Bid fragmentation, which is responsible for cytochrome c release with the subsequent activation of caspase 3. We determined the effect of SP600125 and AdoMet on these apoptotic components. Pretreatment of hepatocyte with SP600125 prevented Bid cleavage induced by ethanol and decreased cytochrome c release from mitochondria be-tween 8 h and 24 h after ethanol exposure. Even though AdoMet did not have an effect on Bid fragmentation, it decreased cytochrome c release from mitochondria (Fig-ure 9). Cytochrome c release contributes to pro-caspase 3 fragmentation, so we observed that cytochrome c release prevention by SP600125 and AdoMet also decreased pro-caspase 3 fragmentation (Figure 10). These results indi-cated that the hepatoprotective effects of SP600125 and AdoMet converge at mitochondrial level, but AdoMet antiapoptotic action mainly lies in an inhibition of the mi-tochondrial apoptotic pathway.

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DISCUSSIONThis study points out that modification of the JNK sig-naling transduction pathway by selective JNK inhibitors and mitochondrial function preservation by AdoMet are among several key factors that must be considered to de-velop successful therapies for ALD treatment. We deter-mined the role of JNK activity in the apoptotic process induced by ethanol in primary hepatocyte cultures and the possible modulator effect of JNK activity by AdoMet, an agent increasingly used in ALD treatment.

JNK activity is related to proapoptotic responses, but it is also linked to antiapoptotic signals and cell prolif-eration[41,42], thus the functions and components of this signaling cascade still remain controversial. We exposed

hepatocytes to 100 mmol/L ethanol, considering previous reports which indicate that ethanol levels in the peripheral blood of some alcoholic patients have been exceeded 100 mmol/L[43]. In the present study, the exposure of primary hepatocyte cultures to 100 mmol/L ethanol promoted JNK activation. We detected that JNK1 basal activity was lower than JNK2 basal activity, while the JNK1 activity increment was higher than JNK2. These findings are in agreement with previous reports[9,44] and strongly suggest that JNK1 activity is related to the proapoptotic func-tion of this signaling pathway activation by ethanol. We determined ethanol JNK activation by measuring c-jun phosphorylation, the most common parameter of JNK activity. In our system, JNK activity was significantly inhib-ited by a 2-h pretreatment with 100 µmol/L SP600125, a concentration previously reported for the selective inhibi-tion of JNK[15]. Indeed, c-jun phosphorylation was signifi-cantly decreased by 100 µmol/L SP600125 in comparison with cells pretreated with 1 g/L DMSO only, as a vehicle control. We measured a decrease in c-jun and not in JNK phosphorylation because previous reports have indicated that SP600125 specifically inhibits c-jun phosphoryla-tion and does not down-regulate p-JNK levels[14,15]. In our study, selective JNK inhibition by SP600125 allowed us to confirm that JNK functioned as a proapoptotic signaling pathway in hepatocyte cultures exposed to 100 mmol/L ethanol for 24 h, since in SP600125-pretreated hepatocyte cultures, DNA ladder and positive TUNEL cells signifi-cantly decreased. Our results, in agreement with previous reports[9,15], indicated that JNK might be a key pathway in-volved in apoptosis induction by ethanol. However, other signaling events must be involved in the apoptotic pathway induced by ethanol, since apoptotic cells in SP600125-pretreated hepatocytes were not completely abolished,

Figure 9 Prevention of cytochrome c release by AdoMet and SP600125. A: Western blot; B: mitochondrial cytochrome c; C: cytosolic cytochrome c densitometric analysis. arepresents a significant decrease (P < 0.05) in cytochrome c release in AdoMet- and SP600125-pretreated hepatocytes in comparison with non-pretreated cells.

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Figure 10 Decrease of ethanol-induced pro-caspase 3 fragmentation by SP600125 and AdoMet. A: Western blot; B: densitometric analysis. aindicates a significant difference (P < 0.05) between SP600125- and AdoMet-pretreated hepatocytes compared to non-pretreated hepatocytes.


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suggesting that JNK activity contributes to but is not the only factor required for the apoptotic process induced by ethanol. MAPK p38 is also a key candidate in this process, since there are several reports indicating that TNF-alpha, which plays a critical role in ALD development, induces p38 activation[45]. Recently, Bid, a pro-apoptotic member of the bcl-2 family, has been reported as a JNK target in different systems[46,47]. We observed that the incubation of primary hepatocyte cultures with 100 mmol/L ethanol induced Bid cleavage, which was decreased by SP600125. These results strongly suggested Bid as a key target of the pro-apoptotic JNK activity induced by ethanol. Even though maximal JNK activity, measured by c-jun phos-phorylation, was observed between 30 min and 4 h after ethanol treatment, Bid fragmentation was not observed until 8 h after ethanol exposure, when c-jun phosphoryla-tion was slightly above control. This slow kinetic of JNK-stimulated Bid cleavage may indicate that several steps separate JNK activation from Bid fragmentation[48]. Since JNK is not a protease, it is likely that one of these steps corresponds to a protease induction by JNK[49,50]. Thus, the exact target of JNK involved in Bid fragmentation is yet to be established[51]. Bid cleavage was also decreased when cultures were exposed to Z-IETD-fmk, a selective caspase 8 inhibitor, indeed it has been reported that Bid is a cas-pase 8 target[52]. Fas-mediated apoptosis has been related to FasL induction by c-jun activation, and downstream to caspase 8 and Bid fragmentation. Our result suggests that Bid fragmentation in the apoptosis process induced by ethanol lies at least in two distinct pathways, and not only depends on JNK activity. In accordance with this re-sult, a previous report indicates that Bid fragmentation by JNK does not depend on transcription, and in addition, SP600125 does not affect Fas-mediated apoptosis[16]. JNK and other kinases that regulate JNK activity are known to be responsive to redox changes[20], and oxidative stress is critically involved in ALD development[22-24]. We observed that treatment of hepatocyte culture with 100 mmol/L ethanol induced a peak of ROS generation between 6 h and 8 h after ethanol exposure, which coincided with a significant decrease in GSH levels. These redox alterations may be resulted from two of the main components of alcohol metabolism, alcohol dehydrogenase (ADH) and cytochrome P450 2E1, since it has been reported under similar culture conditions that 4-methyl pyrazole, an ADH inhibitor, led to the reduction of H2O2 generation[21, 53]. Besides, different non-enzymatic free radical pathways and the mitochondrial dysfunction after the reduction of GSH content are involved in ROS generation induced by etha-nol. Therefore, JNK activation in response to ethanol may be related to the oxidative metabolism of ethanol in the liver and to the resultant alterations in intracellular redox state, primarily reflected by GSH changes. AdoMet, an in-creasingly used agent for liver disease treatment, has wide physiological and pharmacological actions, including its effect on GSH metabolism. However, its hepatoprotective mechanisms have not been completely elucidated[27,54]. We observed that AdoMet, as SP600125, significantly reduced ethanol-induced apoptosis. AdoMet has been identified as a GSH precursor, a major cell antioxidant, and JNK activ-ity has been reported to be down-regulated by antioxi-

dant agents. Thus, our study suggested that AdoMet may down-regulate JNK activity as part of its hepatoprotective mechanism through an antioxidant effect. We found that 4 mmol/L AdoMet was able to prevent GSH decrease in-duced by ethanol in primary hepatocyte cultures and even maintained intracellular GSH levels above those found in non-treated cells. Furthermore, AdoMet pretreatment also diminished ethanol- induced ROS generation. These results indicated that AdoMet might be related to the repletion of GSH levels. There are contradictory results about the effect of AdoMet on GSH levels; while a previ-ous study found no effect of AdoMet on GSH levels[27], present results are in agreement with others which re-ported an increment of GSH levels resulted from AdoMet pretreatment[29,55]. Even though AdoMet exerted a redox state regulating effect, surprisingly, it only decreased c-jun phosphorylation by 25.5% in comparison to the inhibition induced by SP600125, which corresponded to 62.8%. We also tested the effect of AdoMet on Bid fragmentation and did not observe an inhibitory effect of AdoMet until 24 h after ethanol exposure. At this time, JNK activity was below control levels, so it is possible that this decrease was not related to JNK activity. These results indicated that even though AdoMet exerted an antioxidant action, it did not have a significant effect on JNK activity, at least on c-jun or Bid, the measured JNK targets. Thus, we sug-gest that JNK activation may be independent of oxidative stress, in accordance with previously reported results in which cell pretreatment with N-acetyl-cysteine failed to prevent JNK activation[10]. Other reports suggest that JNK activation may be independent of ethanol metabolism, since ethanol activates JNK in HepG2 cells which do not express detectable levels of Cyp2E1 and express low lev-els of alcohol dehydrogenase activity[56]. However, further studies are required to determine the regulatory mecha-nisms of JNK activity independent of ROS generation. Even though AdoMet did not have a significant effect on JNK activity as it was measured by its action on c-jun and Bid, it preserved the mitochondrial metabolic activity as it was measured by the MTT assay. In hepatocytes exposed to ethanol only, the mitochondrial function was significant-ly reduced compared to AdoMet-pretreated hepatocytes. Downstream AdoMet prevented cytochrome c release and pro-caspase 3 fragmentation. In agreement with previous reports which indicated that AdoMet maintained mito-chondrial membrane stability and corrected the reduction of GSH content both in cytosol and mitochondria[29,30,56], these results suggest that the AdoMet protective effect mainly lies at mitochondrial level.

In summary, our results point out that ALD is a complex and multifactorial process, and that the mechanisms involved in ALD development must be thoroughly explored for the design of successful therapeutic strategies. We found that AdoMet, although protected against ethanol apoptosis induction and exerted an antioxidant effect, did not show any inhibitory action on Bid fragmentation, a key proapoptotic target of JNK. This finding is in agreement with the suggestion that the mechanisms which lead to JNK activation are independent of ROS generation. We conclude that the JNK signaling pathway modulation by selective inhibitors


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and mitochondrial integrity maintenance by AdoMet are promising components of therapeutic approaches for ALD.

ACKNOWLEDGMENTSWe are very grateful to Sergio Hernández García for his valuable technical assistance. We are also grateful to Victor Hugo Rosales García for his assistance in the performance of flow cytometry experiments and to the Lab Animal Facility UPEAL, CINVESTAV for the animal handle and care. We also thank Isabel Pérez Montfort for correction of the English version of the manuscript.

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S- Editor Wang J L- Editor Kumar M E- Editor Cao L


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presence and absence of N-acetylcysteine (NAC) and SNAC (56 and 560 µmol/L) and monitored at 234 nm.

RESULTS: Animals in the control group developed mod-erate macro and microvesicular fatty changes in peripor-tal area. SNAC-treated animals displayed only discrete histological alterations with absence of fatty changes and did not develop liver steatosis. The absence of NAFLD in the SNAC-treated group was positively correlated with a decrease in the concentration of LOOH in liver homog-enate, compared to the control group (0.7±0.2 nmol/mg vs 3.2±0.4 nmol/mg protein, respectively, P<0.05), while serum levels of aminotransferases were unaltered. The ability of SNAC in preventing lipid peroxidation was con-firmed in in vitro experiments using LA and LDL as model substrates.

CONCLUSION: Oral administration of SNAC prevents the onset of NAFLD in Wistar rats fed with choline-deficient diet. This effect is correlated with the ability of SNAC to block the propagation of lipid peroxidation in vitro and in vitro .


Key words: Nitric oxide; S -nitroso-N -acetylcysteine; Oxidative stress; Nonalcoholic fatty liver disease

de Oliveira CPMS, Simplicio FI, de Lima VMR, Yuahasi K, Lo-passo FP, Alves VAF, Abdalla DSP, Carrilho FJ, Laurindo FRM, de Oliveira MG. Oral administration of S -nitroso-N-acetylcys-teine prevents the onset of non alcoholic fatty liver disease in rats. World J Gastroenterol 2006; 12(12): 1905-1911


INTRODUCTIONNonalcoholic steatohepatitis (NASH) is considered a par-ticular type of a large spectrum of nonalcoholic fatty liver disease (NAFLD), which includes fat alone and fat with nonspecific inflammation[1,2]. Although several predispos-ing factors such as obesity and diabetes, are related to NAFLD，the pathogenesis of NAFLD and its progres-sion to fibrosis and chronic liver disease are still unclear[3-5]. One of the main hypotheses is that the mechanism of he-patocyte injury in NASH is associated with oxidative stress

Oral administration of S -nitroso-N -acetylcysteine prevents the onset of non alcoholic fatty liver disease in rats

Claudia PMS de Oliveira, Fernanda I Simplicio, Vicência MR de Lima, Katia Yuahasi, Fabio P Lopasso, Venâncio AF Alves, Dulcinéia SP Abdalla, Flair J Carrilho, Francisco RM Laurindo, Marcelo G de Oliveira

Claudia PMS de Oliveira, Vicência MR de Lima, Fabio P Lopasso, Flair J Carrilho, University of São Paulo, School of Medicine, Department of Gastroenterology, São Paulo, SP, Brazil Venâncio AF Alves, School of Medicine, Department of Pathology, São Paulo, SP, Brazil Francisco RM Laurindo, University of São Paulo, Medical School, Heart Institute, InCor, São Paulo, SP, BrazilDulcinéia SP Abdalla, Katia Yuahasi, University of São Paulo, School of Pharmaceutical Sciences, Department of Clinical and Toxicological Analysis, Sao Paulo, SP, BrazilMarcelo G de Oliveira, Fernanda I Simplicio, State University of Campinas, Chemistry Department, Campinas, SP, BrazilSupported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Co-first-authors: Claudia PMS de Oliveira and Fernanda I SimplicioCo-correspondents: Claudia PMS de OliveiraCorrespondence to: Professor Marcelo G. de Oliveira, Instituto de Química, UNICAMP, CP 6154, CEP 13083-970, Campinas, SP, Brazil. [email protected]: +55-19-37883132 Fax: +55-19-37883023Received: 2005-06-30 Accepted: 2005-07-20

AbstractAIM: To evaluate the potential of S -nitroso-N -acetyl-cysteine (SNAC) in inhibition of lipid peroxidation and the effect of oral SNAC administration in the prevention of nonalcoholic fatty liver disease (NAFLD) in an animal model.

METHODS: NAFLD was induced in Wistar male rats by choline-deficient diet for 4 wk. SNAC-treated animals (n=6) (1.4 mg/kg/day of SNAC, orally) were compared to 2 control groups: one (n=6) received PBS solution and the other (n=6) received NAC solution (7 mg/kg/d). Histological variables were semiquantitated with respect to macro and microvacuolar fat changes, its zonal dis-tribution, foci of necrosis, portal and perivenular fibro-sis, and inflammatory infiltrate with zonal distribution. LOOHs from samples of liver homogenates were quanti-fied by HPLC. Nitrate levels in plasma of portal vein were assessed by chemiluminescence. Aqueous low-density lipoprotein (LDL) suspensions (200 µg protein/mL) were incubated with CuCl2 (300 µmol/L) in the absence and presence of SNAC (300 µmol/L) for 15 h at 37 ℃. Extent of LDL oxidation was assessed by fluorimetry. Linoleic acid (LA) (18.8 µmol/L) oxidation was induced by soy-bean lipoxygenase (SLO) (0.056 µmol/L) at 37 ℃ in the


BASIC RESEARCH

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and lipid peroxidation resulting from the imbalance be-tween pro-oxidant and antioxidant chemical species[6]. Such an imbalance is associated with increased β-oxidation of fatty acids by mitochondria, peroxisomes, and cytochrome P450 2E1 (CYP2E1) pathways. These oxidative processes produce free electrons, H2O2, and reactive oxygen species (ROS) while depleting the potent antioxidants, glutathione and vitamin E[1]. The increased levels of free fatty acids present in the fatty liver provide a perpetuating and propa-gating mechanism for oxidative stress via lipid peroxida-tion, with secondary damage to cellular membranes and key organelles such as mitochondria[6]. Lipid peroxidation usually leads to the formation of peroxyl radicals, which are central species in the peroxidation chain reaction. En-zymatic lipid peroxidizing systems include lipoxygenases (LOXs), which are a family of nonheme iron-containing dioxygenases and able to induce enzymatic peroxidation of polyunsaturated fatty acids using atmospheric oxygen (O2) as a second substrate. In contrast to lipid monooxigenases like cytochrome P-450, whose main catalytic activity is the hydroxylation of substrates, LOXs are able to introduce peroxides in lipid substrates, forming reactive fatty acid hydroperoxides (LOOH). In general, LOXs contain an es-sential iron atom, which is present as Fe2+ in the inactive enzyme form. Enzymatic activation occurs through hydro-peroxide-driven oxidation of Fe2+ to Fe3+. Among LOXs, 15-LOX is of particular interest, which can also oxidize esterified fatty acids in biological membranes and lipopro-teins and has been implicated in the pathogenesis of ath-erosclerosis[7-9]. Site-specific oxidation of lipidic substrates can also be performed in model systems when metal ions (Cu(I)/Cu(II)) or Fe(II)/Fe(III)) are used to generate radi-cals in the absence of chelant species[10].

Nitric oxide (NO) can act as a potent inhibitor of the lipid peroxidation chain reaction by scavenging propaga-tory lipid peroxyl radicals and by inhibiting many poten-tial initiators of lipid peroxidation, such as peroxidase enzymes[11]. However, in the presence of superoxide (O2

•

-), NO forms peroxynitrite (OONO-), a powerful oxidant, which is able to initiate lipid peroxidation[12]. An excess of NO is expected to exert a protective effect against lipid peroxidation, while an excess of O2

•-, or equimolar con-centrations of NO and O2

•- are expected to induce lipid peroxidation[13]. Thus, the balance between NO and O2

•- may have important implications in NAFLD, where oxida-tive stress seems to have a pivotal role in the onset and/or progression of the disease[12,13]. NO is believed to coexist in cells with S-nitrosothiols (RSNOs) which are consid-ered endogenous NO carriers and donors in mammals[14]. NO covalently bound to the sulfur atom in RSNOs re-sists oxidant inactivation by oxyhemoglobin and has the same physiological properties of free NO, including its protective action on oxidative stress[15]. RSNOs have been considered potential therapeutic agents in a variety of pa-thologies in which NO may be involved[16] and S-nitroso-N-acetylcysteine (SNAC) is a relatively stable RSNO and a potent vasodilator[17]. SNAC is among the RSNOs, which can be synthesized through the S-nitrosation of the cor-responding free thiol (in this case, N-acetylcysteine, NAC). Free thiols (R-SH) play also an important role in vivo as antioxidants. Hydrogen abstraction from thiol group is

particularly fast compared to hydrogen abstraction from carbon atoms or alkoxyl radicals[18-21]. At physiological pH values, thiyl radicals (R-S•) formed can react with excess thiol anions (R-S-) to give disulphide radical anions (R-SS-R•-), or can dimerize giving rise to inter or intramolecular RS-SR cross-links in a termination process. Compared to free thiols, RSNOs can be more powerful terminators of radical chain-propagation reactions by reacting directly with ROO• radicals, yielding nitro derivatives (ROONO) as end products as well as dimmers RS-SR.

The aim of this study was to evaluate the role of SNAC as an NO donor, in the prevention of NAFLD in an animal model where NAFLD was induced by a choline deficient diet. Our results show, for the first time, that SNAC is able to block the onset of NAFLD in this animal model. This result was correlated with in vitro experiments which have confirmed the ability of SNAC to prevent the oxidation of low-density lipoprotein (LDL) and linoleic acid (LA) as model substrates, by Cu(II) ions and soybean lipoxygenase (SLO), respectively.

MATERIALS AND METHODSMaterialsN-acetyl-L-cysteine (NAC), linoleic acid, sodium nitrite, hydrochloric acid, human lyophilized LDL, soybean lipox-ygenase, sodium dodecil sulfate (SDS), phosphate buffer saline (PBS, pH 7.4) and copper (II) chloride (Sigma, St. Louis, MO) were used in this study. All experiments were carried out using analytical grade water from a Millipore Milli-Q gradient filtration system.

SNAC synthesisSNAC was synthesized through the S-nitrosation of N-acetyl-L-cysteine (Sigma Chemical, St. Louis, MO) in an acidified sodium nitrite solution[17]. Stock SNAC solutions were further diluted in PBS. Solutions were diluted to 2.4 x 10-4 mol/L in PBS (pH 7.4) before administration.

Nitrate quantificationNitrate (NO3

-, a stable metabolite of NO) levels in plasma of portal vein of the animals were assessed by chemilumi-nescence using a Sievers nitric oxide analyzer (NOA-280, Boulder, CO) according to a method described else-where[22]. Higher nitrate concentrations were found in the p lasma of an imals which rece ived SNAC ora l ly (10.8 µmol/L) then intraperitoneally (4.2 µmol/L). This result was used as a criterion to choose oral administration as a protocol to achieve greater SNAC absorption.

Effect of NAC and SNAC on in vitro LDL oxidationOxidation of LDL was induced through the addition of CuCl2 (300 µmol/L) to oxygenated aqueous LDL suspen-sions (200 µg/mL) in the absence and presence of SNAC (300 µmol/L). Aqueous LDL suspensions were prepared by diluting solid LDL to 200 µg protein/mL with EDTA-free PBS and incubated with CuCl2 (300 µmol/L) for 15 h at 37 ℃. The extent of LDL oxidation was assessed by measuring the fluorescence intensity of LDL suspensions. Oxidation of LDL resulted in derivatization of lysine resi-dues of apolipoprotein B by lipid peroxide decomposition


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products, leading to fluorescent free and protein-bound Schiff base conjugates as previously described[23,24]. In all cases, fluorescence spectra of such conjugates were firstly recorded in the range 430-600 nm, in order to character-ize the shape and position of the emission peak. All the spectrofluorimetric measurements were performed using a Perkin-Elmer LS-55 luminescence spectrometer with a temperature-controlled cuvette holder thermostatized at 37℃. Spectra of the solutions were obtained in 1 cm quartz cuvette. The excitation and emission wave-lengths were 360 and 433 nm, respectively. Native LDL (200 µg/mL) served as the control.

Effect of NAC and SNAC on in vitro LA oxidationOxidation of LA was induced through the addition of SLO to aqueous LA dispersions. LA was dispersed in SDS solution (0.01 mol/L). The final LA concentration was 18.8 µmol/L. LA was aliquoted into a quartz cuvette, flushed with O2 for 1 min and SLO (0.056 µmol/L) was added with a syringe to start the oxidation. The oxidation reactions were monitored in the absence or presence of NAC and SNAC (56 and 560 µmol/L) at 37℃ through the increase in absorbance at 234 nm, due to conjugated diene formation. A Hewlett Packard spectrophotometer, model 8453 (Palo Alto, CA, USA) with a temperature-controlled cuvette holder, was used to monitor the spectral changes in the range 200-600 nm in the dark and at 37℃. Spectra of the solutions were obtained in 1 cm quartz cuvette refer-enced against air, under stirring (1 000 r/min). Each point in the kinetic curves of absorbance vs time was the average of two experiments with the error bars expressed by their standard deviations (SD).

AnimalsMale Wistar rats, weighing 300 to 350 g, were housed in cages with a controlled light/dark cycle, receiving free water. Fatty liver was induced in the animals by choline deficient diet for four weeks. The animals were randomly divided into three groups: control group (n = 6) fed with choline deficient diet plus oral administration of vehicle (0.5 mL of PBS), SNAC group (n = 6) fed with choline-deficient diet plus oral administration of SNAC solution (0.5 mL of SNAC solution, reaching 1.4 mg/kg/day), and NAC group (n = 6) fed with choline-deficient diet plus oral administration of NAC solution (0.5 mL of NAC solution, reaching 7 mg/kg per day). After four weeks of treatment, plasma samples were collected, animals were sacrificed, and their livers were collected for histological examination and lipid peroxidation analysis. All procedures for animal experimentation were in accordance to the Helsinki Decla-ration of 1975 and the Guidelines of Animal Experimen-tation from the School of Medicine of the University of São Paulo.

Biochemical analysisSerum alanine aminotransferase (AST), aspartate aminotransferase (ALT), cholesterol and triglycerides were analyzed by standard methods[25].

Histological analysisFragments of liver tissue previously fixed by immersion

in formaldehyde saline (10%) solution were processed and submitted to hematoxylin-eosin (HE) and Masson trichrome staining for histological analysis. Scharlach red (O-tolylazo-o-tolylazo-β-naphthol) fat staining[26] was used for more accurate evaluation of fatty change. Histological variables were blindly semiquantitated from 0 to 4+ with respect to macro and microvacuolar fatty change, its zonal distribution, foci of necrosis, portal and perivenular fibro-sis as well as inflammatory infiltrate with zonal distribu-tion.

Lipid peroxidationSamples of liver homogenates were extracted with a mix-ture of acetonitrile : hexane (4∶10, v/v). The contents were vortexed for 2 min and centrifuged at 2 500 r/min for 10 min for phase separation. The hexane phase con-taining cholesteryl ester derived hydroperoxides (LOOH), was collected and evaporated under nitrogen. The residue was dissolved in methanol : butanol (2∶1, v/v), filtered through a 22 µm Millex filter (Millipore, São Paulo, Brazil) and analyzed by HPLC (Perkin-Elmer series 200, Beacons-field, Buckinghamshire, England) using an LC18DB col-umn (Supelco, Bellefonte, PA, USA). LOOHs were eluted in methanol ∶ butanol 2∶1 (v/v) at a flow rate of 1.0 mL/min through a pump (Perkin-Elmer series 200) and an LC-240 fluorescence detector (Perkin-Elmer) with the excitation source switched off. A solution of 100 mmol/L borate buffer pH 10/methanol 3∶7 (v/v) containing mi-croperoxidase (25 mg/L) was used as the reaction solution for the postcolunm reaction[27]. Peaks were identified using external standards prepared from their respective oxida-tion products as previously described[27] and quantified us-ing the package Turbochrom Navigator software (Perkin-Elmer). Results were expressed as nmol of lipid hydroper-oxides/mg of protein.

Statistical analysisAll data were expressed as mean ± SE or as mean ± SD. Statistical significance was evaluated using the one-way ANOVA test for comparisons among three groups (Control vs NAC vs SNAC-LOOH quantification) and t-test for the comparison between two means (Control vs SNAC - biochemical analysis). P < 0.05 was considered statistically significant.

RESULTSFigure 1 shows the micrographs of liver tissue extracted from animals treated with choline-deficient diet, which re-ceived vehicle or SNAC solutions for four weeks. A mod-erate macro and microvacuolar steatosis in periportal zone could be seen in the control group (Figure 1A) while in the SNAC-treated group the animals did not develop liver steatosis (Figure 1B). Scharlach staining showed a fatty change (positive staining) in the control group (Figure 1C), whereas in the SNAC-treated group no fat change was detected (negative staining) (Figure 1D). In both animal groups, necroinflammatory activity was minimal and no fibrosis was detected. In the NAC-treated group there was a macro and microvacuolar steatosis in periportal zone (data not shown).

de Oliveira CPMS et al. SNAC prevents the onset of NAFLD in rats 1907

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Figure 2 shows that SNAC prevented the rise of LOOH concentration in the liver of the SNAC-treated group, compared to the control group (0.3 ± 0.1 vs 3.2 ± 0.4 nmol/mg protein, respectively). The protective effect of NAC was also expressed by a reduction of hydroperoxide formation that could be seen in the ca. 4.6-fold reduction in LOOH formation (0.7 ± 0.2 nmol/mg protein vs 3.2 ± 0.4 nmol/mg protein, respectively).

On the other hand, the levels of AST and triglycer-ides were increased to a similar extent in the control and SNAC-treated groups. SNAC treatment of the choline-deficient fed rats did not lead to changes in ALT and cho-lesterol levels (Table 1)

Figure 3 shows the emission spectra of human LDL suspension (200 µg/mL) in PBS. The two emission peaks

at ca. 410 and 440 nm (Figure 3A) could be assigned to the partial oxidation of the freshly prepared LDL suspen-sion. It could be seen that these two peaks increased after incubation of LDL with CuCl2 (300 µmol/L) (Figure 3B) reflecting the oxidation of LDL catalyzed by Cu (II) ions. However, incubation of LDL with CuCl2 under the same condition, but in the presence of SNAC (300 µmol/L) completely blocked the growth of the 410 and 440 nm peaks (Figure 3C). In fact, the peak at 440 nm was extin-guished in this case.

Figure 4 shows the effect of SNAC on the kinetics of LA oxidation by SLO. This effect could be evaluated through the analysis of two kinetic parameters: initial rate and extent of the peroxidation reaction until the achieve-ment of the chemical equilibrium. Kinetic curves were ob-

Figure 1 Histological features of liver tissue of rats fed with choline-deficient diet. A: Control group showing a moderate macro and microvacuolar steatosis in periportal zone; B: SNAC-treated animals showing normal liver in periportal zone (hematoxylin-eosin stain-HE); C: Control group showing positive Scharlach staining; D: SNAC-treated animals showing negative Scharlach staining.

A B

C D

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Figure 2 Concentration of hydroperoxides (LOOH) in liver homogenates of the control group, NAC and SNAC-treated animals.

Table 1 Levels of alanine aminotransferase (AST), aspartate aminotransferase (ALT), cholesterol and triglycerides in serum of rats fed with choline-deficient diet (mean±SD)

Normal values in U/L for AST: 10-34; ALT: 10-44; mg/dL: cholesterol and triglyceride: 45-89. 1Control - animals fed with choline-deficient diet. 2SNAC –animals fed with choline-deficient diet and treated daily with oral SNAC administration.

Group Number ofanimals

AST (U/L)

ALT (U/L)

Cholesterol (U/L)

Triglyceride (U/L)

Control1 6 108 ± 3 40 ± 1 36 ± 1 88 ± 3SNAC2 6 95 ± 4 37 ± 8 35 ± 1 70 ± 1


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tained from the corresponding spectral changes in the UV, monitored through the band with maximum at 234 nm. This band was characteristic of conjugated di-enes and could thus be taken as a marker of LA peroxi-dation. While the initial rates of reactions correspond to the inclination of the initial sections of the curves (ca. 10 s), and the extents of reactions corresponded to the absorbance values at the plateaus. It could be seen that both parameters were maximum when LA (18.76 µmol/L) was incubated with SLO (0.056 µmol/L) (Figure 4A). Co-incubation with NAC (560 µmol/L) reduced the extent and rate of oxidation (Figure 4B), but this reduction was much more pronounced in the co-incubation with SNAC at a concentration ten times lower than NAC (56 µmol/L) (Figure 4C). The reduction was further increased in the presence of SNAC (560 µmol/L) (Figure 4D). These ef-fects could also be evaluated in the bar graph of Figure 5, where the initial rates of reaction and the extents of reaction were extracted from the kinetic curves of Figure 4. It could be seen in Figure 5 that both the rates and the extents of reaction in the presence of SNAC were reduced to about half of those obtained in the presence of NAC at a concentration ten times higher.

DISCUSSIONCholine-deficient diet is a classical general model of NAFLD, where Cyp2E1 is up regulated and the animals develop steatosis, steatohepatitis and hepatic fibrosis[28]. The results obtained in this animal model show a strong inhibitory effect of SNAC on fatty change, which is the initial step of NAFLD. The protective effect of SNAC ob-served here could be analyzed according to the suggested role of oxidative stress in the pathology of NAFLD[29-31]. Although the exact role of antioxidants in the prevention of NAFLD is not well established yet, a number of studies have shown that markers of oxidative stress are increased, while levels of endogenous antioxidants (e.g. vitamin E and glutathione, GSH) are decreased in NAFLD[29, 30]. The microsomal enzymes CYPs 2E1 and 4A are believed to be involved in the fatty acid oxidation in the liver of humans with NASH, contributing to the pathogenesis of this dis-ease[31]. In the present case, formation of lipid hydroperox-ides (LOOH), which are one of the main products of the lipid peroxidation process, was observed to be expressively reduced in the liver tissue of the SNAC-treated animals, indicating that SNAC acts as a potent inhibitor of lipid/li-poprotein oxidation. This result is in accordance with the reactivity of NO from SNAC and the ability of NO to block the propagation of radical chain reactions by form-ing nitrated lipid derivatives as end products[32-36].

SNAC-induced inhibition of LDL oxidation by Cu(II) as a model system, was confirmed in the in vitro experi-ments (Figure 3). The emission peaks at 410 and 440 nm in the fluorescence spectra of LDL suspensions were assigned to adduct formation (Schiff bases) between oxi-dation products of the lipid content of LDL particles (mainly malondialdehyde, MDA) and amino groups of the apolipoprotein (mainly Apo-B-100), which are well known markers of LDL oxidation[37,38]. The inhibition of their formation in the co-incubation of LDL with Cu (II) and SNAC, showed that SNAC could block LDL oxidation un-der this condition. The protective effect of SNAC was also confirmed in vitro using LA as a second model compound

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Figure 3 Emission spectra of human LDL (200 mg/mL) suspended in aerated PBS. A: Freshly prepared suspension; B: after incubation with CuCl2 (300 mmol/L) for 15 h; C: after co-incubation with CuCl2 (300 mmol/L) and SNAC (300 mmol/L). The excitation and emission wavelengths were 360 and 433 nm, respectively.

Figure 4 Kinetic curves of linoleic acid (18.76 mmol/L) peroxidation catalyzed by SLO (A) (0.056 mmol/L), SLO co-incubated with NAC (B) (560 mmol/L), SLO co-incubated with SNAC (C) (56 mmol/L) and SLO co-incubated with SNAC (D) (560 mmol/L). Absorbance changes were monitored at 234 nm at 37 ℃.

Times/(s)

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Figure 5 Barr graph showing the extent (Ext) and initial rates (V0) of the peroxidation reaction of linoleic acid (LA) by SLO. Data were extracted from the curves of Figure 4.


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in which peroxidation was catalyzed by SLO (Figures 4 and 5). The co-incubation of LA with SNAC (56 µmol/L) and its correspondent reduced thiol, NAC (560 µmol/L) highlighted the much more potent effect of SNAC in the inhibition of LA peroxidation, once SNAC at a concen-tration ten times lower than NAC exerted a much more important antioxidant effect. The fact that an increase in SNAC concentration to 560 µmol/L did not lead to a proportional reduction in the kinetic parameters associated with LA peroxidation, is probably due to the relatively fast initial steps of LA peroxidation.

As SNAC does not react directly with aldehydes or ketones, the protective effect observed here must be associ-ated with the termination of lipid radical chain propagation reactions, through the inactivation of alkoxyl (LO•) and per-oxyl (LOO•) intermediates, which have already been dem-onstrated to be converted into inactive ROONO products by NO[32-36] in vivo. A general equation for these reactions can be written as:2RS-NO + LO• / LOO•

→LONO / LOONO + RS-SR (1)where RSNO can be any primary S-nitrosothiol and RS-SR is the corresponding oxidized thiol yielded as a dimmer. The same RS-SR dimmers are formed if the RSNOs release NO primarily according to[39]:2 RSNO→RS-SR+2NO (2)

Free NO released in equation 2 is also capable of re-acting with LO•/LOO• species[35], leading to the same ter-mination products of equation 1.

Although NAC (the precursor of SNAC) has also an important antioxidant action due to the ease of hydro-gen abstraction from its thiol group (data not shown), the protective action of SNAC cannot be assigned to its conversion into NAC. Such a reaction does not take place in an oxidative environment. Under such conditions, the anti-oxidant effect of SNAC can be assigned mainly to the lability and reactivity of NO, according to equations 1 and 2. This statement is supported not only by the greater antioxidant action of SNAC compared to NAC in the in vitro experiments with LDL and LA, but also by the in vivo results showing that the daily oral administration of NAC at a concentration five times higher than SNAC, did not prevent the development of liver steatosis in the present animal model and led to a lower reduction in the LOOH level in the liver tissue. The protective action of NAC in this animal model is not entirely dissimilar to that obtained with other more classical anti-oxidants. However, ascorbic acid which reduces liver steatosis in rats on choline-defi-cient diet, is not able to inhibit the onset of this pathol-ogy, and α-tocopherol (vitamin E) does not even reduce fat accumulation in the hepatic tissue in the same animal model[40].

The important protective action of an NO donor in this model suggests that NAFLD can be associated with an impairment of endogenous NO production in the liver. Since the production of endothelium-derived NO has al-ready been demonstrated to be impaired in other diseases related to oxidative stress, like atherosclerosis[41, 42], the ef-fects of NO in NAFLSD can involve other mechanisms in addition to those associated solely with oxidative stress. NO is also known to be a signal transduction mediator and accumulating data suggest that S-nitrosation and nitrosi-

lation reactions performed by NO may be a ubiquitous posttranslational modification involved in signal transduc-tion regulation[43]. The absence of correlation between the reduction of LOOH concentration and the occurrence of macro and microvacuolar steatosis in the NAC-treated group, is an evidence that protective mechanisms other than the inhibition of lipid peroxidation, are operative when SNAC is administered to choline deficient animals. Such mechanisms are probably associated with the bio-chemical/signaling actions of NO and can be specifically linked to the biochemistry of RSNOs. In contrast to other NO donors which are already in widespread clinical use, like organic nitrates, nitrites and sodium nitroprusside, few clinical studies have been reported for RSNOs. Therefore, the use of RSNOs as exogenous NO sources in the treat-ment of NAFLD can bring new perspectives for under-standing the pathogenesis of this disease.

In conclusion, oral administration of SNAC as an exogenous NO source, can block the onset of NAFLD and the reduction of LOOH production in liver tissue as a result of this treatment can be associated with the ability of SNAC to block the lipid peroxidation. These findings have clinical implications, regarding novel therapeutic strategies for the treatment of NAFLD.

ACKNOWLEDGMENTSFIS and CT hold graduate studentships from Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq.

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S- Editor Guo SY L- Editor Wang XL E- Editor Liu WF


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BASIC RESEARCH

Effect of WeiJia on carbon tetrachloride induced chronic liver injury

Pik-Yuen Cheung, Qi Zhang, Ya-Ou Zhang, Gan-Rong Bai, Marie Chia-Mi Lin, Bernard Chan, Chi-Chun Fong,

Lin Shi, Yue-Feng Shi, Jay Chun, Hsiang-Fu Kung, Mengsu Yang

Pik-Yuen Cheung, Qi Zhang, Gan-Rong Bai, Chi-Chun Fong, Lin Shi, Yue-Feng Shi, Meng-Su Yang, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenu, Kowloon, Hong Kong SAR, ChinaYa-Ou Zhang, Life Science Division, Tsinghua University Graduate School at Shenzhen, Tsinghua Campus, University Town, Shenzhen, China Marie Chia-Mi Lin, Institute of Molecular Biology, The University of Hong Kong, Pokfulam, Hong Kong, SAR, ChinaBernard Chan, Jay Chun, LifeTec Enterprise Limited, Central, Hong Kong, SAR, ChinaHsiang-Fu Kung, The Centre for Emerging Infectious Diseases, Faculty of Medicine, The Chinese University of Hong Kong, Sha-tin, N.T., Hong Kong SAR, ChinaSupported by Innovation and Technology Fund of the Hong Kong SAR Government (UIM/101) and the National Hi-Tech 863 Program of the Ministry of Science and Technology of China, 2003AA2Z2052Correspondence to: Professor. Mengsu Yang, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenu, Kowloon, Hong Kong SAR, China. [email protected]: +852-27887797 Fax: +852-27887406Received: 2005-08-18 Accepted: 2005-11-26

AbstractAIM: To study the effect of WeiJia on chronic liver injury using carbon tetrachloride (CCl4) induced liver injury animal model.

METHODS: Wistar rats weighing 180-220g were ran-domly divided into three groups: normal control group (Group A), CCl4 induced liver injury control group (Group B) and CCl4 induction with WeiJia treatment group (Group C). Each group consisted of 14 rats. Liver damage and fibrosis was induced by subcutaneous injection with 40% CCl4 in olive oil at 3 mL/kg body weight twice a week for eight weeks for Groups B and C rats whereas olive oil was used for Group A rats. Starting from the third week, Group C rats also received daily intraperitoneal injection of WeiJia at a dose of 1.25 µg/kg body weight. Animals were sacrificed at the fifth week (4 male, 3 female), and eighth week (4 male, 3 female) respectively. Degree of fibrosis were measured and serological markers for liver fibrosis and function including hyaluronic acid (HA), type IV collagen (CIV), γ-glutamyl transferase (γ-GT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Alpha smooth muscle actin

(α-SMA) and proliferating cell nuclear antigen (PCNA) immunohistochemistry were also performed.

RESULTS: CCl4 induction led to the damage of liver and development of fibrosis in Group B and Group C rats when compared to Group A rats. The treatment of WeiJia in Group C rats could reduce the fibrosis condition sig-nificantly compared to Group B rats. The effect could be observed after three weeks of treatment and was more obvious after eight weeks of treatment. Serum HA, CIV, ALT, AST and γ-GT levels after eight weeks of treatment for Group C rats were 58±22 µg/L (P<0.01), 57±21 µg/L (P<0.01), 47±10 U/L (P<0.01), 139±13 U/L (P<0.05) and 52±21 U/L (P>0.05) respectively, similar to normal control group (Group A), but significantly different from CCl4 induced liver injury control group (Group B). An in-crease in PCNA and decrease in α-SMA expression level was also observed.

CONCLUSION: WeiJia could improve liver function and reduce liver fibrosis which might be through the inhibi-tion of stellate cell activity.


Key words: WeiJia; Carbon tetrachloride; Liver fibrosis

Cheung PY, Zhang Q, Zhang YO, Bai GR, Lin MCM, Chan B, Fong CC, Shi L, Shi YF, Chun J, Kung HF, Yang M. Effect of WeiJia on carbon tetrachloride induced chronic liver injury. World J Gastroenterol 2006; 12(12): 1912-1917


INTRODUCTIONHepatic fibrosis is one of the processes that occur when the liver is damaged through viral activity, toxins, autoim-mune diseases, metabolic disorder or genetic defects. It is a result of chronic liver injury that ultimately leads to cirrho-sis and its complications of portal hypertension, liver fail-ure and hepatocellular carcinoma. Millions of people die each year worldwide[1-5]. Efficient and well-tolerated antifi-brotic drugs are lacking and current treatment of hepatic fibrosis is limited to withdrawal of the noxious agent[6].

Advance in pathophysiology, molecular biology, ge-

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Cheung PY et al. Effect of WeiJia on chronic liver injury 1913

netically engineered animals, liver transplantation etc. has increased our understanding of the molecular mechanism in liver fibrogenesis. Hepatic stellate cell (HSC) was identi-fied as the primary cell type to mediate fibrogenesis which is the major source of extracellular matrix deposition[7-9]. Antifibrotic drug development was focused on fibrogenic cells generating the scarring response in the past decade.

WeiJia is a protein peptide mixture extracted from neonate porcine liver. It is a Category-I new drug on the market approved by the State Food and Drug Adminis-tration (SFDA) for the treatment of severe hepatitis and shows promising results in clinical studies[10-12]. Studies also showed that WeiJia can act as a therapeutic agent in the treatment of cirrhosis[13, 14]. As progressive hepatic fibrosis would lead to cirrhosis, it is likely that WeiJia might also play a role in treatment of hepatic fibrosis. In this study, carbon tetrachloride (CCl4) induced liver injury animal model was used to evaluate the potential of WeiJia as a therapeutic agent for hepatic fibrosis.

