The role of Helicobacter spp. in the ... - Wiley Online Library

FEMS Immunology and Medical Microbiology 44 (2005) 221–225 www.fems-microbiology.org

The role of Helicobacter spp. in the pathogenesis of primary biliary cirrhosis and primary sclerosing cholangitis Sacha Y. Boomkens a, Sjoerd de Rave b, Raymond G.J. Pot b, Herman F. Egberink c, Louis C. Penning a, Jan Rothuizen a, Pieter E. Zondervan d, Johannes G. Kusters b,* b c

a Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, The Netherlands Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, 3015 GD Rotterdam, The Netherlands Department of Infectious Diseases and Immunology, Division of Virology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands d Department of Pathology, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands

Received 20 August 2004; received in revised form 22 October 2004; accepted 3 November 2004 First published online 7 December 2004

Abstract Helicobacter species DNA has been detected in liver tissue of patients affected by primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). To investigate a potential causative relation between Helicobacter species and PBC/PSC, we compared the presence of Helicobacter species-specific DNA in liver tissue of patients with PBC/PSC (n = 18/n = 13) with those of a control group of patients with various liver diseases with known cause (n = 29). A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from paraffin embedded liver tissue. Control patients had hepatitis-B (n = 9), alcoholic cirrhosis (n = 14), or non-cirrhotic metabolic liver disease (n = 6). There was no significant difference between the incidence of Helicobacter spp.-specific DNA in PBC/PSC (9/31; 29%) and the control group (10/29; 34%). Sequence analysis confirmed Helicobacter spp. DNA. Because Helicobacter spp. DNA can be found in approximately one-third of all samples tested, it is unlikely that PSC and PBC are caused by Helicobacter infection. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Helicobacter species; Primary sclerosing cholangitis; Primary biliary cirrhosis; Alcoholic cirrhosis; Hepatitis B virus infection; Metabolic liver diseases

1. Introduction Since the discovery of Helicobacter pylori in 1983 by Warren and Marshall [1], more than 25 other Helicobacter spp. have been detected in stomach, intestinal tract and liver of various mammalian and bird species [2]. Helicobacter infections have been implicated in hepatobiliary disease in mice, ferrets, cats and dogs [3–10]. Primary sclerosing cholangitis (PSC) and primary biliary *

Corresponding author. Tel.: +31 10 463 2982; fax: +31 10 463 2793. E-mail address: [email protected] (J.G. Kusters).

cirrhosis (PBC) are human liver diseases of unknown etiology. PSC is characterized by inflammation, strictures and dilatations of the biliary tree, and PBC is characterized by inflammation and destruction of small bile ducts. Both are cholestatic liver diseases that frequently lead to cirrhosis and liver failure, and are important indications for liver transplantation. Genetic and environmental factors both seem to play a role in the pathogenesis of PSC and PBC [11]. In a recent study Helicobacter-like DNA was detected in the liver of nearly all patients with PSC and PBC, but in only one patient with non-cholestatic liver disease and in none of the normal controls [12]. In contrast, an independent

0928-8244/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2004.11.002

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study failed to detect Helicobacter DNA in such patients [13]. On the other hand, detection of Helicobacter spp. DNA in bile and in hepatobiliary tissue samples has also been reported in patients with hepatobiliary malignancies and bile stones [14–17]. The role of Helicobacter infections in all these benign and malignant hepatobiliary disorders remains controversial [18–21]. Moreover, only in one case of a patient with WilsonÕs disease, who also harbored H. pylori in the stomach, it has been possible to culture H. pylori from liver tissue [22]. Therefore, it was suggested that the organism most likely colonized the already diseased liver as a secondary event and that the hepatic abnormality was necessary for colonization. Because of the general inability to confirm the presence of live bacteria in the liver, it has been suggested that the Helicobacter DNA detected in the above mentioned tissues represent fragmented DNA rather than intact organisms resulting from ‘‘spill-over’’ of macrophage-associated DNA coming from the stomach [23,24]. Given these conflicting results, it is far from certain that Helicobacter is an important factor in the pathogenesis of cholestatic liver diseases. Therefore, we decided to study the possible causative role of Helicobacter spp. infection in the livers of patients with PSC and PBC by comparing them with cirrhotic and noncirrhotic liver diseases of a known non-Helicobacterassociated liver pathology: chronic hepatitis B infection, alcoholic cirrhosis and non-cirrhotic metabolic liver diseases.

