Journalof Hepatoloxy 1999;31: 664-671
Printed in Dmmark
All rights resersed
Munksgnord
Copenhagen
Journal of Hepatology
ISSN 01684278
Are inkctious agents involved in primary biliary cirrhosis?A PCR
approach
Atsushi Tanaka’,
Thomas
I? Prindiville’, Robert Gish2, Jay V. Solnick’, Ross L. Coppe14, Emmet B. Keeffe’,
Aftab Ansari and M. Eric Gershwin”
‘Division of Rheumatology, Allergy and Clinical Immunology and ‘Infectious Diseases, University of Cakfornia at Davis. CA and ‘Call~orniu
Pacz$c Medical Center, San Francisco, CA, USA; 4Department of Microbiology, Monash University, Clayton, Victoriu, Australia; 5Stunford
University Medical Center, Pulo Alto, CA and 6Depurtment qf Pathology, Emory University, School of Medicine, Atlanta, GA, USA
Background/Aims: A variety of data suggest that microbial infections and, in particular, atypical mycobacteria infections, may either initiate and/or be associated with the pathogenesis of primary biliary cirrhosis.
Metkfs:
To address this hypothesis, use was made
of polymerase chain reaction techniques and primers
specific for the 16s rRNA gene of Eubactevia, Archaeabactevia, Mycobacteria and Helicobactev to determine if such sequences were detectable in liver
tissue specimens from 29 patients with primary biliary
cirrhosis. Similar liver tissues from patients with primary sclerosing cholangitis, chronic hepatitis, alcoholic liver disease and otherwise normal donors
were analyzed in parallel. Genomic DNA was extracted from each of these liver tissue specimens using
sterile techniques to avoid possible laboratory contamination. The DNA was subjected to polymerase
chain reaction amplification using bacterial genus spe-
cific primers and the amplified products cloned and
sequenced. Sequence data were analyzed by searching
for homology to existing genes.
Results: Sequences from primary biliary cirrhosis and
control livers corresponded to those found in a variety
of bacteria, but no consensus sequence was found in
primary biliary cirrhosis specimens. Neither Avchaeabacteria nor Mycubacteviu products were detected in
liver specimens of patients with primary biliary cirrhosis, and Helicobactev pylovi DNA was detected in
only one primary biliary cirrhosis patient.
Conclusions: Although bacterial infection, particularly with intracellular organisms, has been suggested
to play a role in the initiation of primary biliary cirrhosis, there is no evidence from this study to suggest
an ongoing chronic infectious process.
N
teins are present within all nucleated cells. Antigenic
mimicry has been hypothesized to account for this
tissue specific pathology, i.e. there may be a number of
molecules of extrinsic origin that contain epitopes
cross-reactive with molecules present in mitochondrial
antigens such as PDC-E2. We have already shown that
there indeed are molecule(s) that cross-react with PDCE2, which are expressed on the apical surface of biliary
epithelial cells selectively in patients with PBC (2). The
origin of these cross-reactive molecules is presently unknown, and the identity of these molecules remains a
critical issue in the study of PBC.
There are, in addition, several other lines of evidence
that implicate a role for infectious agents in the pathogenesis of PBC. Familial PBC (3,4) or geographical
clustering (5) has been reported, suggesting the involve-
ADVANCES
have been made in the study
of the immunopathogenesis
of primary biiiary
cirrhosis (PBC). One of these is the identification of
mitochondrial enzymes, such as the pyruvate dehydrogenase complex-E2 (PDC-E2), as autoantigens in PBC
(1). However, it is not understood why the biliary epithelial cells are the only (or predominant) targets for
autoimmune reaction, even though mitochondrial proUMEROUS
Received 4 February; revised II May; accepted I2 May 1999
Correspondence: M. Eric Gershwin, Division of Rheumatology/Allergy and Clinical Immunology, University of
California at Davis, TB 192, School of Medicine, Davis,
CA 956168660, USA.
Tel: 530 752 2884. Fax: 530 752 4669.
e-mail: [email protected]
664
Key words: Bacteria; Primary biliary cirrhosis; 16s
ribosomal protein.