MATERIALS AND METHODSMaterialsWistar rats, weighing 180-220g were bought from Experi-mental Animal Centre, Daping Hospital, Third Military Medical University, Chongqing. WeiJia was from LifeTec Enterprise Limited, Hong Kong; analytical grade CCl4, alanine aminotransferase (ALT), aspartate aminotrans-ferase (AST), γ-glutamyl transferase (γ-GT) testing kits and DAB were from Sigma (St. Louis, MO, USA); hyaluronic acid (HA) and type IV collagen (CIV) RIA kits were from Shanghai Naval Medical Research Institute; alpha smooth muscle actin (α-SMA) and proliferating cell nuclear anti-gen (PCNA) monoclonal antibodies were from Dako Ltd. (Glostrup, Denmark)

Animal treatment Wistar rats were randomly divided into three groups: nor-mal control group (Group A), CCl4 induced liver injury control group (Group B) and CCl4 induction with WeiJia treatment group (Group C). Each group consisted of 14 rats (8 male, 6 female). Liver damage and fibrosis was in-duced by subcutaneous injection with 40% CCl4 in olive oil at 3 mL/kg body weight twice a week for eight weeks for Group B and C rats whereas olive oil alone was used for Group A rats. Rats were allowed to feed ad libitum. Start-ing from the third week, Group C rats also received daily intraperitoneal injection of WeiJia at a dose of 1.25 µg/kg body weight. All studies were conducted according to the guidelines described in the NIH Guide for the Care and Use of Laboratory Animals.

Sample collectionHalf of the rats (4 male, 3 female) in each group were sacrificed at the fifth week and the other half were sacri-ficed at the eighth week. Amobarbital sodium (0.5%) was injected intraperitoneally into the rats at 4 mL/kg and blood was collected from the heart after anesthesia. Blood (5 mL) was collected and 1.2 mL serum was obtained after centrifugation and stored at 4 ℃ before analysis. Liver tis-sues (1 cm×1 cm×1 cm) from the right liver lobe were dis-

sected and immobilized in 4% paraformaldehyde. Tissue was then embedded in paraffin wax and sectioned (4 µm thick) before analysis.

Serum chemistryLiver fibrosis blood tests for HA and CIV were performed using competitive RIA method. Blood serum level of γ-GT, ALT and AST were measured by standard clinical chemical methods using an automatic analyzer type AL-CYON 300i (Abbott Laboratories Ltd, USA).

Histological examinationTissue was sectioned, haematoxylin and eosin (HE) stain-ing, Van Gieson (VG) staining and immunohistochemistry were performed and examined under light microscope. All histological examinations were performed by experienced pathologist without prior knowledge of the animal treat-ment groups in the study. Images were acquired through Nikon Eclipse E400 (Nikon Corporation, Japan) and ana-lyzed with analySIS 3.0 software.

Degree of fibrosis was measured on HE stained sec-tions. Stage of liver fibrosis was graded with the META-VIR scale, which grades fibrosis on a five-point scale: F0 (no fibrosis), F1 (portal fibrosis without septa), F2 (portal fibrosis with a few septa), F3 (numerous septa without cir-rhosis) and F4 (cirrhosis). METAVIR scale is a widely used scale that has excellent inter-observer reliability[15, 16].

Ballooning degeneration and steatosis for HE stained sections were graded according to a four point scale where Grade 0: negative, Grade (1): up to 33%, Grade (2): 33%-66% and Grade (3): > 66% cells show ballooning de-generation and steatosis[17].

The collagen content of the sections was determined by VG staining. Five random fields were chosen in each section and the amount of total collagen was detected as the area stained by VG and expressed as percentage rela-tive to the total area.

α-SMA and PCNA immunohistochemistry were also performed. Sections were deparaffinized, rehydrated and incubated in 3% hydrogen peroxide at room temperature for 10 min to block endogenous peroxidase. After rinsing with distilled water, sections were incubated in phosphate buffered saline (PBS, 0.01 mol/L, pH 7.4) for 5 min and epitope retrieval was induced with heat in a microwave oven. Non-specific binding sites were blocked with 10% normal goat serum (NGS) / 10% bovine serum albumin (1∶1 dilution) for 30 min at room temperature followed by incubation with monoclonal mouse anti α-SMA or PCNA as primary antibodies at 1∶100 dilution in PBS containing 10% NGS and 0.3% Tween 20 overnight at 4 ℃. Sections were then washed with PBS for 3 times, each 5 min before applying the secondary antibody. Goat anti-mouse anti-body conjugated with horseradish peroxidase (HRP) at 1∶200 dilution in PBS containing 10% NGS was applied and the sections were incubated for 30 min at 37 ℃. Sections were then washed with PBS for 3 times, each 5 min and stained with DAB for 1 to 5 min. Staining was stopped by washing with tap water. Sections were counterstained with haematoxylin, dried and visualized under light microscope.

Expression of α-SMA was determined according to four categories and each category was assigned a number.

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Grade (1): no expression (-, 20=1); Grade (2): individual positive cells expressed in diseased area (+, 21=2); Grade (3): a few positive cells gathered together and expressed in the diseased area (++, 22=4) and Grade (4): wide spread of positive cells (+++, 23=8). Result is expressed in numbers according to different categories.

Expression of PCNA was determined using a double blind method. For each section, 5 random fields at high resolution were chosen and positive cells were recorded by two analysts. Result is expressed as the mean positive cells recorded by the two analysts.

Statistical analysisComparison of the degree of liver fibrosis between samples was performed by WILCOXON method. Other data were analyzed by SPSS11.0 software and reported as mean + standard deviation using one-way ANOVA. Student’s t-test was used for comparison between groups. P values of 0.05 or less are considered statistically significant.

RESULTSWeiJia is an SFDA-approved Category-I new drug for the treatment of severe hepatitis. WeJia showed an overall effi-cacy of 88.9% in relieving symptoms and improving physi-cal conditions of chronic hepatitis patients in a treatment period of six weeks in previous clinical study. Studies also showed that WeiJia can act as a therapeutic agent in the treatment of cirrhosis[13, 14]. As progressive hepatic fibrosis would lead to cirrhosis, it is likely that WeiJia might also play a role in the treatment of hepatic fibrosis. Thus the effect of WeiJia on liver fibrosis was investigated through an animal model in this study.

Rats were induced with CCl4 followed by the treatment with WeiJia. Animals without CCl4 induction or without WeiJia treatment were used as control for comparison. The effect of CCl4 and WeiJia on rat liver fibrosis was deter-mined through histological examination and serological markers test.

Biochemical tests of fibrosisSerum levels of biochemical markers were determined to evaluate the severity of fibrosis. Levels of extracellular ma-trix constituents HA and CIV were measured which wer expected to increase as a result of remodelling and recur-rent scarring in liver fibrogenesis. HA has correlation with stage 3 and 4 fibrogenesis. Together with CIV and other

markers, differentiation of stage 1 and 2 fibrosis from stage 3 and 4 fibrosis can be obtained[18, 19]. HA and CIV serum levels for different treatment groups are shown in Table 1. Significant elevation of serum HA and CIV levels were observed upon CCl4 induction (P < 0.001 vs Group A). Their levels were significantly reduced upon treatment with WeiJia (P < 0.005 vs Group B). Though there was still significant difference between the levels of Group C and Group A rats at the fifth week, there was no apparent dif-ference for CIV level at the eighth week between the two groups, indicating prolonged treatment with WeiJia could alleviate the severity of fibrosis. Decrease of the CIV and HA levels were observed for Group B rats at the 8th wk when compared to their level at the 5th wk indicating some recovery processes took place. However, such recovery processes were not potent enough to revert or alleviate the severity of fibrosis as their enzyme levels were still signifi-cantly higher than that of Group A rats.

Histopathological studyThe great elevation of HA and CIV levels upon CCl4 induction indicated the successful generation of liver fi-brosis animal model. The result was further confirmed by histopathology study. HE staining for sections of normal control group (Group A) showed structural integrity with-out necrosis, inflammation or fibrosis development. VG staining for collagen was negative too. However, CCl4 in-duced liver injury control group (Group B) showed signif-icant fibrosis (P < 0.01) with the loss of structural integ-rity and formation of nodules that lacked a central vein. Steatosis and ballooning degeneration (P < 0.01) were ob-served on the fifth week whereas increased collagen fibres and complete fibrous septa were observed on the eighth week. Nevertheless, CCl4 induction with WeiJia treatment group (Group C) showed that WeiJia could significantly alleviate CCl4 induced alterations as seen in Group B rats. No obvious changes for fibrosis were observed on the fifth week (P > 0.05). However, the condition for balloon-ing degeneration and steatosis were significantly relieved at this stage (P < 0.05). After prolonged treatment, a sig-nificant reduction in inflammation, steatosis (P < 0.05), necrosis, fibrosis (P < 0.01) and collagen fibres (P < 0.05) were observed on the eighth week. Representative HE stained images of sample on the eighth week are shown in Figure 1. There was no apparent difference between male and female rats. The degree of fibrosis, ballooning degeneration and steatosis and collagen expression are summarized in Table 2, Table 3 and Table 4.

Group 5th wk 8th wkHA µg/L CIV µg/L HA µg/L CIV µg/L

A 43 ± 19 19 ± 10 37 ± 17 46 ± 16Bb 324 ± 92 206 ± 70 130 ± 52 136 ± 28Ca 66 ± 20c 74 ± 19c 58 ± 22c 57 ± 211

Table 1 Liver fibrosis serum markers test for rats in different treatment group

n = 7 for each group, aP<0.005 vs Group B, b < 0.001 vs Group A, cP < 0.05 vs Group A, 1P > 0.1 vs Group A.

Group 5th wk 8th wk

F0 F1 F2 F3 F4 F0 F1 F2 F3 F4

A 7 7Bb 3 4 1 6Ca,d 5 2 3 3 1

Table 2 Degree of fibrosis for rats in different treatment group

n = 7 for each group, aP > 0.05 vs Group B at fifth week, bP < 0.01 vs Group A, dP < 0.01 vs Group B at eighth week.


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Liver function testThe effect of CCl4 on liver and the role of WeiJia in treating liver fibrosis were also determined through liver function test. Serum levels of ALT, AST and γ-GT were measured and compared with control groups as shown in Table 5.

Significant increase in serum levels of ALT, AST and γ-GT upon CCl4 induction were observed (P<0.005 Group B vs Group A). A significant decrease in the levels of ALT and AST were observed after WeiJia treatment (P<0.05 Group C vs Group B). However, the decrease was not significant for γ-GT (P > 0.1 Group C vs Group B). This indicates that there was no strong correlation between the degrees of fibrosis and γ-GT as γ-GT is an indicator for primary or metastatic malignancy involving liver[20].

It has been known that ALT and AST are useful serum markers for inflammation and necrosis of the liver. ALT is

especially useful in acute and cholestatic diseases whereas AST is more sensitive in chronic and infiltrative lesion of the liver. Though the levels of these enzymes decreased after WeiJia treatment, they were still significantly different when compared to Group A (P < 0.05), except the levels of ALT and γ-GT at the fifth week. Studies have indicated that the ratio rather than the absolute values of the two en-zymes provides high specificity in monitoring fibrosis[21-23]. By comparing the ratio of the two enzymes, there was no significant difference between rats treated with WeiJia and that from Group A. The results indicate that WeiJia could alleviate the adverse effect on liver function caused by liver injury.

Immunohistochemical studyAs WeiJia treatment could alleviate the effects caused by CCl4 induction, it is important to understand how WeiJia mediates its effect. It was suggested that the proliferative rate of regenerating hepatocytes may be an important pathogenetic factor in chronic liver disease[24]. Recent stud-ies have also shown that HSC are the primary cell type in mediating fibrogenesis[7-9]. Thus the expression of PCNA and α-SMA were determined to evaluate the cell prolifera-tion and HSC activation in liver injury respectively. Results are summarized in Table 6.

Figure 1 Effect of different treatments on rat liver fibrosis induced by CCl4 (HE staining, ×100). A: Normal control Group A rats, treated with vehicle for eight weeks; B: CCl4 induced control Group B rats, treated with CCl4 for eight weeks; C: CCl4 induced and WeiJia treatment Group C rats, treated with CCl4 for two weeks followed by both CCl4 and WeiJia treatment for six weeks.

A

B

C

Group 5th wk 8th wk

0 1 2 3 0 1 2 3

A 7 7Bb 3 4 1 2 4Ca 1 6 4 3

Table 3 Ballooning degeneration and steatosis for rats in different treatment group

n = 7 for each group, aP < 0.05 vs Group B, bP < 0.01 vs Group A.

Group 5th wk 8th wk

A 0 0B b 5.2 ± 1.6 7.3 ± 1.6 C 3.8 ± 0.9a 4.7 ± 1.0c

n = 7 for each group, aP > 0.05 vs Group B, bP < 0.01 vs Group A, cP < 0.05 vs Group B.

Table 4 Percentage collagen content for rats in different treatment groups

Group 5th wk 8th wkALT U/L AST U/L γ-GT U/L ALT U/L AST U/L γ-GT U/L

A 34 ± 12 99 ± 12 13 ± 7 32 ± 9 100 ± 19 15 ± 12Ba 135 ± 24 158 ± 19 45 ± 23 133 ± 34 166 ± 28 74 ± 36C 43 ± 12b,e 118 ± 10b,f 28 ± 19d,e 47 ± 10b,g 139 ± 13c,f 52 ± 21d,f

Table 5 Liver function tests for rats in different treatment group

n = 7 for each group, aP < 0.005 vs Group A, bP < 0.001 vs Group B, cP < 0.05 vs Group B, dP > 0.1 vs Group B, eP > 0.05 vs Group A, fP < 0.01 vs Group A, g P < 0.05 vs Group A.


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Normal hepatocytes are generally quiescent and repli-cate in a limited and regulated manner[25-27]. High prolifera-tive rates were reported in hepatocellular carcinoma, cir-rhosis and acute hepatic failure[28-32]. Nevertheless, recent evidences showed that the replicative activity of hepato-cytes diminishes in advanced cirrhosis in humans and in chronic liver injury in mouse, reaching a state of replicative senescence[33-35].

Only limited expression of PCNA was found in nor-mal control Group A whereas increased PCNA expression was observed upon CCl4 induction in active hepatocytes nuclei. Significant difference was found at the fifth week (P < 0.05, Group B vs Group A). However, expression of PCNA in Group B at the eighth week was not significantly different from that of Group A. PCNA expression in-creased further with WeiJia treatment (P < 0.05 Group C vs Groups A and B).

In normal liver, HSCs are nonparenchymal quiescent cells for vitamin A storage. In pathological conditions as in liver fibrosis, HSCs lose retinoids and synthesize a large amount of extracellular matrix components including col-lagen, proteoglycan and adhesive glycoproteins. Morpholo-gy of these cells also changes from the star-shaped stellate cells to that of fibroblasts or myofibroblasts[36]. α-SMA is a good indicator for HSC activation.

Only limited α-SMA expression was observed in nor-mal control group A. Upon CCl4 induction, increased amount of α -SMA expression by activated HSC was ob-served (P < 0.01 Group B vs Group A). The expression was reduced with WeiJia treatment. However, significant reduc-tion was only observed at the eighth week (P < 0.05 Group C vs Group B). Though WeiJia could reduce α -SMA ex-pression, its level was still significantly different from that of Group A (P < 0.05 Group C vs Group A). The results indicated that WeiJia could mediate the alleviation of CCl4 induced injury through the proliferation of regenerating hepatocytes and the reduction of stellate cell activity.

DISCUSSIONWeiJia is an effective therapeutic agent for severe hepatitis. However, its action mechanism is not clear. WeiJia was also shown to be effective in cirrhosis treatment. Thus we hypothesize that it may also play a role in fibrosis treat-ment. In this study, its effect on liver fibrosis was evaluated through CCl4 induced liver injury animal model. This study also provides some information for understanding the mechanism of WeiJia.

It was found that treatment of WeiJia could relieve CCl4 induced liver necrosis, ballooning degeneration, st-eatosis and inflammation. The effect was significant at an early stage of treatment at the fifth week. An improved liver function was also observed at this stage of treatment. The results suggest that WeiJia protects liver cells from damage induced by CCl4 and the therapeutic effects of WeiJia on severe hepatitis might be related to the protec-tive effect of this medication.

Upon liver injury, the body will attempt to repair the damage through increasing the expression of hepatocyte growth factor (HGF), transforming growth factor beta (TGF-β) and other cytokines to enhance hepatocytes pro-liferation and initiate tissue repairing process. Replicative activity of hepatocytes diminishes in advanced cirrhosis in humans and in chronic liver injury in mouse [33-35]. PCNA expression in Group B was the response of hepatocyte to liver damage. The level of PCNA expression in Group C was significantly higher than that in Group B suggesting that liver cells of rats in Group C have stronger replicative activity. The increase in PCNA expression in WeiJia treated rats with CCl4 induced liver damage demonstrated that WeiJia has protective effect on liver cells.

Uncontrollable remodelling and regeneration would lead to the development of fibrosis due to excessive depo-sition of extracellular matrix. The data from this inves-tigation also showed that WeiJia was effective in fibrosis treatment. In addition to inflammation and necrosis, CCl4 induction also resulted in collagen deposition and liver fibrosis as observed through histopathologic examination. Treatment with WeiJia reduced ballooning degeneration, steatosis and accumulation of collagen. The effect was sig-nificant at an early stage of treatment at the fifth week. An improved liver function was also observed at this stage of treatment with the increase in PCNA expression. Signifi-cant reduction of fibrosis was only observed after a longer period of treatment at the eighth week. Nevertheless, the results indicate that early treatment with WeiJia might prevent the progression of liver injury to fibrosis through increased liver regeneration and reduced liver necrosis.

It is believed that HSC activation is a critical step in hepatic fibrosis. Levels of serological markers (HA, CIV) and α-SMA expression clearly indicated that HSC was acti-vated by CCl4 induced liver injury. WeiJia treatment led to a significant reduction of theses proteins, indicating that the mechanism of WeiJia in reducing hepatic fibrosis may be through the inactivation of HSC.

In conclusion, WeiJia is shown to be an effective thera-peutic agent that could alleviate liver fibrosis through the stimulation of liver regeneration and inhibition of HSC activation.

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PCNA α-SMA PCNA α-SMA

A 17.4 ± 7.0 1.4 ± 0.5 16.6 ± 7.8 1.3 ± 0.5B 27.9 ± 8.3a 4.9 ± 2.3b 21.0 ± 5.4c 4.6 ± 1.5b

Cg 43.4 ± 14.0e 3.7 ± 2.11 31.0 ± 10.0e 2.6 ± 1.0e

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S- Editor Guo SY L- Editor Zhang JZ E- Editor Cao L


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Li-Juan Zhang, Wei-Da Zheng, Mei-Na Shi, Xiao-Zhong Wang, Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China Supported by Science and Technology Fund of Fujian Province, No. c0410025Correspondence to: Xiao-Zhong Wang, Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China. drwangxz @pub6.fz.fj.cnTelephone:+86-591-83357896-8482 Received: 2005-07-05 Accepted: 2005-07-25

AbstractAIM: To study the effects of interleukin-10 (IL-10) on the expression of α-smooth muscle actin (α-SMA), nuclear factor-κB(NF-κB) and Fas/Fas ligand (FasL) in hepatic stellate cells of experimental rats with hepatic fibrosis.

METHODS: Sixty clean SD rats were randomly divided into control group (group N), liver fibrotic group (group C) and IL-10 treatment group (group I). Control group received intraperitoneal injection of saline (2ml•kg-1), twice a week. Fibrotic group was injected intraperitoneally with 50% carbon tetrachloride (CCl4) (2 ml•kg-1), twice a week. IL-10 treatment group was given IL-10 at a dose of 4 µg•kg-1 20 minutes before CCl4 administration from the third week. Hepatic stellate cells (HSCs) were isolated from these rats at the seventh and eleventh weeks during the course of liver fibrosis, respectively. The expression of α-SMA and NF-κB in HSCs was measured by S-P immunohistochemistry. The expression of Fas and FasL mRNA was measured by RT-PCR. Furthermore, liver tissues were harvested from three groups at the same time.

RESULTS: The CCl4- induced experimental rat hepatic fibrosis model was established successfully. The purity of extracted hepatic stellate cells was about 95% and the yield of hepatic stellate cells was 1.2-2.3×106/g liver tissue averagely. The positive expression of α-SMA and NF-κB was 36.5% and 28.5% respectively in group N. The positive levels of α-SMA and NF-κB were increased significantly in group C compared to group N (P<0.01). The positive signals decreased significantly (P<0.05) in group I. In the 11th week, the HSCs of group I became round with visible pyknotic nuclei. The expression of NF-κB in group C was significantly increased in a time-dependentmanner (P<0.01), but there was no difference

in the α-SMA expression (P >0.05). The mRNA of Fas and FasL in group C was significantly increased in a time-dependent manner compared to that in control group. After treated with IL-10, the expression level of Fas and FasL was higher in group I than in group C.

CONCLUSION: The positive expression of α-SMA and NF-κB in hepatic stellate cells is decreased by ectogenic IL-10 in liver fibrosis induced by CCl4. The expression of Fas and FasL is increased in the course of liver fibrosis, and is further increased by IL-10. IL-10 could inhibit the activation of HSCs and cause apoptosis of activated HSCs.


Key words: Liver fibrosis; Hepatic stellate cell; Interleukin-10;α-smooth muscle actin; Nuclear factor-κB; Rat

Zhang LJ, Zheng WD, Shi MN, Wang XZ. Effects of inter-leukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver. World J Gastroenterol 2006; 12(12): 1918-1923


INTRODUCTIONLiver fibrosis is a model of wound-healing responses to chronic liver injury and is the excessive accumulation of extracellular matrix proteins in most types of chronic liver diseases[1]. The cellular and molecular mechanisms of liver fibrosis have greatly advanced since hepatic stellate cells (HSCs) were identified as the main collagen-producing cells in the liver. HSCs are the central event in hepatic fibrosis[2-3]. In normal liver, HSCs reside in the Disse’s space and are the major storage sites of vitamin A. Following chronic injury, HSCs activate or transdifferentiate into myofibroblast-like cells, acquiring contractile and fibrogenic properties, secreting cytokines and expressing α-smooth muscle actin(α-SMA)[4-5]. The mechanisms involved in cytokine secretion by HSCs include activation of the transcription nuclear factor-κB (NF-κB). NF-κB, a key transcription factor induces genes involved in inflammation, responses to infection, and stress[6]. DNA binding activity of NF-κB is demonstrated in activated but not in quiescent HSCs, and activation of HSCs is associated with the nuclear translocation of

BASIC RESEARCH

Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver

Li-Juan Zhang, Wei-Da Zheng, Mei-Na Shi, Xiao-Zhong Wang


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NF-κB. Inhibition of NF-κB by proteosome inhibitors or by adenovirus expressing the IkB superrepressor can markedly blunt cytokine secretion by activated HScs. Using differential display, investigators have shown that intercellular adhesion molecule 1 (ICAM-1) is expressed in HSCs activated in culture or in vivo, but not in quiescent HSCs[7]. The ICAM-1 gene contains a NF-κB binding site and its transcription is stimulated by NF-κB[8,9]. This observation provides functional support for a critical role of NF-κB in the activation of HSCs. As liver injury resolves, the number of activated stellate cells decreases through 1 of 2 potential pathways, namely spontaneous reversion or clearance by apoptosis [10,11]. To date, spontaneous reversion of myofibroblasts to quiescent cells has only been documented in culture, but not in vivo. In contrast, there is evidence that HSCs undergo apoptosis during resolution of liver injury in vivo. Some data demonstrate that apoptosis of HSCs in the course of activation is accompanied with increased expression of FasL by HSCs themselves[12]. Fas/FasL system is the key pathway for apoptosis of HSCs. Our work team found that ectogenic IL-10 could decrease liver fibrosis, but its mechanism still remains unclear. In the present study, α-SMA, NF-κB and Fas/FasL were selected as the targets to study the effects of interleukin-10 on activation and apoptosis of HSCs.

MATERIALS AND METHODSMaterialsSixty clean male Sprague-Dawley rats, weighing 400-500g (provided by Shanghai Experimental Animal Center), were divided randomly into 3 groups. The control group (group N) included 8 rats, the fibrotic group (group C) included 28 rats and the IL-10 intervention group (group I) included 24 rats. All the rats were bred under clean conditions (room temperature: 22℃±2℃; humidity: 55%±5%) in a 12 h light/dark cycle with free access to drinking water and food. High voltage disinfectant animal food was provided by BK Company in Shanghai, China.

ReagentRecombined rat interleukin-10 was provided by Jingmei Biotechnology Corporation. Collagenase type IV and Nycodenz were provided by Sigma Corporation. Pronase E was provided by Merk Corporation. DNase I was provided by Sino-American Biotechnology Corporation. DMEM was provided by Gibco Corporation. Mouse anti-rat α-SMA and NF-κB monoclonal antibodies were provided by Santa Cruz Biotechnology Inc. SP immunocytochemistry kit was provided by American Zymed Company. RNA isolation kit was provided by Gentra Company (USA) and PCR kit was provided by Promega Company. The primers were synthesized by Beijing Sanbo Company.

Animal models The rats of group N were injected intraperitoneally with saline (2ml·kg-1), twice a week. The rats of groups C and I were intraperitoneally injected with 50% CCl4 (dissolved in castor oil) (2ml·kg-1), twice a week. From the third

Zhang LJ et al . Interleukin-10 on activation and apoptosis of hepatic stellate cells 1919

week, the rats of group I were injected intraperitoneally with IL-10 (4μg·kg-1) (dissolved in saline) 20 minutes before they were injected with CCl4

[13]. All injections were performed on Monday and Thursday with the body weights determined before each injection. By the end of the experiment, 12 rats in group C and 8 in group I died. No animals died in group N. In the seventh and eleventh weeks, 3 rats in group N and 5 rats in the other two groups were sacrificed to collect their hepatic stellate cells. The liver tissue was collected from 1 rat in each group at this time point and fixed in 10% formalin and embedded with paraffin.

Isolation and culture of hepatic stellate cellsNonparenchymal cells were isolated from rats of the experimental groups by sequential perfusion with collagenase and pronase E as described previously [14,15]. Buoyant HSCs were separated from the resulting cell suspension by elutriation over a Nycodenz gradient. In brief, the liver of rats was routinely perfused through a portal vein catheter with Ca2+-free D-Hanks solution, then with pronase E and type IV collagenase dissolved in Ca2+

containing D-Hanks solution. The liver was homogenized and incubated with pronase E, type IV collagenase and DNase I dissolved in Ca2+ containing D-Hanks solution for 20 min at 37℃ with constant stiring. This suspension was centrifuged by 11% Nycodenz density gradient centrifugation for 15 minutes at 1500r/min after filtered. The cells were aspirated from the interface, washed twice in DMEM solution, and then resuspended in DMEM containing 20% calf serum. The HSCs were seeded onto plastic tissue culture bottle at 1×106 cells/mL and incubated at 37℃ with 50mL/L CO2 in air.

Immunocytochemistry measurement The HSCs cultured for 24 hours were fixed by 4% paraformaldehyde at 4℃for 24 hours, then incubated in PBS containing 3%H2O2 to remove endogenous perxoidase activity and then in PBS containing 0.1mol/L citrate to saturate nonspecific binding sites. After incubation with 1∶100 mouse anti-rat α-SMA and NF-κB monoclonal antibody, the reaction was carried out with the instant S-P immunocytochemistry reagents. The primary antibody was replaced with PBS as blank contrast. The plates were incubated in a buffer containing 3,3-diaminobenzidine tetrahydrochloride (DAB) and H2O2 to produce a brown reaction product, then imaged under microscope.

Result assessment Two hundred cells were calculated under the microscope. The reactions were graded and scored according to their intensities and percentage of the positive cells as follows: zero score for negative reaction, 1 score for pale yellowish staining, 2 scores for dense yellow staining and 3 scores for brown staining. The eventual result was produced by the two scores for staining intensity and positive cell percentage.

Reverse transcription- polymerase chain reaction (RT-PCR) for Fas and FasL Total RNA was isolated from HSCs using Gentra

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reagent (USA) according to the protocol provided by the manufacturer. The A260/A280 of total RNA was between 1.8 and 2.0. After treated with DNase-I, 1-2μg of total RNA was reversely transcribed into complementary DNA (cDNA) with oligo (dT) using cDNA synthesis kit. Then 2 mL of cDNA product was used as the template to amplify specific fragments in a 25mL reaction system. PCR was performed with an initial denaturation at 94℃ for 5 min, followed by 25 cycles at 94℃ for 45 s, annealing at 55℃ for 30 s (FasL at 58℃), at 72℃for 60 s, with a final extension at 72℃ for 7 min. The primer sequences used are sense: 5’-GAATGCAAGGGACTGATAGC-3’ and antisense: 5’-TGGTTCGTGTGCAAGGC TC-3’ for Fas; sense: 5’-GGAATGGGAAGACACATATGGAACTGC-3’ and antisense: 5’-CATATCTGGCCAGTAGTGCAGTAATTC-3’ for FasL; sense: 5’-GAGCTATGAGCTGCCTGA CG-3’ and antisense: 5’-AGCACTTGCGGTCCAC G A T G - 3 ’ f o r β - a c t i n（6 6 0 b p） ; s e n s e : 5 ’-GAGCTATGAGCTGC CTGACG-3’ and antisense: 5’-AGCACTTGCGGTCCACGATG-3’ for β-actin (410bp).

Electrophoresis and semi-quantitative analysis The PCR products were r un on 2% ag arose g e l electrophoresis and visualized with ethidium bromide staining. The expected product sizes were Fas (414bp), FasL (239bp), β-actin (660bp and 410bp). Bioimagine system was used to detect the density of bands of PCR products. The values of Fas and FasL expression were calculated from percentage of the ratio of band density of PCR products and the band density of β-actin.

Statistical analysis All data were expressed as mean ± SD. The significance for the difference between the groups was analyzed with SPSS10.0 by one-way ANOVA. P < 0.05 was considered statistically significant.

RESULTSAnimal model Liver fibrosis was remarkable during the treatment with CCl4. In the seventh week, steatosis and ballooning degeneration of hepatocytes were obvious, plentiful inflammatory cells infiltrated into the Disse’s space, collagen fibers increased and extended to the parenchyma. In the eleventh week, collagen fibers formed widely, complete fibrous septa were seen and pseudolobular structures were also present occasionally. In the IL-10 treatment group, the CCl4-caused alterations as described

above seemed to be markedly alleviated, less profound steatosis and necrosis were noted in the seventh week, and no distinct change was found in the eleventh week compared to the normal group (Figures 1A, 1B).

Hepatic stellate cell identification HSCs were isolated successfully. The yield of HSCs was 1.2-2.3×106/g liver tissue. HSCs were identified according to their typical autofluorescence at 328-nm excitation wavelength (Figure 2A) and by immunohistochemistry with monoclonal antibody against desmin. Greater than 95% of the isolated HSCs were stellate cells (Figure 2B). The cells’ viability was determined by trypan blue exclusion staining with viability over 90%.

Expression of α-SMA and NF-κB in HSCs The granular positive products of α-SMA were localized in cytoplasm of HSCs, and the NF-κB was in cytoplasm and nuclei. In control group, the expression was weak and pale yellowish, the positive rate of α-SMA and NF-κB was 36.5% and 28.5%, respectively. In group C, the expression increased obviously with the development of liver fibrosis (positive rate was 100% and the granular positive products were dense yellow or brown). The distribution of NF-κB was found mainly in nuclei. In group I, the changes were less pronounced than in group C. Especially in the eleventh week, the HSCs became round with visible pyknotic nuclei, the vigour of cells was weak (Figures 3A-3E). Comparison of α-SMA and NF-κB positive expression levels among 3 groups is shown in Table 1 and in Figures 4A, 4B. In group I, IL-10 treatment resulted in decreased immunoreactivties of α-SMA and NF-κB (P < 0.05). With the development of liver fibrosis, the intensities of NF-κB increased gradually, but that of α-SMA did not change significantly.

Expression of Fas and FasL in HSCs The Fas and FasL mRNA could be measured in HSCs of control g roup. The mRNA of Fas and FasL in fibrotic group was increased in a time-dependent manner compared to that in control group. After treated with IL-10, the expression level of Fas and FasL mRNA was higher in fibrotic group than in control group. The expression of Fas and FasL mRNA was increased in the course of liver fibrosis and was further increased by IL-10 (Figures 4C, 4D). Comparison of Fas and FasL mRNA expression levels among 3 groups is shown in Table 2 and in Figures 4E, 4F.

Figure 1 Liver of rats in groups C (A) and I (B) (week 11, H-E staining, ×100)

A B

Figure 2 Autofluorescence (A) and desmin staining (B) of HSCs.

A B


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DISCUSSIONLiver fibrosis results from the excessive secretion of matrix proteins by HSCs. In normal liver, HSCs are nonparenchymal, quiescent cells whose main function is to store vitamin A[16]. In response to liver injury, HSCs undergo an “activation” process in which they produce cytokines and chemokines, express receptors of cytokines and chemokines, and synthesize ECM [17]. Activation of HSCs is the central event of liver fibrosis, which consists of 2 major phases: initiation and perpetuation[18]. The earliest changes in stellate cells are likely to result from paracrine stimulation by all neighboring cell types, including Kupffer cells, sinusiodal endothelium, etc. Perpetuation of stellate cell activation involves several discrete changes in cell behavior, such as proliferation, chemotaxis, fibrogenesis, contractility, matrix degradation, of which contrac t i l i t y of HSCs may be a major determinant during liver fibrosis. The activated HSCs show common phenotypic features of smooth muscle cells and myofibroblasts, shape of well-developed stress fibers of actin cytoskeleton. The microfilament protein α-SMA has been explored as a marker for activated HSCs. Quiescent cells are negative in vitro or in vivo and activated HSCs are clearly positive[19]. This suggests a close relationship

between α-SMA induction and liver fibrosis. Our data show that α-SMA is expressed in activated hepatic stellate cells in the course of liver fibrosis. After the treatment with IL-10, the expression of α-SMA decreased, indicating that ectogenic IL-10 may release activated HSCs.

NF-κB exists in cytoplasm as an inactive form associated with regulatory proteins called inhibitors of kB (IkB)[20]. Phosphorylation of IkB, an important step in NF-κB activation, is mediated by IkB kinase (IKK). Appropriate stimuli induce selective IkB phosphorylation, which is then degraded by the proteasome pathway. Free NF-κB mig ra tes to nuc le i by v i r tue of i t s nuclear localization signal and induces transcription of multiple kB-dependent genes. Newly synthesized IkB both in cytoplasm and in nuclei inactivates NF-κB. NF-κB regulates the transcription of a number of proinflammatory molecules involved in acute responses to injury and chronic liver inflammation, including TNF-a, IL-6, ICAM-1 etc[21]. The induction of NF-κB during liver regeneration after partial hepatectomy appears to be a required event to prevent apoptosis and allow for normal cell cycle progression. NF-κB is a key transcription factor involved in activation of HSCs[22,23]. Inhibition of the IKK/NF-κB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo

Figure 3 α-SMA in HSCs of group C (A) in week 11 and group I (B) in week 7; NF-κB in HSCs of group C (C) in week 11 and group I (D) in week 7; and changes of HSCs in group I (E) in week 11.

A B

D E

C

Table 1 Positive expression levels of α-SMA and NF-kB in HSCs of 3 groups (mean±SD)

Group N Group C Group I wk 7 wk 11 wk 7 wk 11 wk 7 wk 11

α-SMA 0.64 ± 0.05 0.68 ± 0.07d 2.43 ± 0.03 2.47 ± 0.14b 2.14 ± 0.11 2.09 ± 0.06c

NF-κB 0.43 ± 0.08 0.42 ± 0.05d 2.13 ± 0.09 2.48 ± 0.70a 1.92 ± 0.42 1.62 ± 0.10a

aP<0.05 vs week 7 of group N; bP<0.05 vs week 7 of group C: cP<0.05 vs group C; dP<0.05 vs week 7 of group N.

Table 2 Expression levels of Fas and FasL in HSCs of 3 groups (mean±SD)

group Fas FasL group N group C group I group N group C group I

7th week 0.36 ± 0.02 0.66 ± 0.02 0.74 ± 0.02a 0.34 ± 0.03 0.45 ± 0.03 0.52 ± 0.05a

11th week 0.40 ± 0.01 0.72 ± 0.02a 0.73 ± 0.04b 0.36 ± 0.05 0.62 ± 0.04 0.83 ± 0.04a

aP<0.05 vs groups C and N; bP<0.05 vs week 11 of group C.


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apoptosis in vitro and in vivo. Drugs selectively targeting IKK have potential as antifibrotics[24]. Although in some studies IL-10 has been demonstrated to block NF-κB activation, our data suggest that IL-10 could decrease the NF-κB expression. The molecular target for IL-10-induced inhibition of NF-κB has not been established. There is evidence that IL-10 regulates NF-κB by dual mechanisms[25]. Firstly, IL-10 blocks IKK activity, thus inhibiting phosphorylation and degradation of IkBa. The preserved IkBa continues to bind to NF-κB in cytoplasm, prohibiting NF-κB nuclear translocation and NF-κB-dependent transcription. Secondly, IL-10 can directly block NF-κB DNA binding by a mechanism that is independent of NF-κB nuclear translocation.

Apoptosis associated with liver disease is increasingly viewed as a nexus through which many key pathways converge[26]. Pathologic apoptosis in liver may not only

result from inflammation and fibrosis, but also in turn amplify these responses. In particular, HSCs contribute to apoptosis and inflammation. As the liver injury resolves, apoptosis of activated HSCs may be involved in the reversion of liver fibrosis[27]. Fas is known as an important mediator of apoptosis and acts as an inducer of apoptosis in Fas-expressing cells in response to ligand binding (FasL)[28]. Saile et al[29] reported that resting HSCs display no sign of apoptosis and spontaneous apoptosis becomes detectable in parallel with HSC activation, suggesting that apoptosis might represent an important mechanism terminating proliferation of activated HSCs. They also demonstrated that apoptosis of HSCs in the course of activation is accompanied with increased expression of FasL by the HSCs themselves. The activated HSCs possess more Fas and FasL, compared with HSCs in the resting and transitional phase. The apoptosis of HSCs could largely be

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Figure 4 Expressions of α-SMA (A), NF-κB (B), Fas mRNA (C), FasL mRNA (D), Fas (E), and FasL (F) in HSCs.


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inhibited by blocking Fas, indicating that Fas/FasL system plays a major role in initiation of apoptosis. Thus, driving activated HSCs into apoptosis may be a way to resolve fibrosis. Our data show that with the development of liver fibrosis, the Fas/FasL system mRNA expression increases. In addition, IL-10 could promote the expression of Fas and FasL mRNA-activated HSCs, implying that IL-10 may promote activated HSCs into apoptosis through binding of FasL to Fas on the cell membranes of HSCs.

IL-10 is pleiotropic[30-31] and has multiple effects on diverse cell types. One of the most important properties of IL-10 is its anti-inflammatory action, which restrains the immune response under various stimuli, otherwise the individuals would have deleterious consequences. Evidence of in vivo function of IL-10 indicates that in the absence of IL-10 (in genetically IL-10 deficient animals), an exaggerated inflammatory response can lead to inflammatory states[32]. In our study, the ectogenic IL-10 could alleviate liver fibrosis induced by CCl4 in rats. During the course of liver fibrosis, the positive expression of α-SMA in HSCs was decreased by IL-10, suggesting that activation of HSCs can be inhibited by IL-10. The mechanisms may involve blocking NF-κB activation. At the same time, the expression of Fas and FasL mRNA was further increased by IL-10, suggesting that IL-10 may induce apoptosis of HSCs. In brief, IL-10 could inhibit activation and promote apoptosis of HSCs, which may be related with its mechanism against fibrosis.

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S-EditorGuo SY L-Editor Wang XL E-EditorBai SH


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Alexander Darom, Ilias P Gomatos, Emmanuel Leandros, Emmu Chatzigianni, Dimitris Panousopoulos, Manousos M Konstadoulakis, George Androulakis, Laboratory of Surgical Research, First Department of Propaedeutic Surgery, Athens Medical School, Hippokration Hospital of Athens, 114 Q. Sofia Ave, 11527 Athens, Greece Supported by the Athens University and the Greek Ministry of Health and WelfareCorrespondence to: Ilias P Gomatos MD, Kalvou 24, Old Psichiko, 154 52 Athens, Greece. [email protected] Telephone: +30-210-7486534 Fax: +30-210-6722259 Received: 2005-06-20 Accepted: 2005-08-19

AbstractAIM: To investigate the prognostic significance of PECAM-1 , ICAM-3 and HLA-DR antigens in patients with primary non-Hodgkin's gastric lymphoma.

METHODS: We immunohistochemical ly studied PECAM-1 , ICAM-3 and HLA-DR antigen expression in 36 B-cell MALT-type primary gastric lymphoma patients. Ten non-malignant and ten healthy gastric tissue specimens were used as controls. Clinicopathological and survival data were correlated with the staining results.

RESULTS: HLA-DR antigen expression was detected in 33 gastric lymphoma patients (91.7%) and 6 non-malignant patients (54.5%). PECAM-1 stained tumor cells of 10 patients (27.8%), endothelial cells of 9 patients (25%) and inflammatory infiltrate of 4 patients (40%) with benign gastric disease. ICAM-3 expression was observed on the tumor cells of 17 patients (47.2%), while 5 non-malignant patients (50%) were stained positive as well. None of the healthy controls was stained for any of the genes studied. In the multivariate analysis, HLA-DR antigen and PECAM-1 were proved to be statistically significant independent prognostic factors associated with a favourable and an unfavourable prognosis respectively (P = 0.009 and P = 0.003). In the univariate analysis, PECAM-1 (+)/ICAM-3 (-) and HLA-DR (-)/ICAM-3 (-) patients exhibited a significantly decreased overall survival compared to those with the exactly opposite gene expression patterns (P = 0.0041 and P = 0.0091, respectively). Those patients who were HLA-DR (+)/ICAM-3 (+)/PECAM-1 (-) (n = 8) had a significantly higher survival rate compared to the rest of

the group (n = 24) (P = 0.0289).

CONCLUSION: PECAM-1 , ICAM-3 and HLA-DR are representative markers of tumor expansion potential and host immune surveillance respectively. Their combined use may help us to identify high-risk patients who could benefit from more aggressive therapeutic protocols.