2. Materials and methods 2.1. Patients Patients were selected who underwent liver transplantation between 1989 and 2003, because of the ample availability of liver tissue. Patients were excluded when endoscopic retrograde cholangio-pancreatography (ERCP) was performed, had received antibiotic treatment in the three months prior to transplantation, or when they had recurrent bacterial cholangitis, cholangiocarcinoma, a biliary stent in situ or a prior choledochojejunostomy. The study group consisted of 13 patients with primary sclerosing cholangitis (PSC) and 18 patients with primary biliary cirrhosis (PBC). The control group consisted of 29 patients with liver diseases of known etiology; 14 with alcoholic cirrhosis (ALC), nine with chronic hepatitis B infection (HBV) and six with non-cirrhotic metabolic liver diseases (MET). All diagnoses were based on standard clinical, biochemical and histological criteria. Demographic and clinical characteristics were retrieved from our liver transplantation database and from the patient files.

All samples used in this study were tested blindly in the laboratory. 2.2. DNA isolation DNA was isolated from a total of 60 paraffin embedded liver tissue samples with the Tissue and Hair Extraction Kit and the DNA IQä System (Promega Benelux, Leiden, The Netherlands; Protocol C) using magnetic beads. For each sample, three sections of 10 lm each were used. The surface of all embedded liver sections was approximately 1 cm2. DNA was eluted in the manufacturers elution buffer and stored at 20 °C. A water sample was also included in the DNA isolation procedure to investigate cross contamination of the samples. 2.3. PCR amplification All PCRs were performed in a 50 ll volume with an MJ Research thermal cycler (MJ research Inc., Watertown, MA., USA) Reaction mixtures contained 0.2 lM of each oligonucleotide primer (Isogen Life Science, Maarssen, The Netherlands), PCR buffer (Invitrogen Corporation, Carlsbad, CA, USA), 2.5 U of Platinum Taq polymerase (Invitrogen), 2 mM MgCl2 (Invitrogen) and 250 lM of each nucleotide. Primers HelicoF (5 0 -AACGATGAAGCTTCTAGCTTGCTAG-3 0 ) and HelicoR (5 0 -GTGCTTATTCGTTAGATACCGTCAT3 0 ) based on the 16S rRNA sequences of most, if not all gastric and enterohepatic members of the Helicobacter genus were used to generate a 400-bp amplicon [14]. Five microliters of DNA was used in a first reaction. The resulting amplicons were diluted 10 times and 5 ll of this dilution was used in a second reaction with the same primers under the same conditions. The PCR conditions were the following: an initial denaturation step at 95 °C for 4 min followed by a denaturation step for 30 s at 95 °C, annealing at 50 °C for 30 s, elongation at 72 °C for 1 min. A total of 40 cycles was performed with a final elongation step at 72 °C for 10 min. As controls, chromosomal DNA of H. pylori reference strains 26695 [25] and 1061 [26] was used. In order to exclude that PCR inhibition was the cause of negative PCRs, negative samples were tested for the presence of PCR inhibitors by repeating the procedure after spiking with the lowest detectable amount of H. pylori DNA. This detectable amount was determined by performing a PCR on serial dilutions of H. pylori. Several samples containing water were always used as negative controls to exclude contamination. 2.4. Sequence analysis of the amplicons The generated amplicons were directly sequenced using primers HelicoF and HelicoR. Sequencing was performed with an ABI 3100 genetic analyzer (Applied


Biosystems, Foster City, CA, USA). The resulting nucleotide sequences were compared with the sequences present in the GenBank databases using the BLAST nucleotide program of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov blast) and were aligned and analyzed using Lasergene (DNAStar Inc., Madison, WI, USA) and SECentral (Scientific & Educational Software, Durham, NC, USA) software.