Infectious agents in PBC
ment of environmental factors as etiological agents of
the disease. This is exemplified by the high prevalence
of bacteruria with a very high recurrence rate in females with PBC compared to other forms of chronic
liver disease (6). The sera from patients with PBC have
also been shown to react with both human PDC-E2
and E. coli PDC-E2, and the epitope of E. coli PDCE2 recognized by patient sera maps to a similar lipoyl
domain to that of human PDC-E2 (7). This is not surprising as PDC-E2 is critical in the respiratory chain,
and is well conserved in sequence among various species, from eubacteria to mammals (8). Further, it is
important to note that this cross-reactivity does not
necessarily imply that patients originally reacted to
PDC-E2 of bacterial origin. In addition, it is well
known that granulomas, associated with bacterial infection by organisms such as Mycobacteria, are often
present in the liver of patients with PBC (9). Therefore,
it is possible that the cross-reactive antigens in PBC
might be a bacterial component, and that intracellular
bacterial infection might be closely associated with the
etiology of PBC.
Archaeabacteria, Eubacteria and Eukarya all contain
small subunit rRNA molecules (16s rRNA gene in bacteria and 18s rRNA in eukaryotes). The rRNA gene
contains both conserved and variable sequences
(10,ll). Amplification of the rRNA gene combined
with sequencing is a technique for identifying and
classifying life forms. This method can be used to
avoid the problems associated with identification of
bacteria that do not grow well on conventional growth
media (12), or that are otherwise difficult to classify,
such as the difficulty of utilizing serologic methods to
identify intracellular organisms (13-l 6).
Among all bacteria, Mycobacteria infection has received special attention in terms of the pathogenesis of
PBC. Vilagut et al. demonstrated that the sera from
patients with PBC react with an extract of Mycobacterium gordonae (17), and that antibodies to the M.
TABLE
gordonae 65-kD heat-shock
protein cross-react weakly
with the mitochondrial autoantigens in PBC (18) implicating M. gordonae in the etiology of PBC. Klein et
al. demonstrated that sera from 12 out of 28 patients
with active pulmonary tuberculosis reacted with the
PDC-E2 subunit, whereas only two of 82 patients with
other bacterial and viral infections reacted with the
PBC-specific autoepitopes (19), also emphasizing the
role of mycobacterial infection in PBC. By contrast,
O’Donohue et al. have reported that they failed to confirm the presence of reactivity of PBC patients sera
against M gordonae (20), and also failed to detect
mycobacterial DNA in PBC liver (21). Thus, the role
of Mycobacteria infection in PBC remains controversial.
Helicobacter infection, known as a cause of gastric
inflammation, ulcers, and carcinoma, may play a role
in autoimmune disease. Infection with H. pylori induces autoantibodies reactive with a protein located in
gastric parietal cell canaliculi, H+K+-ATPase (22).
Since H+K+-ATPase is also the major autoantigen in
autoimmune gastritis and pernicious anemia, the initiating role of H. pylori in these diseases has been proposed. It is also suggested that H. hepaticus infection
may play a role in autoimmunity in progressive hepatitis in mice (23). Helicobacter species are detected by
PCR in the bile of patients with chronic cholecystitis
(24) and in patients with primary sclerosing cholangitis
(PSC) (25).
Collectively, these findings provided the rationale for
the studies reported herein. We wished to obtain evidence whether the molecules found on the apical location of biliary epithelial cells of patients with PBC may
arise from cryptic bacterial infection. To do this, we set
out to determine whether bacteria could be detected
in liver specimens from PBC patients, and specifically
whether Mycobacteria or Helicobacter species were
present preferentially in PBC patients compared to
controls.
1
Nucleotide sequences of oligonucleotides used in this study
Eubacteriu-specific universal primers
P3R
PCSB
5’ ATT AGA TAC CCT (G/T/A) GT AGT CC 3’
5’TACCTTGTTACGACTT3’
Archaeubacter-specific
Arch2 1F
Arch958R
5’ TTC CGG TTG ATC C (C/T) G CCG GA 3’
5’ (C/T) CC GGC GTT GA (A/C) TCC AAT T 3’
Mycobucteriu genus-specific primers (for GroEL gene)
TBl
TB2
5’ GAG ATC GAG CTG GAG GAT CCG TAC G 3’
5’ GCG GAT CTT GTT GAC GAC CAG GG 3’
Helicobacter genus-specific primers
H276f
H676r
5’ CTA TGA CGG GTA TCC GGC 3’
S’ATT CCA CCT ACC TCT CCC A 3’
GAPDH primers
GAPDH 5’
GAPDH 3’
5’ ACC ACA GTC CAT GCC ATC AC 3’
5’ TCC ACC ACC CTG TTG CTG TA 3’