Key words: PECAM-1 ; ICAM-3 ; HLA-DR; Non-Hodgkin's gastric lymphoma; Prognosis

Darom A, Gomatos IP, Leandros E, Chatzigianni E, Panou-sopoulos D, Konstadoulakis MM, Androulakis G. Molecular markers (PECAM-1 , ICAM-3 , HLA-DR) determine prognosis in primary non-Hodgkin’s gastric lymphoma patients. World J Gastroenterol 2006; 12(12): 1924-1932


INTRODUCTIONPrimary gastric B-cell non-Hodgkin’s lymphomas are un-common tumors, constituting less than 2% of all primary gastric malignancies[1]. Numerous clinicopathologic stud-ies have identified stage and grade as the most important prognostic factors[1,2]. Still there are a number of patients with favourable stage and grade that exhibit an aggressive phenotype. A second line of molecular prognostic markers has been introduced lately to better describe this clinical entity.

Several different molecules (bcl-2, p53, PCNA, c-fos, c-myc and Ki67) and biologic pathways have been implicated in the initiation and progress of primary non-Hodgkin’s gas-tric lymphoma[3]. Immunohistochemical tracing of these molecules in gastric lymphoma patients has already been employed not only for the diagnosis but for the determina-tion of prognosis as well[4].

HLA-DR antigen is a class II MHC membrane-bound glycoprotein, which plays an important role in the regula-tion of the immune response[5]. Although it is normally expressed exclusively by antigen presenting cells of the im-mune system[6], it shows variable expression in malignan-cies[7,8]. During oncogenesis, it is modified to affect tumor

CLINICAL RESEARCH

Molecular markers (PECAM-1, ICAM-3, HLA-DR ) determine prognosis in primary non-Hodgkin’s gastric lymphoma patients

Alexander Darom, Ilias P Gomatos, Emmanuel Leandros, Emmu Chatzigianni, Dimitris Panousopoulos, Manousos M Konstadoulakis, George Androulakis

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cell behavior by decreasing or enhancing specific anti-tumor immune mechanisms[9].

Cell adhesion molecules are membrane glycoproteins that play a major role in neoplastic disease by interfering with cell-matrix and cell-cell interactions. They are also believed to participate in host immune surveillance by providing antigen non-specific recognition between Th-cells, APCs, Tc-cells, NK-cells and their potential targets. ICAM-3 and PECAM-1 are considered to be representa-tive members of the immunoglobulin Ig superfamily of cell adhesion molecules (CAMs). ICAM-3 is constitutively highly expressed by leukocytes especially in lymphomas and myelomas[10]. It is postulated that it may constitute the critical ligand for the initiation of lymphocyte immune re-sponses [11] with possible antitumoral properties. Tumor ex-pansion is angiogenesis dependent[12,13], a function which is orchestrated by a constant interaction between tumor cells and host cells. This “cross talk” can lead either to cell cycle arrest and tumor regression or to tumor progression[14,15]. PECAM-1 (platelet/endothelial cell adhesion molecule) is considered to be an accurate measure for the assessment of vascular proliferation on tumor sections[12] determining prognosis in a variety of tumors[16,17]. Moreover, PECAM’s-1 expression by immunocompetent cells[11] as well as its regulatory role in the extravasation of leukocytes[18], sug-gests a potential pathway through which lymphoid tumors

Darom A et al . PECAM-1, ICAM-3 and HLA-DR in gastric lymphoma 1925

may escape immune surveillance. Although the above molecules appear to represent

interacting pathways responsible for the antitumoral response and tumor progression, up to day very little information is available on their role in the pathogenesis and prognosis of primary gastric lymphoma[19-21]. The aim of this study was to analyze the immunohistochemical expression of ICAM-3 (CD50), PECAM-1 (CD31) and HLA-DR antigens in a group of patients operated for primary non-Hodgkin’s gastric lymphoma with respect to their clinicopathological characteristics and clinical out-come.

MATERIALS AND METHODSWe retrospectively recruited 36 B-cell MALT-type non-Hodgkin's primary gastric lymphoma patients (76.6%). All of them were diagnosed and treated during the period from 1991 to 1997 at the First Department of Propaedeutic Surgery of the Hippokration Hospital, Athens Medical School. Follow-up time measurements were specifically interrupted by death of the affected individuals from any cause. In this regard, follow-up time ranged between 15 d and 135.5 mo, with a mean of 60.5 ± 38.8 mo and a median of 68.5 mo. Favorable treatment outcome was defined as undetectable disease at the most recent follow-up. Unfavorable treatment outcome was defined as tumor recurrence, either locally or distantly, or death due to the tumor. Four patients had inadequate 5-year survival data and were excluded from the survival analysis. All reported deaths were attributed to the underlying disease, thus the overall survival corresponded to tumor-associated survival. Our study also included 10 gastric tissue specimens of non-malignant origin (Analytically this group consisted of 4 patients with gastritis and 6 patients with non malignant ulcer of the stomach, none of whom was submitted to any kind of surgery) and 10 healthy control tissue specimens. The characteristics of gastric lymphoma patients as well as information regarding adjuvant therapy are summarized in Table 1.

All patients were diagnosed by endoscopic biopsy and preoperatively evaluated by bone marrow biopsy, chest radiography, abdominal CT and indirect laryngoscopy. The mean age of disease presentation was 58.39 ± 15.39 years (median: 59 years).

Staging was performed according to the Japanese Clas-sification of Gastric Carcinoma by the Japanese Gastric Cancer Association[22]. Seventeen patients (47.2%) were stage I, 7 (19.4%) stage II, 6 (16.7%) stage III and 6 (16.7%) stage IV. Histopathologic examination was undertaken according to the working formulation[23]. Fifteen (41.7%) were found to be low grade lymphomas (grade I), 7 (19.4%) intermediate grade lymphomas (grade II) and 14 (38.9%) high grade lymphomas (grade III). The presence of H pylori-associated MALT-type lymphoma was not deter-mined in the majority of cases and this information was not included in our statistical analysis. Ten patients (27.7%) received a total gastrectomy and 20 (55.5%) a subtotal gastrectomy, while there was no information regarding the type of surgery for 6 (16.8%) patients. All patients had macroscopically clear margins, while microscopically

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Table 1 Demographics and clinicopathological characteristics of 36 patients with primary gastric lymphoma

Characteristics Frequency Percentage (%)

Age of diseasepresentation (yr)

58.39 ± 15.39 (median: 59, range: 33-82)

Gender Male 21 58.4 Female 15 41.6Type of Surgery Total gastrectomy 10 27 Subtotal gastrectomy 20 55.5 Unknown 6 17.5Microscopic Margins Positive margins 5 13.9 Negative Margins 31 86.1Stage I 17 47.2 II 7 19.4 III 6 16.7 IV 6 16.7Histologic Grade Low grade 15 41.7 Intermediate grade 7 19.4 High grade 14 38.9Tumor Surface (cm2) 50.04 ± 47.9 (median 29.15,

range 1.5-180)Tumor Thickness (mm) 7.05 ± 4.07 (median: 6,

range:1.5- 15)Tumor Diameter (cm) 6.66 ± 3.8 (median: 5.75,

range: 1-15)Adjuvant Chemotherapy Yes 25 69.4 No 8 22.3 Unknown 3 8.3

involved resection margins were detected in 5 patients (13.9%). Intraoperative staging consisted of biopsies of the liver and any enlarged abdominal lymph nodes. Sple-nectomy was performed only if the spleen was directly invaded.

Adjuvant chemotherapy was administered to 25 pa-tients (69.4%), 3 of whom (8.3%) received a combination of adjuvant chemo/radiotherapy. Eight patients (22.3%) did not receive any kind of supplemental therapy, while no data were retrieved for three more (Table 1).

Immunohistochemistry Immunohistochemical studies were performed on formalin-fixed and paraffin-embedded sections using the streptavidin- biotin- peroxidase method (Novostain Super ABC Novocastra laboratories Ltd, Newcaste, UK) with monoclonal antibodies specific for HLA-DR (DAKO, Glostrup, Denmark, dilution 1:70) and PECAM-1 (Oncogene Research Products, Calbiochem, Boston, dilution 1:50) and ICAM-3 (Oncogene Research Products, Calbiochem, Boston, dilution 1:1000). The assignment was performed on surgical specimens obtained during the surgical excision of the tumors.

The staining technique used was the same as previously described[24, 25]. The sections were deparaffinized in xylene and rehydrated in graded ethanol. Antigenic determinants masked from the formalin-fixation and parafin-embedding were exposed to saponin. Briefly, the endogenous peroxi-dase activity was blocked using a hydrogen peroxide solu-tion. The primary antibody was then applied overnight at 4℃ followed by incubation with a polyvalent antibody for 30 min and then a streptavidin-peroxidase reagent for 30 min at room temperature. Diaminobenzidine tetrahy-drochloride (DAKO) was used as the chromogen, and hematoxylin for counterstaining. Appropriate positive and negative controls were used.

Al l s l ides were evaluated by two independent reviewers. The percentage of HLA-DR (+) tumor cells was estimated, compared to the total area covered by the tumor in 10 randomly selected low power fields (x40). The proportion of HLA-DR (+) stromal cells close to and far from the tumor (macrophages, leukocytes, activated T-cells, necrotic tissue) was also assessed, as well as HLA-DR expression in normal gastric mucosa and stroma, for all the slides containing benign gastric tissue. HLA-DR antigen expression was located mainly in the cytoplasm but on some occasions membrane staining was seen. According to our initial estimation paraffin sections expressing HLA-DR in less than 5% of the tumor were considered negative. Tumor sections in which most of the tumor was found positive for HLA-DR expression in 10 randomly selected low power fields, were characterized as >75% positive.

A semi-quantitative grading system for ICAM-3 and PECAM-1 was used with the following criteria:

Negative (-): no immunoreaction or < 5% of tumor cells stained; (+) : 5-10% of tumor cells stained; (+ +) :10-50% of tumor cells stained; (+ + +) : >50% of tumor cells stained.

Tumor vascularity was assessed using the method desc r ibed by Horak e t a l [12 ] and penfo ld e t a l [26 ] .

Microvessels identified with PECAM-1 staining were counted on three 400 × fields (A=0.302 mm2) within areas of maximum vascularity and the mean microvessel count was calculated for each area.

Statistical analysisData were expressed as mean ±SD. Survival analysis was performed using the Kaplan-Meier method with the log rank test. To determine independent prognostic factors we used the Cox proportional hazards model, which estimates the odds ratio in 95% confidence intervals. Univariate analyses comparing subgroups of patients were performed with the chi-square test (Pearson, Mantel-Haenzel test for linear association). The results of the morphometric examination were studied in various levels of the variables examined by the Student’s t-test or the one-way ANOVA. The Levene test for homogeneity of variance was performed before the application of t-test or ANOVA. Non-parametric tests such as the Mann-Whitney test and the Kruskal –Wallis one-way ANOVA were applied in every case where the requirements of the t-test or the one-way ANOVA were not met. For all statistical tests, P < 0.05 was considered statistically significant.

RESULTSPECAM-1 was immunohistochemically expressed on the tumor cells of 10 gastric lymphoma patients (27.8%) (mean expression = 26.5% ± 8.8% for PECAM-1 positive cells) (Figure 1A). PECAM-1 (+) endothelial cells were found in the vessels of 9 gastric lymphomas (25%) (Figure 1B). The tumor stromal PECAM-1 (+) vessel counts/mm2 varied between 1.987/mm2 and 8.609/mm2 (mean ± SD: 4.6358 ± 2.0679) and were significantly higher (P = 0.027) than PECAM-1 (+) vessel counts located at the tumor margin (mean ± SD: 2.1341 ± 2.7460, range: 0 - 8.609). Similarly, the number of centrally located PECAM-1 (+) vessels/mm2 was significantly higher in gastric lymphoma patients with lymph node involvement (7.285 vs 3.753, P = 0.002). Four gastric lymphoma patients (11.11%) expressed PECAM-1 on their tumoral and neovascular endothelial cells. PECAM-1 was also expressed by the inflammatory infiltrate of 2 (50%) gastritis and 2 (33.33%) non-malig-nant ulcer patients. Nevertheless, its immunohistochemical expression was not detected on the endothelial cells of any of the above patients. PECAM-1 upregulation did not correlate with patients’ age, gender, tumor stage and grade or type of surgical treatment, as well as tumoral diameter, surface or thickness.

ICAM-3 positive staining was observed in 17 gastric lymphoma patients (47.2%) (mean expression = 34.118% ±14.5% for ICAM-3 positive lesions) (Figure 2A). ICAM-3 was also stained in 1 (25%) gastritis and 4 (66.66%) non-malignant ulcer specimens. It was mainly expressed in patients with more advanced local disease as demon-strated by the increased tumor surface (70.074 cm2 vs 29.7 cm2, P = 0.033) and thickness (8.786 mm vs 5.531 mm, P = 0.034) (Table 2). Furthermore, ICAM-3 expression level differed significantly in gastric lymphomas with dif-ferent histologic grade (F = 4.833, P = 0.014) (Figure 2B and Table 3). ICAM-3 expression level correlated with


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increased tumor diameter (r = 0.400, P = 0.028), tumor surface (r = 0.462, P = 0.012), tumor thickness (r = 0.526, P = 0.003) and higher tumor grade (r = -0.362, P = 0.030), but not with age, gender, stage or type of surgical treat-ment.

HLA-DR antigen expression on tumor and peritu-moral stromal cells was observed in 91.7%(n = 33) of gastric lymphoma patients (Figure 2C).The proportion of HLA-DR positive tumor and peritumoral stromal cells was equally distributed in gastric lymphoma patients with dif-ferent stage, grade, gender and type of surgery. HLA-DR was also expressed on stromal cells in 2 (50%) gastritis and 4 (66.6%) non malignant gastric ulcer control cases.

Several patients co-expressed two or more of the markers studied. The patterns of different protein co-expression are demonstrated in Table 4. None of the 10 healthy controls expressed any of the molecules studied.

Survival analysisUnivariate analysis showed that ICAM-3 expression was associated with improved 5-year survival rate (78.6% vs 55.6%, P = 0.1701). PECAM-1 expression on gastric lymphoma tumor cells was associated with significantly decreased 5-year survival rate (28.6% vs 76%, P = 0.0078) (Figure 3A). Although the presence of PECAM-1 (+) ves-sels within the tumor was associated with decreased overall

Figure 1 Positive membrane immunostaining (A) and endothelial staining (B) for PECAM-1 in diffusely growing lymphocytes (magnification 1X400). Gastric glands were identified as negatively stained.

A B

Table 2 Differences in tumor thickness, surface and diameter according to ICAM-3 expression status in patients with non-Hodgkin primary gastric lymphoma (mean±SD)

Table 3 ICAM-3, PECAM-1 and HLA-DR antigen quantitative expressions according to tumor grade (mean±SD)

ICAM-3 Tumor Expression ICAM-3 (+) ICAM-3 (-) P (n = 14) (n = 16)

Mean tumor thickness (mm) 8.79 (± 4.67) 5.53 (± 2.81) <0.05Mean tumor surface (cm2) 70.07 (± 9.18) 29.70 (± 28.96) <0.05Mean tumor diameter (cm) 7.89 (± 7.89) 5.59 (± 2.78) NS1

1 NS: Non-significant.

Gene expression (%) Grade I Grade II Grade III P (n = 15) (n = 7) (n = 14)

ICAM-3 (+) tumor cells

26.67 ±22.09 3.57 ±9.45 11.07± 5.95 < 0.05

PECAM-1 (+) tumor cells

6 ±10.55 10.71±13.36 7.14± 5.28 NS1

HLA-DR (+) tumor cells

40 ±25.98 55.71 ±26.21 31.43± 6.63 NS1

1 NS: Non-significant

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Figure 2 Positive membrane immunostaining for ICAM-3 in diffusely growing lymphocytes (magnification 1X400) (A) and fluctuations of the mean ICAM-3 expression level according to tumor grade (B) and positive intracytoplasm immunostaining for HLA-DR antigen in gastric lymphoma lymphocytes (magnification 1X200) (C) with gastric glands indicated as negatively stained.


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survival rate (55.6% vs 69.6%), this difference was not proved to be statistically significant (P = 0.4067). Neverthe-less, patients who showed PECAM-1 expression both on their endothelial cells (n = 4), presented a decreased overall survival rate compared to the rest of the group (25% vs 71.4%, P = 0.0403).

Different levels of proportional HLA-DR antigen ex-pression were sequentially evaluated for their prognostic value. HLA-DR antigen expression in more than 15% of the tumor cells (n = 26, 72.2%) was associated with an increased 5-year survival rate (75% vs 37.5%, P = 0.0469) (Figure 3B). A corresponding though non-significant increase in the overall survival rate was also observed in those gastric lymphoma patients expressing HLA-DR an-tigen in more than 15% of their peritumoral stromal cells (n = 19, 52.8%) (66.67 % vs 64.29 %, P = 0.8593).

A univariate analysis was also performed to identify high-risk groups of patients with regard to gene co-expression. For the needs of this analysis, the 15% HLA-DR antigen expression level was considered a measure of HLA-DR positivity between tumor cells. Those patients who were PECAM-1(+)/ICAM-3(-) (n = 5) presented a significantly decreased overall survival rate compared to those who were PECAM-1(-)/ICAM-3(+) (n = 12) (20% vs 83.3%, P = 0.0041) (Figure 4A). Furthermore, gastric lymphoma patients who were HLA-DR (+)/ICAM-3(+) (n = 10) presented a significantly improved overall survival rate compared to those who were HLA-DR(-)/ ICAM-3(-) (n = 4) (90% vs 25%, P = 0.0091) (Figure 4B). When all three genes were studied together, all HLA-DR(+)/ICAM-3(+)/PECAM-1(-) gastric lymphoma patients (n = 8) were alive 5 years postoperatively (100% 5-year survival rate), compared to a 54.2% survival rate for the rest of the group (n = 24) (P = 0.0289) (Figure 5).

Univariate analysis revealed that patients´ gender, tu-mor stage, histologic grade and marginal status were not associated with their overall survival rate as demonstrated in Table 5.

To identify the independent prognostic factors that would predict survival, multivariate analysis was per-formed. The analysis included HLA-DR antigen and PECAM-1 upregulation (which was proved to be statisti-cally significant prognostic markers in univariate analysis),

age of the patients, tumor stage and tumor grade. Four patients were excluded from the process due to inadequate 5-year survival data. Multivariate Cox regression analysis revealed that PECAM and HLA-DR antigen expressions were the only statistically significant independent prog-nostic variables in the group of gastric lymphoma patients (P = 0.005 and P = 0.016, respectively). Similar results were obtained when PECAM-1 and HLA-DR antigen expres-sions were the only covariates submitted to multivariate analysis (P = 0.003 and P = 0.009, respectively). The results of multivariate analysis are depicted in Table 6 and Table 7.

DISCUSSIONPrimary non-Hodgkin’s gastric lymphoma represents a rare malignant tumor comprising 2-5% of all cases of malignant gastric tumor[27, 28]. The 5-year survival rate ranges between 57%[29] and 96% for IE and IIE patients[30], while it has been considered as low as 25% when all stages are grouped together. Several molecular markers like P27, cyclin E[31] and bcl-6[32] have been recently assessed for their prognostic value. Still, most of them have no independent prognostic value.

In malignant lymphoma patients, clinical outcome and prognosis appear to depend largely on host immune response and vascular invasion. The current study attempted to clarify the prognostic value of HLA-DR antigen, ICAM-3 and PECAM-1 since they represent specific markers of regional immune reactions, cell-cell interactions and transendothelial migration.

HLA-DR antigen expression is a marker of host immune response in human malignant neoplasms. Its expression in tumor cells has been reported to be related with a favorable prognosis in patients with different types of cancer such as breast cancer[33] and squamous cell laryngeal carcinoma[34]. In our multivariate analysis, HLA-DR antigen expression was found to be a statistically significant independent prognostic factor associated

Table 4 ICAM-3 , PECAM-1 and HLA-DR antigen expressions in gastric lymphoma tumor cells 1

Table 5 Five-year survival rate according to patients´ gender, tumor stage, histologic grade and microscopic resection margins

Gene expression Frequency Percentage (%)

ICAM-3(+)/PECAM-1(+)/HLA-DR tumor(+) 4 11.1ICAM-3(+)/PECAM-1(+)/HLA-DR tumor(-) 1 2.8ICAM-3(+)/PECAM-1(-)/HLA-DR tumor(-) 4 11.1ICAM-3(+)/PECAM-1(-)/HLA-DR tumor(+) 8 22.2ICAM-3(-)/PECAM-1(+)/HLA-DR tumor(+) 4 11.1ICAM-3(-)/PECAM-1(+)/HLA-DR tumor(-) 1 2.8ICAM-3(-)/PECAM-1(-)/HLA-DR tumor(+) 10 27.8ICAM-3(-)/PECAM-1(-)/HLA-DR tumor(-) 4 11.1

1While PECAM-1 and ICAM-3 were positive when more than 5% of the tumor cells were stained positive, HLA-DR antigen positivity depicted in Table 4 represents the 15% cut-off level used in our survival analysis.

Characteristics Number 5-yr survival P of cases rate (%)

Gender NS1

Male 20 65 Female 12 66.7Stage NS I 16 62.5 II 9 66.7 III 1 100 IV 6 66.7Histologic grade NS Low 13 76.9 Intermediate 7 71.4 High 12 50Microscopic margins NS Positive 5 60 Negative 27 66.7

1 NS: Non-significant.


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with a favorable clinical outcome (P = 0.0199). This is in agreement with previous reports[33, 34]. The expression of HLA-DR antigen in tumor cells has a modulating effect on the host immune response, possibly by helping in the presentation of tumor associated antigens to T-lymphocytes[35], thus enabling the immune system to inhibit cancer growth. Regarding peritumoral stroma cells, the presence of tumor infiltrating leukocytes (TIL) invading the site of malignancy, suggests that the immune

system is capable of reacting with tumors. The presence of such immune infiltrates has been documented in numerous reports and is often associated with improved prognosis[36,

37]. In our primary gastric lymphoma group of patients, HLA-DR antigen expression in peritumoral stromal cells was associated with an increased (though non-significant) survival rate.

Cell adhesion molecules are membrane glycoproteins that play a major role in neoplast ic disease. Four primary groups of cell adhesion molecules have been described including the integrin receptor family, the immunoglobin superfamily, selectins and cadherins[38]. PECAM-1 constitutes a significant representative of the immunoglobulin superfamily and has been applied with considerable accuracy as an angiogenesis marker in several kinds of human neoplasia[16, 17, 39, 40].

A significant number of gastric lymphoma patients expressed PECAM-1 in their tumor cells (n = 10, 27.8%). Since PECAM-1 is constitutively expressed not only on the surface of endothelial cells, but on platelets, leukocytes, monocytes, neutrophils and selected T cell subsets as well[11], our findings can be attributed to the lymphoid origin of the malignancy. PECAM-1 positive staining on gastric lymphoma tumor cells was found to be a statistically significant independent prognostic factor, associated with unfavorable prognosis (P = 0.0029). Although there are several reports[17, 39] correlating increased angiogenesis with an unfavorable prognosis, this is the first report

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B Figure 3 Five- year survival according to PECAM-1 (A) and HLA-DR antigen (B) expression (HLA-DR posit ive patients had expression of HLA-DR antigen in more than 15% of their tumor cells).

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B Figure 4 Decreased 5 –year survival rate in PECAM-1(+)/ICAM-3(-) (A), and increased 5-year survival rate in HLA-DR(+)/ICAM-3(+) (B) gastric lymphoma patients.

Figure 5 One hundred percent 5-year survival rate in HLA-DR(+)/ICAM-3(+)/PECAM-1(-) gastric lymphoma patients.


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on the clinical outcome of gastric lymphoma patients based on PECAM-1 expression by tumor cells. As a key participant in cell adhesion cascade, PECAM-1 can lead to the extravasation of leukocytes[18], constituting a possible regulator of the metastatic process in lymphoid tumors. Its presence on the surface of tumor cells can be therefore associated with altered cellular adhesivity, enabling malignant cells to dissociate from their primary sites, leading to tumor growth and metastasis[41-43].

The detection of PECAM-1 (+) vessels within the tumor was related to an unfavorable, though non-significant clinical outcome. This difference reached statistical significance only for gastric lymphoma patients (n = 4) who showed PECAM-1 expression both in their tumor cells and in their endothelial cells (P = 0.0403). Although the fact that PECAM-1 expression in tumor endothelial cells has been associated with unfavorable prognosis in several tumors[2, 16, 17, 40, 44-46], tumors with PECAM-1 (+) endothelia exhibit an extended[47] overall survival rate.

Vascular density at the tumor margins is significantly lower than that within tumor stroma[12]. Additionally, gastric lymphoma patients with lymph node involvement present a significantly higher number of PECAM-1 (+) stromal vessel counts (7.285 vs 3.753, P = 0.002). It appears that the density of PECAM-1 (+) stromal microvessels/mm2 correlates with the lymphatic metastatic pathway. Several studies have reported that PECAM-1 is a non-specific angiogenesis marker, which stains both lymphatic (weaker staining) and blood vessel endothelial cells[48, 49]. New proliferating capillaries in the tumor stroma have fragmented basement membranes[50], while endothelial cells at the tips of growing capillaries secrete collagenases and plasminogen activators [51]. These properties facilitate the metastatic process and may explain the association between tumor stromal vessel counts and lymph node metastasis as observed in our study.

Antigen-dependent and/or independent interactions between target cells and lymphocytes are required for the initiation of a specific immune response. In addition to antigen-specific interactions, “accessory” cell-surface molecules, ICAMs[52,53], mediate an antigen-independent, non-specific adhesion between the reactant cells, which is considered the primary step in activation of lymphocytes[54]. These events often take place in a host against malignant cells. Recent work has indicated that resting leukocytes express a third ligand, ICAM-3, which appears to be the major ligand for LFA-1 in initiating phases of immune response[10,55]. Although it is poorly investigated, ICAM-3 upregulation has been well documented in lymphoid tumors[56,57]. In our gastric

lymphoma group of patients, ICAM-3 upregulation was associated with a non- significantly improved 5-year survival rate. The fact that ICAM-3 expression level is associated with increased tumor surface and thickness provides evidence that its regulation is directly proportional to the tumor burden.

Furthermore, ICAM-3 expression when studied in combination with PECAM-1 and/or HLA-DR, improved their prognostic accuracy. From our univariate analysis, three distinct gene expression patterns were distinguished. Two of them [PECAM-1(+)/ICAM-3(-) and HLA-DR(-)/ ICAM-3(-)] were associated with a significantly decreased 5-year survival rate, when compared to the exactly opposite gene expression patterns [ PECAM-1(-)/ICAM-3(+) with P = 0.0041 and HLA-DR(+)/ICAM-3(+) with P = 0.0091, respectively]. Furthermore, gastric lymphoma patients who were HLA-DR (+) / ICAM-3 (+) / PECAM-1 (-) had 100% 5-year survival rate. Similar results have been report-ed by Hosch et al[58], who observed that the co-expression of HLA class I molecules and ICAM-1 is a significant predictor of increased disease-free survival in patients with primary esophageal carcinomas. It appears that the lack of expression for both HLA-DR and ICAM-3 [HLA-DR(-)/ICAM-3(-)] in gastric lymphoma patients is associated with a compromised host immune response against the tumor as well as with an unfavourable prognosis compared to those who were HLA-DR(+)/ICAM-3(+) (n = 10, P = 0.0091). On the contrary, gastric lymphomas expressing both HLA-DR and ICAM-3 but not PECAM-1 have not only a more potent host immune surveillance, but a negative expansion potential and a growth disadvantage as well.

In our study, PECAM-1 and HLA-DR antigen expres-sions were proved to be statistically significant independ-ent prognostic factors in a group of patients with primary B-cell MALT-type non-Hodgkin’s gastric lymphoma, sug-gesting that upregulation of both PECAM-1 and HLA-DR antigen is closely related to the clinical phenotype exhibited by the affected cases. Furthermore, it appears that ICAM-3, PECAM-1 and HLA-DR represent com-plementary biologic pathways associating host immune surveillance with non-specific intercellular interactions and endothelial transmigration. Their combined study in gastric lymphoma patients may amplify their prognostic accuracy and provide a better description of the biologic behaviour of these tumors.

ACKNOWLEDGMENTSThe authors are indebted to the Athens University and the Greek Ministry of Health and Welfare for sponsoring this study.

Table 6 Multivariate Cox regression analysis I (Age, grade, stage, PECAM-1 and HLA-DR antigen expression were included)

Variable P value Exp (B) 95% CI for Exp (B)

Age NS 1.0085 0.9541-1.0660Stage NS 2.0919 0.2317-18.8828Grade NS 0.2272 0.0475-1.0855PECAM-1 tumor expression P < 0.01 19.9490 2.7865-142.8178HLA-DR tumor expression P < 0.05 0.1373 0.0258-0.7299

Table 7 Multivariate Cox regression analysis II (Only PECAM-1 and HLA-DR antigen expressions were included, since they were the only statistically significant factors found in the univariate analysis)

Variable P value Exp (B) 95% CI for Exp (B)

PECAM-1 tumor expression P < 0.01 10.3520 2.2196-48.2812HLA-DR tumor expression P < 0.01 0.1308 0.0286-0.5989


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Sri Prakash Misra, Vatsala Misra, Marisha Dwivedi, Department of Gastroenterology and Pathology, Moti Lal Nehru Medical College, Allahabad 211 001, IndiaCorrespondence to: S P Misra, MD, DM, FRCP, FICP, FACG, FNASc, Professor, Department of Gastroenterology, Motilal Nehru Medical College, Allahabad 211 001, India. [email protected]: +91-532-2600087 Fax: +91-532-2611420Received: 2005-06-11 Accepted: 2005-07-15

AbstractAIM: To assess the causes of ileocecal mass in patients with amebic liver abscess.

METHODS: Patients with amebic liver abscess and ileocecal mass were carefully examined and investigated by contrast-enhanced CT scan followed by colonoscopy and histological examination of biopsy materials from lesions during colonoscopy.

RESULTS: Ileocecal masses were found in seventeen patients with amebic liver abscess. The cause of the mass was ameboma in 14 patients, cecal tuberculosis in 2 patients and adenocarcinoma of the cecum in 1 patient. Colonic ulcers were noted in five of the six (83%) patients with active diarrhea at presentation. The ileocecal mass in all these patients was ameboma. Ulcers were seen in only one of the 11 (9%) patients without diarrhea. The difference was statistically significant from the group with diarrhea (P < 0.005).

CONCLUSION: Ileocecal mass is not an uncommon finding in patients with amebic liver abscess. Although, the ileocecal mass is due to ameboma formation in most cases, it should not be assumed that this is the case in all patients. Colonoscopy and histological examination of the target biopsies are mandatory to avoid missing a more sinister lesion.


Key words: Amebiasis; Ameboma; Colon; Colonoscopy; Diagnosis

Misra SP, Misra V, Dwivedi M. Ileocecal masses in patients with amebic liver abscess: Etiology and management. World J Gastroenterol 2006; 12(12): 1933-1936


INTRODUCTIONIntestinal amebiasis occurs throughout the world[1]. It is estimated that out of all people infected with E. histolytica, 40 million develop colitis or extra-intestinal abscesses and of these 40 000 die annually[1]. Extra-intestinal amebiasis is also not uncommon and the liver is the most common extra-intestinal organ to be involved[1-3]. A post-mortem study reported that the liver is involved in 37% of cases[4]. Involvement of the colon is also common in patients with amebic liver abscess (ALA)[5, 6]. A recent study showed that colonic ulcers are observed in 55% of patients with ALA. These ulcers are more common in patients with active diarrhea or history of diarrhea in the recent past[6]. Some of the patients with ALA also have a palpable ileocecal mass on abdominal examination. The cause of the ileocecal mass in these patients still remains unclear.

Formation of an ameboma has been noted to be an uncommon complication of amebiasis[7-18]. It has been estimated that ameboma occurs in only about 1.5% of all cases[8]. Although there have been recent reports dealing with the problem of ameboma[14, 17], in regions where ALA is not very common, ameboma has been confused with cancer of the colon[9, 13, 14, 18]. However, in regions where ALA is common, the reverse may happen and if diagnostic facilities are not widely available as in the case of several developing countries, cancer of the colon or tuberculosis may be confused with ameboma, especially in patients with concurrent ALA and its clinical signs and symptoms of high fever, local pain and tenderness. We therefore report our findings in patients with amebic liver abscess(es) and associated cecal mass. We believe that this is the largest collection of patients with ameboma.

MATERIALS AND METHODSConsecutive patients with ALA having a palpable ileocecal mass were studied. The diagnosis of ALA was made on the basis of the clinical features of fever, right upper abdominal pain, tender hepatomegaly with a typical ultrasonic finding of a single or multiple hypoechoeic lesion(s) in the liver and antiamebic antibody titer >160 in serum (immunofluorescent assay).

A detailed clinical history was obtained from all patients and recorded. Patients were asked specifically about loose stools (three or more in a day for more than 48 h), with or without blood during the last eight weeks. Whole stool samples were observed and three fresh samples were examined for the presence of trophozoites and cysts of E. histolytica.

Ileocecal masses in patients with amebic liver abscess: Etiology and management

Sri Prakash Misra, Vatsala Misra, Manisha Dwivedi


CLINICAL RESEARCH

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Contrast-enhanced CT scan was performed for all patients and apart from the abscess in the liver, the ileocecal area was carefully studied.

Colonoscopy was carried out in all but one patient within two days of instituting anti-amebic treatment after stool samples were obtained for microscopic examination. In one patient colonoscopy could be performed on the fifth day of instituting anti-amebic therapy.

Colonic preparation was done for all patients using a polyethylene glycol-electrolyte-based solution (Peglec, Tablets India Ltd, Chennai). The procedure was performed under conscious sedation with intravenous diazepam (5-10 mg) and pentazocine (25-50 mg). The findings observed during colonoscopy were recorded. The site, size, number and presence of inflammation around the ulcers were recorded if ulcers were noted. Multiple biopsies were obtained from the ulcer margins for histopathological examination. When the cecum was reached, the area was carefully inspected and multiple biopsies were obtained from any lesion that was encountered.

Patients who were too sick, those who did not give consent, those with ruptured ALA, those requiring urgent needle or catheter drainage and those with severe comorbid illnesses, were excluded from the study. Patients who received anti-amebic treatment during the preceding eight weeks were also excluded.

The protocol for the study was cleared by the Ethical Committee of the Hospital and a detailed informed consent was obtained from all participating patients.

RESULTS A total of 17 patients with ALA and ileocecal mass were

studied. There were 16 males and one female. The mean (SD) age of these patients was 37.9 (9.9) years (range 24 to 62 years). Two patients presented with active lower gastrointestinal bleeding.

Contrast-enhanced CT scan demonstrated rounded hypodense lesions in the liver due to ALA (Figure 1). In all the patients, thickening of the cecal wall was noted (Figure 2A). Lymphnodal enlargement was not evident and a definite diagnosis could be made on the basis of the CT findings in none of the patients.

Diarrhea was present in six patients. The other 11 patients did not complain of diarrhea. Ulcers were noted in the colon of five of the six (83%) patients with active diarrhea. The ileocecal mass in all these patients was due to ameboma formation (Figure 2B). Ulcers were seen in only one of the 11 (9%) patients without diarrhea. The difference between the two groups was statistically significant (P < 0.005, Fisher’s exact test).

At colonoscopy, sixteen patients showed mass lesions in the cecum. The patient who underwent colonoscopy on the fifth day of starting anti-amebic therapy showed evidence of amebic liver abscess (Figure 3A) and thickening of the cecal wall on contrast-enhanced CT scan (Figure 3B), but colonoscopy showed multiple round and oval, deep ulcers of varying sizes confined to the cecum and the adjoining ascending colon (Figure 4A). Colonic biopsies obtained from the ulcers showed trophozoites of E. histolytica (Figure 4B).

The colonoscopic appearance in all patients, except for one who underwent delayed examination, showed mass lesions in the cecum and the colonoscopic appearance was similar in all of them. The final diagnosis of ileocecal mass was made after histological examination of the tissue obtained at colonoscopy. The final diagnosis was ameboma in 14 patients, ileocecal tuberculosis in two patients (Figure 4C) and adenocarcinoma of the cecum in one patient.

The patient with cancer of the colon was a 32-year-old man who was admitted due to massive lower GI bleeding. At the time of hospitalization, the pulse rate was 160/min. The systolic blood pressure was 70 mm Hg. The hemoglobin was 5 g/dL. The patient was in shock and managed with intravenous fluids and blood transfusion. After haemodynamic stability was achieved, emergency colonoscopy was performed, which showed that the bleeding originated from the ascending colon. The bleeding stopped spontaneously on the next day. An ultrasound examination of the abdomen showed a large hypoechoeic

Figure 1 Hypodense round les ions in the liver due to amebic liver abscess.

Figure 2 Contrast-enhanced CT scan of the abdomen showing the ileocecal mass with thickening of the cecal wall (A) and histological examination of the biopsies showing trophozoites of E. histolytica (B) (H & E X 800).

BA

Figure 3 Contrast-enhanced CT scan of the abdomen with the presence of ALA in the liver (A) and ileocecal mass (B).

BA


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R

5 cm

5 cm

10 cm

←

Distance 6.9 cmDistance 7.23 cm

R

10 cm

Distance 2.92 cm

lesion in the left lobe of the liver, consistent with the diagnosis of amebic liver abscess. CT scan showed similar findings. In addition to the lesion in the liver, there was thickening of the wall of the cecum. No other abnormality was detected. Aspiration of the liver abscess yielded an anchovy sauce-like material. Cytological examination of the aspirate revealed acute inflammatory cells. No amebic trophozoites or malignant cells were seen. Indirect hemagglutination test for presence of antibody against E. histolytica was positive in titres of 1:160.

Repeat colonoscopy following bowel preparation showed irregular, nodular friable growth in the cecum (Figure 5). Multiple biopsies were obtained from the growth. Histological examination of the biopsies revealed adenocarcinoma. The patient was advised to undergo right hemi-colectomy but did not consent to be operated. The amebic liver abscess responded to needle aspiration and metronidazole.

A 62- year old lady who was diagnosed to have ALA five months ago received anti-amebic therapy at another hospital and was reported to have been cured of the disease. She however, visited our hospital with diarrhea, high fever and a mass in the right lower quadrant. Ultrasonography and CT scan of the abdomen showed features of ALA. CT scan also showed thickening of the cecal wall. Colonoscopy showed a friable nodular mass in the cecum. A provisional diagnosis of cancer of the cecum was made. Histological examination of biopsies obtained from the cecal mass during colonoscopy, however, showed

trophozoites of E. histolytica and the mass was finally diagnosed as an ameboma.

All the patients received metronidazole. Nine patients required needle aspiration of ALA. All patients were cured of ALA. The ileocecal masses due to ameboma formation disappeared after treatment with metronidazole in all the patients. Repeat colonoscopy did not reveal any abnormality in 12 patients and mild hyperemia in the remaining two patients with ameboma. The two patients with ileocecal tuberculosis received anti-tuberculous treatment and were well during follow-up. The cecal masses were no longer palpable after follow-up of 6 and 7 months respectively.

DISCUSSIONAlthough ALA is rare in developed countries, it is not an infrequent disease in developing countries. However, the occurrence of ileocecal mass due to ALA is rare, except for a case report[11]. If an ileocecal mass is detected in a patient with ALA, an ameboma should be diagnosed.

It has been estimated that out of all cases with amebiasis, ameboma formation occurs in only about 1.5% of patients[8]. However, it is important to note that in regions where ALA is not very common, ameboma has been confused with cancer of the colon[9, 13, 14, 18]. More importantly, in regions where ALA is common, the reverse may happen and cancer of the colon or tuberculosis may be confused with ameboma, especially in patients with concurrent ALA and its clinical signs and symptoms of high fever, local pain and tenderness. This was evident in one patient in this series who had carcinoma of the cecum but was clinically thought to have an ameboma due to the young age and concurrent occurrence of ALA. Two other patients in this series were clinically thought to have ameboma of the cecum on the basis of the colonoscopic findings, but showed tuberculosis of the cecum on histological examination of colonoscopic biopsies. In an elderly patient, a clinical diagnosis of cecal cancer was considered and it was only on histological examination of the biopsies that the diagnosis of ameboma could be made.

It is generally considered that ameboma formation occurs due to untreated or partially treated amebic colitis[1,7]. However, in the present series, only one patient

Figure 4 Colonoscopy findings in a patient showing deep round and oval ulcers in the cecum (A), histological examination of the biopsies obtained from ulcers in the cecum showing the presence of trophozoites of E. histolytica (B) and from a patient with amebic liver abscess and cecal mass showing the presence of multiple epithelioid cell granulomas and Langhans giant cells (C) (H & E X 40).

A CB

Figure 5 Colonoscopic appearance of the mass lesion in a patient with cecal cancer.

Misra SP et al . ALA and ileocecal mass 1935

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had a history of anti-amebic treatment five months ago for ALA. None of the other patients received any anti-amebic treatment in the recent past. Moreover, all of them had evidence of ALA at the time when they were found to have an ileocecal mass, suggesting that ameboma formation can occur even in the acute or subacute phase.

CT findings of cecal ameboma with pericolonic lessions have been reported recently[17]. The ileocecal valve shows a large abscess within the mesentry causing compression of the adjacent terminal ileum and cecum. Even then a differential diagnosis of lymphoma, neoplasm, enterocolitis and inflammatory bowel disease are considered. It was reported that due to concentric thickening of the cecal wall and mass like appearance, a clinical diagnosis of ameboma could only be established after histopathological examination of colonoscopic biopsies[14]. It is therefore evident that all patients with ileocecal masses, even with concurrent ALA, should undergo colonoscopy and biopsies should be obtained from any suspicious lesions in order not to miss tuberculosis or cancer.

Although in the present series, we encountered only amebomas, cancer of the cecum and tuberculosis as the cause for the ileocecal mass, lymphomas as well as enterocolitis, acute appendicitis, Crohn’s disease, abscess and fungal infections should also be considered as a probable diagnosis.

The simultaneous occurrence of ALA and colonic tuberculosis in this part of the world is not surprising because both diseases are commonly encountered in this region[6, 19-21]. This situation may hold true for most developing countries and the two diseases may also occur simultaneously even in developed countries, especially in immigrants, people living in confined areas and patients with immunodeficiency virus infection. The ileocecal mass in patients with cecal tuberculosis is due to proliferative lesions in the cecum resulting in a tumor-like presentation[19]. The cause of the lesions could not be therefore diagnosed on the basis of CT or colonoscopic findings.

As noted in an earlier study[6], the frequency of colonic ulcers is higher in patients with active diarrhea at presentation as opposed to those who had no diarrhea at the time of admission. What was interesting to note is that, in all patients with active diarrhea, the ileocecal mass is due to ameboma formation. Whether active diarrhea in such patients signifies ameboma can only be ascertained if a larger number of patients with similar presentation are studied.