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P = 0.009). Also, more immigrants from outside Western Europe were found in the control group (7 in the control group versus 1 in the cholestatic disease group; P = 0.024). In the cholestatic disease group there were more patients with known duodenal ulcers or Helicobacter-associated gastritis (6 versus 0 in the control group, P = 0.012). The ages in the two groups were similar (P = 0.568), with a median age of 52 years (range 23–66 years).

2.5. Statistics 3.2. PCR amplification with 16S rRNA primers Non-parametric tests were used for all comparisons; the Fisher exact test for categorical and the Mann– Whitney U-test for continuous variables. Multivariate testing was done by logistic regression analysis. A pvalue of 0.05 or lower was considered significant. All statistical analyses were performed with the SPSS 11.0.1 Software (SPSS Inc. USA).

3. Results 3.1. Study population From our liver transplantation database we carefully selected 31 patients with cholestatic liver diseases (PSC and PBC) with the strict selection criteria as described. As controls we selected 29 patients with liver diseases (HBV, ALC and MET), in which Helicobacter spp. are not associated with the cause of these diseases. These patients were selected with the same strict selection criteria as the patients with cholestatic liver diseases. Unfortunately we were unable to select healthy individuals as controls, since these were not available. The study group consisted of 30 males and 30 females. The male/female ratio was higher in the controls than in the group of patients with cholestatic liver diseases (20/9 versus 10/21,

A total of 60 paraffin embedded formalin fixed liver tissue samples were tested by PCR for the presence of Helicobacter spp.-specific DNA. From these 60 samples, 19 (32%) were positive in the PCR. All generated amplicons had the expected size of 400 bp, whereas the positive and negative controls were indeed positive and negative, respectively. This indicates that contaminations in the PCR procedure are highly unlikely. Repeated testing in a blinded fashion gave identical results. Furthermore, spiking of the negative samples with the lowest detectable concentrations of Helicobacter DNA to investigate PCR inhibition, showed that no inhibition had occurred. The number of positive samples in the cholestatic liver disease group (PSC and PBC) was 9/31 (29%), whereas in the control group (HBV, ALC and MET) 10/29 (34%) samples were positive. The amount of positive samples per disease group is depicted in Table 1. In brief, the percentage of positive samples in the disease groups ranges from 28% to 36%. There was no significant difference between the cholestatic liver disease group and the control group (P = 0.783), and, as a result, there were no significant differences between the different diseases groups. Sex, ethnic background and known presence of duodenal ulcer or helicobacter-associated gastritis

Table 1 The number of Helicobacter-specific PCR positive samples Disease type

Total no PCR pos (% of total)

Closest homology PCR fragments (% of PCR pos) H. pylori a

H. pylori ‘‘liver 3’’b

H. pametensisc

PSC + PBC PSC PBC

9/31 (29%) 5/13 (38%) 4/18 (22%)

4/9 (44%) 2/5 (40%) 2/4 (50%)

5/9 (66%) 3/5 (60%) 2/4 (50%)

0/9 (0%) 0/9 (0%) 0/9 (0%)

ALC + HBV + MET ALC HBV MET

10/29 (34%) 5/14 (36%) 3/9 (33%) 2/6 (33%)

4/10 (40%) 2/5 (40%) 1/3 (33%) 1/2 (50%)

5/10 (50%) 2/5 (40%) 2/3 (66%) 1/2 (50%)

1/10 (10%) 1/5 (20%) 0/3 (0%) 0/2 (0%)

a >98% homology to H. pylori reference strain 26695 (GenBank Accession number NC_00915), and several other H. pylori strains that share the same 16S rRNA sequence in this tested region. b >98% homology to H. pylori ‘‘liver 3’’ (GenBank Accession number AF142585) and several other H. pylori strains that share the same 16S rRNA sequence in this tested region but differ from the reference strain 26695 at two fixed positions (see text). c >98% homology to H. pametensis (GenBank Accession number AF302105).