universal primers
665
A. Tanaka et al.
Materials and Methods
Liver specimens
Fifty-five liver specimens were subjected to analysis. Explanted liver
specimens collected at the time of transplantation
included a total of
29 patients with PBC. eight patients with PSC, six patients with
chronic hepatitis C, four patients with chronic hepatitis B, and one
patient with alcoholic liver disease. In addition, liver specimens were
also taken from seven healthy individuals who were supposed to be
donors for transplantation
but their livers could not be used for various clinical reasons. All liver specimens were obtained in the same
way. The liver specimens were collected from explanted livers in the
operation theater and frozen at -70°C until use.
721 bp +
DNA extraction
Liver specimens were processed in a laminar flow hood in a separate
laboratory,
where PCR is not performed,
using meticulous
aseptic
techniques. They were cut into small pieces, about 50-100 mg, with a
sterile disposable
scalpel, placed in an 1.5 ml eppendorf
tube, and
thawed in 80 ~1 of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.8). A
tube containing
no specimen was processed with each sample batch
to control for inadvertent
contamination
by exogenous nucleic acids.
Pretreated
glass beads (25-50 pm) were added to each tube and the
tube was vigorously shaken for 60 s twice in a mini bead-beater
(Biospec Products,
Bartlesville, OK, USA) to disrupt the potential bacterial cell wall. Thereafter,
DNA was purified using QIAamp Tissue
Kit (QIAGEN, Basel, Switzerland).
The quality and quantity of purified DNA was determined by gel electrophoresis
and by measurement
of 0DZ6,,.
PCR ampliJicution of the potential bacterial gene
We used two sets of universal primers for Eubucteria as well as Archaeabacter, and two sets of Helicobacter- and Mycobacteria-genus
specific primers. In addition, we also performed PCR using GAPDHspecific primers to exclude the possibility that inhibitory
materials
may be present in template DNA prepared from livers that inhibit
PCR reaction. The sequences of the primers utilized in this study are
shown in Table 1. The sequences of these primers were derived from:
Eubacteria (26), Archaeabacteria
(27), Helicobacter (28), and the sequences for oligonucleotides
for Mycobacteria and GAPDH were determined
using the NCBI database.
Half a microgram
of the extracted DNA from each liver specimen was used as template in a PCR
amplification.
The reaction mixture (25 ~1) was composed
of the
DNA template, 0.2 mM of each deoxynucleoside
triphosphate
(Ultrapure dNTP set, Pharmacia
Biotech, Uppsala, Sweden), 2.5 U of
AmpliTaqTM
Gold DNA Polymerase
and 2.5 pl of GeneAmp@
1OXPCR buffer containing
1.5 mM MgClz (PE Applied Biosystems,
Foster City, CA, USA). Pfu DNA Polymerase (Stratagene,
La Jolla,
CA, USA) was used instead of AmpliTaq Gold DNA Polymerase for
the amplification
of the 16s rRNA gene using universal Eubacteriu
primers to avoid misincorporation
of nucleotides into PCR products.
Primers at a concentration
of 0.2 PM were utilized, except for the
Archaeabacteria-specific
primers which were degenerate primers and
used at 2 PM. The samples in 0.2 ml tubes were subjected to PCR in
a thermal cycler (DeltaCycler
II System, Ericomp, San Diego, CA,
USA). The PCR conditions
utilized for the universal Eubacteria
primers included an initial denaturation
at 94°C for 10 mitt, followed
by 35 cycles at 94°C for 30 s, at 50°C for 30 s and at 72°C for 1 min
30 s. For the other primers the PCR conditions were the same except
for the annealing temperature:
55°C for the universal Archaeabacteriu
primers and the Helicobacter-genus
specific primers, 65°C for the Mycobacteria-genus
specific primers and GAPDH primers. Five microliters of each reaction mixture were then subjected to electrophoresis
on 1% agarose gel stained with ethidium bromide.