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Lauren M Aleksunes, José E Manautou, University of Connecticut, Department of Pharmaceutical Sciences, Unit 3092, Storrs, CT, United StatesMichael Goedken, University of Connecticut, Department of Pathobiology, Unit 3089, Storrs, CT, United StatesCorrespondence to: Dr. José E. Manautou, Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269-3092, United States. [email protected]: +1-860-4863852 Fax: +1-860-4865792Received: 2005-08-19 Accepted: 2005-08-31

AbstractAIM: To investigate the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in human liver specimens obtained from patients with liver damage due to acetaminophen (APAP) overdose or primary biliary cirrhosis (PBC).

METHODS: NQO1 activity was determined in cytosol from normal, APAP and PBC liver specimens. Western blot and immunohistochemical staining were used to determine patterns of NQO1 expression using a specific antibody against NQO1.

RESULTS: NQO1 protein was very low in normal human livers. In both APAP and PBC livers, there was strong induction of NQO1 protein levels on Western blot. Correspondingly, significant up-regulation of enzyme activity (16- and 22-fold, P < 0.05) was also observed in APAP and PBC livers, respectively. Immunohistochemical analysis highlighted injury-specific patterns of NQO1 staining in both APAP and PBC livers.

CONCLUSION: These data demonstrate that NQO1 protein and activity are markedly induced in human livers during both APAP overdose and PBC. Up-regulation of this cytoprotective enzyme may represent an adaptive stress response to limit further disease progression by detoxifying reactive species.


K e y w o r d s : NQO1 ; Qu i none ox i d o r edu c t a s e ; Acetaminophen; APAP; Primary biliary cirrhosis; PBC; Nrf2; Diaphorase

Aleksunes LM, Goedken M, Manautou JE. Up-regulation of

NAD(P)H quinone oxidoreductase 1 during human liver injury. World J Gastroenterol 2006; 12(12): 1937-1940


INTRODUCTION Although NAD(P)H quinone oxidoreductase 1 (NQO1) has been historically associated with generation of hydroquinones from reactive quinones, additional substrates including nitrosoamine, nitro and azo chemical moieties have been identified. NQO1 is also capable of scavenging superoxide anions generated during oxidative stress and regenerating reduced forms of protective endogenous antioxidant compounds.

There is very low expression of NQO1 mRNA and protein in normal human livers, with slightly greater mRNA levels observed in biliary epithelial cells[1, 2]. NAD(P)H quinone oxidoreductase 2 (NQO2) mRNA is greater in hepatocytes compared to NQO1 mRNA and is in turn thought to play a more critical role in maintaining low levels of quinones in hepatocytes[2]. Consequently, it has been suggested that human NQO1 does not play a major role in hepatic xenobiotic metabolism under normal conditions[3].

Instead, NQO1 may be more important during periods of hepatic oxidative stress and damage. NQO1 mRNA protein and activity are markedly increased in mouse liver following bile duct ligation, a model of obstructive cholestasis (José Manautou, unpublished observations). Similar elevations in rodent NQO1 mRNA also occur after exposure to centrilobular hepatotoxicants such as acetaminophen (APAP), carbon tetrachloride and bromobenzene[4, 5]. Although different regions of the liver lobule are injured in cholestasis and drug-induced hepatocellular necrosis, oxidative stress contributes to the pathogenesis of both diseases. We hypothesize that up-regulation of NQO1 may represent a common response to liver injury with an oxidative stress component.

The present study was undertaken to determine if NQO1 expression and activity are similarly altered in two types of human liver disease, injury by APAP overdose and primary biliary cirrhosis (PBC). The results showed that protein levels and activity of human hepatic NQO1 were elevated during APAP overdose and PBC. Immunohistochemical analysis highlighted injury-specific patterns of NQO1 staining. APAP livers showed loss of

Up-regulation of NAD(P)H quinone oxidoreductase 1 during human liver injury

Lauren M Aleksunes, Michael Goedken, José E Manautou


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centrilobular cells and NQO1 staining in midzonal and periportal hepatocytes. Conversely, PBC livers exhibited prominent staining of hyperplastic biliary epithelium and hepatocytes at the periphery of cirrhotic nodules.

MATERIALS AND METHODSChemicals 2,6-dichlorophenol-indophenol (DCPIP), dicumarol, sucrose, Tris-hydrochloride, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) were all obtained from Sigma Chemical Co. (St. Louis, MO).

Human liver specimens Archival samples of frozen and formalin-fixed, paraffin-embedded adult livers (normal, primary biliary cirrhosis and APAP overdose) were obtained from the Liver Tissue Procurement and Distribution System at the University of Minnesota which was funded by NIH Contract #N01-DK-9-2310. Diagnosis was first established by the University of Minnesota and confirmed by histological examination of tissue sections at the University of Connecticut in a blinded fashion. Patient characteristics are provided in Table 1.

NQO1 protein expression Livers were homogenized in sucrose-Tris buffer (0.25 mol/L sucrose, 10 mmol/L Tris–HCl, pH 7.4) and centrifuged at 105 000 r/min for 60 min at 4°C. The resulting supernatant contained the cytosolic fraction. Protein concentrations were determined by the method of Lowry using Bio-Rad protein assay reagents (Bio-Rad Laboratories, Hercules, CA). Anti-NQO1 monoclonal antibodies (clones A180 and B771) were kindly provided by David Ross (University of Colorado, Health Science Center, Denver, CO). Cytosolic proteins (40 µg protein/lane) were electrophoretically resolved using polyacrylamide gels (12% resolving, 4% stacking) and transblotted overnight at 4ºC onto PVDF-Plus membrane (Micron Separations, Westboro, MA). Immunostaining was performed as previously described[1]. NQO1 protein-antibody complexes were detected using an ECL chemiluminescent kit (Amersham Life Science,

Arlington Heights, IL) and exposed to Fuji medical X-ray film (Fisher Scientific, Springfield, NJ). Equal protein loading was confirmed by Coomassie blue staining of blots (data not shown). Antibody specificity was confirmed using 2.5 μg recombinant human NQO1 (rNQO1)(Sigma Chemical Co. St. Louis, MO) as a positive control.

NQO1 activity assay NQO1 act iv i ty was ca lcu la ted by measur ing the colorimetric oxidation of NADPH to NADP+ using DCPIP as substrate. The disappearance of NADPH was measured at 600 nm for over 1 min as described by Ernster with modifications by Benson[6, 7]. NQO1 activity was measured in 1-mL reactions (27°C) containing liver cytosol, 200 µmol/L NADPH, 40 µmol/L DCPIP and Tris-HCl buffer (25 mmol/L Tris-HCl, pH 7.4, 0.7 mg/mL bovine serum albumin). Parallel reactions were performed in the presence of 20 µmol/L dicumarol. The rate of dicumarol-sensitive NQO1 activity was determined as the difference between the uninhibited and dicumarol-inhibited rates and was normalized to total cytosolic protein as previously described[6].

NQO1 immunohistochemistry Immunohistochemistry was performed on tissue sections cut from archival paraffin blocks as previously described[1]. Negative control staining was performed by incubating the sections without primary antibody. The sections were photographed on an Olympus BX50 microscope equipped with a QImaging MicroPublisher 3.3 RTV camera. Images were acquired with QCapture Pro software.

Statistical analysis NQO1 activity was presented as mean nmol reduced DCPIP/min/mg protein ± SE (n = 3-5). Differences in NQO1 activity between normal livers and either APAP or PBC livers were determined using Student’s t test. P < 0.05 compared to normal livers was considered statistically significant.

RESULTSNQO1 expression and activity in normal human liver were very low as previously described[1]. Immunoblot analysis demonstrated marked induction of NQO1 protein in liver cytosol isolated from APAP and PBC specimens (Figure 1A). This increase in protein corresponded to a significant up-regulation of NQO1 activity from 3 ± 2 nmol DCPIP/min/mg protein in normal livers to 45 ± 16 nmol DCPIP/min/mg protein (P < 0.05) and 62 ± 32 nmol DCPIP/min/mg protein (P < 0.05) in APAP and PBC livers, respectively (Figure 1B).

Immunohistochemical staining demonstrated minimal NQO1 expression in hepatocytes and biliary epithelium of normal liver tissue (Figures 2A and 2B). Histology of APAP livers showed centrilobular necrosis and cell loss with lymphocytic accumulations and minimal biliary hyperplasia (Figures 2C and 2D). NQO1 staining was cytosolic and concentrated in midzonal and portal hepatocytes surrounding regions of centrilobular cell loss.

Table 1 Characteristics of patients

Patient Gender Age Diagnosis 1 F 54 Normal liver 22 F adult Normal liver 3 M 21 Normal liver 4 M 20 Normal liver 5 M 18 Normal liver 61 F 33 Fulminant hepatic-failure (APAP-induced) 71 F 35 Fulminant hepatic-failure (APAP-induced) 81 M 15 Fulminant hepatic-failure (APAP-induced) 91 F 55 Primary biliary cirrhosis101 F 62 Primary biliary cirrhosis111 F 63 Primary biliary cirrhosis

1 Specimen collected at transplantation2 Age not provided


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Conversely, sections from PBC livers exhibited necrosis, fibrosis and nodular regeneration with lymphocytic-histiocytic infiltration and mild bile stasis (Figures 2E and 2F). Hepatocytes on the periphery of nodules were stained positive for cytosolic NQO1 protein. Positive staining was also observed in hyperplastic biliary epithelium in PBC livers (Figures 2G and 2H). No signal was detected in negative control sections (data not shown).

DISCUSSIONThis is the first report documenting strong induction of NQO1 protein and activity in livers from individuals with two very different types of liver injury; exposure to toxic amounts of APAP or chronic PBC. Drug-induced liver injury is the most common cause of acute liver failure, with APAP exposure accounting for more than 40% of cases[8]. Ingestion of supratherapeutic amounts of APAP results in degeneration and necrosis of centrilobular hepatocytes. Conversely, PBC is a progressive disease primarily attributed to autoimmunity. Chronic inflammation and destruction of bile ducts during PBC cause biliary hyperplasia, marked fibrosis, and cirrhosis in later stages. The specificity of NQO1 up-regulation during liver damage is unknown. Increased NQO1 expression in APAP and PBC livers is similar to that in non-viral human hepatocellular carcinoma[9]. Conversely, livers from patients with type B or C hepatitis-induced hepatocellular carcinoma, focal nodular hyperplasia or cholangiocarcinoma do not demonstrate altered NQO1 levels[2, 10]. Together, these and previously reported data suggest differential regulation of NQO1 in human liver disease of varying etiologies.

Transcription factor NFE-2-related factor 2 (Nrf2) is the key regulatory pathway for NQO1 expression. Oxidative stress causes translocation of Nrf2 to the nucleus and binding to antioxidant response elements in the promoter region of numerous detoxification genes, including NQO1[11]. Subsequent gene activation leads to production of NQO1 and other proteins involved in cytoprotection. With the use of Nrf2-null mice, NQO1

expression and induction have been shown to be Nrf2-dependent[12]. Activation of the Nrf2 signaling pathway during different types of human liver disease may be responsible for NQO1 up-regulation in drug-induced damage, cirrhosis and carcinoma.

As an Nrf2 target gene, NQO1 plays a role in cellular antioxidant defense beyond general drug metabolism, which includes dismutation of superoxide[13]. Therefore, NQO1 may be a scavenger of superoxide anions that are generated during both APAP toxicity and PBC[14]. Similarly, NQO1 also limits oxidative stress and lipid peroxidation by converting endogenous antioxidants (such as vitamin E) back to their more active, reduced forms[13, 15].

Loss of NQO1 function heightens susceptibility of rodents and humans to a number of diseases as demonstrated in transgenic mice lacking NQO1 and humans with a naturally-occurring NQO1 polymorphism. NQO1-null mice exhibit increased susceptibility to menadione-induced hemolytic anemia while patients possessing a C → T substitution at residue 609 of NQO1 have lower levels of NQO1 protein and exhibit an increased risk of benzene toxicity, urothelial tumors, acute myelogenous leukemia, and numerous additional cancers[13, 16, 17].

Since the pathogenesis of APAP hepatotoxicity is

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Figure 1 NQO1 protein (A) and activity (B) in cytosolic fractions from normal, APAP and PBC human liver specimens. The data are presented as nmol reduced DCPIP/min/mg protein ± SE (n = 3-5, aP < 0.05 vs normal liver specimens).

Figure 2 Immunoperoxidase staining of NQO1 in normal (A, B), APAP (C, D), and PBC (E, F) human liver specimens as well as in PBC hyperplastic biliary epithelium (G, H).

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Aleksunes LM et al . Nqo1 up-regulation in human liver injury 1939

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mediated by an electrophilic quinone, NQO1 might possess the ability to convert the APAP metabolite back to parent compound[18]. Limited data suggest that inhibition of NQO1 by dicumarol potentiates APAP toxicity in mice[19]. Conversely, administration of Nrf2 activating compounds, such as oltipraz, protects hamsters against APAP hepatic damage by inducing detoxification pathways including NQO1[20, 21].

The relative constitutive level of hepatic NQO1 among rodents and humans may also explain in part the different susceptibility to APAP hepatotoxicity across species. NQO1 activity is markedly higher in rat liver compared to mouse and human livers. Along the same line, rats exhibit a greater resistance to APAP-induced liver damage compared to mice and humans. Although additional variations in APAP metabolism between rodents and humans are known, differences in basal NQO1 activity among these species may also be a determinant of susceptibility to APAP.

In conclusion, NQO1 is upregulated in APAP–induced and cholestatic human hepatic injury, suggesting that this Nrf2 gene may limite the progression of these liver diseases.

ACKNOWLEDGEMENTS The authors thank David Ross and David Siegel for providing the monoclonal NQO1 antibody and procedure for NQO1 immunohistochemistry and Jonathan Maher for technical assistance. Lauren Aleksunes is a Predoctoral Fellow Howard Hughes Medical Institute.

REFERENCES1 Siegel D, Ross D. Immunodetection of NAD(P)H:quinone oxi-

doreductase 1 (NQO1) in human tissues. Free Radic Biol Med 2000; 29: 246-253

2 Strassburg A, Strassburg CP, Manns MP, Tukey RH. Differen-tial gene expression of NAD(P)H:quinone oxidoreductase and NRH:quinone oxidoreductase in human hepatocellular and biliary tissue. Mol Pharmacol 2002; 61: 320-325

3 Ross D, Kepa JK, Winski SL, Beall HD, Anwar A, Siegel D. NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotec-tion, bioactivation, gene regulation and genetic polymor-phisms. Chem Biol Interact 2000; 129: 77-97

4 Aleksunes LM, Slitt AM, Cherrington NJ, Thibodeau MS, Klaassen CD, Manautou JE. Differential expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride. Toxicol Sci 2005; 83: 44-52

5 Heijne WH, Slitt AL, van Bladeren PJ, Groten JP, Klaassen CD, Stierum RH, van Ommen B. Bromobenzene-induced hepato-toxicity at the transcriptome level. Toxicol Sci 2004; 79: 411-422

6 Benson AM, Hunkeler MJ, Talalay P. Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc Natl Acad Sci U S A 1980; 77: 5216-5220

7 Ernster L. DT-Diaphorase. Methods Enzymol 1967; 10: 309-3178 Lee WM. Acute liver failure in the United States. Semin Liver

Dis 2003; 23: 217-226 9 Cresteil T, Jaiswal AK. High levels of expression of the

NAD(P)H:quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin. Biochem Pharmacol 1991; 42: 1021-1027

10 Iizuka N, Oka M, Yamada-Okabe H, Hamada K, Nakayama H, Mori N, Tamesa T, Okada T, Takemoto N, Matoba K, Ta-kashima M, Sakamoto K, Tangoku A, Miyamoto T, Uchimura S, Hamamoto Y. Molecular signature in three types of hepatocel-lular carcinoma with different viral origin by oligonucleotide microarray. Int J Oncol 2004; 24: 565-574

11 Nioi P, McMahon M, Itoh K, Yamamoto M, Hayes JD. Identifi-cation of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence. Biochem J 2003; 374: 337-348

12 Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P, Kensler TW. Sensitivity to carcinogenesis is in-creased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci U S A 2001; 98: 3410-3415

13 Ross D. Quinone reductases multitasking in the metabolic world. Drug Metab Rev 2004; 36: 639-654

14 Siegel D, Gustafson DL, Dehn DL, Han JY, Boonchoong P, Berliner LJ, Ross D. NAD(P)H:quinone oxidoreductase 1: role as a superoxide scavenger. Mol Pharmacol 2004; 65: 1238-1247

15 Siegel D, Bolton EM, Burr JA, Liebler DC, Ross D. The reduc-tion of alpha-tocopherolquinone by human NAD(P)H: qui-none oxidoreductase: the role of alpha-tocopherolhydroqui-none as a cellular antioxidant. Mol Pharmacol 1997; 52: 300-305

16 Radjendirane V, Joseph P, Lee YH, Kimura S, Klein-Szanto AJ, Gonzalez FJ, Jaiswal AK. Disruption of the DT diaphorase (NQO1) gene in mice leads to increased menadione toxicity. J Biol Chem 1998; 273: 7382-7389

17 Siegel D, Anwar A, Winski SL, Kepa JK, Zolman KL, Ross D. Rapid polyubiquitination and proteasomal degradation of a mutant form of NAD(P)H:quinone oxidoreductase 1. Mol Pharmacol 2001; 59: 263-268

18 Powis G, See KL, Santone KS, Melder DC, Hodnett EM. Qui-noneimines as substrates for quinone reductase (NAD(P)H: (quinone-acceptor)oxidoreductase) and the effect of dicumarol on their cytotoxicity. Biochem Pharmacol 1987; 36: 2473-2479

19 Lee SM, Cho TS, Kim DJ, Cha YN. Protective effect of etha-nol against acetaminophen-induced hepatotoxicity in mice: role of NADH:quinone reductase. Biochem Pharmacol 1999; 58: 1547-1555

20 Davies MH, Schnell RC. Oltipraz-induced amelioration of ac-etaminophen hepatotoxicity in hamsters. II. Competitive shunt in metabolism via glucuronidation. Toxicol Appl Pharmacol 1991; 109: 29-40

21 Davies MH, Schamber GJ, Schnell RC. Oltipraz-induced ame-lioration of acetaminophen hepatotoxicity in hamsters. I. Lack of dependence on glutathione. Toxicol Appl Pharmacol 1991; 109: 17-28



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Giardia lamblia infection in patients with irritable bowel syndrome and dyspepsia: A prospective study

Barbara Grazioli, Giovanni Matera, Costanza Laratta, Giuseppina Schipani, Giovanni Guarnieri, Ester Spiniello, Maria Imeneo, Andrea Amorosi, Alfredo Focà, Francesco Luzza

Barbara Grazioli, Giuseppina Schipani, Giovanni Guarnieri, Maria Imeneo, Francesco Luzza, Dipartimento di Medicina Sperimentale e Clinica, Cattedra di Gastroenterologia; Università di Catanzaro “Magna Graecia”, Catanzaro, Italy Giovanni Matera, Costanza Laratta, Ester Spiniello, Alfredo Focà, Dipartimento di Scienze Mediche, Istituto di Microbiolo-gia; Università di Catanzaro “Magna Graecia”, Catanzaro, Italy Andrea Amorosi, Dipartimento di Medicina Sperimentale e Cli-nica, Cattedra di Anatomia Patologica; Università di Catanzaro “Magna Graecia”, Catanzaro, Italy Correspondence to: Francesco Luzza, MD, Dipartimento di Medicina Sperimentale e Clinica, Cattedra di Gastroenterologia, Università di Catanzaro “Magna Graecia”, Via T Campanella 115 - 88100 Catanzaro, Italy. [email protected] Telephone: +39-961-771859 Fax: +39-961-772885Received: 2005-07-04 Accepted: 2005-07-28

AbstractAIM: To evaluate the prevalence of Giardia lamblia (G. lamblia ) infection in patients with irritable bowel syndrome (IBS) and dyspepsia and to establish which is the most accurate test to diagnose the infection in this setting.

METHODS: One hundred and thirty-seven patients who consecutively attended the Outpatient Gastroenterology Clinic for the first time between January 2002 and De-cember 2003 due to symptoms of IBS and/or dyspepsia were recruited. All patients underwent clinical evaluation, first-step haematology and chemistry tests, serologic as-says for celiac disease, lactose-H2 breath test, abdominal ultrasonography, and esophagogastroduodenoscopy. Helicobacter pylori status was evaluated. In patients with symptoms of IBS older than 45 years, colonoscopy was also performed. In all patients, duodenal biopsies and stool samples were examined for trophozoites and cysts of G. lamblia by several methods.

RESULTS: G. lamblia was identified in 9 patients. The following diagnoses were also made: IBS (100/137, 73%), functional dyspepsia (62/137, 45%), organic dyspepsia (33/137, 24%), and lactose intolerance (75/137, 55%). A significant association was found between giardiasis and H pylori infection (χ2 = 6.632, OR = 12.4, CI = 1.5-68.1). There were no symptoms that reliably allowed the recognition of giardiasis. Direct search of the parasite in duodenal biopsy and stool sample examinations gave concordant results in all cases while histological examination of duodenal

biopsies displayed a low sensitivity (e.g., 22.2%).

CONCLUSION: In this consecutive series, diagnosis of G. lamblia infection accounted for 6.5% of patients with IBS and dyspepsia. Duodenal biopsies for diag-nosis of giardiasis may be unnecessary if stool sample examination is performed.


Key words: Dyspepsia; Giardiasis; H pylori ; Irritable bowel syndrome

Grazioli B, Matera G, Laratta C, Schipani G, Guarnieri G, Spiniello E, Imeneo M, Amorosi A, Focà A, Luzza F. Giardia lamblia infection in patients with irritable bowel syndrome and dyspepsia: A prospective study. World J Gastroenterol 2006; 12(12): 1941-1944


INTRODUCTIONGiardia lamblia is a cosmopolitan parasite with worldwide distribution and the most common protozoan isolated from gastrointestinal tract[1]. The prevalence of infection varies from 2%-7% in industrialized countries to 40% in tropical/subtropical regions with poor sanitation and hygienic conditions[2-5]. Giardiasis typically occurs following the ingestion of water or foods contaminated with fecal material containing cysts, and the infective dose may be as low as 10 cysts. The clinical aspects of giardiasis are largely nonspecific. The infection can be asymptomatic or patients may present with extraintestinal symptoms, such as fever, maculopapular rashes, pulmonary infiltrates, lymphadenopathy, polyarthritis and urticaria. However, the most common symptoms are diarrhea, abdominal pain, bloating, flatulence and weight loss resulting from malabsorption[6]. Since these symptoms may overlap those of patients with other gastrointestinal disorders[7-9], it is not clear when the infection should be suspected. Classically, diagnosis of giardiasis is performed by microscopic examination of stool samples and/or duodenal biopsies and further methods include immunocromathography and immunofluorescence on stool samples. There are

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few studies comparing the diagnostic proficiency of the methods using either stool or duodenal biopsy samples. The aim of this study was to evaluate the prevalence of G. lamblia infection in patients with irritable bowel syndrome (IBS) and dyspepsia and to establish the most accurate test for its diagnosis in this setting.

MATERIALS AND METHODSPatient recruitmentFour hundred and thir ty-five consecutive patients attending our hospital for the first time between January 2002 and December 2003 on two days of the week (Monday and Wednesday) due to symptoms of IBS who satisfied Rome II criteria[10] and/or dyspepsia were considered for the study. Patients with alarm features (such as weight loss, dysphagia, vomiting, bleeding), familiarity for gastrointestinal neoplasia and inflammatory bowel disease were excluded as well as patients who took nonsteroidal anti-inflammatory drugs and proton pump inhibitors in the last 15 days and antibiotics in the last 30 days. Patients with symptoms lasting more than one year were also excluded. Finally, 137 patients (48 M, 89 F; median age 39 years, range 19-77 years) were enrolled. All patients underwent routine blood investigations, including erythrocyte sedimentation rate, C-reactive protein, thyroid hormones, antigliadin, antiendomysial and antitransglutaminase antibodies. Furthermore, urinalysis, microscopic examination of stools, esophagogastroduod-enoscopy with duodenal biopsy, evaluation of Helicobacter pylori (H pylori) infection, abdominal ultrasonography, and lactose-H2 breath test were performed. Patients with symptoms of IBS older than 45 years (n = 46) underwent also colonoscopy.

Laboratory tests for G. lamblia infectionThe presence of the parasite was evaluated by three different approaches: direct search of the parasite in the duodenal biopsy samples with or without use of fixation and dyes, histological examinations of other aliquots of the above bioptic samples, and parasitological evaluations of stool samples.Search of the parasite in duodenal biopsy samples Duodenal biopsies were obtained during the upper gastrointestinal endoscopy, immediately submitted to microbiology laboratory where they were put into a sterile plastic container filled with 10 mL of sterile saline. The samples were processed within 2 h of arrival in the laboratory. Wet mount preparations were obtained by cutting the original biopsy into 0.5 mm pieces on a glass slide, with the help of a razor blade and a plastic pipette tip. Then such pieces were suspended in 2 drops of sterile saline and a cover glass was put on them. Microscopic examination for motile organisms was carried out using low and high dry power (× 200 and × 400). Permanent stains were prepared as reported above. However the biopsy pieces were air dried, fixed and the giemsa (Giemsa Plus, Trend Scientific) or trichrome staining (Trichrome Stain, Scientific Device Laboratory Inc., Des Plaines, IL, USA) were carried out following the manufacturer’

s instructions. Also the vital staining with acridine orange was used on unfixed specimens examined by confocal microscopy to help identify the parasite and address both the mobility and some details of its cytostructure.Histology of bioptic samples Duodenal biopsies obtained during the upper gastrointestinal endoscopy were also submitted to the Pathology laboratory, where the specimens where paraffin-included, stained with hematoxylin-eosin and examined for Giardia trophozoites as well as for inflammatory infiltrate particularly of plasma cellular type.Stool parasitology Liquid or semi liquid fecal specimens were immediately submitted to the microbiolog y laboratory, a wet mount with saline and Lugol’s iodine (Dobel l react ive) was prepared and microscopic examination for motile organisms and Giardia cysts was carried out using low and high dry power (× 200 and × 400). Moreover, both liquid and solid fecal samples were processed using the formalin/ether enrichment method followed by microscopy for Giardia cysts. Coproantigens were also evaluated in the stool specimens by immunochromatographic technique (Xpect, Remel Europe, Dartford Kent, UK), an EIA method (Prospect, Remel Europe, Dartford Kent, UK), and commercial direct fluorescent antibody assay (Merifluor, Meridian Bioscience Inc., Cincinnati, USA). Laboratory tests for Giardia coproantigens were carried out and the results were evaluated following the manufacturer’s instructions. The microbiologist (GM) and pathologist (AA) were blinded to the clinical and laboratory data.

H pylori statusPatients were classified as H pylori positive when the urease quick test (Yamanouchi Pharma, Milan, Italy) was positive. Patients underwent C-13 urea breath test (Cortex, Milan, Italy) when the urease quick test was negative and were considered free of the infection when the two tests were both negative for H pylori.

Statistical analysisThe relation between presence of giardiasis and H pylori infection was evaluated with χ2test and odds ratio (OR). Data were given together with 95% confidence interval (CI).

RESULTSG. lamblia infection was found in 9 of the 137 patients (6 .5%). According to the c l in ica l and laborator y evaluations, the following diagnoses were also made: IBS, lactose intolerance, functional and organic dyspepsia, colon diverticulosis, celiac disease, inflammatory bowel disease, gastric cancer, colon cancer, and collagenous colitis (Figure 1). All patients with giardiasis (7 M, 2 F; median age: 38 years, range 26-61 years) had nonspecific (mostly mild) symptoms largely overlapping those complained by patients without giardiasis (Table 1). Indeed, according to the clinical and laboratory findings, these patients were considered affected also by other functional and organic disorders (Table 1). In patients with giardiasis, blood examination was normal with only one patient


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showing a moderate eosinophilia (1.190/µL; normal range: 0-500/µL). Direct search of the parasite in duodenal biopsy (Figures 2A, 2B) and stool sample (microscopic) examination (Figures 2C, 2D) were concordantly positive (9 of 137 patients) and negative (128 of 137 patients) for G. lamblia, while histological evaluation of duodenal biopsy was positive in only two cases (2 of 137 patients) and negative in the remaining (135 of 137 patients) (Table 2). Using as reference the former two tests, the sensitivity and specificity of the histological examination of duodenal biopsy for the diagnosis of giardiasis were 22.2% and 100%, respectively. Fifty-eight patients (42%) were infected with H pylori (18 M, 40 F; median age 40 years, range 20-65 years). A significant association was found between giardiasis and H pylori infection. Eight of 58 patients (14%) with H pylori infection had giardiasis while 1 of 79 (1%) without H pylori infection had giardiasis (χ2 = 6.632, OR = 12.4, CI = 1.5-68.1).

DISCUSSION This study aimed at investigating the impact of G. lamblia infection on patients who complained of symptoms referring to IBS and dyspepsia. In order to prevent information and observational bias, we properly excluded patients who have had a previous gastroenterological consultation, those with symptoms lasting more than one year and those who had been recently taking drugs

that might interfere with complaints and laboratory investigations. Furthermore, to minimize the presence of serious illness, patients complaining of alarm symptoms were excluded. In the consecutive series of 137 patients with symptoms of IBS and/or dyspepsia, the prevalence of giardiasis was 6.5% (9/137). Possibly due to the selection of a symptomatic study population, this figure is slightly higher than the average in the general population in a Western country[2-5]. Nevertheless, no comparisons can be made with the incidence of giardiasis in the asymptomatic population coming from our geographical area. Furthermore, an important finding of this prospective study is that no symptoms could reliably discriminate patients with giardiasis from those without the parasite. Giardias is may be present in pat ients with other gastrointestinal disorders as observed in this study, it seems that symptoms of one disease may overcome/overlap those of the other. Nevertheless, the prevalence of giardiasis in patients suffering from IBS and/or dyspepsia can be considered as the cause of 1BS and/or dyspepsia. Another important finding of this study involves the diagnostic procedures to be taken into account when dealing with G. lamblia infection. The data showed that histological examination of duodenal biopsies for G. lamblia was unsuitable due to an unacceptable rate of false negative results (e.g., 22.2% sensitivity). At the same time, it has been shown that stool examination is as accurate as the direct search of the parasite in the duodenal samples.

Table 1 Characteristics of 9 patients with G. lamblia infection among the 137 complaining of symptoms of irritable bowel syndrome (IBS) and/or dyspepsia (FD: functional dyspepsia; OD: organic dyspepsia) or displaying lactose intolerance (LI)

Patient Age (yr) Sex Symptoms IBS LI FD OD

1 61 M Abdominal pain, constipation/diarrhea + - + -2 38 F Abdominal pain, diarrhea, pirosis + + - +3 33 M Abdominal pain, diarrhea + - - -

4 26 F Abdominal pain and bloating, constipation + + - -5 48 M Abdominal pain, diarrhea + + - -6 47 M Abdominal pain, diarrhea, pirosis + - + -7 37 M Abdominal pain and bloating, diarrhea + - - +8 26 M Abdominal pain and bloating, diarrhea + + + -9 39 M Abdominal pain, diarrhea + - - -

Table 2 Comparison of the laboratory tests for G. lamblia infection in the study population (n = 137)

Patient Age (yr) Sex Direct search induodenal biopsy

Stool sampleexamination

Histology ofduodenal biopsy

1 61 M + + -2 38 F + + -3 33 M + + +4 26 F + + -5 48 M + + -6 47 M + + -7 37 M + + +8 26 M + + -9 39 M + + -

In the remaining 128 patients, all the three tests were negative for the presence of G lamblia.

Figure 1 Diagnostic categories in the 137 patients complaining of symptoms of irritable bowel syndrome (IBS) and/or dyspepsia (FD: functional dyspepsia; OD: organic dyspepsia). LI: lactose intolerance; Other diagnoses included: colon diverticulosis (n = 3), celiac disease (n = 2), inflammatory bowel disease (n = 1), gastric cancer (n = 1), colon cancer (n = 1), and collagenous colitis (n = 1).

120

100

80

60

40

20

n = 9 n = 100 n = 75 n = 62 n = 33 n = 9 (6.5) (73) (66) (45) (24) (6.5) (%)

IBS

LIFD

OD

G lambliainfection

Otherdiagnoses

Grazioli B et al. Contrast-enhanced computed tomographic colonograph 1943

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Consequently, when a skilled microbiologist and appropriate techniques are available, duodenal biopsy seems unnecessary in accurate identification of G. lamblia, thus invasive and expensive tools such as upper gastrointestinal endoscopy could be avoided. This study also showed that giardiasis was significantly associated with H pylori infection, possibly reflecting that the two infections share a number of risk factors[11]. This association may have several clinical implications in regard to the transmission mode[12,13], the possibility of a synergy in metronidazole resistance[14], and the experimental evidence of a common pathogenesis scenario, leading to gastrointestinal metaplasia[15].

Figure 2 Confocal microscopy images (A and B) of G. lamblia trophozoite (x1000) in duodenal bioptic sample, using acridine orange as a vital stain. A: a dorsal projection showing the peculiar tear-shaped cell with flagella. B: an unusual flank projection shows the nuclei on the back of the ventral disk. C: G. lamblia cysts (x200) microscopic images after a wet mount with Lugol’s iodine of fecal specimens. D: G. lamblia cysts (x200) in stool sample following formalin/ether enrichment of filtered samples and staining with a direct fluorescent antibody.

A B

C D

In conclusion, the prevalence of giardiasis in patients suffering from symptoms of IBS and dyspepsia is non-negligible, and duodenal biopsies for the parasite may be unnecessary if stool examination is performed.

REFERENCES1 Adam RD. The biology of Giardia spp. Microbiol Rev 1991; 55:

706-7322 Meyer EA. The epidemiology of Giardiasis. Parasitol Today

1985; 1: 101-1053 Flanagan PA. Giardia--diagnosis, clinical course and epidemi-

ology. A review Epidemiol Infect 1992; 109: 1-224 Scotti S, Pettoello Mantoani M, Polito G, Carlomagno F, Cop-

pola A, di Martino L. [Giardia duodenalis infections in pedia-trics: our series]. Infez Med 1996; 4: 35-40

5 Odoi A, Martin SW, Michel P, Holt J, Middleton D, Wilson J. Determinants of the geographical distribution of endemic giardiasis in Ontario, Canada: a spatial modelling approach. Epidemiol Infect 2004; 132: 967-976

6 Garcia LS. Diagnostic medical parasitology. American Society for microbiology. Washington D.C: 2001; 36-49

7 D'Anchino M, Orlando D, De Feudis L. Giardia lamblia infec-tions become clinically evident by eliciting symptoms of irrita-ble bowel syndrome. J Infect 2002; 45: 169-172

8 Carr MF Jr, Ma J, Green PH. Giardia lamblia in patients un-dergoing endoscopy: lack of evidence for a role in nonulcer dyspepsia. Gastroenterology 1988; 95: 972-974

9 Rana SV, Bhasin DK, Vinayak VK. Lactose hydrogen breath test in Giardia lamblia-positive patients. Dig Dis Sci 2005; 50: 259-261

10 Thompson WG, Longstreth GF, Drossman DA, Heaton KW, Irvine EJ, Müller-Lissner SA. Functional bowel disorders and functional abdominal pain. Gut 1999; 45 Suppl 2: II43-II47

11 Moreira ED Jr, Nassri VB, Santos RS, Matos JF, de Carvalho WA, Silvani CS, Santana e Sant'ana C. Association of Helico-bacter pylori infection and giardiasis: results from a study of surrogate markers for fecal exposure among children. World J Gastroenterol 2005; 11: 2759-2763

12 Ashbolt NJ. Risk analysis of drinking water microbial con-tamination versus disinfection by-products (DBPs). Toxicology 2004; 198: 255-262

13 Leclerc H, Schwartzbrod L, Dei-Cas E. Microbial agents as-sociated with waterborne diseases. Crit Rev Microbiol 2002; 28: 371-409

14 Land KM, Johnson PJ. Molecular basis of metronidazole resist-ance in pathogenic bacteria and protozoa. Drug Resist Updat 1999; 2: 289-294

15 Patterson MM, Schrenzel MD, Feng Y, Fox JG. Gastritis and intestinal metaplasia in Syrian hamsters infected with Helico-bacter aurati and two other microaerobes. Vet Pathol 2000; 37: 589-596



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Clinical characteristics of a group of adults with nodular lymphoid hyperplasia: A single center experience

Alberto Rubio-Tapia, Jorge Hernández-Calleros, Sagrario Trinidad-Hernández, Luis Uscanga

Alberto Rubio-Tapia, Jorge Hernández-Calleros, Luis Uscanga, Department of Gastroenterology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran (INNSZ), Mexico City, MexicoSagrario Trinidad-Hernández, Department of Pathology,

Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran (INNSZ), Mexico City, MexicoCorrespondence to: Alberto Rubio Tapia, MD, Department of Gastroenterology, INNSZ, Vasco de Quiroga 15, Tlalpan 14000, Distrito Federal, México. [email protected]: +52-55-55733418 Fax: +52-55-56066222Received: 2005-08-04 Accepted: 2005-08-24

abstractAIM: To describe the clinical and histological charac-teristics of a group of adults with small-bowel nodular lymphoid hyperplasia (NLH).

METHODS: Patients were searched for five years in pathology records of our institution. The biopsy mate-rial was reassessed using strict histopathological criteria. Clinical data were obtained from medical records.

RESULTS: Small-bowel NLH was diagnosed in 18 cases. The female: male ratio was 2∶1. The most frequent symptoms were diarrhea (72%), involuntary weight loss (72%) and abdominal pain (61%). Nine patients (50%) had immunodeficiency. Small-bowel bacterial overgrowth was found in three (17%) cases. At small-bowel NLH diagnosis, three (17%) had associated lymphoma: two intestinal and one extra-intestinal lymphomas. In two patients with villous atrophy and anti-endomysial anti-bodies the diagnosis of celiac disease was established. Giardia lamblia infection was found in only one patient with hypogammaglobulinemia (Herman’s syndrome).

CONCLUSIONS: NLH is uncommon in adult patients. Associated diseases are immunodeficiency and lymphoid tissue malignancies.


Key words: Nodular lymphoid hyperplasia; Hypogamma-globulinemia; Lymphoma

Rubio-Tapia A, Hernández-Calleros J, Trinidad-Hernández S, Uscanga L. Clinical characteristics of a group of adults with nodular lymphoid hyperplasia: A single center experience. World J Gastroenterol 2006; 12(12): 1945-1948


INTRODUCTIONN o d u l a r l y m p h o i d hy p e r p l a s i a ( N L H ) o f t h e gastrointestinal (G1) tract is characterized by markedly hyperplasic, mitotically active germinal centers with well-defined lymphocyte mantles[1] . The nodules are found in mucosa and/or submucosa anywhere in the gastrointestinal tract and may resemble small adenomatous polyps on gross examination[2,3]. Approximately 20% of adults with common variable immunodeficiency (CVI) are found to have NLH. It has been suggested that NLH is a risk factor for both intestinal and extra intestinal lymphoma[4]. It has also been reported in patients with human immunodeficiency virus infection[5] . In children, NLH tends to have a benign course and usually regresses spontaneously but in adults it is rare and poorly described. In a previous report by Canto et al[6] NLH was diagnosed in 11 Mexican subjects. In five of them a diagnosis of Herman’s syndrome was established. Patients were collected from the pathology database of our institute from 1973 to 1988. Since this time, an increased number of duodenal biopsies have been taken for the approach of patients with chronic diarrhea who were referred to our institution from different places. In this work we described the clinical characteristics of 18 new patients with NLH seen at the Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”, a National Institute of Health located in Mexico City, which is a referral center for gastrointestinal diseases.

MATERIALS AND METHODSPatientsFrom January 1998 to December 2002, patients with the histological diagnosis of NLH were identified from the pathology database. Medical records were reviewed and a pathologist (STH) reassessed slides or paraffin-embedded biopsies. NLH was considered when the following histological criteria were observed: hyperplasic lymphoid follicles, mitotically active germinal centers with well-defined lymphocytes mantles, and lymphoid follicles localized at mucosa and/or submucosa.

Histology The original duodenal mucosal biopsy specimens were

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used for reassessment. The histopathological evaluation was performed on hematoxylin-eosin-stained samples, including counting and localization of lymphoid follicles, type and severity of inflammation in lamina propria, and presence of villous atrophy. If the quality of the original sections was too poor for the reassessment, new sections from the original paraffin wax-embedded biopsy blocks were made.

Clinical and laboratory dataAge, duration and type of symptoms, body mass index (BMI) and associated diseases were evaluated. The following tests were recorded at the time of diagnosis: hemoglobin, mean cell volume, total proteins, albumin, globulins, β-carotene, electrolyte profile, prothrombin time, partial thromboplastin time, human immunodeficiency virus antibody, liver enzymes, stool analysis for ova and parasites, microscopic examination and culture of sterile collected duodenal fluid.

Statistical analysisClinical and histopathological data were expressed as absolute and/or relative frequencies and mean±SD. The Mann-Whitney U or χ2 tests were used to compare groups. P < 0.05 was considered statistically significant. SPSS software (version 10) was used.

RESULTSPatientsEighteen out of 550 duodenal biopsies had the histological diagnosis of NLH. After re-evaluation of the histological material all of them were included in the analysis. Twelve were women and 6 were men, yielding a female: male ratio of 2:1. Their median age at diagnosis was 41 years (18 to 76 years). Duration of symptoms from the onset to diagnosis varied from 1 to 48 mo (median = 18 mo). The median body mass index at diagnosis was 19 kg/m2 (11 to 26 kg/m2).

Clinical dataDiarrhea and weight loss were the predominant symptoms in 13 (72%) patients. A mean of 6 ± 4 bowel movements per day and a weight loss of 11.6 ± 6.9 kg in the last six

months were presented. Eleven (61%) had abdominal pain (Table 1).

Histopathology A mean of 2.5 (range 2-6) hyperplasic lymphoid follicles was observed in a single field 4 × magnification with light microscopy. Hyperplasic lymphoid follicles were localized in mucosa of 13 cases (69%) and in mucosa and submucosa of five cases (31%) (Figure 1). Patients with 3 or more hyperplasic follicles had lower β-carotene levels (46.7 ± 25 mg/dL vs 108 ± 42.6 mg/dL, P < 0.05) and mixed localization of follicles (mucosa and submucosa) (8 vs 1, P < 0.05) than patients with ≤ 2 follicles. Subjects with mixed localized lymphoid follicles showed a statistically significant lower hemoglobin (11.9 ± 1.4 g/L vs 14 ± 13 g/L, P = 0.03) and serum β-carotene (44 ± 21.9 mg/dL vs 111.2 ± 38.7 mg/dL, P < 0.02) as well as a tendency to prolong thromboplastin time (17.3 ± 7.2 vs 11.5 ± 2.5, P < 0.07) than those with exclusively mucosa-localized follicles. Villous atrophy (partial or total) was observed in seven cases (38%) and it was associated with weight loss (7 vs 2, P < 0.02), lientery (4 vs 0, P < 0.07) and low globulin level (2.3 ± 0.87 g/L vs 3.2 ± 5.2 g/L, P < 0.06). This group had also lower hemoglobin (12.3 ± 1.3 g/L vs 14.2 ± 14 g/L, P < 0.03) and cholesterol serum level (170 ± 38.1 mg vs 200 ± 17.1 mg, P < 0.03) than those without atrophy (Table 2).

