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Dong-Feng Chen, Lu Hu, Ping Yi, Wei-Wen Liu, Dian-Chun Fang, Hong Cao, Department of Gastroenterology, Daping Hospital,Third Military Medical University, Chongqing 400042, China Supported by the National Natural Science Foundation of China, No. 39970039 Correspondence to: Dr. Dong-Feng Chen, Department of Gastroenterology, Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China. dfchen9@hotmail.com Telephone: +86-23-68757362 Fax: +86-23-68813806 Received: 2005-08-01 Accepted: 2007-01-23
Abstract AIM: To study whether H pylori locate in the gallbladder mucosa of patients with chronic cholecystitis.
METHODS: Using Warthy-Starry (W-S) silver stain and immunohistochemistry stain with anti-H pylori antibodies, we screened paraffin specimens in 524 cases of cholecystitis. H pylori urease gene A (HPUA) and H pylori urease gene B (HPUB) were analyzed by polymerase chain reaction (PCR) in the fresh tissue specimens from 81 cases of cholecystitis.
RESULTS: H pylori-like bacteria were found in 13.55% of the gallbladders of the cholecystitis patients using W-S stain. Meanwhile, bacteria positive for H pylori antibodies were also found in 7.1% of the gallbladders of patients with cholecystitis by immunohistochemistry. Of 81 gallbladders, 11 were positive for both HPUA and HPUB, 4 were positive for HPUA only and 7 were positive for HPUB only.
CONCLUSION: H pylori exist in the gallbladders of patients with chronic cholecystitis.
© 2007 The WJG Press. All rights reserved.
Key words: H pylori; Gallbladder mucosa; Chronic cholecystitis; Polymerase chain reaction; H pylori urease gene
Chen DF, Hu L, Yi P, Liu WW, Fang DC, Cao H. H pylori exist in the gallbladder mucosa of patients with chronic cholecystitis. World J Gastroenterol 2007; 13(10): 1608-1611
http://www.wjgnet.com/1007-9327/13/1608.asp
INTRODUCTION H pylori closely correlate with chronic gastritis[1-5], peptic ulcer[6-8], gastric carcinoma and malignant lymphoma of gastric mucosa-related lymphoid tissues (MALToma)[9-11]. Recently, it has been found that H pylori have certain relationship to some diseases in the organs besides the stomach and duodenum[12]. However, it is still unclear whether H pylori have any correlation with adjacent structures of the stomach[13], the liver and the gallbladder[14,15]. In the present study, we compared the detection rate of H pylori in the stomachs of patients with chronic cholecystitis using Warthy-Starry (W-S) silver stain, immunohistochemistry and polymerase chain reaction (PCR) and showed that H pylori exist in the gallbladders.
MATERIALS AND METHODS Subjects A total of 142 patients who had chronic cholecystitis confirmed by pathologic examination since 1995 in our hospital and received gastroscopy and H pylori urease test were included. Randomly chosen patients without diseases of the gallbladder, who received gastroscopy due to symptoms of digestive tract were used as control group. The paraffin sections of 524 cholecystitis specimens were stained with W-S to observe H pylori-like bacteria. From fresh gallbladder specimens of 81 patients receiving cholecystectomy due to cholelithiasis and chronic cholecystitis in our hospital, DNA was extracted for PCR amplification.
Methods Paraffin specimens of cholecystitis were sliced into 4 µm sections, stained with W-S and sealed with D.P.X for an optic microscopic observation for the existence of H pylori-like bacteria. Immunohistochemistry stain of H pylori was made using streptovitacin peroxidase (SP). Anti-H pylori antibody (Dako Corporation) was diluted to 1:10. At the same time, PBS was used as blank control, normal blood serum as negative control and the sections of gastric mucosa with positive H pylori as positive control. PCR amplification of H pylori urease genes A and B was carried out on 81 specimens. The gallbladder was placed in a prepared bottle for DNA extraction from gallbladder mucosa and frozen at -20˚C. The procedures of PCR amplification were as follows: (1) Primer synthesis of urease gene A (HPUA) 1 and HPUA2 was based on nucleic acids 304-714 of urease gene A, with an amplification portion of 411 bp. While the primers of urease gene B (HPUB) 1 and HPUB2 were designed according to nucleic acids 1971-2102 of urease gene B, with amplification part of 132 bp. These two primers maintained a high specificity and primer arrays that were HPUA1: 5'-GCCAATGGTAAATTAGTT-3'; HPUA2: 5'-CTCCTTAATTGTTTAC-3'; HPUB1: 5'-TGGGATTAGCGAGTATGT-3'; HPUB2: 5'-CCCATTTGACTCAATG-3', respectively, the primers of which were synthesized by Shanghai Bioengineering Corporation. (2) Reaction systems and parameters were as described previously[16]; (3) Gel electrophoresis analysis of the products of PCR amplification was performed under ultraviolet light and compared with the marker.
Statistical analysis The data were processed with Chi-square test and P < 0.05 was considered a significant difference.