We obtained specimens from human gastric biopsies known to contain Helicobacter pylori by histochemistry,
and also from rhesus macaque large bowel infected with Mycobacterium avium as positive controls. In addition, we spiked 10 ng of Helicobacter pylori and Mycobacteria gordonae DNA into non-infected
liver tissue. We then used
serial dilutions of DNA extracted from these tissues as positive controls.
666
Fig. I A. PCR using Eubacteria-sprc$c
universal primers.
PCR products were migrated in 1% ugurose gel, and stained
with ethidium bromide. M: 100 bp ladder (Promegu, Madison WI, USA), the thicker band corresponds to 500 bp.
Lane 1: E. coli DNA, 10 ng, Lane 2: 1 ng, Lane 3: 100 pg,
Lane 4: 10 pg. Lane 5: I pg. Lane 6: 100 fg. Lane 7: 10
fg. Lane 8: I fg, Lane 9: 0.1 fg. Lane 10: sample control, no
liver specimens in DNA extraction. Lane II: PCR control
without uny template. M: 100 bp ladder. Note that no bands
are visible in lane 10 and II. B. PCR using Eubacteriaspecific universal primers. PCR products were migruted in
1% agarose gel, and stained with ethidium bromide. M: 100
bp ladder. Lane I-9: patients with PBC. Lane 10: a patient
with PSC. Lane 1 I: a patient with chronic heputitis. Lanes
12-13: normal individuuals. Lune 14: E. coli DNA, 10 pg.
Lane 15: sample control, no liver specimens in DNA extraction. Lane 16: PCR control without any template. M: 100
bp ladder. Note thut no bands are visible in 1ane 12, 13, 15
and lane 16.
DNA sequencing oj PCR products
PCR products were subcloned into pCR@ 2.1 Vector and transformed
using the Original
TA Cloning@ Kit (Invitrogen,
Carlsbad,
CA,
USA). PCR reactions were used to confirm whether the colonies contain the transformants.
The colonies were picked by a sterile toothpick and directly transferred
to a 0.2 ml tube containing
the PCR
reaction mixture with 0.2 PM of M 13 forward (5’ GTA AAA CGA CGG CCA GT 3’) and M 13 reverse (5’ GGA AAC AGC TAT GAC CAT G 3’) primers, 0.2 mM of each deoxynucleoside
triphosphate, 0.25 U of AmpliTaq -rM Gold DNA Polymerase
and 1 nl of
GeneAmp@ IOXPCR buffer containing
I .5 mM MgClz (PE Applied
Biosystems,
Foster City, CA. USA). Colonies with inserted DNA
Infectious agents in PBC
were cultured overnight in 3 ml LB media containing 50 &ml of
ampicillin. The plasmids were purified by QIAGEN Plasmid Mini
Kit (QIAGEN, Basel, Switzerland). DNA sequencing was carried out
using an automated sequencer, and homology searches were performed by NCBI database by using BLAST analysis (http://
www.ncbi.nlm.nih.gov/BLAST/).
Results
Detection and classtjication of Eubacteria
First, we examined for the presence of Eubacteria in
the liver specimens. The sensitivity of our techniques
was demonstrated by the fact that as little as 10 fg of
DNA extracted from Escherichia coli utilized as a positive control was readily detected. In addition, data obtained also confirmed that the two negative controls
(one was a sample control which consisted of the solution utilized for DNA extraction without any liver
specimens and was used as a template, and the other
was a PCR control without any template) did not give
any PCR products (Fig. 1A). We used liver DNA as
templates from nine patients with PBC as well as four
controls, including one PSC, one chronic hepatitis and
two normal individuals. We detected distinct bands in
all specimens examined, except for normal individuals,
by PCR reaction using Pfi DNA polymerase (Fig. 1B).
One hundred and twenty-seven clones from PBC
livers and 10 clones from control livers were cloned and
sequenced. The sequencing data were compared with
the NCBI database, and the results are shown in Table
TABLE 2
Sequencing analysis of 16s rRNA gene amplified by the universal Eubacteria primers from primary biliary cirrhosis (PBC) and control livers
Liver specimes
PSC=primary
Number of
sequenced
clones
Identified Eubacteria
Frequency
Bacillus brevis
Brevibacillus formosi
315
215
Acidovorax temperans
Pseudomonas sp.