Associated diseasesNLH was associated with hypogammaglobulinemia in 9 (50%) patients, CVI in 8 cases and selective IgA deficiency

Figure 1 Histological (A) and endoscopic (B) characteristics of NLH. Hyperplasic follicles with active germinal centers localized at mucosa and submucosa, Brunner’s glands at center and a pseudo-polypoid pattern of duodenum mucosa were observed.

A B

Table 1 Symptoms and signs of patients with NLH

Symptom or sign n (%)Diarrhea 13 (72)Involuntary weight loss 13 (72)Abdominal pain 11 (61)Lymphadenopathy 4 (22)Pallor 4 (22)Chronic cough 2 (11)Edema 2 (11)Arthralgia 1 (6)Neuropathy 1 (6)


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in 1 case. In this group the mean level of globulins was 1.6 ± 3.5 g/L which was quite different from that in subjects with normal globulins (3.2 ± 4.5 g/L, P < 0.001). Giardia lamblia infection was found in only one case. E. coli, Streptococcus sp, and Klebsiella sp were respectively isolated from duodenal fluid in three (17%) (106 bacterial colonies per milliliter). These patients showed a markedly prolonged prothrombin time (18.5 ± 7.4 s vs 11.4 ± 1.9 s, P < 0.014). Anti-endomysium antibodies were tested in 16 subjects and two (12.5%) were positive. Both had villous atrophy thus the diagnosis of celiac sprue was established. Lymphoma was found in three (17%) cases: one with nodular sclerosing-Hodgkin´s disease on the mediastinum, one with jejunum non-Hodgkin’s lymphoma (diffuse large B-cell lymphoma) and one with gastric lymphoma. Patients with lymphoma were older (58 ± 10 years old) and only one of them presented diarrhea as a predominant symptom. No AIDS-associated NLH was found in this series (Table 3).

DISCUSSIONNodular l ymphoid hyperp las ia (NLH) i s a we l l -characterized condition frequently associated with increased risk of gastrointestinal tumors, mainly gastrointestinal lymphoma. As a pathological entity it seems to be rare. It has been described in immunocompromised and immunocompetent subjects, normal children and adults and patients with AIDS[1-5]. This series represent the whole experience of the National Institute of Health in Mexico, which is a tertiary referral center for gastrointestinal diseases. In previous works Canto et al[6] and Castañeda et al[7] have described some cases with NLH seen at the same institution. In these two papers 12 patients are included in a period of 18 years contrasting with the 18 cases found by us in only a 5-year period. This difference could be explained by the selection criteria used. While Canto et al[6] selected the cases based on the radiological and histological findings. We included only the cases who met the strict histological criteria from a large database of subjects with duodenal biopsy. On the other hand, upper GI endoscopy with biopsy and duodenal fluid aspirate was performed in each patient with chronic diarrhea. NLH was found in 18 cases (4%). This

low frequency can be explained by our selection criteria. Again we only included subjects with a duodenal biopsy with or without small bowel radiology. It was reported that NLH is most frequently located in jejunum-ileum[1-3]. The pathogenesis of NLH is unknown but is likely the result of an accumulation of plasma-cell precursors due to a maturational defect in the development of B-lymphocytes in order to compensate for functionally inadequate intestinal lymphoid tissue[8]. The role of functionally inadequate intestinal lymphoid tissue is suggested by the common occurrence of hypogammaglobulinemia and recurrent infections (particularly by Giardia lamblia)[9]. Nine patients had disgammaglobulinemia, but we were able to demonstrate Giardia lamblia infection only in one. It should be noted that we only used stool analysis for ova and parasites and microscopic duodenal fluid examination. It is possible that some cases could be missed because we did not perform fecal ELISA for giardia-specific antigen, which is the most sensitive and specific method for giardia detection[10]. Another plausible explanation is the high frequency of auto-medication used in our patients. Mexican subjects with chronic diarrhea are empirically treated with antibiotics including metronidazole. Histological diagnosis of NLH can demonstrate the hyperplasic lymphoid follicles with mitotically active germinal centers at mucosa and/or submucosa. The number of follicles is not a diagnostic criterion[1,2]. We found a mean of 2.5 hyperplasic lymphoid follicles in a single 4× magnification field. Patients with three or more localized hyperplasic follicles in mucosa or submucosa showed markers of poor intestinal absorption such as low serum hemoglobin and β-carotene and a marked prolonged prothrombin time, suggesting that fat malabsorption may in turn lead to deficiencies of fat-soluble vitamins. These patients also had chronic diarrhea and a marked weight loss. The association between NLH and other malignant and benign diseases has been clearly described[9,12-15]. In our series, seven cases had CVI and one had selective IgA def ic iency. CVI is a heterogeneous for m of immunodeficiency associated with decreased serum immunoglobul in levels, recur rent s inopulmonary infections, gastrointestinal disorders, and increased frequency of malignancies[12,13]. The association between NLH, hypogammaglobulinemia and Giardia lamblia infection is known as Herman’s syndrome[8]. We only found one case with disgammaglobulinemia, NLH and Giardia lamblia infection. The only real difference with

Table 3 Disorders found in patients with NLH

Disorder nHypogammaglobulinemia 9Lymphoma 3Intestinal bacterial overgrowth 3Celiac sprue 2Giardiasis 1Graves disease 1MNGIE 1Chronic pancreatitis 1Pancreas divisum 1

MNGIE: mitochondrial neurogastrointestinal encephalomyopathy.

Table 2 Findings in duodenal mucosa biopsy specimens from patients with NLH

Abnormality n (%)Localization of lymphoid follicles• Mucosa 13 (69)• Mucosa and submucosa 5 (31)Inflammatory changes in lamina propriaChronic 18 (100)• Lymphocytes-plasma cells 10 (53)• Plasma cells 7 (38)• Lymphocytes 1 (9)Acute and chronic 2 (15)Villous atrophy 7 (38)• Partial 5 (23)• Total 2 (15)Increased intraepithelial lymphocytes 2 (15)Crypt hyperplasia 1 (9)

Rubio-Tapia A et al. Nodular lymphoid hyperplasia 1947

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Canto’s work was the low detection of giardiasis in our series, a feature which is needed to establish the diagnosis of Herman’s syndrome. The risk of malignancy has been well recognized in subjects with NLH[14]. Three of our cases had lymphoma. Lymphoma has been reported in patients with and without immunodeficiency[15,16]. The link between the extra intestinal lymphoma and NLH is less clear. We found one patient with Hodgkin’s disease localized on the mediastinum. Jonsson et al[4] have reported a case of extra intestinal lymphoma associated with NLH. Hyperplasic tissue completely disappeared after chemotherapy with remission of lymphoma and then reappeared at relapse. To the best of our knowledge, no relationship between NLH and celiac disease has been reported. In our series, intestinal atrophy, increased intraepithelial lymphocytes and anti-endomysium antibodies were found in two patients. One of them was asymptomatic on gluten-free diet, and complete resolution of anatomical changes was found in duodenal-specimens taken two months later. In the other case, no clinical response was found six weeks after implementation of a gluten-free diet. This patient lost her follow-up and we do not know her current histological status. Moderate to severe intestinal atrophy and increased intraepithelial lymphocytes have been reported in patients with CVI known as “pseudo-celiac” pattern[17]. In contrast to the typical changes seen in patients with celiac disease, CVI cases showed mild inflammatory infiltrate at the lamina propria with normal enterocyte maturation. We identified partial villous atrophy in five patients with negative anti-endomysium antibodies. Perhaps these 5 patients correspond to the pseudo-celiac pattern previously described.

REFERENCES1 RambaudJC, De Saint-Louvent P, Marti R, Galian A, Mason

DY, Wassef M, Licht H, Valleur P, Bernier JJ. Diffuse follicular lymphoid hyperplasia of the small intestine without primary immunoglobulin deficiency. Am J Med 1982;73: 125-132

2 RanchodM, Lewin KJ, Dorfman RF. Lymphoid hyperplasia of the gastrointestinal tract. A study of 26 cases and review of the literature. Am J Surg Pathol 1978;2: 383-400

3 Case records of the Massachusetts General Hospital. Weekly

clinicopathological exercises. Case 8-1997. A 65-year-old man with recurrent abdominal pain for five years. N Engl J Med 1997; 336: 786-793

4 JonssonOT, Birgisson S, Reykdal S. Resolution of nodular lymphoid hyperplasia of the gastrointestinal tract following chemotherapy for extraintestinal lymphoma. Dig Dis Sci 2002; 47: 2463-2465

5 Levendoglu H, Rosen Y. Nodular lymphoid hyperplasia of gut in HIV infection. Am J Gastroenterol 1992; 87: 1200-1202

6 Canto J, Arista J, Hernández J. [Nodular lymphoid hyper-plasia of the intestine. Clinico-pathologic characteristics in 11 cases]. Rev Invest Clin 1990; 42: 198-203

7 Castañeda-Romero B,Díaz-Caldelas L,Galván-Guerra E, Sixtos S, Arista J, Uscanga L. [Intestinal lymphoid nodular hy-perplasia in a patient with acquired dysgammaglobulinemia, chronic diarrhea, and bacterial overgrowth syndrome]. Rev Gastroenterol Mex 1993; 58(3): 225-228

8 Hermans PE, Diaz-Buxo JA, Stobo JD. Idiopathic late-onset immunoglobulin deficiency. Clinical observations in 50 pa-tients. Am J Med 1976; 61: 221-237

9 deWeerthA, Gocht A, Seewald S, Brand B, van Lunzen J, Seitz U, Thonke F, Fritscher-Ravens A, Soehendra N. Duode-nal nodular lymphoid hyperplasia caused by giardiasis infec-tion in a patient who is immunodeficient. Gastrointest Endosc 2002; 55: 605-607

10 VesyCJ, Peterson WL. Review article: the management of Giardiasis. Aliment Pharmacol Ther 1999; 13: 843-850

11 AlSamman M, Zuckerman MJ, Mohandas A, Ting S, Hoff-pauir JT. Intestinal nodular lymphoid hyperplasia in a patient with chronic diarrhea and recurrent sinopulmonary infections. Am J Gastroenterol 2000; 95: 2147-2149

12 Cooper MD, Schoerder HW. Primary immune deficiency diseases. In: Kasper DL, ed. Harrison´s Principles of Internal Medicine, 16th ed. McGraw-Hill, 2005: 1939-1947

13 LaiPing SoA, Mayer L. Gastrointestinal manifestations of primary immunodeficiency disorders. Semin Gastrointest Dis 1997; 8: 22-32

14 RyanJC. Premalignant conditions of the small intestine. Semin Gastrointest Dis 1996; 7:88-93

15 MatuchanskyC, Touchard G, Lemaire M, Babin P, Demeocq F, Fonck Y, Meyer M, Preud’Homme JL. Malignant lymphoma of the small bowel associated with diffuse nodular lymphoid hyperplasia. N Engl J Med 1985; 313: 166-171

16 CastellanoG, Moreno D, Galvao O, Ballestín C, Colina F, Mollejo M, Morillas JD, Solís Herruzo JA. Malignant lym-phoma of jejunum with common variable hypogammaglobu-linemia and diffuse nodular hyperplasia of the small intestine. A case study and literature review. J Clin Gastroenterol 1992; 15: 128-135

17 Washington K, Stenzel TT, Buckley RH, Gottfried MR. Gastroin-testinal pathology in patients with common variable immunodefi-

S-EditorGuo SY L-Editor Wang XL E-EditorBi L


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Hyperlactatemia in patients with non-acetaminophen-related acute liver failure

Pilar Taurá, Graciela Martinez-Palli, Julia Martinez-Ocon, Joan Beltran, Gerard Sanchez-Etayo, Jaume Balust, Teresa Anglada, Antoni Mas, Juan-Carlos Garcia-Valdecasas

Pilar Taurá, Graciela Martinez-Palli, Julia Martinez-Ocon, Joan Beltran, Gerard Sanchez-Etayo, Jaume Balust, Teresa Anglada, Department of Anesthesiology, Hospital Clinic, Barcelona, Spain Antoni Mas, Department of Hepatology, Hospital Clinic, Barcelona, SpainJuan-Carlos Garcia-Valdecasas, Department of Surgery, Hospital Clinic, Barcelona, SpainCorrespondence to: Pi lar Taurá, MD, Department of Anesthesiology, Liver Transplant Unit, Hospital Clínic. Villarroel 170, Barcelona University, E-08036 Barcelona,Spain. [email protected]: +34-93-2275558 Fax: +34-93-2275454 Received: 2005-03-08 Accepted: 2005-08-26

AbstractAIM: To characterize hyperlactatemia in patients with non-acetaminophen acute liver failure (ALF) in an attempt to clarify the mechanisms implicated and the role as a prognosis factor.

METHODS: In the setting of liver transplantation, 63 consecutive patients with non-acetaminophen acute liver failure were studied in relation to tissue oxygenation, hemodynamic and metabolic parameters. Before and after transplantation, the number of infected patients and outcome were registered.

RESULTS: Acute ALF showed higher levels of lactate than subacute ALF (5.4 ± 1 mmol/L versus 2.2 ± 0.6 mmol/L, P = 0.01). Oxygenation parameters were within the normal range. Lactate levels showed good correlation with respiratory quotient (r = 0.759, P < 0.005), mean glucose administrat ion (r = 0.664, P = 0.01) and encephalopathy (r = 0.698, P = 0.02), but not with splanchnic arteriovenous difference in PCO2, pH and the presence of infection (P = 0.1). Portal vein lactate was higher (P < 0.05) than arterial and mixed venous lactate, suggesting its production of hyperlactatemia in the intestine and spleen. The presence of infection was an independent predictor of survival.

CONCLUSION: Hyperlactatemia is not a prognosis factor due to byproduct of the overall acceleration in glycolysis.


Key words: Hyperlactatemia; Non-acetaminophen acute liver failure; Splanchnic hypoperfusion; Acute liverfailure

Taurá P, Martinez-Palli G, Martinez-Ocon J, Beltran J, Sanchez-Etayo G, Balust J, Anglada T, Mas A, Garcia-Valdecasas JC. Hyperlactatemia in patients with non-acetaminophen-related acute liver failure. World J Gastroenterol 2006; 12(12): 1949-1953


INTRODUCTIONUp to now hyperlactatemia, a common finding in the setting of acute liver failure (ALF), has been attributed mainly to severe circulatory disturbance, abnormal vasomotor tone, plugging of small vessels by platelets and/or interstitial edema[1] and it has been proposed as a bad prognosis factor. Several studies suggest that splanchnic circulation abnormalities during ALF may result in inadequate distribution of blood flow and occlusion of the microvasculature, leading to tissue hypoperfusion, although oxygen delivery is increased in a macro circulation[2,3]. Inadequate balance between splanchnic oxygen delivery and demand results in splanchnic hypoxia of tissues that consume oxygen avidly[4]. In this condition, as cellular oxygen concentrations decrease, ATP concentrations cannot be maintained by oxidative phosphorylation. In an attempt to maintain cellular function, the cells shift over to anaerobic glycolysis with accumulation of lactic acid and hydrogen ions as ATP is hydrolyzed. However, other mechanisms may be implicated in hyperlactatemia in the context of ALF. Although lactate is the end-product of anaerobic glycolysis and blood concentrations rise in response to hypoxia, well-oxygenated tissues may also produce lactate due to aerobic glycolysis[5]. On the other hand, since the liver is the principal organ responsible for removal of whole body lactate clearance, hyperlactatemia may be the result of a deficit of lactate clearance by the insufficient liver. We undertook the present study to characterize hyperlactatemia in patients with non-acetaminophen ALF who were considered candidates for liver transplantation in an attempt to clarify the mechanisms implicated in high

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lactate levels as well as the role as a prognosis factor.

MATERIALS AND METHODSPatientsA to t a l o f 63 pa t i en t s w i th d i a gnos i s o f non -acetaminophen ALF were admitted to the Liver Intensive Therapy Unit in our hospital and submitted to liver transplantation. All patients were prospectively evaluated and included in the study. Four of them were considered hyperacute, 39 acute while 20 subacute hepatic failure based on the criteria of the Kings College Hospital Group[6]. This prospective study was approved by the Clinic Hospital Research Ethic Committee. Informed consent for the study was obtained from each patient or patient’s family.

General managementAll patients were managed in the Liver Intensive Therapy Unit, in a standard manner[7]. They were maintained on 10%-20% glucose solution infusion (keeping blood glucose levels between 90 and 120 mg/dl). Prophylaxis for upper gastrointestinal bleeding and close microbiological surveillance (all patients received norfloxacin and nystatin) were performed. Coagulation profile was corrected only in the presence of overt bleeding. Those in grade III and IV coma (43 patients) were treated with standard sedation and mechanical ventilation. An epidural transducer (Ladd Research Laboratories, Burlington, VT) was inserted into the epidural space to enable early recognition and treatment of intracranial hypertension (>25 mmHg), mannitol (0.5 to 1 mg/kg) for over 30 min and pentobarbital infusion as a following step were used to control intracranial pressure (ICP). Systemic arterial hypotension (systolic <100 mmHg) was managed by dopamine infusion according to the hemodynamic conditions in an attempt to maintain cerebral perfusion pressure (CPP) (>60 mmHg). No artificial liver support techniques were used and no patient was treated with N-acetylcysteine or epoprostenol.

Anesthesia and hemodynamic managementIn 20 patients who remained awake (encephalopathy grades I and II), anesthesia was induced following our standard policy. All patients were mechanically ventilated (Servo 900C, Siemens) and the fraction of inspired oxygen was adjusted to achieve an arterial PaO2 of 180 - 220 mmHg. Ventilatory parameters were regulated to maintain the end tidal CO2 around 32 mmHg and PaCO2 below 35 mmHg. In all patients vasopressor dopamine at 3 µg/kg was started at the beginning of the surgical procedure and maintained through the transplant and increased to 8 µg/kg if necessary to achieve a mean arterial pressure greater than 75 mmHg. After graft reperfusion, arterial hypotension was treated by intravenous bolus of 10 µg of epinephrine. Fiberoptic pulmonary artery floatation catheter (7.5 French, Edwards Laboratories, Irvine, Calif.) was introduced through the right internal jugular vein and an arterial catheter (18 gauge, Arrow, Reading, PA) was placed via the left radial artery. At the beginning of the surgical procedure portal vein catheter (Certofix ®

Mono S330 16G Braun) was placed by introducing it through a branch of the superior mesenteric vein and advanced to the portal vein until it was felt, in order to register portal pressure and to obtain blood samples for oxygen and metabolic parameters in splanchnic area. Not all patients received portal vein cannulation because it was not considered technically possible by the surgeon in 11 patients (4 with subacute ALF and 7 with acute ALF).

Variables analyzed in the setting of LTxBefore the surgical transplant procedure was started, the following parameters were measured. Mean arterial pressure (MAP, mmHg), cardiac index (CI, L/min/m2) and systemic vascular resistance index (SVRI, dyn.sec.cm-5.m2) were obtained. Systemic parameters including oxygen content difference [D(a-v)O2, ml/dl], oxygen delivery (DO2,

mL/min/m2), oxygen consumption (VO2, mL/min/m2), oxygen extraction ratio (VO2/DO2, %), mixed venous/arterial gradient of PCO2 (VACO2, mmHg), arterial/mixed venous gradient of pH (AVpH, U ) as well as respiratory quotient (RQ) were calculated. Splanchnic parameters including arterial/portal venous oxygen content difference [D(a-v)O2, ml/dl], oxygen extraction ratio (VO2/DO2), portal veno/arterial gradient of PCO2 (VACO2, mmHg), and arterial/portal venous gradient of pH (AVpH, U) were obtained. All these variables were calculated following standard formulas. Plasma lactate levels were measured with three blood samples simultaneously drawn from arterial catheter (La), the distal part of pulmonary catheter (Lv) and portal vein catheter (Lp). Blood lactate level was determined using a Kodak Ektachem 700XR (Rochester, NY. USA) analyzer[8]. The need of catecholamine administration and the mean glucose administration in the last 48 hours before transplant were recorded. The need of vasopressor drugs administered during the transplant procedure was also registered. The degree of encephalopathy and intracranial pressure were recorded.

Explanted liver pathologyWeight and structural characteristics of all explanted livers were studied.

Infectious complicationsDiagnosis of infection was made by the presence of the white blood cell count greater than 12×109/L or less than 4×109/L, the presence of immature neutrophils, temperature higher than 38 ℃ or lower than 36 ℃ and microbiological confirmation. Also, chest infection was confirmed by radiology. All these parameters were recorded daily during the ICU admission before transplantation and the ten following days. The number of infected patients and episodes and pathogens microbiologically confirmed was registered, respectively.

ICU stay and immediate outcomeThe mean stay in ICU during the first admission of patients after transplantation and immediate outcome (first admission in the hospital) were recorded.


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Statistical analysisStatistical analysis was performed using two-sided paired t-test for comparison of paired data and two-sided unpaired t-test for comparison of groups. The Bonferroni correction test was applied as appropriate. Categorical data were compared with the chi-square test for relationship between encephalopathy and infection. Correlation between lactate and the hemodynamic and oxygenation variables was assessed by linear regression analysis. Variables reaching significance in the univariate analysis between survivors and non-survivors were included in the multivariate analysis. Multivariate analysis was carried out by stepwise logistic regression analysis to determine discriminants of survival. All values were expressed as mean±SD. P < 0.05 was considered statistically significant.

RESULTSDemographic and clinical characteristics of the patients are shown in Table 1. No patient needed blood transfusion before the transplant.

Systemic hemodynamic and oxygenation parametersS y s t e m i c h e m o d y n a m i c p a r a m e t e r s s h owe d a hyperdynamic circulatory status with high CI (4.53 ± 1.4 L/min/m2) and low SVRI (1029±420 dyn.sec.cm-5.m2). Oxygenation parameters remained between normal ranges (DO2 623 ± 36 mL/min/m2, VO2 96.4 ± 21 mL/min/m2 and VO2/DO2 18.4 ± 3.1%).

Blood lactate levelsThe production of lactate in the intestine or in the spleen (Table 2), in accordance with Murphy et al[9], was suggested by the increased difference between portal, arterial and mixed venous lactate (P < 0.05). Acute ALF showed significant higher levels (acute: 5.4 ± 1 mmol/L, subacute: 2.2 ± 0.6 mmol/L, P = 0.01). Lactate levels did not correlate with any of the hemodynamic or oxygenation parameters studied, except for the respiratory quotient (Figure 1 A). The grade of encephalopathy and the amount of glucose administered 48 hours before transplant (Figure 1 B and Figure 1 C) correlated well with blood lactate levels.

Splanchnic oxygenation parameters The splanchnic VACO2 and AVpH levels were in normal range and did not correlate with plasma lactate levels (r = 0.203 and r = 0.164, respectively). No differences were found (Table 2) when we compared patients with high (acute ALF) and low lactate levels (subacute ALF). In order to maintain hemodynamic stability, only five patients with acute ALF needed administration of dopamine prior to the transplant. Only one of the patients demonstrated a high level of lactate (8.2 mmol/L). There was no significant correlation between lactate levels and the dosages of dopamine and epinephrine (r = 0.020 and r = 0.13 respectively) through the transplant. The total dose of epinephrine administered after graft reperfusion in patients with low (subacute ALF) and high lactate level (acute ALF) was similar in both groups (42.6 ± 12 µg and 54.1 ± 18 µg, respectively).

Explanted liver pathologyNo relationship was found between lactate levels and liver weight. Interestingly, although the rate of massive necrosis was similar, liver weight of subacute ALF patients was significantly lower than that of acute ALF patients (762 ± 22 g and 932 ± 38 g, P < 0.05).

Infectious complicationsTwenty-one patients (33.3%) were infected in the perioperative period. Bacterial infection was found in 16 patients (25.4%), fungal infection in 5 patients (7.9%) and viral infection in 2 patients (3.1%). The incidence of infectious episodes of acute (13 patients, 30.2%) and subacute (8 patients, 40%) hepatic failure showed no difference. The level of lactic acidosis (Figure 2) no correlated with the presence of infection (P = 0.1). Sixteen patients were infected before the transplant, 11 of them requiring mechanical ventilation because of pulmonary infection (confirmed on chest radiograph and microbiologically), showed significantly higher arterial lactate levels compared with mixed venous lactate level (5.2 ± 1.1 and 4.5 ± 0.8 mmol/L, P = 0.03), suggesting lactate production within the lungs.

ICU stay and immediate outcome of patients with ALFDuring the stay in ICU, 13 patients died (8 patients with acute and 5 subacute liver failure). Bleeding was not controlled in 2 patients. Furthermore, 3 patients needed

Table 1 Clinical and demographic characteristics of the patients

Age (yr) 32.7 ± 11Sex (M/F) 26/37

Etiology (n, %) Viral hepatitis 18 (30) Cryptogenic (non-A, non-B, non-C)

32 (52)

Drug toxicity 7 (11) MAOi 3 Rifampin+Isoniacid 2 Isoflurane 1 α metil-dopa 1Metabolic disease 4 (6)

Encephalopathy I-II (subacute form: 20 p) 20 (31.7) III-IV (acute form:39 p) (hyperacute form: 4 p.)

43 (68)

Table 2 Plasma lactate levels at different sites (mean±SD)

Total (n = 52)

Acute (n = 36)

Subacute (n = 16)

P

La (mmol/L) 4.1 ± 1.8 (1.1-15.2) 5.4 ± 1 2.2 ± 0.6 0.01Lv (mmol/L) 4.3 ± 2 (1.4-16.3) 4.7 ± 2 2.4 ± 1.4 0.03Lp (mmol/L) 5.3 ± 1.1a (2.1-17.6) 6.8 ± 1.8a 2.9 ± 0.9 <0.01pH (units) 7.36 ± 0.07 (7.28-7.42) 7.33 ± 0.02 7.36 ± 0.04 NSVACO2(mmHg) 12.4 ± 7 (8.3-13.4) 13.8 ± 5 9.6 ± 9 NSAvpH (units) 0.06 ± 0.03 (0.02-0.07) 0.08 ± 0.02 0.07 ± 0.04 NS

La: arterial lactate; Lv: mixed venous lactate; Lp: portal vein lactate; L(a-p): transplanchnic arterial/portal difference of lactate; VA CO2: portal/arterial gradient of CO2; AVpH: arterial/portal gradient of pH; aP < 0.05 vs La, Lv and Lp.

Taurá P et al. Mechanisms implicated in hyperlactatemia 1951

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retransplantation because the primary graft did not function and one of them died after retransplantation because of infection complications. The mean stay in ICU was 14.6 days (range 4 - 38 d). The ICU stay correlated well with infection complications (P < 0.01) but not with plasma lactate levels. The mean plasma lactate level was similar in nonsurvivors and survivors (4.3 ± 0.7 mmol/L and 3.9 ± 10 mmol/L, respectively, (P = 0.69). Univariate analysis showed that infectious complications (71.4% vs 22.4%, P = 0.02) and subacute ALF (30% vs 18.6%, P = 0.08) were the potential risk of non-survivors compared to survivors. Multiple logistic regression revealed that the presence of infection was the only discriminator of survival. Encephalopathy g rade and splanchnic VACO2 and AVpH did not significantly improve the prediction of immediate survival.

DISCUSSION Hyperlactatemia observed in the context of non-acetaminophen ALF seemed to be related to aerobic glycolysis but not to tissue hypoperfusion. Additionally, the incidence of infection in these patients, which appeared as an independent predictor of survival, did not correlate with the degree of hyperlactatemia. It is important to emphasize that the foremost cause

of ALF in our series was acute viral hepatitis that differs substantially in other series. Since acetaminophen exerts a direct toxic effect on cellular respiration, this type of cytotoxicity may be different from other types of ALF. Currently, the presence of hyperlactatemia in patients with ALF is assumed as the consequence of underlying overt tissue hypoxia. In these patients, oxygen delivery may be impaired. Combined measurement of VACO2 and AVpH may serve as a good indicator of tissue hypoxia with a closer relation to cardiac output than blood lactate, because it is less affected by changes in fuel substrate utilization or enzymatic alterations [10-12]. In our study no differences were found in splanchnic VACO2 and AVpH between patients with high (acute ALF) and low lactate levels (subacute ALF). I f hype r l a c t a t emia i s a t t r ibu t ab l e to t i s sue hypoperfusion, the increase in oxygen delivery should reduce lactate levels. However, several reports[13-15] have failed to identify this evidence in patients with ALF due to acetaminophen overdose. In our patients selected to receive liver transplantation, plasma lactate levels did not correlate with hemodynamic or oxygenation parameters, except for RQ and the amount of glucose administered, suggesting that aerobic glycolysis is responsible of lactate hyperproduction. Since patients with ALF may exhibit hyperinsulinemia due to pancreatic hypersecretion and/or decreased hepatic clearance of insulin, they need glucose infusion in order to maintain blood glucose level. Previous studies[16-18] showed that infusion of glucose results in a dose-dependent rise in splanchnic lactate levels. Moreover, peripheral tissues of cirrhotic patients produce an exaggerated lactate production in response to glucose administration[19]. In our study, 7 patients who did not require glucose administration (subacute liver failure), showed normal lactate levels (<1.3 mmol/L). A complete round of the Cori cycle is proton-neutral because the H+ produced by lactate from glucose is subsequently removed during synthesis of glucose from lactate in the liver. In the context of liver failure, the possibility to handle lactate to glucose (gluconeogenesis) is handicapped and consequently the possibility to develop “lactic acidosis” without the presence of high muscular lactate overproduction is not significant. In our patients the arterial blood pH stayed within normal ranges (7.28 to 7.41). If hyperlactatemia is not related

Figure 2 Arterial lactate levels in 21 infected and 40 non-infected patients. Box plot indicates that there is no significant difference between the infected and non-infected patients.

9

8

7

6

5

4

3

2

1

0

Lact

ate

leve

l (m

mol

/L)

n = 40 21 Non-infection Infection

Figure 1 Correlation between arterial lactate level (nmol/L) and respiratory quotient (n = 61) (A), arterial lactate level (nmol/L) in patients with different grade of encephalopathy (n = 61) (B), correlation between arterial lactate level (nmol/L) and total amount of glucose (g) administered 48 hours before the transplant (C).

r = 0.759P < 0.005

16

14

12

10

8

6

4

2

0

Lact

ate

leve

ls (

mm

ol/L

)

0.4 0.6 0.8 1.0 1.2 1.4 1.6 Respiratory quotient

A

r = 0.698P = 0.02

Lact

ate

leve

ls (

mm

ol/L

)

10

9

8

7

6

5

4

3

2

1

0N = 6 14 9 32 1 2 3 4 Encephalopathy

B

r = 0.664P = 0.01

16

14

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2

0

Lact

ate

leve

ls (

mm

ol/L

)

C

0 100 200 300 400 500 Glucose administered (g)


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to the mortality, infection, or operative hemodynamic management difficulty, the usage of dichloroacetate (which acts by stimulating pyruvate dehydrogenase) is not a priority unless its benefit to avoid postoperative severe alkalosis (lactate metabolism by the graft consume H+) is considered. However, it seems that this treatment fails to attenuate metabolic alkalosis after transplant[20]. Clemmesen et al[5] have suggested a hypermetabolic condition secondary to an excessively high glycolysis in relation to the small liver mass, but we have not shown any correlation between liver weight and splanchnic lactate level in patients with ALF. Wendon et al[21] demonstrated that there is a significant correlation between arterial lactate and survival in acetaminophen related ALF, but in our study no relationship was found between plasma lactate and outcome. Inadequate splanchnic blood flow and tissue hypoperfusion could contribute to bacterial translocation and sepsis, however in these patients several factors can predispose to infection such as comatose state, steroid therapy, mechanical ventilation and intravenous catheters. Patients with ALF are susceptible to infection as a result of multiple immunologic defects (excessive cytokine production from cells, such as monocytes and macrophages in response to a number of stimuli including bacterial lipopolysaccharide)[22]. In our patients the Gram-positive organisms were predominant. The presence of Gram-negative organisms such as pseudomonas was not uncommon, showing a high incidence in this group of patients. These results suggest that selective bowel decontamination can be used in the treatment of such patients[23]. We did not find any correlation between lactate levels and infection incidence although there is evidence that cytokines may promote augmented production of lactate[24] by several organs, enhancing cellular glucose uptake and glycolytic metabolism. However, in agreement with other studies[25], arterial lactate is higher than venous mixed lactate in patients with pulmonary infection. In summary, the presence of high plasma lactate levels in patients with non-acetaminophen ALF, is not related to tissue hypoperfusion and is not a prognostic factor in the treatment of ALF.

REFERENCES1 Makin AJ, Hughes RD, Williams R. Systemic and hepatic

hemodynamic changes in acute liver injury. Am J Physiol 1997; 272: G617-G625

2 Bihari D, Gimson AE, Lindridge J, Williams R. Lactic acidosis in fulminant hepatic failure. Some aspects of pathogenesis and prognosis. J Hepatol 1985; 1: 405-416

3 Bihari D, Gimson AE, Waterson M, Williams R. Tissue hypoxia during fulminant hepatic failure. Crit Care Med 1985; 13: 1034-1039

4 Clemmesen JO, Gerbes AL, Gülberg V, Hansen BA, Larsen FS, Skak C, Tygstrup N, Ott P. Hepatic blood flow and splanchnic oxygen consumption in patients with liver failure. Effect of high-volume plasmapheresis. Hepatology 1999; 29: 347-355

5 Clemmesen JO, Høy CE, Kondrup J, Ott P. Splanchnic metabolism of fuel substrates in acute liver failure. J Hepatol 2000; 33: 941-948

6 O'Grady JG, Schalm SW, Williams R. Acute liver failure: redefining the syndromes. Lancet 1993; 342: 273-275

7 Castells A, Salmerón JM, Navasa M, Rimola A, Saló J, Andreu H, Mas A, Rodés J. Liver transplantation for acute liver failure: analysis of applicability. Gastroenterology 1993; 105: 532-538

8 Kropf J, Marx AM, Hildebrandt J, Gressner AM. Practical implications of coexistent different technologies in clinical chemical laboratories. Solid phase chemistry and conventional analysis. Eur J Clin Chem Clin Biochem 1991; 29: 675-683

9 Murphy ND, Kodakat SK, Wendon JA, Jooste CA, Muiesan P, Rela M, Heaton ND. Liver and intestinal lactate metabolism in patients with acute hepatic failure undergoing liver transplantation. Crit Care Med 2001; 29: 2111-2118

10 Ducey JP, Lamiell JM, Gueller GE. Arterial-venous carbon dioxide tension difference during severe hemorrhage and resuscitation. Crit Care Med 1992; 20: 518-522

11 Bakker J, Vincent JL, Gris P, Leon M, Coffernils M, Kahn RJ. Veno-arterial carbon dioxide gradient in human septic shock. Chest 1992; 101: 509-515

12 Johnson BA, Weil MH. Redefining ischemia due to circulatory failure as dual defects of oxygen deficits and of carbon dioxide excesses. Crit Care Med 1991; 19: 1432-1438

13 Harrison PM, Wendon JA, Gimson AE, Alexander GJ, Williams R. Improvement by acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure. N Engl J Med 1991; 324: 1852-1857

14 Wendon JA, Harrison PM, Keays R, Gimson AE, Alexander GJ, Williams R. Effects of vasopressor agents and epoprostenol on systemic hemodynamics and oxygen transport in fulminant hepatic failure. Hepatology 1992; 15: 1067-1071

15 Wendon JA, Harrison PM, Keays R, Gimson AE, Alexander G, Williams R. Arterial-venous pH differences and tissue hypoxia in patients with fulminant hepatic failure. Crit Care Med 1991; 19: 1362-1364

16 Shulman GI, Lacy WW, Liljenquist JE, Keller U, Williams PE, Cherrington AD. Effect of glucose, independent of changes in insulin and glucagon secretion, on alanine metabolism in the conscious dog. J Clin Invest 1980; 65: 496-505

17 Myers SR, Biggers DW, Neal DW, Cherrington AD. Intraportal glucose delivery enhances the effects of hepatic glucose load on net hepatic glucose uptake in vivo. J Clin Invest 1991; 88: 158-167

18 Bratusch-Marrain PR, Waldhäusl WK, Gasić S, Korn A, Nowotny P. Oral glucose tolerance test: effect of different glucose loads on splanchnic carbohydrate and substrate metabolism in healthy man. Metabolism 1980; 29: 289-295

19 Leatherdale BA, Chase RA, Rogers J, Alberti KG, Davies P, Record CO. Forearm glucose uptake in cirrhosis and its relationship to glucose tolerance. Clin Sci (Lond) 1980; 59: 191-198

20 Shangraw RE, Winter R, Hromco J, Robinson ST, Gallaher EJ. Amelioration of lactic acidosis with dichloroacetate during liver transplantation in humans. Anesthesiology 1994; 81: 1127-1138

21 Bernal W, Donaldson N, Wyncoll D, Wendon J. Blood lactate as an early predictor of outcome in paracetamol-induced acute liver failure: a cohort study. Lancet 2002; 359: 558-563

22 Rolando N, Wade J, Davalos M, Wendon J, Philpott-Howard J, Williams R. The systemic inflammatory response syndrome in acute liver failure. Hepatology 2000; 32: 734-739

23 Salmerón JM, Titó L, Rimola A, Mas A, Navasa MA, Llach J, Ginès A, Ginès P, Arroyo V, Rodés J. Selective intestinal decontamination in the prevention of bacterial infection in patients with acute liver failure. J Hepatol 1992; 14: 280-285

24 Douzinas EE, Tsidemiadou PD, Pitaridis MT, Andrianakis I, Bobota-Chloraki A, Katsouyanni K, Sfyras D, Malagari K, Roussos C. The regional production of cytokines and lactate in sepsis-related multiple organ failure. Am J Respir Crit Care Med 1997; 155: 53-59

25 Routsi C, Bardouniotou H, Delivoria-Ioannidou V, Kazi D, Roussos C, Zakynthinos S. Pulmonary lactate release in patients with acute lung injury is not attributable to lung tissue hypoxia. Crit Care Med 1999; 27: 2469-2473


Taurá P et al. Mechanisms implicated in hyperlactatemia 1953

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RAPID COMMUNICATION

Clinical features of hepatopulmonary syndrome in cirrhotic patients

Amir Houshang Mohammad Alizadeh, Seyed Reza Fatemi, Vahid Mirzaee, Manoochehr Khoshbaten, Bahman Talebipour, Afsaneh Sharifian, Ziba Khoram, Farhad Haj-sheikh-oleslami, Masoomeh Gholamreza-shirazi, Mohammad Reza Zali

Amir Houshang Mohammad Alizadeh, Seyed Reza Fatemi, Vahid Mirzaee, Manoochehr Khoshbaten, Bahman Talebipour, Afsaneh Sharifian, Ziba Khoram, Mohammad Reza Zali, Research Center for Gastroenterology and Liver Disease, Shaheed Beheshti University of Medical Sciences, Tehran, IranFarhad Haj-sheikh-oleslami, Masoomeh Gholamreza-shirazi, Cardiologist, Shaheed Beheshti University of Medical Sciences, Tehran, IranCorrespondence to: Amir Houshang Mohammad Alizadeh, Research Center for Gastroenterology and Liver Disease, Shaheed Beheshti University of Medical Sciences, 7th floor, Taleghani Hospital, Yaman Str., Evin, 19857 Tehran, Iran. [email protected]: +98-21-22418871 Fax: +98-21-22402639 Received: 2005-02-06 Accepted: 2005-08-26

AbstractAIM: To evaluate the frequency, c l in ica l and paraclinical features of hepatopulmonary syndrome (HPS) and to determine their predictive values in di-agnosis of this syndrome in patients in Iran.

METHODS: Fifty four cirrhotic patients underwent contrast enhanced echocardiography to detect intra-pulmonary and intracardiac shunts by two cardiolo-gists. Arterial blood oxygen, O2 gradient (A-a) and orthodoxy were measured by arterial blood gas (ABG) test. The patients positive for diagnostic criteria of HPS were defined as clinical HPS cases and those manifesting the intrapulmonary arterial dilation but no other criteria (arterial blood hypoxemia) were defined as lHPS cases. HPS frequency, sensitivity, positive and negative predictive values of clinical and paraclinical features were studied.

RESULTS: Ten (18.5%) and seven (13%) cases had clinical and subclinical HPS, respectively. The most common etiology was hepatitis B. Dyspnea (100%) and cyanosis (90%) were the most prevalent clinical features. Dyspnea and clubbing were the most sen-sitive and specific clinical features respectively. No significant relationship was found between HPS and splenomegaly, ascites, edema, jaundice, oliguria, and collateral veins. HPS was more prevalent in hepatitis B. PaO2 < 70 and arterial-alveolar gradient had the highest sensitivity in HPS patients. Orthodoxy speci-

ficity was 100%.

CONCLUSION: Clubbing with positive predictive val-ue (PPV) of 75% and dyspnea with negative predic-tive value (NPV) of 75% are the best clinical factors in diagnosis of HPS syndrome. PaO2 < 70 and P (A-a) O2 > 30 and their sum, are the most valuable negative and positive predictive values in HPS patients.


Key words: Hepatopulmonary syndrome; Cirrhosis; Contrast enhanced echocardiography

Mohammad Alizadeh AH, Fatemi SR, Mirzaee V, Khoshbaten M, Talebipour B, Sharifian A, Khoram Z, Haj-sheikh-oleslami F, Gholamreza-shirazi M, Zali MR. Clinical features of hepatopulmonary syndrome in cirrhotic patients. World J Gastroenterol 2006; 12(12): 1954-1956


INTRODUCTION Ascites by elevating the diaphragm and confounding the ventilation/perfusion might lead to mild hypoxemia in most patients due to chronic hepatic involvement, not regarding the etiology. When cirrhotic patients have no sign of any cardiovascular diseases, severe hypoxemia(PO2 < 60mmHg) strongly recommends hepatopulmonary syndrome[1-3]. Hepatopulmonary syndrome is one of the pulmonary complications of cirrhosis which affects the treatment and disease prognosis and is a factor for arterial blood oxygen reduction. The diagnosis of this syndrome is confirmed by presence of cirrhosis in liver biopsy, absence of cardiovascular diseases, arterial blood oxygen reduction found in arterial blood gas (ABG) tests and pulmonary vein dilation in imaging[4]. Many studies have been performed on evaluating the prevalence, etiology, clinical features, early diagnosis, treatment and prognosis of this syndrome worldwide. The aim of this study was to evaluate the clinical and paraclinical characteristics and their predictive values in diagnosis of this syndrome.