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were not related to the PCR results, neither in univariate nor in multivariate tests. 3.3. Sequence analysis of the amplicons All 19 amplicons from the positive PCRs were subjected to sequencing and all were homologous to Helicobacter spp.-specific DNA sequences (Table 1). However, the amplicons were either most closely related to (i) the 26695 H. pylori reference strain, or (ii) to the sequence deposited by Avenaud et al. detected in liver tissue of human patients with HCC (deposited at GenBank; accession number AF142585; H. pylori ‘‘liver 3’’), and (iii) a single sample which was most closely related to H. pametensis. In addition to some non-conserved mutations, the samples from strains under (i) and (ii) all differed by two basepairs from the 400bp PCR product at positions 91 (G–A) and 129 (T–C).

4. Discussion Recently, Helicobacter species DNA has been detected in bile and in hepatobiliary tissue samples of humans and other mammals [5–10], and it has also been reported in patients with hepatobiliary malignancies [14]. However, whether Helicobacter species play a role in the cholestatic liver diseases primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) is still unclear. In one study [12] Helicobacter DNA has been detected in these diseases, while in another, these sequences were absent [13]. Therefore, we investigated whether Helicobacter species might be the cause of cholestatic liver diseases. We have tested the presence of Helicobacter species-specific DNA in liver tissue of patients with PSC and PBC. As controls we selected patients with liver diseases with a known, non-Helicobacter related origin. Of the total of 60 well-defined samples tested in this study, 19 (32%) were positive in a Helicobacter genus 16S rRNA-based PCR. In the cholestatic disease (PSC and PBC) group 9 out of 31 (29%) were positive, whereas in the control (HBV, ALC and MET) group 10 of the 29 (34%) samples were positive. Because the Helicobacter spp.-specific DNA sequences of the amplicons differ from the positive control used in this study, and the many other controls used in this study, it is unlikely that the positive samples resulted from contamination either during sampling of the biopsies, storage, isolation or the amplification procedures of the samples. The number of positive samples in our patients with cholestatic liver diseases is lower than those reported by others for PSC and PBC [12]. This difference can be explained by the rigorous exclusion criteria applied in our study, aimed at minimizing the risk of false-positives and false-negatives. The meaning of the presence of Helicobacter DNA in liver tissue samples remains uncertain. The positive PCRÕs

may be the result of spill-over of degraded DNA carried by macrophages coming from the stomach through the enterohepatic circulation. However, none of the patients with duodenal ulcers or Helicobacter-associated gastritis were positive in the PCR on liver tissue. Also, of the patients positive in the PCR on liver tissue, none had duodenal ulcers or Helicobacter-associated gastritis. Therefore, our data do not support this possibility. What our study does indicate is that Helicobacter DNA can be detected in the liver of patients with cholestatic and noncholestatic liver diseases. While the cause of PSC and PBC is unknown, the controls all had liver diseases of known etiology unrelated to Helicobacter infections. The fact that no difference was found between the two groups argues against a role for Helicobacter in the pathogenesis of PSC and PBC. It could well be that Helicobacter spp. do colonize the liver, and that diseased and immunocompromized individuals are being colonized more easily. Regardless of the type of the disease approximately one-third of all samples tested in this study was positive for Helicobacter spp.-specific DNA by means of PCR. Moreover, no significant difference in the prevalence between the control group and the cholestatic disease group, neither between the separate disease groups was found. These percentages correspond to the incidence of H. pylori infections in a European population [27]. Therefore, we were unable to find a relation between the type of disease and the presence of Helicobacter-specific DNA sequences. From our results, we can conclude that Helicobacter species, most likely do not play a causal role in the development of the cholestatic liver diseases PBC and PSC.

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