Hydrogenophaga palleronii
9113
2113
2113
6
Flavobacterium aquatile
Beta proteobacterium 87
416
216
PBC4
7
Herbaspirilkm seropedicae
Xylophilus ampelinus
317
417
PBC-5
29
Porphyrobacter tepidarius
Xanthomonas hyacinthi
Xanthomonas sp.
Pseudomonas lemoignei
Xanthomonas axonopodis
8129
7129
2129
2129
10129
PBC-6
24
Janthinobacterium lividum
Unidentified Eubacterium
Leptothrix sp.
Phyllobacterium rubiacearum
Myxococcus xanthus
Stenotrophomonas sp.
Xanthmonas albilineans
PBC-7
22
Leptothrix sp.
Zoogloea ramigera
Acinetobacter junii
Alcaligenes defragrans
2122
5122
IO/22
5122
PBC-8
16
Leptothrix sp.
Flexibacter ferrugineum
Janthinobacterium Iividum
Xanthomonas axonopodis
10116
2l16
2l16
2l16
PBC-9
5
Xanthomonas axonopodis
Pseudomonas pseudoalcaligenes
315
2l5
Chronic hepatitis
5
Xanthomonas axonopodis
Arthrobacter ilicis
Leptothrix sp.
315
115
l/5
PSC
5
Janthinobacterium Iividum
Xanthomonas axonopodis
l/5
415
PBC-1
5
PBC-2
13
PBC-3
7124
U24
3124
6124
1124
3f24
2l24
sclerosing cholangitis.
667
A. Tanaka
et al.
tested derived from PBC livers (Fig. 2). All the DNA
templates from control livers also gave distinct bands
of the expected size (data not shown).
452
bp -+
Fig. 2. PCR using primers specific for GAPDH. PCRproducts were migrated in 1% ugurose gel, and stained with ethidium bromide. M: 100 bp ladder. Lanes 1-18: putients with
PBC. Lane 19: sample control, no liver specimens in DNA
extraction. Lane 20: PCR control without uny templute.
M: 100 bp ladder.
1234567M
937
Fig. 3. PCR using Archaeabacteria-specific
universal
primers. PCR products were migrated in I’% agarose gel,
and stained with ethidium bromide. Lane 1: IO0 pg of Haloferax volcanii DNA. Lane 2: 10 pg. Lune 3: I pg. Lane 4.
100 fg. Lane 5: 10 fg. Lane 6: I j& Lane 7: negative control
(PCR products with no template). M: 100 bp ladder. Note
that there is a faint band in lane 6.
Detection of Archaeabacteria
We tried to detect the 16s rRNA gene of Archaeabacteria in the liver specimens. We detected as little as 1
fg of Haloferax volcanii DNA as positive control (Fig.
3). However, no Archaeabacteria
DNA was detected
in any of the 18 PBC liver specimens and 13 control
specimens analyzed (Table 3). We also performed PCR
using 1:10 and 1: 100 diluted template DNA from PBC
livers in case of the presence of inhibitory materials of
PCR in the template, but no PCR products were detected (data not shown).
of Mycobacteria and Helicobacter
Gastric biopsy specimens from Helicobacter pylori-infected individuals and large bowel specimens from
Mycobacterium
aviun?-infected monkey demonstrated
positive results for Helicobacter and Mvcobucteria, respectively (Fig. 4 and 5). The sensitivity of the PCR
technique utilized for both Helicobacter and Mycobacteria sp. were determined to be 10 fg (Fig. 4 and 5),
using serial dilutions of DNA extracted from non-infected liver tissue spiked with 10 ng of Helicobacter
pylori and Mycobacteriu
gordonae DNA. Helicobacter
16s rRNA gene was detected in only one of 29 PBC
livers, and none of the 26 control liver specimens
showed evidence for this organism. Furthermore, sequencing analysis of the positive specimen revealed
that H. pylori was present (Table 3). No Mycobacteria
DNA was found in any of the 29 PBC livers or the 26
control liver specimens (Table 3). Diluted template
DNA failed to produce any PCR products using either
Mycobacteria
or Helicobacter genus-specific primers
(data not shown).