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Mohammad Alizadeh AH et al. Hepatopulmonary syndrome in cirrhotic patients 1955

MATERIALS AND METHODS This study was performed in 54 randomly chosen cirrhotic patients referred to Gasteroenterology Department of Taleghani Hospital in 2004. In the patients who entered the study, cirrhosis was confirmed by biopsy, clinical and paraclinical evaluations. Echocardiography and pulmonary function tests were done for all patients and plain chest x-ray was taken. Those with cardiovascular and known respiratory diseases were excluded from this study. The patients with ascites underwent large volume paracentesis. These 54 pat ients underwent contrast enhanced echocardiography performed by two cardiologists from Cardiovascular Department of Talaghani Hospital. The procedure was performed by injecting agitated saline into patient’s right hand cubital vein. Left and right sides of the heart were evaluated by echo after 5 beats. Presence of opacity after 5 beats in the left heart was determined as intrapulmonary shunt. If opacity was present immediately after injection, it was a sign of intracardiac shunt. ABG test was performed in patients at supine. After one hour in vertical position, the oxygenation saturation, arterial blood oxygen, (A-a) O2 gradient and orthodoxy were evaluated. Physical examination was performed to detect clinical features including clubbing in fingers and toes, central and peripheral cyanosis, presence of spider angioma, telangiectasia, jaundice, collateral veins in abdomen, ascites, consciousness, splenomegaly, dyspnea, peripheral edema, palmar erythema, oliguria or anuria and pleural effusion for the underlying etiology. All patients were tested for hepatitis B, hepatitis C, biliary, autoimmune, metabolic, cardiac, alcoholic and idiopathic etiologies. Complete blood count (CBC), liver function test (LFT), creatinine, prothrombin time (PT), partial thromboplastin time (PTT), albumin and other routine tests were measured in all patients. Ascitic fluid was tested for protein, albumin and white blood cells. The patients presenting the three diagnostic criteria of hepatopulmonary syndrome, including hepatic cirrhosis, arterial blood deoxygenation (PO2 < 80 mmHg) and intrapulmonary arterial dilation were defined as clinical hepatopulmonary cases. Those presenting intrapulmonary arterial dilation but no other two criteria (arterial blood hypoxemia) were defined as subclinical hepatopulmonary cases. The study was carried out in accordance with the Helsinki Declaration and approved by the Ethics Committee of the Research Center for Gastroenterology and Liver Disease, Shaheed Beheshti University of Medical Sciences. The data were presented by descriptive statistics. The variables were compared by c2 test. Sensitivity, specificity, positive and negative predictive values of clinical and paraclinical features in diagnosis of hepatopulmonary syndrome were evaluated. P < 0.05 was considered statistically significant.

RESULTSAmong the 54 patients who participated in the study, 10(18.5%) met the clinical hepatopulmonary syndrome criteria and 7(13%) with intrapulmonary arterial dilation (but no other criteria) were defined as subclinical

hepatopulmonary cases. Most frequent age group was 71-80 years. Figure 1 shows the age distribution. The most common etiology was HBV, but there was no significant relation. Figure 2 shows the prevalence of HPS etiologies. By paying attention to the frequency of clinical features in HPS patients, dyspnea (100%) and cyanosis (90%) were the most prevalent. Dyspnea and clubbing were the most sensitive and specific clinical features. Table 1 presents the characteristics and diagnostic values of signs and symptoms in HPS patients. No significant relation was found between splenomegaly, ascites, edema, jaundice, oliguria, collateral veins and hepatopulmonary syndrome. Table 2 presents the patients with or without hepatopulmonary syndrome. HPS was more common in class C. Table 3 presents the diagnostic value of arterial blood gas in HPS. PaO2 and arterial–alveolar oxygen gradients were most sensitive in diagnosis of HPS. Orthodoxy specificity was 100%.

DISCUSSION Hepatopulmonary syndrome includes the triad of liver

Table 1 Characteristics and diagnostic values of signs and symptoms in hepatopulmonary syndrome

Symptomsand signs

Frequencyin HPS (%) Sensitivity

(%)Specificity (%)

PPV(%)

NPV (%)

PPos Neg

Cyanosis 90 15.9 90 80.1 60 97 <0.0001Clubbing 80 9.1 80 90.9 75 95 <0.0001Dyspnea 100 25 100 75 50 100 <0.0001Palmar erythema 80 29.5 80 70.5 38 94 0.003Spider angioma 80 29.5 80 70.5 38 94 0.005

Table 2 Child class in hepatopulmonary syndrome and subclinical patients

Child classification HPS positive HPS negative TotalClass A 0 5 5Class B 2 27 29Class C 8 12 20Total 10 44 54

12

10

8

6

4

2

021-30 31-40 41-50 51-60 61-70 71-80 P = 0.249, Age group (yr)

HPS PosHPS Neg

Figure 1 Age distribution in patients with hepatopulmonary syndrome.

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HPS: hepatopulmonary syndrome, PPV: positive predictive value, NPV: negative predictive value.

disease, arterial blood deoxygenation and pulmonary vein dilation. Although the mortality of this syndrome is high, its influence on patient survival is unknown. In our study, the prevalence of hepatopulmonary syndrome was 18.5% and the prevalence of pulmonary vein dilation was 13%. Our findings are compatible with those of other studies in this field[1-5]. In our study, age and Child class C were significantly associated with HPS, suggesting that this syndrome is related with development of cirrhosis. Studies have shown a relation between HPS and cyanosis, clubbing and orthodoxy, although spider angioma is significantly related to intrapulmonary vein dilation[1]. The same results were observed in our study also. Cyanosis, clubbing and orthodoxy had positive and negative predictive values of 75% and 100% in hepatopulmonary syndrome. Previous studies showed that the most common underlying etiologies of HPS were cryptogenic cirrhosis and cirrhosis due to hepatitis B[3,4]. The reported positive predictive values were 37% and 53% for (A-a) O2 gradients and 93% and 94% for PaO2

[3,4]. Orthodoxy has been

reported to be 88%, but in our study it was 66% and no significant statistical relation was found[6-7]. These clinical results strongly lead to diagnosis of HPS and the above tests can be used in screening patients. In conclusion, hepatopulmonary syndrome and intrapulmonary vein dilation are relatively frequent in patients with portal hypertension. Clubbing with the highest positive predictive value (75%) and dyspnea with the highest negative predictive value (100%) are the best clinical features in HPS patients. Further studies are needed to confirm our results.

REFERENCES1 Lima BL, França AV, Pazin-Filho A, Araújo WM, Martinez

JA, Maciel BC, Simões MV, Terra-Filho J, Martinelli AL. Frequency, clinical characteristics, and respiratory parameters of hepatopulmonary syndrome. Mayo Clin Proc 2004; 79: 42-48

2 Schenk P, Fuhrmann V, Madl C, Funk G, Lehr S, Kandel O, Müller C. Hepatopulmonary syndrome: prevalence and predictive value of various cut offs for arterial oxygenation and their clinical consequences. Gut 2002; 51: 853-859

3 H i r a H S , K u m a r J , T y a g i S K , J a i n S K . A s t u d y o f hepatopulmonary syndrome among patients of cirrhosis of liver and portal hypertension. Indian J Chest Dis Allied Sci 2003; 45: 165-171

4 Anand AC, Mukherjee D, Rao KS, Seth AK. Hepatopulmonary syndrome: prevalence and clinical profile. Indian J Gastroenterol 2001; 20: 24-27

5 Schenk P, Schöniger-Hekele M, Fuhrmann V, Madl C, Silberhumer G, Müller C. Prognostic significance of the hepatopulmonary syndrome in patients with cirrhosis. Gastroenterology 2003; 125: 1042-1052

6 Mimidis KP, Vassilakos PI, Mastorakou AN, Spiropoulos KV, Lambropoulou-Karatza CA, Thomopoulos KC, Tepetes KN, Nikolopoulou VN. Evaluation of contrast echocardiography and lung perfusion scan in detecting intrapulmonary vascular dilatation in normoxemic patients with early liver cirrhosis. Hepatogastroenterology 1998; 45: 2303-2307

7 Aller R, Moya JL, Moreira V, Boixeda D, Cano A, Picher J, García-Rull S, de Luis DA. Diagnosis of hepatopulmonary syndrome with contrast transesophageal echocardiography: advantages over contrast transthoracic echocardiography. Dig Dis Sci 1999; 44: 1243-1248

Table 3 Diagnost ic va lue of arter ia l b lood gases in hepatopulmonary syndrome

Laboratoryresults

Frequency (%) Sensitivity

(%)Specificity (%)

PPV (%)

NPV (%)

P

Pos Neg

Pa02 < 70 and(A-a)02>30

100 4.5 100 92 85 100 < 0.0001

Pa02 < 70 100 4.5 100 95 83 100 < 0.0001a02 < 65 70 4.5 70 98 77 95 < 0.0001Pa02 < 60 30 4.5 30 92 66 87 0.039(A-a)02>15 100 75 100 25 23 10 0.1(A-a)02>20 100 63.6 100 36 26 100 0.24(A-a)02>30 100 31.8 100 70 88 89 0.001(A-a)02>40 90 11.4 90 88 65 75 < 0.0001Orthodoxy>10 60 0 60 100 15 93 < 0.001

HPS PosHPS Neg

Figure 2 Causes of liver disease in patients with hepatopulmonary syndrome.

14

12

10

8

6

4

2

0Hepatitis B Cryptogenic Hepatitis C AIH Biliary Metabolic PBC Alcoholic



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P = 0.980

Etiology


Seroprevalence of Helicobacter pylori in dyspeptic patients and its relationship with HIV infection, ABO blood groups and life style in a university hospital, Northwest Ethiopia

Feleke Moges, Afework Kassu, Getahun Mengistu, Solomon Adugna, Berhanu Andualem, Takeshi Nishikawa, Fusao Ota

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Feleke Moges, Afework Kassu, Department of Microbiology and Parasitology, Gondar College of Medicine and Health Sciences, University of Gondar, EthiopiaGetahun Mengistu, Department of Internal Medicine, Faculty of Medicine, Addis Ababa University, Ethiopia Solomon Adugna, Department of Biochemistry, Gondar College of Medicine and Health Sciences, University of Gondar, Ethiopia Berhanu Andualem, Department of Biology, Faculty of Applied Science, University of Gondar, EthiopiaTakeshi Nishikawa, Hokkaido University of Education, Sapporo Campus, Division of Medicine and Nursing, JapanAfework Kassu, Fusao Ota, Department of Preventive Environment and Nutrition, Institute of Health Biosciences, The University of Tokushima, JapanSupported by University of GondarCorrespondence to: Feleke Moges, Department of Microbiology and Parasitology, Gondar College of Medicine and Health Sciences, University of Gondar, PO Box 196, Gondar, Ethiopia. [email protected]: +251-8-110174-250 Fax: +251-8-111479Received: 2005-06-22 Accepted: 2005-06-22

AbstractAIM: To determine the prevalence of Helicobacter pylori (H pylori ) among dyspeptic patients and to assess the relationship between H pylori infection, blood group, HIV infection and life style of the patients.

METHODS: In a hospital-based cross-sectional study, patients attending Outpatient Department of University of Gondar Hospital were enrolled. Socio-demographic information was collected using questionnaires. Serum was analyzed for anti-H pylori IgG antibodies using a commercial kit. HIV serostatus was determined by enzyme-linked immunosorbent assay (ELISA). Blood grouping was performed by slide agglutination tests.

RESULTS: A total of 215 dyspeptic patients were included in the study. One hundred and sixteen patients (54%) were females and 99 (46%) were males. Anti-H pylori IgG antibodies were detected in sera of 184 (85.6%) patients. The prevalence was significantly higher in patients aged 50 years and above. Twenty point five percent of the patients were found to be seropositive for HIV. No significant association was found between sex, ABO blood groups, consumption of spicy diets, socio-economic status and seropositivity for H pylori . However,

alcohol consumption was significantly associated with H pylori serology.

CONCLUSION: The prevalence of H pylori infection is associated with a history of alcohol intake and older age. The effect of different diet, alcohol and socioeconomic status as risk factors for H pylori infection needs further study.


Key words: H pylori ; Blood group; HIV; Life style

Moges F, Kassu A, Mengistu G, Adugna S, Andualem B, Nishikawa T, Ota F. Seroprevalence of Helicobacter pylor i in dyspeptic patients and its relationship with HIV infection, ABO blood groups and life style in a university hospital, Northwest Ethiopia. World J Gastroenterol 2006; 12(12): 1957-1961


INTRODUCTIONHelicobacter pylori (H pylori), a flagellate Gram negative rod, infects over half of the world’s population[1] and plays a pivotal role in the etiology of a number of gastroduodenal diseases and development of gastric malignancy[2-4]. H pylori has been recognized as grade one carcinogen[5] and the National Institute of Health Consensus Development Conference Statement recommended that all patients who are found to have gastric or duodenal ulceration and concurrent H pylori infection should receive treatment aimed at eradicating the bacterium[6]. Low socio-economic status, low level of education, consumption of alcohol, gender and occupation are the risk factors for H pylori infection[7]. In addition, blood group antigens, which mediate attachment of H pylori to gastric mucosa, are associated with a risk of developing peptic ulcer and gastric cancer[1]. About 95% of patients with duodenal ulcers and perhaps 80% of patients with peptic ulcers are infected with this bacterium and its eradication greatly diminishes the recurrence of these ulcers[8]. However, there have been very few studies on the prevalence of H pylori in Ethiopian patients[9,10]. Therefore,

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the aim of this study was to determine the prevalence of H pylori among dyspeptic patients and to assess the relationship between H pylori infection, blood group, HIV infection and life style of patients attending Gondar University Hospital in Northwest Ethiopia.

MATERIALS AND METHODSThe study was conducted at the Hospital of Gondar University, Northwest Ethiopia, from February 2003 to June 2003. Patients who presented with complaints of dyspepsia were included in the study. Institutional ethical clearance was obtained from the Research and Publication Committee of Gondar University. Socio-demographic and relevant clinical information was collected by a questionnaire. A senior internist examined the patients and filled the questionnaires. The variables included sex, age, ethnicity, smoking, stress, nutrition, alcohol consumption, exercise and socio-economic factors. After informed consent was obtained from each patient and appropriate pre-test counseling was provided about 5mL of venous blood was collected in vacutainer tubes and sera were separated by centrifugation immediately after clotting. Serological status of H pylori infection was tested with commercial H pylori serology kits (HEXAGON H.PYLORI, Germany) following instructions of the manufacturer. The kit was used for the detection of H pylori IgG antibodies in serum. It could detect anti-H pylori antibodies with a highsensitivity (97%) and specificity (95%)

when compared with ELISA (HEXAGON H.PYLORI, Germany). Blood grouping was performed by slide agglutination test using monoclonal anti-A, anti-B, anti-AB and anti-D (Rho) antibodies. Testing for the presence of HIV antibodies was determined by ELISA following the manufacturer’s instructions (Vironostica HIV Uni-Form II plus O, Organon Teknika, Boxtel, The Netherlands). HIV serology test was carried out anonymously with all clinical and laboratory data identified by a code number. Data entry and analysis were performed using SPSS version 10 statistical package. χ2 test was used to compare the categorical data and logistics regression was used to avoid the confounder effect and to calculate the risk ratio. Odds ratio (OR) and 95% confidence interval (CI) were used to measure the strength of association. P < 0.05 was considered statistically significant.

RESULTSA total of 215 dyspeptic patients were included in the study. Table 1 shows the socio-demographic characteristics of the study population in relation with H pylori serostatus. The mean age of the subjects was 35 years (range 14-74 years). A substantial majority (55.8%) of the patients were young adults aged 20-40 years. Females constituted 54% of the patients. Fifty three percent of the patients were farmers followed by students (13.5%). Regarding the marital status, 62.8% were married, 18.1% were singles and 10.2% were divorced. Fifty nine point one percent of the

Table 1 Association between socio-demographic characteristics and seropositivity for antibodies against H. pylori infection in dyspeptic patients (Northwest Ethiopia, 2003)

Characteristics n Serum anti-HP+[ n (%)] Serum anti-HP- [n (%)] OR (95% CI) P value

Sex 0.2 Male 99 88 (88.9) 11 (11.1) 1 Female 116 96 (82.8) 20 (17.2) 0.6 (0.272,1.32)Age group (yr) 0.2 <20 22 18 (81.8) 4 (18.2) 1 20-29 62 51 (82.3) 11 (17.7) 1.03 (0.29, 3.65) 30-39 58 47 (81.0) 11 (19.0) 0.95 (0.27, 3.37) 40-49 32 27 (84.4) 5 (15.6) 1.20 (0.28, 5.08) 50-59 27 27 (100.0) 0 (0.0) 2205.4 (0.0, 4.7E+19) 60-69 10 10 (100.0) 0 (0.0) 2205.4 (0.0, 1.5E+3) 70-79 4 4 (100.0) 0 (0.0) 2205.4 (0.0, 5.6E+45)Occupation 0.69 Farmer 114 97 (85.1) 17 (14.9) 1 House wife 14 12 (85.7) 2 (14.3) 1.05 (0.22,5.12) Govt employee 20 18 (90) 2 (10.0) 1.57 (0.34, 7.43) Student 29 24 (82.8) 5 (17.2) 0.84 (0.28, 2.51) Unemployed 14 14 (100.0) 0 (0.0) 639.67 (0.0, 3.6E+39) Merchant 6 5 (83.3) 1 (16.7) 0.87 (0.09, 7.97) Soldier 2 2 (100.0) 0 (0.0) 639.67 (0.0, 1.5E+39) Others 16 12 (75.0) 4 (25.0) 0.52 (0.15, 1.82)Address Rural 127 109 (85.8) 18 (14.2) 1 Urban 88 75 (85.2) 13 (14.8) 0.95 (0.44, 2.06)Marital status 0.7 Married 135 117 (86.7) 13 1 Widowed 13 11 (84.6) 2 (15.4) 0.85 (0.17, 4.13) Divorced 22 18 (81.8) 4 (18.2) 0.69 (0.21, 2.28) Single 39 32 (82.1) 7 (17.9) 0.70 (0.27, 1.83)Underage 6 6(100.0) 0(0.0) 206.42 (0.0, 1.1E+15)

HP+= Helicobacter pylori positive ; HP- = Helicobacter pylori negative.


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patients came from Gondar town and the remaining 48.1% came from rural areas. Among the patients, 184 (85.6%) were found to be positive for anti-H pylori antibodies. The sex specific prevalence in males was 88.9% which was not significantly different from that of females (82.8%). The prevalence of H pylori was significantly higher in patients aged 50 years and above (100%) when compared to patients aged < 50 years (82.2%) (χ2 = 8.54, γ = 1, P < 0.05). H pylori seroprevalence was not associated with residence, marital status, occupation and ethnic group of the patients (P > 0.05) (Table 1). The prevalence of HIV among the patients was about 20.5% (44/215). The HIV seropositivity was not associated with H pylori seropositivity as only 19.6% (36/184) of the patients who were positive for H pylori were also positive for HIV while 25.8% (8/31) patients who were negative for H pylori were positive for HIV (Table 2). Analysis of demographic factors for HIV infection showed a significant association by residence (χ2 = 5.78, P < 0.01) and occupation (χ2 = 24.78, γ = 7, P < 0.001) of the dyspeptic patients (Table 3)

Analysis of blood group showed that 201 (93.5%) of the total subjects were rhesus positive (Rh+) and 14 (6.5%) were rhesus negative (Rh-). Among the total subjects blood

group O was the most common blood group (43.3%) in the patients followed by blood groups B (28.4%), A (22.3%) and AB (6.0%), respectively. However, no statistically significant association was seen between H pylori infection and blood group of the patients (Table 4). History of diet, alcohol consumption and socio-economic status were obtained from the patients. It was observed that seropositivity for anti-H pylori antibodies was significantly associated with history of alcohol consumption (OR = 2.78, 95% CI: 1.19-6.5, P < 0.01) (Table 5). Although there was a higher prevalence of H pylori infection in those with low socioeconomic status than in those having average income, the association was not statistically significant. Likewise, no statistically significant interaction was observed in the prevalence of H pylori infection and consumption of coffee and spicy foods (Table 5).

DISCUSSIONThe prevalence of H pylori IgG antibody among dyspeptic patients was 85.6%. This finding is higher than an earlier report from 136 patients with non ulcer dyspepsia from Addis Ababa, showing a prevalence of 65% for H pylori[9]. However it is comparable with a recent report from blood donors in Addis Ababa, where a seroprevalence of 89% has been observed[10]. When compared to studies from other countries like England, France, Scandinavia, Italy, Belgium and USA, the seroprevalence found in this study is much higher[11]. This may be explained by the association between H pylori and low socioeconomic status as evidenced by unsafe drinking water[12] and other factors like low educational level which would have an impact on personal hygiene and environmental sanitation[13]. Prevalence of H pylori increases with age. It is interesting to note that in patients aged 50 years and above its prevalence is 100%. Similar results have been reported in

Table 3 Demographic factors for HIV infection in dyspeptic patients attending Gondar University Hospital (Northwest Ethiopia, 2003)

Characteristics HIV serostatus PSeropositive n (%) Seronegative n (%)

Male 19 (19.2) 80 (80.8) P = 0.6Sex OR= 0.87

(95% CI 0.42-1.77) Female 25 (21.6) 91 (78.4) χ2 = 0.18Age group (yr) 10-29 21 (25.3) 62 (74.7) P = 0.3 30-49 17 (18.7) 74 (81.3) χ2 = 2.23 ≥ 50 6 (14.6) 35 (85.4) γ = 2

Residence P< 0.01 Rural 19 (15.0) 108 (85.0) OR = 0.45

(95% CI 0.21-0.92) Urban 25 (28.4) 63 (71.6) χ2 = 5.78Occupation Farmer 18 ( 15.8) 96 (84.2) P< 0.001 Housewife 3 (21.4) 11 (78.6) χ2 = 24.78 Gov’t employee 2 (10.0) 18 (90.0) γ = 7 Students 4 (13.8) 25 (86.2) Unemployed 8 (57.1) 6 (42.9) Merchant 4 (66.7) 2 (33.3) Soldier 0 2 (100) Others 5 (31.3) 11 (68.7)

Table 4 Relationship between ABO blood groups and seropositivity of antibodies against Helicobacter pylori infection

Blood group Serum anti HP+[n (%)] Serum anti HP- [n (%)]

O 76 (84.4) 14 (15.6)B 59 (88.1) 8 (11.9)A 41 (85.4) 7 (14.6)AB 8 (80) 2 (20)Total 184 (85.6) 31 (14.4)

Table 5 Relationship between Helicobacter pylori infection and life style in dyspeptic patients attending Gondar University Hospital (Northwest Ethiopia, 2003)

Serum anti-HP+ n (%)

Serum anti-HP- n (%) P value

Yes 128 (90.1) 14 (9.9) < 0.01Alcohol intake χ2 = 7.05 No 56 (76.7) 17 (23.3) γ = 1 Coffee 24 (82.8) 5 (17.2) = 0.2Diets Spicy food 108 (89.3) 13 (10.7) χ2 = 3.15 Others 52 (80) 13 (20) γ = 2

Table 2 H pylori and HIV co-infection in dyspeptic patients attending Gondar University Hospital (Northwest Ethiopia, 2003)

H pylori Total n (%) StatisticsSeropositive

n (%)Seronegative

n (%)HIV seropositive 36 (19.6) 8 (25.8) 44 (20.5) P = 0.4 HIV seronegative 148 (80.4) 23 (74.2) 171 (79.5) χ2 = 0.63

Moges F et al. Seroprevalence of H pylori , HIV and ABO blood groups 1959

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other studies indicating the high frequency of infection in the elderly[13]. This tendency is believed to be attributable to the environmental factors specific to these age group rather than aging[13]. The lack of significant association between sex, residence of patients and H pylori is in line with previous reports from elsewhere[14,15]. In the present study, more than one fifth of the patients (20.5%) were found to be seropositive for HIV and the seroprevalence of HIV in H pylori-infected patients was also very high (19.6%). This high prevalence of HIV in dyspeptic patients reflects the severe magnitude of HIV infection in the general population in Northwest Ethiopia. Recent studies showed that 51.4% tuberculosis patients[16] and 5% cataract patients[17] were seropositive for HIV in the region substantiating our present observation. Co-infection with HIV can debilitate the defense mechanism of patients and increase morbidity and mortality. Blood group O is associated with duodenal ulcer disease, while gastric ulcer and gastric carcinoma are associated with blood group A[18]. Since the identification of H pylori, no pathogenic mechanism has been identified to support this earlier finding. Lewis B blood group antigen has recently been shown to function as a receptor for H pylori adhesins, mediating bacterial adherence to the gastric epithelial surface, which is essential for bacterial colonization[19]. Furthermore, substitution of the Lewis B antigen with blood group A and B determinants results in failure of H pylori binding[19]. Reduced exposure of the Lewis B epitope in persons of blood groups A and B could result in lower H pylori infection rates and a predominance of H pylori infection in persons of blood group O[20]. This is consistent with the reported association of blood group O with duodenal ulcer disease, but at variance with the association of blood group A with gastric ulcer and carcinoma, which is also related with H pylori infection. The present study did not demonstrate any significant difference in H pylori serological status of dyspeptic patients with varying blood groups, which is consistent with similar studies from other countries[18,20-22]. Histories of alcohol (local alcoholic drinks like “Tella”, “Teji” “Araki” and beer) consumption appears to be a risk factor for H pylori infection, which is in line with study from Finland[23]. However, different results have been reported in other countries[24]. The reason for this contradictory result might be due to the difference in the type of alcoholic beverages consumed and the life time history of alcohol consumption. Socioeconomic status does not appear to be a risk factor for H pylori infection, although a higher proportion of patients with low income are found to be positive for H pylori antibody. This result is in line with reports form Zambia and United Kingdom[25,26]. Spicy foods and coffee are considered to be risk factors for H pylori infection. However, no statistically significant interaction was observed in the present study, but it has been reported in Japan[27]. In conclusion, the prevalence of H pylori infection is very high and associated with history of alcohol intake and older age. Different diet, alcohol and socioeconomic status as risk factors for H pylori infection need further study.

ACKNOWLEDGMENTSThe authors acknowledge the Research and Publication Office of University of Gondar for financing and thank Drs. Moges Tiruneh, Yenew Kebede and Mr. Andargachew Mulu from Department of Medical Microbiology for their valuable comments and Mr. Getu Degu for statistical consultation. Our thanks also go to Mrs. Birhanemeskel Tegene, Abebaw Getahun and Tessema Zewudie for their technical assistance.

REFERENCES1 MontecuccoC, Rappuoli R. Living dangerously: how Helico-

bacter pylori survives in the human stomach. Nat Rev Mol Cell Biol 2001; 2: 457-466

2 HowdenCW. Clinical expressions of Helicobacter pylori in-fection. Am J Med 1996; 100: 27S-32S; discussion 32S-34S

3 MarshallBJ. Helicobacter pylori. Am J Gastroenterol 1994; 89: S116-S128

4 Mégraud F. Resistance of Helicobacter pylori to antibiotics. Aliment Pharmacol Ther 1997; 11Suppl1: 43-53

5 International Agency for Research on Cancer. Schistosomiasis, Liver flukes and Helicobacter pylori. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, Lyon: IARC 1994: 6

6 NIH Consensus Conference. Helicobacter pylori in peptic ulcer disease. NIH Consensus Development Panel on Helico-bacter pylori in Peptic Ulcer Disease. JAMA 1994; 272: 65-69

7 MalatyHM, Kim JG, Kim SD, Graham DY. Prevalence of He-licobacter pylori infection in Korean children: inverse relation to socioeconomic status despite a uniformly high prevalence in adults. Am J Epidemiol 1996; 143: 257-262

8 CalamJ. Clinical Science of Helicobacter pylori infection: Ul-cers and NSAIDS. Farthing M.JG and Patchett S.E.eds. In Heli-cobacter infection. Panther publishers, India.1998: 55-62

9 TsegaE, Gebre W, Manley P, Asfaw T. Helicobacter pylori, gastritis and non-ulcer dyspepsia in Ethiopian patients. Ethiop Med J 1996; 34: 65-71

10 DestaK, Asrat D, Derbe F. Seroprevalence of H. pylori infec-tion among health blood donors in Addis Ababa, Ethiopia. Ethiop J Health Sci 2002; 12: 109-116

11 Pounder RE, Ng D. The prevalence of Helicobacter pylori infection in different countries. Aliment Pharmacol Ther 1995; 9Suppl2: 33-39

12 Klein PD, Graham DY, Gaillour A, Opekun AR, Smith EO. Water source as risk factor for Helicobacter pylori infection in Peruvian children. Gastrointestinal Physiology Working Group. Lancet 1991; 337: 1503-1506

13 Epidemiology of, and risk factors for, Helicobacter pylori in-fection among 3194 asymptomatic subjects in 17 populations. The EUROGAST Study Group. Gut 1993; 34: 1672-1676

14 YamashitaY, Fujisawa T, Kimura A, Kato H. Epidemiology of Helicobacter pylori infection in children: a serologic study of the Kyushu region in Japan. Pediatr Int 2001; 43: 4-7

15 Karari EM, Lule GN, McLigeyo SO, Amayo EO. Endoscopic findings and the prevalence of Helicobacter pylori in chronic renal failure patients with dyspepsia. East Afr Med J 2000; 77:406-409

16 KassuA, Mohammad A, Fujimaki Y, Moges F, Elias D, Me-konnen F, Mengistu G, Yamato M, Wondmikun Y, Ota F. Se-rum IgE levels of tuberculosis patients in a tropical setup with high prevalence of HIV and intestinal parasitoses. Clin Exp Immunol 2004; 138: 122-127

17 KassuA, Mekonnen A, Bekele A, Abseno N, Melese E, Moges F, Wondmikun Y, Ota F. HIV and syphilis infection among elderly people in northwest Ethiopia. Jpn J Infect Dis 2004; 57: 264-267

18 SmithAW, Aathithan S, Power EG, Abdulla Y. Blood group


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antigens and Helicobacter pylori infections. Lancet 1994; 343: 543

19 BorénT, Falk P, Roth KA, Larson G, Normark S. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. Science 1993; 262: 1892-1895

20 NivY, Fraser G, Delpre G, Neeman A, Leiser A, Samra Z, Scapa E, Gilon E, Bar-Shany S. Helicobacter pylori infection and blood groups. Am J Gastroenterol 1996; 91: 101-104

21 LoffeldRJ, Stobberingh E. Helicobacter pylori and ABO blood groups. J Clin Pathol 1991; 44: 516-517

22 HenrikssonK, Uribe A, Sandstedt B, Nord CE. Helicobacter pylori infection, ABO blood group, and effect of misoprostol on gastroduodenal mucosa in NSAID-treated patients with rheumatoid arthritis. Dig Dis Sci 1993; 38: 1688-1696

23 PaunioM, Höök-Nikanne J, Kosunen TU, Vainio U, Salaspuro M, Mäkinen J, Heinonen OP. Association of alcohol consump-tion and Helicobacter pylori infection in young adulthood and early middle age among patients with gastric complaints. A

case-control study on Finnish conscripts, officers and other military personnel. Eur J Epidemiol 1994; 10: 205-209

24 Brenner H,Rothenbacher D, Bode G, Adler G. Relation of smoking and alcohol and coffee consumption to active Heli-cobacter pylori infection: cross sectional study. BMJ 1997; 315: 1489-1492

25 McLaughlinNJ, McLaughlin DI, Lefcort H. The influence of socio-economic factors on Helicobacter pylori infection rates of students in rural Zambia. Cent Afr J Med 2003; 49: 38-41

26 ParsonsHK, Carter MJ, Sanders DS, Winstanley T, Lobo AJ. Helicobacter pylori antimicrobial resistance in the United Kingdom: the effect of age, sex and socio-economic status. Ali-ment Pharmacol Ther 2001; 15: 1473-1478

27 Machida-Montani A, Sasazuki S, Inoue M, Natsukawa S, Shaura K, Koizumi Y, Kasuga Y, Hanaoka T, Tsugane S. Asso-ciation of Helicobacter pylori infection and environmental fac-tors in non-cardia gastric cancer in Japan. Gastric Cancer 2004; 7: 46-53

S-EditorWang J L-Editor Wang XL E-EditorBi L

Moges F et al. Seroprevalence of H pylori , HIV and ABO blood groups 1961

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CONCLUSION: The gastric mucosal protective effect by EA at the acupoints of SMFY and GMFY was related to the expression variance of ITF, indicating certain merid-ian specificity exists. It could be one proof for the TCM theory “Relative particularity between SMFY and stomach”.


Key words: EA; Relative particularity between Stomach Meridian of Foot-Yangming; Gastric mucosal damage; Stress; Intestinal trefoil factor; Gene expression

Li XP, Yan J, Yi SX, Chang XR, Lin YP, Yang ZB, Huang A, Hu R. Effect of electroacupuncture on gastric mucosal intestinal trefoil factor gene expression of stress-induced gastric mucosal injury in rats. World J Gastroenterol 2006; 12(12): 1962-1965


INTRODUCTIONAcupunc ture i s one o f the t r ad i t iona l Ch inese medicine(TCM) therapeutic techniques that can be traced back at least 2500 years, gaining popularity in the West as an alternative and complementary therapeutic interven-tion[1-4]; while “Relative particularity between Foot Yang-ming meridian and stomach”, is one of important TCM theories. Such theory was proved by our previous research results[5-7]: acupuncture at acupoints of Sibai (ST 2), Liang-men (ST 21), and Zusanli (ST36), could regulate gastric motion and gastric secretion. Recently, a group of new peptides have been discovered, called trefoil factor family (TFF) or trefoil peptides because of their uniquely distinc-tive cysteine-rich “three-leaf ” secondary structure[8], which probably protects these peptides from the degradation by luminal acid and proteases within the gastrointestinal tract[9]. Intestinal trefoil factor (ITF) belongs to the grow-ing family of trefoil peptides[10]. Most of the researches to date have revealed important roles for ITF in protection and repair against injury to the gastrointestinal mucosa[11-14]. To our knowledge, there existed still no evidence of ITF gene expression in stress-induced gastric mucosal lesion in rats by the treatment of acupuncture. In our present study, ITFmRNA was detected to probe gastric


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Effect of electroacupunture on gastric mucosal intestinal trefoil factor gene expression of stress-induced gastric mucosal injury in rats

Xi-Ping Li, Jie Yan, Shou-Xiang Yi, Xiao-Rong Chang, Ya-Ping Lin, Zong-Bao Yang, Ai Huang, Rong Hu

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Xi-Ping Li, Jie Yan, Shou-Xiang Yi, Xiao-Rong Chang, Ya-Ping Lin, Zong-Bao Yang, Ai Huang, Rong Hu, Institute of Acupuncture and Moxibustion, Hunan College of Traditional Chinese Medicine, Chang'sha 410007, Hunan Province, ChinaSupported by the National Natural Science Foundation of China, No. 90209023 Correspondence to: Professor Jie Yan, Institute of Acupuncture and Moxibustion, Hunan University of Traditional Chinese Medi-cine, Chang'sha 410007, Hunan Province, China. [email protected] Telephone: +86-731-5381159 Received: 2005-08-10 Accepted: 2005-12-06

Abstract AIM: To investigate electroacupunture(EA) at the acu-points of Stomach Meridian of Foot-Yangming(SMFY), Gallbladder Meridian of Foot-Yangming(SMFY) on gastric mucosal intestinal trefoil factor (ITF) gene expression detection in stress-induced rats with gastric mucosal le-sion, and to explore the regulatory mechanism and sig-nificance of EA-related gastric mucosal protective effect.

METHODS: Forty rats were randomly divided into 4 groups: Blank group, Model group, Model group＋EA at acupoints of SMFY group(“SMFY group”), and Model group＋EA at acupoints of GMFY group(GMFY group). All rats (except blank group) were made model by water immersion and restraint stress (WRS). Then the gastric mucosa tissue in each rat was taken off after assessment of gastric mucosal lesion index(GUI), and the expres-sion of ITF mRNA of the tissues was detected by reverse transcription-polymerase chain reaction(RT-PCR) method.

RESULTS: Compared with Model group(54.3 ± 1.34), the GUI value in SMFY group (31 ± 2.21) decreased significantly(P < 0.01), so did that in GMFY group (39.8 ± 1.62, P < 0.05), meanwhi le GUI value in SMFY group was significantly lower than in GMFY g roup(P < 0 .01 ) . Compared w i th Mode l g roup (0.65 ± 0.01), EA had a tendency to improve the expres-sion of gastric mucosal ITFmRNA gene: such tendency existed in GMFY group (0.66 ± 0.01) but with no signfi-cant difference(P > 0.05), in SMFY group(0.76 ± 0.01) with an extremely obvious difference (P < 0.01), further-more the expression in SMFY group was significantly higher than in GMFY group (P < 0.01).

Li XP et al. Effect of electroacupunture on gastric mucosa protection 1963

mucosal protective mechanism of the factor, and to prove the classical TCM theory “Relative particularity between Stomach Meridian of Foot-Yangming(SMFY) and stomach”.

MATERIALS AND METHODS

ReagentsTrizol reagent was obtained from Invitrogen Co. (USA). One tube RT-PCR kit was from Promega Co. (USA). Prim-ers for rat ITF and GAPDH were designed by ourselves in accordance with gene sequence in GenBank, synthesized and purified by Gibco BRL Biological Engineering Co. All other reagents were analytically pure.

Animals Forty spraque-dawley rats weighing 180-250 g, male and female mixture, were used. They were housed three to four per cage at temperature 22 ℃ ± 2 ℃ with a 12/12 h light/dark (0:00 a.m. to 20:00 p.m.) cycle under controlled environment. Rats were fed standard laboratory chow, and water was given ad libitum. The rats were kept for 7 d in laboratory for habituation. This study was carried out ac-cording to the protocol approved by the Ethical Commit-tee of Hunan University of Traditional Chinese Medicine, Changsha, China.

Animal groups and model All rats were randomly divided into 4 groups(ten rats in each). The design of the experimental animal groups is shown in Table 1.

Acupuncture methodAcupoints location was defined by reference of rat-acu-point-atlas[15] and analogy to human body. According to the induction stated above, three pairs of acupoints consisting of Sibai (ST 2), Liangmen (ST 21), Zusanli (ST36), in the Foot Yangming Meridian, were designed, which represent acupoints of different level(head, trunk, limb); thus 3 pairs of acupoints of the Foot Shaoyang Meridian in the same horizontal level were selected: Yangbai(GB 14), Riyue(GB 24), Yanglingquan(GB 34). Acupoint location: Sibai acu-point, at the depression of the infraorbital foramen; Li-angmen acupoint, at intersection of the midline between anterior midline and midclavicular line and the middle horizontal line of omphalos and xiphoid. Pairs of stainless-steel needles of 0.25 mm in diameter were inser ted into the acupoints stated above of experimental rabbits(Groups C and D). The needles were connected to the output of an electronic pulse generator, a medical EA stimulator (Model G6805-1, made by Shanghai Medical Electro-apparatus Factory, China), which achieves intermittent-and-irregular wave(intermittent wave:4 Hz, irregular wave:20 Hz, intensity of 6-15 V, the depth of acupuncture of 0.5 cm, constant time of 20 min), while there was a light vibration in the rats’ lower limbs.

Induction of gastric mucosal lesion indexSeven days after corresponding treatment, each rat was immobilized in a restraint cage and immersed for

10 h to the height of the xiphoid in a water bath kept at 20 ℃ ± 1 ℃, and then sacrificed under anesthesia with 10% urethane intraperitoneally (1 ml/100 g). The abdomen was opened, and the stomach was removed, opened along the great curvature and the gastric mucosa was carefully examined under a dissection microscope to determine gastric mucosal lesion index(GUI) by the score systems suggested by Guth[16]. Briefly, the stomach mucosa was examined with magnifier for the presence of erosions and scored as follows: (1) for small, round hemorrhagic erosion; (2) hemorrhagic erosions less than 1 mm in length; (3) hemorrhagic erosions between 1-2 mm in length; (4) hemorrhagic erosions between 2-3 mm in length; (5) hemorrhagic erosions longer than 4 mm in length. The score value was multiplied by 2 when the width of erosion was larger than 1 mm. The extent of the lesion (lesion index) is expressed as the sum of the length of these breaks per stomach.

RNA extractionAfter the treatment stated above, each rat’s gastric mucosal tissue was collected and put in freezing-and-storing tubes and kept in the nitrogen tank quickly. Five samples of each group were selected randomly for experiment. Expression of EGFR mRNA was evaluated with RT-PCR. Total RNA was isolated from gastric mucosal samples using a guanidinium isothiocyanate/phenol chloroform single step extraction kit from Stratagene(Gibco BRL, USA), precipitated in ethanol and resuspended in sterile RNAase-free water for storage at -80 ℃ until use. Total RNA was quantified spectrometrically at 260 nm, and the quality of isolated RNA was analysed on agarose gels under standard conditions.

Reverse transcription reactionTotal RNA (10 µL, about 0.5 µg/sample) was reverse transcribed (RT) using ol igo(dT)18 primers 1 µL (30 pmol/L), 5×RT buffer 4 µL(Promega Co.), dNTPs(10 mmol/L) 1 µL, RNasin(20 MU/µL, Promega Co. Madison, America) 0.5 µL, M-MULV reverse transcriptase (200 MU/µL, Promega Co., Madison, America) 1 µL, and DEPC water 2.5 µL in a 20 µL reverse transcription reaction system, and such system was performed at 42 ℃ for 30~60 min, then cooled and centrifuged for several seconds so that target mRNA of total RNA sample was transcribed into target cDNA.

Polymerase chain reaction (PCR) An aliquot of the same RT product from each sample (1/20

Table 1 Design of experimental animal groups

Group/code Treatment Group A: Blank group Untreated rats as normal control groupGroup B: Model group having no EA or other treatment for 7 d, then

WRS rat model was established.Group C: SMFY group After EA at points of SMFY for 7 d, WRS rat

model was establishedGroup D: GMFY group After EA at points of GMFY for 7 d, WRS rat

model was established

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of the total volume) was used in the PCR amplification reactions for ITF and GAPDH. The PCR reaction system compound contained 4 µL cDNA, 10×PCR buffer(Promega Co., Madison, America )5 µL, dNTPS (10 mmol/L) 1 µL, oligonucleotide primers sense/antisense (10 mmol/L) 1 µL (the related primer sequence as stated below), Taqase(5 MU/µL Promega Co.) 1 µL, ddH2O 32 µL in a total volume of 50 µL. Reaction mixtures were incubated for predenaturation at 94 ℃ for 2 min, followed by 35 cycles for ITF (denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 1 min, and extension at 72 ℃ for 1 min) and 35 cycles for GAPDH (denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s, and extension at 72 ℃ for 30 s), and a final extension at 72 ℃ for 5 min.