Detection
Discussion
In the studies reported here, we first attempted to detect and identify the profile of Eubacteria and Archaeabacteria present in the liver tissue of patients with PBC
2. The identified clones were randomly distributed
among PBC. Although four clones, Xanthornonas
axonopodis, Janthinobacterium
lividurn, Leptothrix sp.,
and Pseudomonas pseudoalcaligenes,
were found in two
PBC specimens, these clones were also detected in control specimens. Thus, we failed to identify “consensus”
and specific Eubacteria in PBC livers.
Detection
of GAPDH
gene
PCR reactions using GAPDH-specific primers produced the expected 452 bp band in all DNA templates
668
TABLE
3
Summary
teria
of PCR for Archaeahactrria.
Arclueahacteria
Helicohmterirr
Mycohacreria
Helicobncteria
PBC (n=29)
Controls
0129
1/29*
O/29
0126
0126
O/26
* Sequence analysis revealed that Helicobactericr pylwi
in only one patient with PBC.
and Mycohac-
(n=26)
was detected
Infectious agents in PBC
12345678M
400
bp +
Fig. 4. PCR
using Helicobacter
genus-specific
primers.
PCR products were migrated in 1% agarose gel, and stained
with ethidium bromide. Lane 1: 100 pg of DNA from a
human gastric biopsy which was positive for Helicobacter
pylori confirmed by histochemistry. Lane 2: I:102 dilution
of DNA extractedfrom
non-infected liver tissue spiked with
10 ng of Helicobacter pylori DNA (corresponding to 100
pg of Helicobacter pylori DNA). Lanes 3-7: l:l@ to 1:107
dilution, IO pg to I fg DNA. Lane 8: negative control (PCR
products with no template). M: 100 bp ladder. Note that
there is a faint band in lane 6.
specimens, and should not be detected in control specimens. We failed to identify any bacteria that fulfill such
criteria.
We also failed to detect any Archaeabacteria
and
Mycobacteria
DNA in the PBC liver specimens, and
detected Helicobacter DNA in only one PBC specimen.
It is well known that some biological materials, such
as blood or feces, may have PCR-inhibiting effects
(32). Thus the failure to detect Archaeabacteria
DNA
may be caused by the presence of the PCR-inhibiting
materials. However, it is unlikely in this study that biological materials in liver DNA inhibited the amplification of bacterial DNA by Taq polymerase for several
reasons, First, we detected distinct bands with the same
DNA templates using the housekeeping GAPDH-specific primers. Second, even dilutions of 1: 10 or 1: 100
of the DNA templates did not produce any PCR products. Serial dilution suppresses the inhibitory effect of
PCR-inhibiting
materials such as glycoprotein
or
glycolipid, but PCR amplification is not affected by a
1:10 or 1:100 dilution. Third, we obtained positive results using liver tissues spiked with as little as 10 fg of
Helicobacter or Mycobacteria
DNA.
One caveat in these studies is the possibility of spuri-
12345678M
and controls, using PCR amplification of the 16s
rRNA gene. It has been previously noted that contamination of exogenous bacterial DNA into template
DNA or PCR reagents represents a significant issue in
the interpretation of data using this methodology (2931). To minimize contamination by exogenous bacterial DNA, we obtained liver specimens as soon as
possible during liver transplantation, transferred them
into sterile containers, cryopreserved these at -70°C
and subsequently extracted DNA using sterile conditions. Since there were no PCR products using universal 16s rRNA gene specific primers in either the
sample control or the PCR control, we believe that
bacterial contamination is low or non-existent. In addition, we failed to detect any bacterial origin PCR
products in normal liver specimens, which supports
this view. Thus, it is likely that the identified bacteria
listed in Table 2 are truly present in the liver tissues of
PBC patients and controls. The identity of the cloned
sequences was 100% of the published sequences. There
seemed to be, however, no individual or distinct bacterial species present in the majority of PBC liver specimens. Our hypothesis was that the bacteria which
might be associated with pathogenesis of the disease
should be detected in all or most of PBC liver tissue
626
bp +
Fig. 5. PCR using Mycobacteria genus-specific primers.
PCR products were migrated in 1% agarose gel, and stained
with ethidium bromide. Lane 1: 100 pg of DNA from rhesus
macaque large bowel infected with Mycobacterium avium.