PCR primer design By use of the relatively quantitative method to measure ITF-mRNA, rat GAPDH was selected as internal control substance. The primer sequences and sizes of amplification products are as follows: ITF sense, 5’-ACAACCCTGCTGCTGGTCCT -3’; antisense, 5’-TCTGTCTCTTGCAGAGGTTTG-3’ (212 base pairs); GAPDH sense, 5’-TGCTGAGTATGTCGTGGAGTC-3’; antisense: 5’- AAGGCCATGCCAGTGAGCTTC-3’ (426 base pairs).

RT-PCR product electrophoresis Five microl i ter PCR products were ana lyzed on 10 g/L agarose gel containing ethidiumbromide with TBE buffer at 80 V for 40 min and photographed under UV illumination. The band intensities were quantified by densitometry. ITF and GAPDH PCR products were respectively 212 base pairs(Figure 1), and 426 base pairs(Figure 2). ITF and GAPDH were determined by computer-assisted densitometric scanning. Signals were quantified by density analysis of the digital images using Eagle Eye Ⅱ image software (Stratagene Co.) and ITF/GAPDH quotient indicated the relative expression of ITF.

Experiments were performed in triplicate. Statistical analysisThe data were expressed as mean±SD of 10 rats per group. Comparison between groups was assessed using one-way analysis of variance (ANOVA) on ranks. Differences were considered statistically significant if the P value was less than 0.05. Software SPSS 10.0 was used in all statistical tests.

RESULTSGastric mucosal injury condition and GUIThere were some dots and strips injury detected by the magnifier(10×). The GUI in Model group was highest, while in Blank group lowest, with significant difference between them (P < 0.01). It demonstrated the ulcer model was successful. Compared with Model group, GUI in SMFY group and GMFY group reduced significantly (P < 0.05 or P < 0.01), and the GUI in SMFY group was lower than that in GMFY group(P < 0.01, Table 2).

Effect of ITF-mRNA expression on gastric mucosal tissueThe expression of ITF mRNA using RT-PCR was detected in the intact gastric mucosa of Blank group as a weak signal but it was well-defined among other groups: Model, SMFY, and GMFY groups(P < 0.01). Compared with Model group, EA at acupoints of SMFY group could upregulate significantly the expression of ITF-mRNA expression in gastric mucosal tissue(P < 0.01), while there did not exist difference of expression between GMFY group and model group(P > 0.05), but obvious difference between SMFY group and GMFY group was found (P < 0.01, Table 2).

DISCUSSIONAccording to classical TCM theory, SMFY, running from head, via chest and abdomen, along anterior lateral lower limb to foot, is a crucial meridian for its good bidirectional modulation of digestive diseases. Previously, we studied the effect of single acupoint of SMFY on gastric function as well as the whole SMFY’s functional mechanism. Acupuncture at acupoints of head and face, trunk, or lower limb (such as acupoint “Sibai”, “Tianshu”, “Liangmen”, “Zusanl i” , “Shangjuxu”) , could produce cer ta in ameliorative effect through the following mechanisms: augmentation of gastric antrum anrea, reinforcement of

1 2 3 1 2 3 1 2 3 1 2 3 marker

B(Model group) A(Blank group) D(GMFY group) C(SMFY group)

212 bp 200 bp300 bp

Figure 1 Electrophoresis of ITFmRNA RT-PCR product in gastric mucosal tissue (1, 2 and 3 are randomly selected from each group).

1 2 3 1 2 3 1 2 3 1 2 3 marker

B(Model group) A(Blank group) D(GMFY group) C(SMFY group)

426 bp500 bp

Figure 2 Electrophoresis of GAPDH mRNA RT-PCR product in gastric mucosal tissue (1, 2, and 3 are randomly selected from each group).

Table 2 Effect of EA on GUI and ITF-mRNA expression in gastric mucosal tissue (mean ± SD)

G GUI score (n = 10) ITF mRNA/GAPDH mRNA (n = 5)A(Blank group) 0.3 ± 0.48 0.45 ± 0.01B(Model group) 54.3 ± 1.34b 0.65 ± 0.01b

C(SMFY group) 31 ± 2.21bd 0.76 ± 0.01bd

D(GMFY group) 39.8 ± 1.62bcf 0.66 ± 0.01bf

bP < 0.01 vs blank group, cP < 0.05 vs Group B, dP < 0.01 vs Group B, fP < 0.01 vs Group C.


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pressure power of gastric pyloric sphincter, promotion or inhibition of related gastrointestinal peptide secretion[17-19]. All of these have provided experimental evidence for the theory “Relative particularity between SMFY and stomach”. T he cy topro tec t ive func t ions in p ro tec t ing gastrointestinal tract against ongoing damage may be accomplished in several ways, and evidences for participation in both the early phase of epithelial repair known as restitution (marked by increased cell migration but no proliferation), and in the subsequent, protracted phase of glandular renewal (marked by proliferation, differentiation and migration) have been published[20-22]. ITF is one of the most recently described members of the trefoil peptide family, a peptide of 59 amino acids, expressed normally by mucus secretory cells of the small and large intestine[9,23]. It is secreted onto the luminal surface of the gastrointestinal tract, and may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those that may act at the basolateral pole of the epithelium[11,24]. This study assessed for the first time ITF expression by RT-PCR analyses in rat gastric mucosa after exposure to water immersion and restrained stress. It showed that expression of ITF in gastric mucosa was enhanced shortly after the stress, leading us to hypothesize that this process might be mediated by ITF. Meanwhile, it was proved that EA had a tendency to improve the expression of gastric mucosal ITFmRNA gene, and such expression of SMFY group was strongly higher than model group and GMFY group, indicating that the expression discrepancy of ITFmRNA may be the underlying mechanism of different effect of EA at acupoints of SMFY and that of GMFY, thus it could be one proof for the TCM theory “Relative particularity between SMFY and stomach”.

REFERENCES1 ShermanKJ, Cherkin DC, Eisenberg DM, Erro J, Hrbek A,

Deyo RA. The practice of acupuncture: who are the providers and what do they do? Ann Fam Med 2005; 3: 151-158

2 EisenbergDM, Cohen MH, Hrbek A, Grayzel J, Van Rompay MI, Cooper RA. Credentialing complementary and alternative medical providers. Ann Intern Med 2002; 137: 965-973

3 Cherkin DC, Deyo RA, Sherman KJ, Hart LG, Street JH, Hrbek A, Davis RB, Cramer E, Milliman B, Booker J, Mootz R, Barassi J, Kahn JR, Kaptchuk TJ, Eisenberg DM. Characteris-tics of visits to licensed acupuncturists, chiropractors, massage therapists, and naturopathic physicians. J Am Board Fam Pract 2002; 15: 463-472

4 LuW. Acupuncture for side effects of chemoradiation therapy in cancer patients. Semin Oncol Nurs 2005; 21: 190-195

5 ChangX, Yan J, Yi S, Lin Y, Yang R. The affects of acupunc-ture at sibai and neiting acupoints on gastric peristalsis. J Tra-dit Chin Med 2001; 21: 286-288

6 YueZH, Yan J, Chang XR, Lin YP, Yi SX, Cao XP, Shen J. [Ef-fects of cake-separated moxibustion on ultrastructures of en-dothelial cells of aorta in the rabbit of hyperlipemia]. Zhongguo Zhen Jiu 2005; 25: 64-67

7 LiuJH, Yan J, Yi SX, Chang XR, Lin YP, Hu JM. Effects of elec-troacupuncture on gastric myoelectric activity and substance P in the dorsal vagal complex of rats. Neurosci Lett 2004; 356: 99-102

8 Katoh M. Trefoil factors and human gastric cancer (review). Int J Mol Med 2003; 12:3-9

9 TaupinD, Podolsky DK. Trefoil factors: initiators of mucosal healing. Nat Rev Mol Cell Biol 2003; 4: 721-732

10 PodolskyDK. Mechanisms of regulatory peptide action in the gastrointestinal tract: trefoil peptides. J Gastroenterol 2000; 35Suppl12: 69-74

11 KindonH, Pothoulakis C, Thim L, Lynch-Devaney K, Podol-sky DK. Trefoil peptide protection of intestinal epithelial bar-rier function: cooperative interaction with mucin glycoprotein. Gastroenterology 1995; 109: 516-523

12 Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science 1996; 274: 262-265

13 KanaiM, Mullen C, Podolsky DK. Intestinal trefoil factor in-duces inactivation of extracellular signal-regulated protein ki-nase in intestinal epithelial cells. Proc Natl Acad Sci U S A 1998; 95: 178-182

14 Xu LF, Li J, Sun M, Sun HW. Expression of intestinal trefoil factor, proliferating cell nuclear antigen and histological changes in intestine of rats after intrauterine asphyxia. World J Gastroenterol 2005; 11: 2291-2295

15 LinWZ, Wang P. Shiyan Zhenjiu Xue. 1th edition. Shanghai: Shanghai Science and technology Publishing Press, 1999: 280-290

16 GuthPH, Aures D, Paulsen G. Topical aspirin plus HCl gas-tric lesions in the rat. Cytoprotective effect of prostaglandin, cimetidine, and probanthine. Gastroenterology 1979; 76: 88-93

17 ChangXR, Yan J, Li YP, Yi SX, Liu H. Influence of Puncture-ing points of Foot-Yangming Channel on the content of moti-lin and gastrin of plasma in health person. Zhongguo Zhongxiyi Jiehe Xiaohua Zazhi 2001; 9:69-70

18 YanJ, Yang RD , Yi SX, Chang XR, Lin YP. Study on regularity of multimeridians controlling a same organ from the protective action of acupuncture at different acupoints on gastric mucosa in the rabbit. Zhongguo Zhenjiu 2004; 24: 579-583

19 YiSX, Lin YP, Yan J, Chang XR, Yang Y. Effect of electroacu-puncture on gastric motility, substance P (SP) and motilin (MTL) in rats. Shijie Huaren Xiahua Zazhi 2001; 9:284-287

20 NieSN, Qian XM, Wu XH, Yang SY, Tang WJ, Xu BH, Huang F, Lin X, Sun DY, Sun HC, Li ZS. Role of TFF in healing of stress-induced gastric lesions. World J Gastroenterol 2003; 9:1772-1776

21 PodolskyDK. Mucosal immunity and inflammation. V. Innate mechanisms of mucosal defense and repair: the best offense is a good defense. Am J Physiol 1999; 277: G495-G499

22 WrightNA. Aspects of the biology of regeneration and repair in the human gastrointestinal tract. Philos Trans R Soc Lond B Biol Sci 1998; 353: 925-933

23 SandsBE, Podolsky DK. The trefoil peptide family. Annu Rev Physiol 1996; 58: 253-273

24 Fernandez-EstivarizC, Gu LH, Gu L, Jonas CR, Wallace TM, Pascal RR, Devaney KL, Farrell CL, Jones DP, Podolsky DK, Ziegler TR. Trefoil peptide expression and goblet cell number in rat intestine: effects of KGF and fasting-refeeding. Am J Physiol Regul Integr Comp Physiol 2003; 284: R564-R573

S-EditorPan BR L-Editor Zhu LH E-EditorBi L

Li XP et al. Effect of electroacupunture on gastric mucosa protection 1965

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Alba Rocco, Pasquale Borriello, Debora Compare, Patrizia De Colibus, Loredana Pica, Gerardo Nardone, Department of Clinical and Experimental Medicine, Gastroenterology, University “Federico II”, Naples, Italy Alessandro Iacono, Department of Biomorphological and Func-tional Science, Pathology; University “Federico II”, Naples, ItalySupported by grants from the Italian Ministry of University and Research (MURST) to the Department of Clinical and Experimen-tal Medicine, University Federico II, Naples, ItalyCorrespondence to: Professor Gerardo Nardone, Department of Clinical and Experimental Medicine, Gastroenterology Unit, Uni-versity “Federico II”, via S. Pansini n. 5, 80131 Naples, Italy. [email protected] Telephone: +39-81-7464293 Fax: +39-81-7464293Received: 2005-08-23 Accepted: 2005-10-12

AbstractBrunner’s gland adenoma (BGA）is a very rare benign tumour of the duodenum, which is usually asymptomatic and discovered incidentally at endoscopy. Occasionally, this lesion may be large, causing upper gastrointestinal haemorrhage or intestinal obstruction. The case had a large Brunner’s gland adenoma, presenting melena that was managed by endoscopic excision.


Key words: Brunner’s gland adenoma; Endoscopic resec-tion; Giant brunneroma

Rocco A, Borriello P, Compare D, De Colibus P, Pica L, Iaco-no A, Nardone G. Large Brunner’s gland adenoma: Case report and literature review. World J Gastroenterol 2006; 12(12): 1966-1968


INTRODUCTIONBrunner’s glands, described by the anatomist Brunner in 1688, are submucosal mucin-secreting glands. They are predominantly localized in the duodenal bulb and proximal duodenum and progressively decrease in size and number in the distal portions. Brunner’s glands exert a physiologi-cal “anti-acid” function by secreting alkaline fluid com-posed of mucin which protects the duodenal epithelium


CASE REPORT

Large Brunner’s gland adenoma: Case report and literature review

Alba Rocco, Pasquale Borriello, Debora Compare, Patrizia De Colibus, Loredana Pica, Alessandro Iacono,

Gerardo Nardone

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from the acid chime of the stomach[1]. Furthermore, they produce and secrete “enterogastrone”, an enteric hormone inhibiting gastric acid secretion.

Brunner’s gland adenoma (BGA), firstly described by Curveilheir in 1835, is a benign tumour arising from the Brunner’s glands that exceptionally may evolve towards a malignant transformation[2-4]. BGA is an extremely rare tu-mour with an estimate incidence of 0.008% reported in a single series of 215 000 autopsies[1]. At present, < 200 cases have been reported in the world medical English literature.

Here, one case of large BGA managed by endoscopic resection is reported together with a review of the perti-nent literature.

CASE REPORTA 58-year old female was referred to our Gastroenterol-ogy Unit due to episodes of epigastric discomfort over the past few months. Past medical history included appendi-cectomy performed during childhood, cholecystectomy for gallstones performed 4 years previously and a self-limiting single episode of melena occurred two months previously. The patient denied weight loss or use of non steroidal anti-inflammatory drugs (NSAIDs).

Clinical examination and routine blood tests were normal. Esophago-gastro-duodenoscopy (EGDS) dem-onstrated normal esophagus and stomach. A large pedun-culated polyp, 3 cm × 4 cm in size, completely occupying the duodenal bulb was found. Healing microerosions were present on the mucosa. Histological examination of the multiple biopsy specimens obtained during EGDS, sug-gested “Brunner’s gland hyperplasia” in a context of mild chronic gastritis without evidence of Helicobacter pylori (H pylori) infection.

Abdominal CT-scan confirmed the presence of a poly-poid mass originating in the mucosa of the duodenal bulb and extending to the second portion of the duodenum for about 4 cm, without evidence of duodenal wall infiltration (Figure 1). Endoscopic resection was carried out to remove the duodenal mass. Histological examination revealed closely packed Brunner’s glands and ducts embedded in a fibrous stroma with a moderate degree of lymphocyte and monocyte infiltration (Figures 2A and 2B). A diagnosis of Brunner’s adenoma was made. During the 6-month follow-up, the patient remained symptom-free and no further epi-sodes of melena occurred. EGDS did not find any residual lesion.

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Rocco A et al. Brunner’s gland adenoma 1967

DISCUSSION BGA has a tendency to be predominant in the fifth or sixth decade of life with equal gender distribution [5]. Clinical presentation is variable. However, the majority of cases are asymptomatic or present with non specific, vague symptoms such as abdominal pain or discomfort, nausea or bloating. BGA is usually an incidental finding during imaging studies or EGDS[5].

In symptomatic patients, the most common clini-cal presentations are gastrointestinal bleeding (37%) and obstructive symptoms (37%)[5]. Gastrointestinal bleeding manifests in the majority of cases as chronic loss of blood with iron deficiency and anaemia[6]. Less frequently, when erosion or ulceration of the tumour occurs, patients can present with melena or haematemesis. These findings are usually described in BGA occurring beyond the first por-tion of the duodenum, probably because these lesions are subjected to more stress and vascular damage from gas-trointestinal motility[6]. In our case, the patient referred to our unit for a self-limiting episode of melena and showed a large polyp at EGDS with a short peduncle localised in the duodenal bulb with an ulcer scar on the surface.

Aetiology and pathogenesis of BGA still remain to be elucidated. Due to the “anti-acid” function of Brunner’s glands, it has been postulated that an increased acid secre-tion could stimulate these structures to undergo hyperpla-sia[7]. Franzin et al [8] have reported an association between BGA and hyperchlorhydria in patients with chronic gastric erosions and duodenal ulcers, but Spellberg et al[9] have not found regression of the lesion with acid secretion inhibi-tors[9]. A second hypothesis suggests that this lesion is of inflammatory origin due to the presence of a dense inflam-matory cell infiltration[10]. Since lymphocytes are usually present in the normal submucosa of the intestinal tract, the presence of inflammatory foci in the BGA is not suf-ficient to sustain the “inflammatory hypothesis”. Finally, it has been suggested that H pylori infection may play a role in the pathogenesis of BGA. In a recent study involving 19 100 subjects, H pylori infection was found in five out of seven (71%) BGA cases[11]. In our patient, H pylori infec-tion was not found. The extreme rarity of BGA and the high prevalence of H pylori infection in general popula-tion do not allow us to draw a clear pathogenetic link. At

present, the most accredited pathogenetic hypothesis re-mains that BGA is a duodenal dysembryoplastic lesion or hamartoma[12].

The duodenal bulb is the most frequent localization of BGA (57%) [13]. In the majority of cases, these lesions develop into a polypoid mass, usually pedunculated (88%), being 1 to 2 cm in size[14] while few cases reaching several centimetres as the ”giant BGA” have been reported[6,15-18]. On the other hand, lesions < 1 cm are referred to as Brun-ner’s gland hyperplasia[19].

Diagnosis of BGA is not always easy at present. Ra-diological findings (X-ray and computed tomography) are often non specific[12]. Indeed, the duodenal filling de-fect can mimic several other lesions, such as leiomyoma, lipoma, lymphoma, aberrant pancreatic tissue or carcinoid tumours[20]. Computed tomography is useful only to con-firm the absence of extra-luminal extension of BGA[20]. Diagnosis can be obtained by histological examination of the excised mass. Traditional endoscopy of pinching biopsies is usually negative since the biopsy forceps are unable to reach the tumoral tissue localized completely in the submucosa layer [21]. In our case, diagnosis was made on endoscopic biopsy samples of duodenal mass, because Brunner’s gland hyperproliferation was extended beyond muscularis mucosae reaching lower portion of duodenal villi (Figure 2).

Endoscopic or surgical removal of BGA has been suggested to prevent the development of complications (haemorrhage, severe anaemia, obstruction or intussuscep-tion). Endoscopic polypectomy represents the ideal ap-proach, which is more cost-effective and less invasive of the abdominal surgery [22, 23]. However, the success depends on site and size of the BGA and presence of a peduncle. Several cases of successful endoscopic resections have been reported [12, 24-26]. In our case, even though the lesion was large (3 cm × 4 cm) with a short peduncle occupying entirely the duodenal bulb (Figure 1), endoscopic polypec-

ADDOME

R

WL=50

0.0D(224.256)

-15.00mm/r HP3.0

→

Figure 1 Evidence of polypoid mass in duodenal bulb extending to second duodenal portion (arrow).

A

B

Figure 2 Gland hyperproliferation extending beyond m u s c o l a r i s m u c o s a e reaching lower p o r t i o n o f d u o d e n a l v i l l i with irregular and squat profi le of these structures (A) (H&E, original m a g n i f i c a t i o n X4) and detail of interface between Brunner’s gland proliferation and duodenal mucosa w i t h a b s e n c e o f m u s c u l a r i s m u c o s a e a n d irregular profile o f v i l l i ( B ) (E&E , o r i g i na l m a g n i f i c a t i o n X20).

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tomy was effective for the treatment of BGA, without complication and recurrence of the lesion during the 6-month follow-up.

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stine. A review of experience with 115 cases including a report of a rare case of malignant hemangio-endothelioma. Am J Surg 1962; 103: 358-365

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11 KovacevićI, Ljubicić N, Cupić H, Doko M, Zovak M, Troskot B, Kujundzić M, Banić M. Helicobacter pylori infection in patients with Brunner’s gland adenoma. Acta Med Croatica 2001; 55: 157-160

12 GaoYP, Zhu JS, Zheng WJ. Brunner’s gland adenoma of duo-denum: a case report and literature review. World J Gastroen-terol 2004; 10: 2616-2617

13 WaldenDT, Marcon NE. Endoscopic injection and polypec-tomy for bleeding Brunner’s gland hamartoma: case report and expanded literature review. Gastrointest Endosc 1998; 47: 403-407

14 NakanishiT, Takeuchi T, Hara K, Sugimoto A. A great Brun-ner’s gland adenoma of the duodenal bulb. Dig Dis Sci 1984; 29: 81-85

15 RhinerR, Meyenberg A, Aeberhard P, Moschopulos M. [Large Brunner’s adenoma of the duodenal bulb--a case report]. Swiss Surg 1997; 3: 13-16

16 Smirnov OA, Serezhin BS, Nikonova OA. [Brunner’s gland adenoma of the duodenal bulb]. Arkh Patol 1995; 57: 71-73

17 deSilvaS, Chandrasoma P. Giant duodenal hamartoma consi-sting mainly of Brunner’s glands. Am J Surg 1977; 133: 240-243

18 BästleinC, Decking R, Voeth C, Ottenjann R. Giant Brun-neroma of the duodenum. Endoscopy 1988; 20: 154-155

19 MaglinteDD, Mayes SL, Ng AC, Pickett RD. Brunner’s gland adenoma diagnostic considerations. J Clin Gastroenterol 1982; 4: 127-131

20 MerineD, Jones B, Ghahremani GG, Hamilton SR, Bayless TM. Hyperplasia of Brunner glands: the spectrum of its radio-graphic manifestations. Gastrointest Radiol 1991; 16: 104-108

21 PerezA, Saltzman JR, Carr-Locke DL, Brooks DC, Osteen RT, Zinner MJ, Ashley SW, Whang EE. Benign nonampullary duo-denal neoplasms. J Gastrointest Surg 2003; 7: 536-541

22 ParkJH, Park CH, Park JH, Lee SJ, Lee WS, Joo YE, Kim HS, Choi SK, Rew JS, Kim SJ. [The safety and usefulness of endo-scopic polypectomy for treatment of Brunner’s gland adeno-mas]. Korean J Gastroenterol 2004;43:299-303

23 BaladasHG, Borody TJ, Smith GS, Dempsey MB, Richardson MA, Falk GL. Laparoscopic excision of a Brunner’s gland hamartoma of the duodenum. Surg Endosc 2002; 16: 1636

24 KhawajaHT, Deakin M, Colin-Jones DG. Endoscopic removal of a large ulcerated Brunner’s gland adenoma. Endoscopy 1986; 18: 199-201

25 JansenJM, Stuifbergen WN, van Milligen de Wit AW. Endo-scopic resection of a large Brunner’s gland adenoma. Neth J Med 2002; 60: 253-255

26 Rajagopalan S, al-Arrayed S, Dhiman RK. Brunner’s gland polyp with upper gastrointestinal bleeding managed by endo-scopic polypectomy: a report of two cases. Trop Gastroenterol 2003; 24: 133-134

S-Editor Wang J L-Editor Wang XL E-EditorCao L



Metastasis of hepatocellular carcinoma to the small bowel manifested by intussusception

Hyun Soo Kim, Jung Woo Shin, Gyu Yeol Kim, Young Min Kim, Hee Jeong Cha, Yoong Ki Jeong, In Du Jeong,

Sung-Jo Bang, Do Ha Kim, Neung Hwa Park

Hyun Soo Kim, Jung Woo Shin, In Du Jeong, Sung-Jo Bang, Do Ha Kim, Neung Hwa Park, Division of Gastroenterology, Department of Internal Medicine, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, KoreaGyu Yeol Kim, Department of Surgery, University of Ulsan Col-lege of Medicine, Ulsan University Hospital, Ulsan, KoreaYoung Min Kim, Hee Jeong Cha, Department of Pathology, University of Ulsan College of Medicine, Ulsan University Hospi-tal, Ulsan, KoreaYoong Ki Jeong, Department of Radiology, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, KoreaCorrespondence to: Jung Woo Shin, MD, Ulsan University Hos-pital 290-3 Jeonha-dong, Dong-gu, Ulsan 682-714, Korea. [email protected]: +82-52-2508806 Fax: +82-52-2518235Received : 2005-10-17 Accepted : 2005-11-18

AbstractHepatocellular carcinoma (HCC) is a so highly invasive tumor that metastasizes hematogenously and lymphog-enously to distant site. Frequent sites are lung, regional lymph node, bone, and adrenal gland. But metastasis to the gastrointestinal (GI) tract is rare, and most com-mon site is stomach. Metastasis to the small intestine is extremely rare. Moreover, metastatic HCC of the small bowel causing intussusception has not been reported un-til now. Here, we report a case of metastasis of HCC to the small bowel manifested by intussusception.


Key words: Hepatocelluar carcinoma; Small bowel me-tastasis; Intussusception

Kim HS, Shin JW, Kim GY, Kim YM, Cha HJ, Jeong YK, Jeong ID, Bang SJ, Kim DH, Park NH. Metastasis of hepatocellular carcinoma to the small bowel manifested by intussuscep-tion. World J Gastroenterol 2006; 12(12): 1969-1971


INTRODUCTIONHepatocellular carcinoma (HCC) is a highly malignant tumor with frequent metastasis. Extrahepatic metastases

have been reported in 25 to 65 percent of autopsy series, with lung, bones, and regional lymph nodes as the most commonly involved sites[1-5]. Metastasis of HCC to the gastrointestinal (GI) tract is rare and most common site of metastasis is reported stomach by direct invasion of tumor[4,5]. The distant hematogenous metastasis of HCC to the small intestine is extremely rare[6].

The majority of adult intussusceptions arising in the small bowel are due to benign lesions, and metastatic melanoma is the most frequent malignancy causing small bowel intussusception[7]. Intussusception caused by the metastatic HCC to the small bowel has not been reported until now. We report a case of metastasis of HCC to the small bowel manifested by intussusception.

CASE REPORTA 65-year-old man was referred to emergency room with complaint of sudden onset, cramping periumbilical pain for 2 d. The pain was continuous with frequent exacerba-tion, and was associated with nausea and vomiting. The patient had been assigned a diagnosis of hepatitis B virus associated cirrhosis 3 years previously. Two months before, he was diagnosed as HCC with multiple bones and region-al lymph nodes metastases according to the large enhanced hepatic mass on computed tomography scan with elevated level of serum alpha-fetoprotein (629 µg/L). On physical examination, the abdomen was distended, with provocable pain in the periumbilical area and increased bowel sound. Complete blood cell count revealed anemia (Hb 72 g/L) and leukocytosis (15.1×109/µL). Liver function tests and renal function test were not remarkable. Plain radiography of abdomen demonstrated presence of marked distension of bowel loops with air fluid level and absence of colonic gas, which were known as a characteristic finding of small bowel obstruction (Figure 1). Computed tomography scan showed ill defined hepatic mass with regional lymph node enlargement and a “target mass” lesion in the right lower abdomen (Figure 2). Abdominal ultrasound showed 10 cm long intussusception in the mid small bowel level (Figure 3A) and a small mass which was suspected of lead-ing point of intussusception was also observed at the head of intussusception (Figure 3B).

After five days of conservative management, segmental small bowel resection and end-to-end anastomosis was performed. At surgery, intussusceptional segment was lo-cated at the level of jejunum 110 cm distal from the Treitz

CASE REPORT

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ligament and 8 cm sized protruding mass was grown into the lumen of small intestine within intussusceptional seg-ment. Other three masses which measured 0.5 cm, 0.8 cm, and 3 cm respectively were also grown into the lumen at the level of jejunum 170 cm distal from the Treitz liga-ment.

Macroscopic examination of the surgical specimen showed multiple luminal protruding submucosal mass

with extension to the subserosa (Figure 4). The largest mass measured 3.0 cm × 2.0 cm × 0.8 cm. In microscopic examination, the atypical epithelial cells were poorly differ-entiated carcinoma with hepatoid feature of the trabecular pattern (Figure 5). There tumor cells were positive for alpha-fetoprotein and cytokeratin 18 on the immunohis-tochemical staining (Figure 6). These evidences supported HCC metastasis to small bowel.

Figure 1 Plain radiography of abdomen showed stepladder appearance.

Figure 2 Computed tomography scan showed a “target mass” lesion (arrow) in the right lower abdomen, representing the intussuscepted mesenteric fat and vessel.

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Figure 3 Abdominal ultrasound showed, A: “pseudo kidney sign” at the body of intussusception, B: Lobulated mass lesion at the head of intussusception, which was suspected of leading point of intussusception.

A B

Figure 4 Surgical specimen showed that mass arising from submucosa grown into lumen and had dimpling portion on the top of tumor.

Figure 5 Histological examination demonstrated a poorly differentiated adenocarcinoma with hepatoid feature of trabecular pattern (HE X 100).

CK-10 AFP

Figure 6 Strongly positive staining for alpha-fetoprotein and cytokeratin 18 (Immunoperoxidase × 100).


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DISCUSSIONHCC is so highly invasive that extrahepatic metastasis is frequently observed [2,5]. The most common site of me-tastasis is lung, followed by regional lymph node, adrenal gland and bone. HCC metastasis to gastrointestinal tract is a rare condition, being found only in 4-12% of cases in autopsy series[5]. These metastatic lesions are usually as-ymptomatic, and most are discovered on postmortem ex-amination[8]. Reports of premoterm diagnosis of GI tract involvement in HCC are extremely rare and are associated with poor prognosis[8,9]. Chen et al reported that median survival following the diagnosis of GI tract involvement in HCC was only one month[8].

The modes of metastasis of HCC are known as he-matogenous, lymphatic spread and local infiltration[8].

The presumed mode of metastasis of HCC to GI tract is mostly direct invasion to adjacent GI tract via adhesion to the serosal side by bulky mass. Lin et al. reported that nine of eleven patients with HCC represented direct invasion of HCC to adjacent GI tract[9]. In Park’s series, the mode of spread was direct invasion from a contiguous HCC (66.7 %), hematogenous metastasis (17%), peritoneal seed-ing (6%)[10]. Characteristic features of HCC with direct involvement of GI tract were large, located at the subcap-sular area, and had exophyting growth pattern[9,10]. HCC has a high incidence of venous invasion and thrombosis formation. Hematogenous spread of HCC to the GI tract may be caused by tumor thrombi via the portal system and disseminated by hepatofugal portal spread to the GI tract. In previous reports, most of patients with hematogenous metastasis of HCC to the GI tract showed main portal vein thrombosis[8,9]. But in some cases, vascular involve-ment was not observed. In our case, the presumed mode of metastasis to small intestine was hematogenous spread. However despite of bulky mass with multiple bone metas-tases, main portal vein thrombosis was not observed.

The most commonly involved site of the GI tract in HCC was reported to be the stomach[9]. The small intes-tine can often be involved in tumors such as melanoma, carcinoma of the lung, cervix of the uterus, the breast, and colon[11,12]. HCC metastasis to small intestine was rarely reported. Few cases with the involvement of the duode-num have been reported, but in those cases, the duodenum was directly invaded by tumor in the liver[6, 8-10]. In our case, HCC metastasized to jejunum by hematogenous spread and formed multiple polypoid masses.

Metastatic tumors in the small intestine predominantly involve the bowel wall, the subserosa, the serosa or the mesentery. But submucosal involvement is rare. According to Farmer and Hawk, metastatic masses to small bowel are divided into three types: (1) spherical mesenteric masses encroaching on or extension into the intestine, (2) intra-mural masses, often with ulceration; and (3) bulky polyp-oid masses extending into the bowel lumen [11]. Polypoid lesions are resulted from mucosal or submucosal involve-ment rather than intramural involvement. In the present case, histological examination showed that polypoid mass sprouted from submucosa and extended to serosa. This finding suggested that the main metastatic site of HCC

was the submucosa of the small intestine. Intussusception, although common in children, is rare

in adults and represents 1% of cause of small bowel ob-struction in adult [13]. Adult intussusception presents with a variety of acute, intermittent, and chronic symptoms mimicking partial small bowel obstruction[14]. Thus, the diagnosis of intussusception is difficult to make before surgery and computed tomography scan is the most use-ful diagnostic tool. In distinction to intussusception in children, intussusceptions in adults are mainly resulted from organic lesion. The most common cause of benign lesion was postoperative band and malignant lesions were mainly consisted of metastatic tumor[13]. In previous re-port, melanoma was the most common tumor of small intestine and lymphoma, followed by sarcoma, squamous cell carcinoma[13]. In our case, cause of small bowel intus-susception was diagnosed as metastatic HCC by patho-logic examination.

We reported an extremely rare case of metastatic HCC to small intestine, which caused small bowel intussuception in adult. Up to our knowledge, this is the first case report of small bowel intussuception resulting from HCC metas-tasis.

REfERENCES1 Edmondson HA, Steiner PE. Primary carcinoma of the liver:

A study of 100 cases among 48,900 necropsies. Cancer 1954; 7: 462-503

2 Kay CJ. Primary hepatic cancer: review of 96 case. Arch Intern Med 1964; 113:46-53

3 Sung JL, Wang TH, Yu JY. Clinical study on primary carci-noma of the liver in Taiwan. Am J Dig Dis 1967; 12: 1036-1049

4 Anthony PP. Primary carcinoma of the liver: a study of 282 cases in Ugandan Africans. J Pathol 1973; 110: 37-48

5 Nakashima T, Okuda K, Kojiro M, Jimi A, Yamaguchi R, Sakamoto K, Ikari T. Pathology of hepatocellular carcinoma in Japan. 232 Consecutive cases autopsied in ten years. Cancer 1983; 51: 863-877

6 Yang PM, Sheu JC, Yang TH, Chen DS, Yu JY, Lee CS, Hsu HC, Sung JL. Metastasis of hepatocellular carcinoma to the proximal jejunum manifested by occult gastrointestinal bleed-ing. Am J Gastroenterol 1987; 82: 165-167

7 Gayer G, Zissin R, Apter S, Papa M, Hertz M. Pictorial re-view: adult intussusception-a CT diagnosis. Br J Radiol 2002; 75:185-190

8 Chen LT, Chen CY, Jan CM, Wang WM, Lan TS, Hsieh MY, Liu GC. Gastrointestinal tract involvement in hepatocellular carcinoma: clinical, radiological and endoscopic studies. En-doscopy 1990; 22: 118-123

9 Lin CP, Cheng JS, Lai KH, Lo GH, Hsu PI, Chan HH, Hsu JH, Wang YY, Pan HB, Tseng HH. Gastrointestinal metastasis in hepatocellular carcinoma: radiological and endoscopic studies of 11 cases. J Gastroenterol Hepatol 2000; 15: 536-541

10 Park MS, Kim KW, Yu JS, Kim MJ, Yoon SW, Chung KW, Lee JT, Yoo HS. Radiologic findings of gastrointestinal tract in-volvement in hepatocellular carcinoma. J Comput Assist Tomogr 2002; 26: 95-101

11 Farmer RG, Hawk WA, Metastatic tumors of the small bowel. Gastroenterology 1964; 47:496-504

12 de Castro CA, Dockery MB, Mayo CW. Metastatic tumors of the small intestines. Surg Gynecol Obstet 1957; 105:159-165

13 Azar T, Berger DL. Adult intussusception. Ann Surg 1997; 226: 134-138

14 Begos DG, Sandor A, Modlin IM. The diagnosis and manage-ment of adult intussusception. Am J Surg 1997; 173: 88-94

S- Editor Guo SY L- Editor Zhang JZ E- Editor Cao L

Kim HS et al. Small bowel intussusception by metastatic HCC 1971

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Teresa Casanovas-Taltavull, Ana Berrozpe, Sara Jordan, Department of Gastroenterology , Hospital Universitari de Bellvitge, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Barcelona, SpainJosepa Ribes, F Xavier Bosch, Epidemiology and Cancer Registration Unit, Institut Català d’Oncologia, Hospital Universitari de Bellvitge, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Barcelona, SpainAurora Casanova, Department of Microbiology, Hospital Universitari de Bellvitge, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Barcelona, SpainConcha Sancho, Carles Valls, Department of Radiology. Hospital Universitari de Bellvitge, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Barcelona, SpainSupported by FIS (Fondo Investigación Sanitaria) 94/1635 for the following project: Epidemiological survey of HBV carriers. Mortality and risk factors for the development of chronic liver disease and from the Instituto de Salud Carlos III of the Spanish Government (grants RTICCC C03/09 & RTICESP C03/10).Correspondence to: Dr. Teresa Casanovas-Taltavull, Department of Gastroenterology, Hospital Universitari de Bellvitge, IDIBELL, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Barcelona, Spain. [email protected]: +34-93-2607909 Fax: +34-93-2607603Received: 2005-08-09 Accepted: 2005-09-10

AbstractLittle is known about the long-term survivors of acute arsenic intoxication. We present here a clinical case report of a man with chronic hepatitis B virus (HBV) infection who developed hepatocellular carcinoma four years after acute arsenic poisoning. HBsAg was detected in serum in 1990 when he voluntarily donated blood. In 1991, the patient suffered from severe psychological depression that led him to attempt suicide by massive ingestion of an arsenic-containing rodenticide. He survived with polyneuropathy and paralysis of the lower limbs, and has been wheelchair-bound since then. During participation in a follow-up study conducted among HBV carriers, abdominal ultrasound detected a two-centimeter liver mass consistent with hepatocellular carcinoma. The tumor was confirmed by computed tomography (CT) and magnetic resonance image (MRI). Because of his significant comorbidity, the patient received palliative treatment with transarterial lipiodol chemoembolization (TACE) on three occasions (1996, 1997 and 1999). At his most recent visit in May 2005, the patient was asymptomatic, liver enzymes were normal and the tumor

was in remission on ultrasound.


Key words: HBV carriers; Occult HBV; HCC therapy; Arsenic intoxication

Casanovas-Taltavull T, Ribes J, Berrozpe A, Jordan S, Casanova A, Sancho C, Valls C, Bosch FX. Patient with hepa-tocellular carcinoma related to prior acute arsenic intoxica-tion and occult HBV: Epidemiological, clinical and therapeu-tic results after 14 years of follow-up. World J Gastroenterol 2006; 12(12): 1972-1974


INTRODUCTIONArsenic is a recognized human carcinogen established by the International Agency for Research on Cancer[1]. Chronic arsenic intoxication has been associated with skin lesions (such as hyperpigmentation, hyperkeratosis and carcinoma) and liver disease (non-cirrhotic portal hypertension, hepatocellular carcinoma and angiosarcoma). Moreover, chronic arsenic exposure is associated with a higher frequency of diabetes mellitus, ischemic heart disease and hypertension[2-4] . Epidemiological studies mainly involving cohorts of chronic arsenic-exposed subjects suggest that chronic arsenic intoxication increases the risk of developing liver cancer as compared with non-exposed populations[5,6] .

Patients who survive the initial effects of acute arsenic exposure may develop peripheral neuropathy and encephalopathy[7]. Hepatic injury is uncommon but is associated with severe liver disease and high mortality[8]. Little is known about the long-term survivors of acute arsenic intoxication.

We present here a clinical case report of a Caucasian man with chronic hepatitis B virus (HBV) infection who developed hepatocellular carcinoma four years after acute arsenic poisoning.

CASE REPORTThe man born in 1947 was obese with no HBV carriers

CASE REPORT

Patient with hepatocellular carcinoma related to prior acute arsenic intoxication and occult HBV: Epidemiological, clinical and therapeutic results after 14 years of follow-up

Teresa Casanovas-Taltavull, Josepa Ribes, Ana Berrozpe, Sara Jordan, Aurora Casanova, Concha Sancho, Carles Valls, F Xavier Bosch

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in his family and no previous hepatitis or parenteral drug use. He was a heavy smoker and alcohol consumer before 1987. HBsAg was detected in serum in 1990 when he voluntarily donated blood.

In 1978 he had self-limited cardiac arrhythmia because of stress and in 1991 arrhythmia due to atrial fibrillation that reverted with cardioversion. In 1991, the patient suffered from severe psychological depression that led him to attempt suicide by massive ingestion of an arsenic-containing rodenticide. He developed acute renal failure and generalized paralysis and was hospitalized for 11 days in the intensive care unit, requiring assisted ventilation and chelation therapy with dimercaprol. He survived with polyneuropathy and paralysis of the lower limbs, and has been wheelchair-bound since then. In 1994 and 1995 he suffered from several transient ischemic attacks that were managed with chronic decoagulation therapy. In 2000 diabetes was diagnosed. He also required long-term treatment for sleep apnea-hypoapnea syndrome with automatic continuous positive airway pressure.

In November 1995 he was invited to participate in a follow-up study conducted in HBV carriers[9]. The viral serology results were negative for HBsAg, anti-HBs, HBeAg, IgM-anti-HBc, and positive for anti-HBe, IgG-anti-HBc. Serum HBV-DNA was negative by PCR technique, while anti-HDV, anti-HCV and anti-HIV were all negative. ALT was 1.36 µKat/mL (normal <0.5 µKat/mL), AST and alpha-fetoprotein were normal. Abdominal ultrasound detected a pattern suggestive of chronic liver disease and a liver mass two centimeters in diameter consistent with hepatocellular carcinoma. The tumor was confirmed by computed tomography (CT) scanning and magnetic resonance imaging. Because of his comorbidity, the patient was not considered eligible for liver transplant or hepatectomy. Palliative treatment with transarterial lipiodol chemoembolization (TACE) was performed on three occasions (1996, 1997 and 1999) (Figure 1) with clinical, analytical and imaging follow-up studies (Figure 2). At his last visit in May 2005, he was asymptomatic, liver enzymes were normal and the tumor was in remission on ultrasound imaging.

Casanovas-Taltavull T et al . Result therapy in HCC, related to HBV and arsenic 1973

DISCUSSIONTo the best of our knowledge, this patient is the first known case of liver tumor developed after acute arsenic exposure. Standard chelat ion treatment for acute intoxication could not remove arsenic from intracellular sites because of its lipophobic nature, suggesting that this treatment cannot protect cells from long-term clinical consequences[10]. As has been described, the presence of intracellular arsenic may be related to the development of diabetes, arterial hypertension, ischemic arterial disease and liver cancer in such patients. It should also be taken into consideration that intraindividual variability in arsenic methylation can increase susceptibility to arsenic-induced cancer[11].

Given the fact that the patient was obese and had occult HBV infection (his anti-HBc and anti-HBe positive status could indicate integration of the virus in the host genome) prior to arsenic intake, we are not sure that some hepatic injury is not due to these risk factors[12,13] . The presence of these risk factors may enhance the hepatic lesion and the development of liver cancer.

Angiosarcoma is the type of liver tumor most often related with chronic arsenic exposure[5]. In our case, histological confirmation of the liver tumor could not be performed due to the anticoagulant treatment (Sintrom). However, the features of the tumor on CT and TACE are consistent with hepatocellular carcinoma, particularly when the tumor characteristics are considered after lipiodol uptake during and after TACE[14]. Even though the most effective therapy for hepatocellular carcinoma is liver transplantation or partial hepatectomy[15], our patient remains asymptomatic after nine years of TACE treatment. This fact is consistent with the increasing survival rates of patients receiving this palliative treatment[16].

In conclusion, this is the first patient with occult HBV infection who developed hepatocellular carcinoma after acute arsenic exposure, and his tumor remitted after palliative treatment with TACE. The clinical implication of our observation is that periodical liver studies should be undertaken in patients who survive after acute arsenic exposure.

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Figure 1 Non-contrast CT one month after arterial chemoembolization with iodized oil (Lipiodol) and doxorubicine emulsion. CT shows a 2-cm nodule with dense Lipiodol enhancement in segment VI (arrow), consistent with hepatocellular carcinoma.

Figure 2 Follow-up contrast-enhanced CT five years later from 1995 shows a small hypervascular lesion (arrow) in segment VI consistent with residual tumor. Tumoral Lipiodol retention was absent and the size of the lesion was significantly decreased.

10 cm

REFERENCES1 Some drinking-water disinfectants and contaminants, includ-

ing arsenic. IARC Monogr Eval Carcinog Risks Hum 2004; 84: 1-477

2 Chen CJ, Wang CJ. Ecological correlation between arsenic level in well water and age-adjusted mortality from malignant neoplasms. Cancer Res 1990; 50: 5470-5474

3 Centeno JA, Mullick FG, Martinez L, Page NP, Gibb H, Long-fellow D, Thompson C, Ladich ER. Pathology related to chron-ic arsenic exposure. Environ Health Perspect 2002; 110 Suppl 5: 883-886

4 Nevens F, Fevery J, Van Steenbergen W, Sciot R, Desmet V, De Groote J. Arsenic and non-cirrhotic portal hypertension. A report of eight cases. J Hepatol 1990; 11: 80-85

5 Regelson W, Kim U, Ospina J, Holland JF. Hemangioendo-thelial sarcoma of liver from chronic arsenic intoxication by Fowler’s solution. Cancer 1968; 21: 514-522

6 Chen CJ, Chuang YC, Lin TM, Wu HY. Malignant neoplasms among residents of a blackfoot disease-endemic area in Tai-wan: high-arsenic artesian well water and cancers. Cancer Res 1985; 45: 5895-5899

7 Goebel HH, Schmidt PF, Bohl J, Tettenborn B, Krämer G, Gutmann L. Polyneuropathy due to acute arsenic intoxication: biopsy studies. J Neuropathol Exp Neurol 1990; 49: 137-149

8 Vantroyen B, Heilier JF, Meulemans A, Michels A, Buchet JP, Vanderschueren S, Haufroid V, Sabbe M. Survival after a lethal dose of arsenic trioxide. J Toxicol Clin Toxicol 2004; 42: 889-895

9 Bosch FX, Ribes J, Cléries R, Díaz M. Epidemiology of hepato-cellular carcinoma. Clin Liver Dis 2005; 9: 191-211, v

10 Kalia K, Flora SJ. Strategies for safe and effective therapeu-tic measures for chronic arsenic and lead poisoning. J Occup Health 2005; 47: 1-21

11 Steinmaus C, Yuan Y, Kalman D, Atallah R, Smith AH. Intra-individual variability in arsenic methylation in a U.S. popula-tion. Cancer Epidemiol Biomarkers Prev 2005; 14: 919-924

12 Pollicino T, Squadrito G, Cerenzia G, Cacciola I, Raffa G, Craxi A, Farinati F, Missale G, Smedile A, Tiribelli C, Villa E, Raimondo G. Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection. Gastroenterology 2004; 126: 102-110

13 Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Over-weight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med 2003; 348: 1625-1638

14 Valls C, Cos M, Figueras J, Andía E, Ramos E, Sánchez A, Serrano T, Torras J. Pretransplantation diagnosis and stag-ing of hepatocellular carcinoma in patients with cirrhosis: value of dual-phase helical CT. AJR Am J Roentgenol 2004; 182: 1011-1017

15 Figueras J, Ramos E, Ibáñez L, Valls C, Serrano T, Rafecas A, Casanovas T, Fabregat J, Xiol X, Torras J, Baliellas C, Jaurrieta E, Casais L. [Surgical treatment of hepatocellular carcinoma. Long term results]. Med Clin (Barc) 2002; 118: 410-414

16 Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma: Chemoembolization improves survival. Hepatology 2003; 37: 429-442



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Srdjan P Djuranovic, Milan M Spuran, Nada V Kovacevic, Milenko B Ugljesic, Dragutin M Kecmanovic, Marjan T Micev, Institute for Digestive Diseases, Clinical Center of Serbia, K. Todorovica St No 6, 11000 Belgrade, Serbia and MontenegroCorrespondence to: Srdjan P Djuranović, MD, PhD, Institute for Digestive Diseases, Clinical Center of Serbia, K.Todorovića St. No 6, 11000 Belgrade, Serbia and Montenegro. [email protected] Telephone: + 381-11-3615587 Fax: + 381-11-3615587Received: 2005-05-30 Accepted: 2005-06-08

AbstractMucinous cystadenoma of the appendix is a rare condition and represents one of the three entities with the common name mucocele of the appendix. It is characterized by a cystic dilatation of the lumen with stasis of mucus inside it. Histopathologically mucocele is divided into three groups: focal or diffuse mucosal hyperplasia, mucinous cystadenoma and mucinous cystadenocarcinoma. This condition is often associated with other neoplasia, especially adenocarcinoma of the colon and ovaries. We here describe a 57 year old male patient who presented with abdominal discomfort, constipation, fresh blood in stool and frequent urination. He had a big cystadenoma of the appendix associated with adenocarcinoma of the colon and hepatocellular carcinoma of the liver. The patient underwent right haemicolectomy, sigmoid colon resection and segmental resection of the liver. Now 3 years later he has no evidence of disease relapse. According to this, we stress the need of accurate preoperative diagnosis and intraoperative exploration of the whole abdomen in these patients.


Key words: Mucocele; Appendiceal cystadenoma; Colon carcinoma; Hepatocellular carcinoma

Djuranovic SP, Spuran MM, Kovacevic NV, Ugljesic MB, Ke-cmanovic DM, Micev MT. Mucinous cystadenoma of the appendix associated with adenocarcinoma of the sigmoid colon and hepatocellular carcinoma of the liver: Report of a case. World J Gastroenterol 2006; 12(12): 1975-1977


INTRODUCTIONMucocele of the appendix is a common name for three different entities with similar clinical presentations. Its main characteristic is cystic dilatation of the appendiceal lumen with mucus inside it. Focal or diffuse mucosal hyperplasia and mucinous cystadenoma are of benign nature, but could lead to complications due to rupture, invasion to adjacent organs or recurrence. Mucinous cystadenocarcinoma is a malignant disease and pseudomyxoma peritonei is its worst complication. On the other hand, this condition is often associated with other intra-abdominal neoplasia. According to this, it is necessary to apply strict oncologic principles for resection in order to minimize the possible complications. A correct preoperative diagnosis may help to avoid iatrogenic rupture during surgery and missing the possible associated intra-abdominal tumors. We describe here a case of correct preoperative diagnosis of big appendiceal mucinous cystadenoma associated with adenocarcinoma of the sigmoid colon and hepatocellular carcinoma of the liver.

CASE REPORTThe patient was male, 57 years old with pain in ileo-cecal region for 6 mo prior to administration. He had abdominal discomfort, constipation, fresh blood in stool and frequent urination. On physical examination he had palpable tumor mass in the lower right quadrant of abdomen, enlarged liver and subicterus of sclera. Laboratory findings showed inflammatory syndrome with sideropenic normocytic anemia, elevated alkaline phosphatase, carcinoembryonic antigen, carboanchidratic 19-9 antigen and alpha-feto protein. He had negative markers for hepatotropic viruses (B and C). Transabdominal sonography showed the presence of a large bilocular cystic tumor in the right lower quadrant of abdomen with outlined capsule and maximal dimensions of 106 mm × 74 mm, slightly enlarged liver with focal hyperechogenous tumor in the 6th and 7th liver segments (maximal diameter of 67 mm) and “pseudokidney” sign in the left lower quadrant of abdomen. CT scan displayed tumor of the right liver lobe (Figure 1). Barium enema showed extra luminal compression and medial displacement of cecum and terminal ileum with appendix not filled with the contrast and 4-cm long tubular stenosis of the proximal part of

Mucinous cystadenoma of the appendix associated with adenocarcinoma of the sigmoid colon and hepatocellular carcinoma of the liver: Report of a case

Srdjan P Djuranovic, Milan M Spuran, Nada V Kovacevic, Milenko B Ugljesic, Dragutin M Kecmanovic, Marjan T Micev


CASE REPORT

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sigmoid colon (Figure 2). According to that we suspected that it was the mucocele of the appendix with neoplasia of the sigmoid colon and hepatic tumor. The patient underwent right haemicolectomy with ileo-transverso termino-lateral anastomosis and sigmoid colon resection with colo-recto termino-terminal anastomosis as well as paraaortal and paracaval lymphadenectomy. Segmetal resection of the liver (the 6th and 7th segments) was done and the whole tumor was resected. Histology showed big mucocele of the appendix caused by mucinous cystadenoma with severe displasia (Figure 3 and Figure 4), adenocarcinoma of the sigmoid colon (T3,N1,M0,L1,V0; Dukes C; Astler-Coller C-2) and carcinoma hepatis hepatocellulare (well-differentiated alveolar type). Adjuvant therapy with 5-fluorouracile and Leucovorine was administrated in five cycles. Now, three years later, the patient is doing well and has gained 17 kilograms with no evidence of disease relapse and his laboratory findings including tumor markers are within the normal range.

DISCUSSIONMucocele of the appendix is a descriptive term that implies a dilated appendiceal lumen caused by abnormal accumulation of mucus. Mucocele is found in 0.2-0.3% of all appendectomy specimens. The male:female ratio is 1:4 and the mean age of patients is around 55 years[1,2]. Post-imflammatory obstruction was initially thought to be the main reason for the majority of mucoceles, but recent evidence suggests that neoplasia is the basis of most of these cases. The understanding of the nature of appendiceal mucocele and its terminology has been

greatly changed since the initial description of Rokitansky in 1842[3] and Higa et al[4] in 1973. Appendiceal mucocele can be histologically divided into three groups: focal or diffuse mucosal hyperplasia without epithelial atypia, mucinous cystadenoma with some degree of epithelial atypia and mucinous cystadenocarcinoma. A large series of hyperplasia were found in 24.7% cystadenoma, 63% cystadenocarcinoma and 12.3 % of all cases[1,2,4]. Our case could be regarded as mucinous cystoadenoma with severe dysplasia.

Peritoneal (and/or retroperitoneal and pleural) pseudomyxoma is regarded as a worst complication of this disease which could have metastatic characteristics in case of mucinous adenocarcinoma. It is well known that this entity is often associated with colorectal carcinoma (11-20%), ovarian mucinous cystadenoma, uterine myoma, carcinoma of the pancreas and carcinoid tumor of the appendix[4]. Our patient is interesting because of the two associated tumors while one of them (hepatocellular carcinoma) has not so far been mentioned in literature in association with mucinous cystadenoma.

Clinical symptomatology of these patients is not specific. Abdominal pain is present in 64% of the patients and palpable ileo-cecal mass in 50% of them. Disease course is asymptomatic in 25% of the patients even when they have large tumors. Urinary infection and haematuria

Figure 1 Abdominal CT showing presence of the right lobe tumor of the liver.

Figure 2 Barium enema displaying extraluminal c o m p r e s s i o n a n d m e d i a l d i s l o c a t i o n of the cecum due to cystadenoma of the appendix and tubular stenosis of the sigmoid c o l o n d u e t o t h e adenocarcinoma.

Figure 3 Cystadenoma mucinosum appendicis with obvious dysplastic epithelial lining and focally evident mucinous cytoplasmatic production (H&E, 64x).

Figure 4 Cystadenoma mucinosum appendices with small extracellular mucinous deposit just beneath focally eroded adenomatous epithelium (H&E, 13x).


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10 cm

1

12

21

R

3

L/U -1024/ 3071

SD 1 3.4Area 1 0.02Mean 2 49.0SD 2 9.9Area 2 0.02Mean 3 33.8SD 3 2.6Area 3 0.02

Mean 1 44.6

Di 1

Di 2An 1

An 2

6.7

544.3

1.42

are often associated (20%)[5]. Intestinal obstruction caused by intussusception and intestinal bleeding are rare complications[6]. Our patient had a long history of abdominal pain, blaunting, subicterus and urinary discomfort, constipation and rectal bleeding. Some of these symptoms are due to associated tumors.

Laboratory findings are usually non specific, but the elevation of tumor marker level often indicates neoplastic origin and/or associated tumors, which was seen in our patient.

Correct and complete preoperative diagnosis was rare in the past, but modern techniques of imaging allow us today to recognise most of the complications and associated conditions[7]. Sonography usually shows a cystic encapsulated lesion with liquid content adjacent to cecum. CT/NMR scan shows a low density, encapsulated, thin-walled mass that does not contain contrast medium and communicates directly with the cecum. Other solid or cystic abdominal and peritoneal tumors could also be visualised by these methods. Barium enema could point out a failure of the appendix to fill with contrast medium and signs of the extra luminal compression in the ileo-cecal region[8,9]. The "sign of volcano" is a pathognomonic colonoscopy finding[8,9]. We could not perform total colonoscopy due to neoplastic stenosis of the sigmoid colon.

Fine needle aspiration biopsy is not recomended because of great risk of pseudomyxoma dissemination.

Classical surgical approach is the best therapy option with intraoperative abdominal cavity exploration because of the already stressed associated tumors. Appendectomy is advised for focal or diffuse mucosal hyperplasia and cystadenoma when the appendiceal base is intact. Cecal resection is performed for cystadenoma with a large base and right colectomy is recommended for cystadenocarcinoma. Other surgical procedures depend upon the existence of associated tumors. In cases of disseminated pseudomyxoma peritonei, ultrasonic surgical aspirator can be used[10, 11]. Laparoscopic approach to cystadenoma of the appendix is safe if surgery can be performed without grasping the lesion and if the specimen is removed through the abdominal wall using a bag[12].

The five-year survival rate is 100% in cases of benign mucocele and about 45% in malignant cases. Radiotherapy, chemothe r apy and new the r apeu t i c moda l i t i e s (radioimmunotherapy and matrix metalloproteinase inhibitors) have still to be proven by prospective analysis[13].

In conclusion, accurate preoperative diagnosis and intraoperative exploration of the whole abdomen can improve the prognosis of patients with appendiceal mucocele.

ACKNOWLEDGMENTSThe authors thank Professor Božina Radević, PhD, surgeon, Institute for Cardiovascular Diseases Dedinje, Belgrade and Professor Ivan Boričić, PhD, pathologist, Institute for Pathology, Belgrade, for their kind help.

REFERENCES1 Kim SH, Lim HK, Lee WJ, Lim JH, Byun JY. Mucocele of the

appendix: ultrasonographic and CT findings. Abdom Imaging 1998; 23: 292-296

2 Krebs TL, Daly BD, Wong-You-Cheong JJ, Grumbach K. Gen-eral case of the day. Mucinous cystadenocarcinoma of the ap-pendix. Radiographics 1998; 18: 1049-1050

3 Rokitansky CF. A manual of pathological anatomy. Vol.2. English translation of the Vienna edition (1842). Philadelphia: Blancard and Lea, 1855: 89

4 Higa E, Rosai J, Pizzimbono CA, Wise L. Mucosal hyperplasia, mucinous cystadenoma, and mucinous cystadenocarcinoma of the appendix. A re-evaluation of appendiceal “mucocele”. Cancer 1973; 32: 1525-1541

5 Minni F, Petrella M, Morganti A, Santini D, Marrano D. Giant mucocele of the appendix: report of a case. Dis Colon Rectum 2001; 44: 1034-1036

6 Jones CD, Eller DJ, Coates TL. Mucinous cystadenoma of the appendix causing intussusception in an adult. Am J Gastroen-terol 1997; 92: 898-899

7 Scotté M, Laquerrière A, Riff Y, Majerus B, Manouvrier JL, Leblanc I, Michot F, Hémet J, Ténière P. [Appendiceal muco-celes]. Pathophysiology and therapeutic indications J Chir (Paris) 1994; 131: 303-312

8 Madwed D, Mindelzun R, Jeffrey RB Jr. Mucocele of the ap-pendix: imaging findings. AJR Am J Roentgenol 1992; 159: 69-72

9 Isaacs KL, Warshauer DM. Mucocele of the appendix: com-puted tomographic, endoscopic, and pathologic correlation. Am J Gastroenterol 1992; 87: 787-789

10 Keating JP, Frizelle FA. Use of ultrasonic surgical aspirator in operative cytoreduction of pseudomyxoma peritonei. Dis Co-lon Rectum 2000; 43: 559-560

11 Zagrodnik DF 2nd, Rose DM. Mucinous cystadenoma of the appendix: diagnosis, surgical management, and follow-up. Curr Surg 2003; 60: 341-343

12 Navarra G, Asopa V, Basaglia E, Jones M, Jiao LR, Habib NA. Mucous cystadenoma of the appendix: is it safe to remove it by a laparoscopic approach? Surg Endosc 2003; 17: 833-834

13 Stevens KJ, Dunn WK, Balfour T. Pseudomyxoma extraperito-nei: a lethal complication of mucinous adenocarcinoma of the appendix. Am J Gastroenterol 1997; 92: 1920-1922


Djuranovic SP et al . Mucinous cystadenoma of the appendix 1977

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Ugur Unluturk, Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, TurkeySercan Aksoy, Gulten Tekuzman, Hacettepe University Institute of Oncology, Ankara, Turkey Ozlem Yonem, Yusuf Bayraktar, Hacettepe University Faculty of Medicine, Department of Internal Medicine, Unit of Gastroenterology, Ankara, Turkey Correspondence to: Dr. Sercan Aksoy, Hacettepe University Institute of Oncology, 06100, Sıhhiye, Ankara, Turkey, [email protected]: +90-312-305 2937 Fax: +90-312-324 2009Recevied: 2005-09-30 Accepted: 2005-11-10


Unluturk U, Aksoy S, Yonem O, Bayraktar Y, Tekuzman G. Cytomegalovirus gastritis after rituximab treatment in a non-Hodgkın’s lymphoma patient. World J Gastroenterol 2006; 12(12): 1978-1979


To The ediTorRituximab is a chimeric monoclonal antibody against CD20 antigen expressed on most B cells and is used for the treatment of malignant lymphomas expressing the CD20 antigen[1]. It temporarily eliminates normal B-lym-phocytes without a substantial decrease in serum immuno-globulin levels[2]. Recently, several serious viral infections have been reported in association with rituximab use. Cy-tomegalovirus (CMV) is one of the agents that may be a cause of morbidity and mortality in immunocompromised individuals[2, 3]. In the face of exogenous or endogenous causes of immunosuppression, cytomegalovirus can result in retinitis, colitis, pneumonitis, or encephalitis. Presented here is a case of a patient with non-Hodgkin’s lymphoma (NHL) who developed gastritis and enterocolitis due to CMV infection after treatment with rituximab.

A 65-year old female patient was admitted with com-plaints of epigastric pain and diarrhea. Prior to her admis-sion she was treated for diffuse large cell type NHL, stage IIIB bulky disease with six courses of a regimen consisting of cyclophosphamide, doxorubicin, vincristine and predni-sone (CHOP) and rituximab, and a good partial response was achieved. Due to the presence of bulky disease at presentation，she also received radiotherapy (RT) to the parailiac and inguinal regions at a dose of 3 000 centigray

(cGy), one month after chemotherapy. Two weeks after radiotherapy, she presented with epigastric pain and diar-rhea without blood and mucous. Microscopic examina-tion of stool specimens was unremarkable. Her blood and stool cultures were negative. Her chest X-ray was also normal. Initial investigations revealed normal hemoglobin concentration and platelet count as well as mild leucopenia. The number of white blood cells was 3.6×109/L (3.0×109/L neutrophils). Serum levels of glucose, amylase, liver transaminases, urea and electrolytes were normal. Based on the preliminary diagnosis of radiation enteritis, antidi-arrheal therapy was initiated. The patient did not benefit from this, and her epigastric pain aggravated. A comput-erized tomography scan of the abdomen revealed gastric wall thickening as well as findings consistent with colitis of the ascending and transverse colon. Subsequent upper gastrointestinal endoscopic evaluation demonstrated mul-tiple linear exudative gastric ulcers, the largest being 4 cm in diameter, from which multiple biopsy specimens were obtained. Histological examination revealed hyperchro-matic epithelial cells with nuclear viral inclusions (owl’s eye). These cells were positive for monoclonal antibodies against CMV. However, peripheral blood CMV viral load was negative. She failed to respond to acid suppression therapy and her clinical condition deteriorated further. Repeat upper gastrointestinal endoscopy and histological examination of biopsy specimens were consistent with the previous results. Consequently, treatment with intravenous gancyclovir (2×5 mg/kg) was initiated with a diagnosis of CMV gastritis and enterocolitis. Dramatic clinical im-provement was achieved within one week and treatment was continued for 3 weeks. Her symptoms resolved com-pletely and a subsequent endoscopy only showed signs of healing ulcers.

Pelvic or abdominal RT is a known cause of acute en-teritis characterized by abdominal cramping and diarrhea in approximately 50% of treated patients, and the incidence is higher with concomitant chemotherapy[4]. Acute intestinal side effects of RT occur at doses of approximately 1000 cGy. Since the curative doses for most abdominal or pel-vic tumors range between 5 000 and 7 000 cGy, enteritis is more often than not likely to occur[5]. Our patient received 3000cGy of RT and probably developed RT-induced en-teritis. However, antidiarrheal therapy with oral opiates failed to alleviate the patient’s complaints, which only re-solved after anti-CMV therapy.

Cytomegalovirus enterocolitis should be considered

in the differential diagnosis in patients receiving rituximab

LETTERS TO THE EDITOR

Cytomegalovirus gastritis after rituximab treatment in a non-Hodgkın’s lymphoma patient

Ugur Unluturk, Sercan Aksoy, Ozlem Yonem, Yusuf Bayraktar, Gulten Tekuzman

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whose symptoms fail to resolve after conventional anti-diarrheal therapy, irrespective of peripheral blood CMV viral load.

reFereNCeS1 CzuczmanMS, Grillo-López AJ, White CA, Saleh M, Gordon

L, LoBuglio AF, Jonas C, Klippenstein D, Dallaire B, Varns C. Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy. J Clin Oncol 1999; 17: 268-276

2 Suzan F , Ammor M, Ribrag V. Fatal react ivat ion of cytomegalovirus infection after use of rituximab for a post-transplantation lymphoproliferative disorder. N Engl J Med

Unluturk U et al . Cytomegalovirus gastritis after rituximab treatment 1979

2001; 345: 10003 Goldberg SL, Pecora AL, Alter RS, Kroll MS, Rowley SD,

Waintraub SE, Imrit K, Preti RA. Unusual viral infections (progress ive mul t i foca l l eukoencepha lopathy and cytomegalovirus disease) after high-dose chemotherapy with autologous blood stem cell rescue and peritransplantation rituximab. Blood 2002; 99: 1486-1488

4 BensonAB3rd, Ajani JA, Catalano RB, Engelking C, Kornblau SM, Martenson JA Jr, McCallum R, Mitchell EP, O’Dorisio TM, Vokes EE, Wadler S. Recommended guidelines for the treatment of cancer treatment-induced diarrhea. J Clin Oncol 2004; 22: 2918-2926

5 PerezCA, Brady LW, eds.: Principles and Practice of Radiation Oncology. 3rd ed. Philadelphia, Pa: Lippincott-Raven Publishers, 1998

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ACKNOWLEDGMENTS

Acknowledgments to Reviewers of World Journal of Gastroenterology

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Many reviewers have contributed their expertise and time to the peer review, a critical process to ensure the quality of World Journal of Gastroenterology. The editors and authors of the articles submitted to the journal are grateful to the following reviewers for evaluating the articles (including those were published and those were rejected in this issue) during the last editing period of time.

Kyoichi Adachi, MD Department of Gastroenterology and Hepatology, Shimane University, School of Medicine Shimane, 89-1 Enya-cho, Izumo-shi Shimane 693-8501, Japan

Yasuji Arase, MDDepartment of Gastroenterology, Toranomon Hospital, 2-2-2Toranomonminato-ku, Tokyo 105-8470, Japan

Giovanni Barbara, ProfessorInternal Medicine and Gastroenterology, University of Bologna, St.Orsola Hospital - Building No. 5Via Massarenti, 9 - 40138, Bologna 40138, Italy

Julio Horacio Carri, ProfessorInternal Medicine – Gastroenterology, Universidad Nacional de Córdoba, Av.Estrada 160-P 5-Department D, Córdoba 5000, Argentina

Julio Horacio Carri, ProfessorInternal Medicine – Gastroenterology, Universidad Nacional de Córdoba, Av.Estrada 160-P 5-Department D, Córdoba 5000, Argentina

Jun Cheng, ProfessorDean Assistant, Beijing Earth Altar Hospital Dean 13 Earth Altar Park, Anwai Avenue, East District, Beijing 100011, China

Andrew Seng Boon Chua, MDDepartment of Gastroenterology, Gastro Centre Ipoh, 1, lorong Rani, 31, lebuhraya Tmn Ipoh, Ipoh Garden South, IPOH 30350, Malaysia

Christoph F Dietrich, MDInnere Medizin 2, Caritas-Krankenhaus, Uhlandstr. 7, Bad Mergentheim97980, Germany

Bart Rik De Geest, DrCenter for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, Leuven 3000, Belgium

Edoardo G Giannini, Assistant ProfessorDepartment of Internal Medicine, Gastroenterology Unit, Viale Benedetto XV, no. 6, Genoa, 16132, Italy

David Y Graham, ProfessorDepartment of Medicine, Michael E. DeBakey VAMC, Rm 3A-320 (111D), 2002 Holcombe Blvd, Houston, TX 77030, United States

David Y Graham, ProfessorDepartment of Medicine, Michael E. DeBakey VAMC, Rm 3A-320 (111D), 2002 Holcombe Blvd, Houston, TX 77030, United States

Hohenberger Werner Hohenberger, ProfessorChirurgische Klinik und Poliklinik, Krankenhausstrasse 12, Erlangen D-91054, Germany

Xiao-Long Ji, ProfessorInstitute of Nanomedicine, Chinese Armed Police General Hospital, 69 Yongding Road, Beijing 100039, China

Aydin Karabacakoglu, Dr, Assistant ProfessorDepartment of Radiology, Meram Medical Faculty, Selcuk University, Konya 42080, Turkey

Elias A Kouroumalis, ProfessorDepartment of Gastroenterology, University of Crete, Medical School, Department of Gastroenterology, University Hospital, PO Box 1352, Heraklion, Crete 71110, Greece

Patricia F Lalor, DrLiver Research Laboratory, Room 537 Institute of Biomedical Research, Divsion of Medical Science, University of Birmingham, Birmingham B15 2TT, United Kingdom

Alex B Lentsch, PhD, Associate ProfessorDepartment of Surgery, Division of Trauma and Critical Care, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati OH 45267-0558, United States

Masatoshi Makuuchi, ProfessorDepartment of Surgery, Graduate School of Medicine University of Tokyo, T Hepato-Biliary-Pancreatic Surgery Division Tokyo 113-8655, Japan

Stephan Miehlke, PhDMedical Department I, Technical University Hospital, Fetscher Str. 74, Dresden 01307, Germany

Kazunari Murakami, ProfessorDepartment of General Medicine, Oita University, 1-1 Idaigaoka, Hasama, Oita 879-5593, Japan

Shotaro Nakamura, MDDepartment of Medicine and Clinical Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

Bo-Rong Pan, ProfessorDepartmant of Oncology, Xijing Hospital, Fourth Military Medical University, No.1, F. 8, Bldg 10, 97 Changying East Road, Xi’an 710032, Shaanxi Province,China

Amado Salvador Peña, ProfessorDepartment of Pathology, Immunogenetics, VU University Medical Centre, De Boelelaan 1117, PO Box 7057, Amsterdam 1007 MB, The Netherlands

Bashkim Resuli, MDDepartment of Internal Medicine and University Service of Gastrohepatology, University Hospital Center “ Mother Theresa”, Medical Faculty of Tirana, Tirana, Albania

Stephen M Riordan, Associate ProfessorGastrointestinal and Liver Unit, The Prince of Wales Hospital, Barker Str, Randwick 2031, Australia and University of New South Wales, Sydney, Australia

Richard A Rippe, DrDepartment of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7038, United States

Luis Rodrigo, ProfessorGastroenterology Service, Hospital Central de Asturias, c/ Celestino Villamil, s.n., Oviedo 33.006, Spain

Michiie Sakamoto, ProfessorDepartment of Pathology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

Nathan Subramaniam, DrMembrane Transport Laboratory, The Queensland Institute of Medical Research 300 Herston Road, Herston, Brisbane, QLD 4006, Australia

Wei Tang, MD, EngD, Assistant ProfessorH-B-P Surgery Division, Artificial Organ and Transplantation Division, Department of surgery, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan

Simon D Taylor-Robinson, MDDepartment of Medicine A, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0HS, United Kingdom

Paul Joseph Thuluvath, ProfessorDepartment of Gastroenterology and Hepatology, The Johns Hopkins Hospital, 1830 E. Monument St, Baltimore MD 21205, United States

Hitoshi Togashi, Associate ProfessorDepartment of Gastroenterology, Course of Internal Medicine and Therapeutics, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan

Yvan Vandenplas, Professor Department of Pediatrics, AZ-VUB, Laarbeeklaan 101, Brussels 1090, Belgium

Yvan Vandenplas, ProfessorDepartment of Pediatrics, AZ-VUB, Laarbeeklaan 101, Brussels 1090, Belgium

Jian-Ying Wang, ProfessorUniversity of Maryland School of Medicine, Baltimore VA Medical Center (112), 10N. Greene St, Baltimore, MD 21201, United States

Yuan Yuan, ProfessorCancer Institute of China Medical University, 155 North Nanjing Street, Heping District, Shenyang 110001, Liaoning Province, China

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TextFor most article types, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RESULTS and DISCUSSION, and should include in appropriate Figures and Tables. Data should be presented in the body text or in Figures and Tables, but not in both.

IllustrationsFigures should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each fi gure on a separate page. No detailed legend should be involved under the fi gures. This part should be added into the text where the fi gures are applicable. Digital images: black and white photographs should be scanned and saved in TIFF format at a resolution of 300 dpi; color images should be saved as CMYK (print fi les) but not as RGB (screen-viewing fi les). Place each photograph in a separate fi le. Print images: supply images of size no smaller than 126 mm×76 mm printed on smooth surface paper; label the image by writing the Figure number and orientation using an arrow. Photomicrographs: indicate the original magnifi cation and stain in the legend. Digital Drawings: supply fi les in EPS if created by freehand and illustrator, or TIFF from photoshops. EPS fi les must be accompanied by a version in native fi le format for editing purposes. Existing line drawings should be scanned at a resolution of 1200 dpi and as close as possible to the size where they will appear when printed. Please use uniform legends for the same subjects. For example: Figure 1 Pathological changes of atrophic gastritis after treatment. A: ...; B: ...; C: ...; D: ...; E: ...; F: ...; G: ...

TablesThree-line tables should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each table. No detailed legend should be included under the tables. This part should be added into the text where the tables are applicable. The information should complement but not duplicate that contained in the text. Use one horizontal line under the title, a second under the column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted.

Notes in tables and illustrationsData that are not statistically significant should not be noted. aP<0.05, bP<0.01 should be noted (P>0.05 should not be noted). If there are other series of P values, cP<0.05 and dP<0.01 are used. Third series of P values can be expressed as eP<0.05 and fP<0.01. Other notes in tables or under illustrations should be expressed as 1F, 2F, 3F; or some other symbols with a superscript (Arabic numerals) in the upper left corner. In a multi-curve illustration, each curve should be labeled with ●, ○, ■, □, ▲, △, etc. in a certain sequence.

AcknowledgmentsBrief acknowledgments of persons who have made genuine contributions to the manuscripts and who endorse the data and conclusions are included. Authors are responsible for obtaining written permission to use any copyrighted text and/or illustrations.

REFERENCESCoding systemThe author should code the references according the citation order in text in Arabic numerals, put references codes in square brackets, superscript it at the end of citation content or the author name of the citation. For those citation content as the narrate part, the coding number and square brackets should be typeset normally. For example, Crohn’s disease (CD) is associated with increased intestinal permeability[1,2]. If references are directly cited in the text, they would be put together with the text, for example, from references [19,22-24 ], we know that... When the authors code the references, please ensure that the order in text is the same as in reference part and also insure the spelling accuracy of the fi rst author’s name. Do not code the same citation twice.

PMID requirementPMID roots in the abstract serial number indexed by PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed). The author should supply the PMID for journal citation. For those references that have not been indexed by PubMed, a printed copy of the fi rst page of the full reference should be submitted. The accuracy of the information of the journal citations is very important. Through reference testing system (http://www.aushome.cn/cgi-bin/index.pl), the authors and editor could check the authors name, title, journal title, publication date, volume number, start page, and end page. We will interlink all references with PubMed in ASP fi le so that the readers can read the abstract of the citations online immediately.

Style for journal referencesAuthors: the fi rst author should be typed in bold-faced letter. The surname of all authors should be typed with the initial letter capitalized and followed by their name in abbreviation (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR). Title of the cited article and italicized journal title (Journal title should be in its abbreviation form as shown in PubMed), publication date, volume number (in black), start page, and end page [PMID: 11819634] Note: The author should test the references through reference testing system (http://www.aushome.cn/cgi-bin/index.pl)

Style for book referencesAuthors: the fi rst author should be typed in bold-faced letter. The surname of all authors should be typed with the initial letter capitalized and followed by their name in abbreviation (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR) Book title. Publication number. Publication place: Publication press, Year: start page and end page.

FormatStandard journal article (list all authors and include the PubMed ID [PMID] where applicable)1 Das KM, Farag SA. Current medical therapy of infl ammatory bowel dis-

ease. World J Gastroenterol 2000; 6: 483-489 [PMID: 11819634]2 Pan BR, Hodgson HJF, Kalsi J. Hyperglobulinemia in chronic liver disease:

Relationships between in vitro immunoglobulin synthesis, short lived sup-pressor cell activity and serum immunoglobulin levels. Clin Exp Immunol 1984; 55: 546-551 [PMID: 6231144]

3 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologic effect of Jianpi Yishen decoction in treatment of Pixu-diarrhoea. Shijie Huaren Xiaohua Za-zhi 1999; 7: 285-287

Books and other monographs (list all authors)4 Sherlock S, Dooley J. Diseases of the liver and billiary system. 9th ed. Ox-

ford: Blackwell Sci Pub, 1993: 258-296Chapter in a book (list all authors)5 Lam SK. Academic investigator’s perspectives of medical treatment for

peptic ulcer. In: Swabb EA, Azabo S. Ulcer disease: investigation and basis for therapy. New York: Marcel Dekker, 1991: 431-450

Electronic journal (list all authors)6 Morse SS. Factors in the emergence of infectious diseases. Emerg Infect

Dis serial online, 1995-01-03, cited 1996-06-05; 1(1):24 screens. Available from: URL: http//www.cdc.gov/ncidod/EID/eid.htm

Inappropriate referencesAuthors should always cite references that are relevant to their article, and avoid any inappropriate references. Inappropriate references include those that are linked with a hyphen and the difference between the two numbers at two sides of the hyphen is more than 5. For example, [1-6], [2-14] and [1, 3, 4-10, 22] are all considered as inappropriate references. Authors should not cite their own unrelated published articles.

Statistical dataPresent as mean ± SD or mean ± SE.

Statistical expressionExpress t test as t (in italics), F test as F (in italics), chi square test as χ2 (in Greek), related coeffi cient as r (in italics), degree of freedom as γ (in Greek), sample number as n (in italics), and probability as P (in italics).

UnitsUse SI units. For example: body mass, m (B) = 78 kg; blood pressure, p (B) = 16.2/12.3 kPa; incubation time, t (incubation) = 96 h, blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/L; blood CEA mass concentration, p (CEA) = 8.6 24.5 μg/L; CO2 volume fraction, 50 mL/L CO2 not 5% CO2; likewise for 40 g/L formaldehyde, not 10% formalin; and mass fraction, 8 ng/g, etc. Arabic numerals such as 23, 243, 641 should be read 23 243 641.

The format about how to accurately write common units and quantum is at: http://www.wjgnet.com/wjg/help/15.doc

AbbreviationsStandard abbreviations should be defi ned in the abstract and on fi rst mention in the text. In general, terms should not be abbreviated unless they are used repeatedly and the abbreviation is helpful to the reader. Permissible abbreviations are listed in Units, Symbols and Abbreviations: A Guide for Biological and Medical Editors and Authors (Ed. Baron DN, 1988) published by The Royal Society of Medicine, London. Certain commonly used abbreviations, such as DNA, RNA, HIV, LD50, PCR, HBV, ECG, WBC, RBC, CT, ESR, CSF, IgG, ELISA, PBS, ATP, EDTA, mAb, can be used directly without further mention.

ItalicsQuantities: t time or temperature, c concentration, A area, l length, m mass, V volume.Genotypes: gyrA, arg 1, c myc, c fos, etc.Restriction enzymes: EcoRI, HindI, BamHI, Kbo I, Kpn I, etc.Biology: Helicobacter pylori, H pylori, E coli, etc.

SUBMISSION OF THE REVISED MANUSCRIPTS AFTER ACCEPTEDPlease revise your article according to the revision policies of WJG. The revised version including manuscript and high-resolution image fi gures (if any) should be copied on a fl oppy or compact disk. Author should send the revised manuscript, along with printed high-resolution color or black and white photos, copyright transfer letter, the fi nal check list for authors, and responses to reviewers by a courier (such as EMS) (submission of revised manuscript by e-mail or on the WJG Editorial Offi ce Online System is NOT available at present).

Language evaluation The language of a manuscript will be graded before sending for revision. (1) Grade A: priority publishing; (2) Grade B: minor language polishing; (3) Grade C: a great deal of language polishing; (4) Grade D: rejected. The revised articles should be in grade B or grade A.

Copyright assignment formIt is the policy of WJG to acquire copyright in all contributions. Papers accepted for publication become the copyright of WJG and authors will be asked to sign a transfer of copyright form. All authors must read and agree to the conditions outlined in the Copyright Assignment Form (which can be downloaded from http://www.wjgnet.com/wjg/help/9.doc).

Final check list for authorsThe format is at: http://www.wjgnet.com/wjg/help/13.doc

Responses to reviewersPlease revise your article according to the comments/suggestions of reviewers. The format for responses to the reviewers’ comments is at: http://www.wjgnet.com/wjg/help/10.doc

Proof of fi nancial supportFor paper supported by a foundation, authors should provide a copy of the document and serial number of the foundation.

Publication feeAuthors of accepted articles must pay publication fee.EDITORIAL and LETTERS TO THE EDITOR are free of change.


World Journal of Gastroenterology standard of quantities and unitsNumber Nonstandard Standard Notice

1 4 days 4 d In fi gures, tables and numerical narration 2 4 days four days In text narration 3 day d After Arabic numerals 4 Four d Four days At the beginning of a sentence 5 2 hours 2 h After Arabic numerals 6 2 hs 2 h After Arabic numerals 7 hr, hrs, h After Arabic numerals 8 10 seconds 10 s After Arabic numerals 9 10 year 10 years In text narration 10 Ten yr Ten years At the beginning of a sentence 11 0,1,2 years 0,1,2 yr In fi gures and tables 12 0,1,2 year 0,1,2 yr In fi gures and tables 13 4 weeks 4 wk 14 Four wk Four weeks At the beginning of a sentence 15 2 months 2 mo In fi gures and tables 16 Two mo Two months At the beginning of a sentence 17 10 minutes 10 min 18 Ten min Ten minutes At the beginning of a sentence 19 50% (V/V) 500 mL/L 20 50% (m/V) 500 g/L 21 1 M 1 mol/L 22 10 μM 10 μmol/L 23 1N HCl 1 mol/L HCl 24 1N H2SO4 0.5 mol/L H2SO4 25 4rd edition 4th edition 26 15 year experience 15- year experience 27 18.5 kDa 18.5 ku,18 500u or Mr18 500 28 25 g.kg-1/d-1 25 g/(kg·d) or 25 g/kg per day 29 6900 6 90030 1000 rpm 1 000 r/min 31 sec s After Arabic numerals 32 1 pg·L-1 1 pg/L33 10 kilograms 10 kg 34 13 000 rpm 13 000 g High speed; g should be in italic and suitable conversion. 35 1000 g 1 000 r/min Low speed. g cannot be used. 36 Gene bank GenBank International classifi ed genetic materials collection bank 37 Ten L Ten liters At the beginning of a sentence 38 Ten mL Ten milliliters At the beginning of a sentence 39 umol μmol 40 30 sec 30 s41 1 g/dl 10 g/L 10-fold conversion42 OD260 A260 “OD” has been abandoned. 43 One g/L One microgram per liter At the beginning of a sentence 44 A260 nm A260 nm A should be in italic. bP<0.05 aP<0.05 In Table, no note is needed if there is no significance instatistics: aP<0.05, bP<0.01 (no note if P>0.05). If there is a second set of P value in the same table, cP<0.05 and dP<0.01 are used for a third set: eP<0.05, fP<0.01.45 *F=9.87, §F=25.9, 1F=9.87,2F=25.9, Notices in or under a table #F=67.4 3F=67.446 KM km kilometer 47 CM cm centimeter 48 MM mm millimeter 49 Kg, KG kg kilogram 50 Gm, gr g gram 51 nt N newton 52 l L liter 53 db dB decibel 54 rpm r/min rotation per minute 55 bq Bq becquerel, a unit symbol 56 amp A ampere 57 coul C coulomb58 HZ Hz 59 w W watt 60 KPa kPa kilo-pascal 61 p Pa pascal 62 ev EV volt (electronic unit) 63 Jonle J joule 64 J/mmol kJ/mol kilojoule per mole 65 10×10×10cm3 10 cm×10 cm×10 cm66 N·km KN·m moment 67 x±s mean±SD In fi gures, tables or text narration 68 Mean±SEM mean±SE In fi gures, tables or text narration 69 im im intramuscular injection 70 iv iv intravenous injection 71 Wang et al Wang et al. 72 EcoRI EcoRI Eco in italic and RI in positive. Restriction endonuclease has its prescript form of writing.73 Ecoli E.coli Bacteria and other biologic terms have their specific expression.74 Hp H pylori 75 Iga Iga writing form of genes76 igA IgA writing form of proteins77 -70 kDa ~70 ku

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