Lane 2: 1:1@ dilution of DNA extractedfrom
non-infected
liver tissue spiked with 10 ng of Mycobacteria gordonae
DNA (corresponding to 100 pg of Mycobacteria gordonae
DNA). Lanes 3-7: 1:103 to 1:107 dilution, 10 pg to 1 fg
DNA. Lane 8: negative control (PCR products with no
template). AI: 100 bp ladder. Note that there is a band in
lane 6.
669
A. Tanaka et al.
sequences being generated as a consequence of the
error-prone polymerase activity of Tuq polymerase.
Thus, it is possible that there may be mutations introduced into the cloned PCR products. To avoid this
problem, we used Pfu DNA polymerase for the amplification of 16s rRNA gene, which is known to have the
lowest error rate of any thermostable DNA polymerases. In the case of 16s sequences, scattered mutations introduced in this way might lead to misassignment of sequences, but only to a closely related species.
Thus, if there was a predominant contaminating bacteria, even in the presence of such mutations, one
might expect to discern a predominant genus. No such
observation was made.
We detected Helicobacter DNA in one out of 29 liver
tissues from PBC patients and none of the liver tissues
from the other control patients. The detected clone was
found to be Helicobacter pylori by sequencing. The difference in detection of Helicobacter, however, between
PBC and controls was not statistically significant and
we believe that the presence of Helicobacter pylori in
one PBC liver specimen may be due to chance alone.
Fox et al. reported that Helicobacter species were detected in 13 out of 23 patients with chronic cholecystitis and five out of eight patients with PSC (24,25),
though not all of these were confirmed to be Helicobatter species by sequencing method. Their results
could be interpreted as evidence for the fact that Helicobacter infection in patients with cholestatic disease
might be a secondary phenomenon associated with an
intrahepatic accumulation of bile. Our results, demonstrating that Helicobacter was not detected in another
cholestatic disease, PBC, indicates that Helicobacter infection is not always associated with cholestasis and
may be closely related with the pathogenesis of PSC or
chronic cholecystitis.
It is also noteworthy that no Mycobacteria DNA
was detected in liver specimens from the PBC patients.
Since it has been reported that sera of patients with
PBC reacts with Mycobacteria protein (17), Mycobacteria infection, especially Mycobacteria gordonae, has
been regarded as one potential candidate for the involvement of an infectious agent in the pathogenesis of
PBC. Based on our current findings, we believe that
the reactivity of PBC sera in their report was not
caused by the presence of anti-Mycobacteria gordonae
antibodies, but by the cross-reactivity between autoantigens in PBC and proteins derived from Mycobacteriu
gordonae. Our detection system is capable of detecting
as little as 10 fg of Mycobacteria gordonae DNA
spiked in non-infected liver tissue. Although there
might be less than 10 fg of Mycobacteriu gordonae in
the liver with PBC, it is very unlikely that such a low
ous
670
amount of bacteria could produce proteins which continuously stimulate anti-Mycobacteria antibody production. Also, we note that the annealing temperature
depends on the Tm of primers. The primer set we used
the Tm value was 70°C for the 5’ primer and 65°C for
the 3’ primer. Thus we chose 65°C as the annealing
temperature. In fact a number of bands corresponding
to non-specific products were seen when the annealing
temperature was set at 50°C and 55°C. Taken together,
this could lead one to conclude that Mycobacteria infection plays very little if any role in the etiology of
PBC. We suggest that the data and conclusions presented here should be interpreted with appropriate
caution, as in our studies we examined liver quite late
in the course of disease. The positive detection of bacteria would have required that any involved organism
be chronically present for many years. In this regard,
it would be most interesting to repeat these studies in
patients diagnosed with PBC relatively recently and/or
during a pre-clinical phase. Unfortunately, however, it
is very difficult to obtain such samples for logistical
reasons and because PBC may have a very long preclinical phase.
In conclusion, we failed to detect any bacteria which
seemed to be present specifically in PBC liver specimens. Although the possibility of bacterial infection,
particularly with intracellular organisms, is an attractive hypothesis in PBC, there is, at least based on the
data presented here and the technology applied, no evidence based on utilization of PCR techniques to suggest an ongoing infectious process. The state of affairs
at the time of induction of the disease process remains
unknown.
Acknowledgements
Supported in part by NIH grant DK 39588.
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