RESULTS H pylori in the stomach of patients with chronic cholecystitis
There were 142 patients with chronic cholecystitis
including 13 with gastric ulcer detected by gastroscopy, 8 with
duodenal ulcer and 121 with chronic gastritis, of which 76 had bile
reflux. A total of 123 randomly chosen patients with digestive
symptoms who were examined by gastroscopy and without gallbladder
diseases were used as control, of which 6 cases were with gastric
ulcer, 7 with duodenal ulcer, 2 with gastric carcinoma and 108 with
chronic gastritis. Bile reflux occurred in 36 cases in the control
group. In both groups, the detection rates (61/123 vs 81/142)
of H pylori in the stomach were similar, but the detection
rate of the bile reflux in the cholecystitis group was significantly
higher than that in the control group (36/123 vs 76/142 P
< 0.01, Table 1). There was no significant difference with
respect of detection rate of
W-S silver stain of the filed cholecystitis paraffin Under optic microscopes, in contrast with the yellow staining of the gastric mucosa, there could be seen bended, curved and spiral brown bacteria in the epithelial cells of the gastric mucosa that were used as positive control. Using W-S silver stain, H pylori-like bacteria could be seen in 71 (13.6%) out of 524 cholecystitis specimens under optic microscopes, showing curved, pole-like, bended, spiral and fusiform bacteria, and also some spherical shaped bacteria. In 34 specimens, besides positive H pylori-like bacteria, other bacteria could be seen (Figure 1).
Immunohistochemical observation of anti-H pylori antibody
After 71 specimens positive for H pylori-like
bacteria were stained immunohistochemically with anti-H pylori
antibody, bacteria positive for H pylori antibody could
be seen in only 37 cases (52.1%) under optic microscopes. That is to
say, bacteria positively stained with anti-
PCR amplification of HPUA and HPUB Of 81 cholecystectomy specimens, positive amplification zone of HPUA or HPUB was seen in 22 (27.2%), including 11 (13.6%) with positive amplification zones of HPUA and HPUB, 4 with positive HPUA only and 7 with positive HPUB only. However, in 6 gastric mucosa specimens as positive control, the two primers had positive amplification zones of HPUA and HPUB (Figure 3).
DISCUSSION
By means of gastroscopy, we have found that the
incidence rate of bile reflux in patients with chronic cholecystitis
was significantly higher than that in the control group. It
indicates that patients with chronic cholecystitis are apt to bile
regurgitation, which occurs in 80% of patients with chronic
cholecystitis while in only 32% of normal persons[17,18].
H pylori are sensitive to bile salts, especially the
unconjugated bile salts that exert poisonous effect on H pylori.
The latter cannot live in an environment with bile salts[19].
We also found that in the stomachs of patients with chronic lithic
cholecystitis and a bile reflux, H pylori were present in up
to 46.1% (35/76), higher than 44.4% (16/36) in patients without bile
reflux. It indicates that a high incidence rate of H pylori
infection still existed in the stomach although there was a bile
reflux. The results of our study are consistent with that of
Caldwell et al[20], who studied the infection rate
of gastritis and H pylori in patients with chronic calculus
cholecystitis before and after cholecystectomy and found that the
duodenogastric reflux increased after cholecystectomy. The gastritis
was aggravated and the infection rate of H pylori in the
stomach increased from preoperative 32% to postoperative 68%, with a
significant difference (P < 0.05). Therefore, they concluded
that H pylori could live in the basic condition and even
aggravate gastritis. Our study indicates that the patients with
chronic lithic cholecystitis were apt to bile regurgitation;
however, there was still a high infection rate of H pylori in
their stomachs, suggesting that a kind of
In 524 chronic cholecystitis specimens, 71 showed
positive bacteria by W-S silver stain, while only 37 had positive
bacteria by immunohistochemistry stain using anti-H pylori
antibodies. It suggests that W-S silver stain to screen H pylori
in gallbladder specimens is suitable for an initial selection. In
patients with chronic calculus cholecystitis, the infection rate of
H pylori was 7.1% (37/524), a rather low level, which was
confirmed by immunohistochemistry using anti-H pylori
antibodies. After the fresh specimens of 81 patients with chronic cholecystitis treated with cholecystectomy were amplified by PCR with specific primers of HPUA and HPUB, the positive amplification zone existed in 27.2% (22/81), significantly higher than the positive rate (13.6%) detected by W-S silver stain and that (7.1%) by immunohistochemistry stain using H pylori antibodies. This is partly because the template DNA amplified by PCR was extracted from tissues (0.5 cm × 0.5 cm) of gallbladder mucosa and the PCR with a high sensitivity had a high detection rate of H pylori. In the meantime, the PCR was conducted to detect DNA of H pylori; therefore, under many conditions, the positive amplification zone would appear only if H pylori DNA existed in the gallbladder mucosa, whether the H pylori were dead or the remains from previous infections. In the present study, in specimens with positive PCR amplification, 11 were positive for HPUA and HPUB; while in the control group, 6 gastric mucosa specimens infected by H pylori were positive for HPUA and HPUB. It proved that H pylori exist in the gallbladder. However, it still remains unclear whether H pylori are present in the gallbladder of patients with positive HPUA or HPUB, whether the gallbladder is infected by H pylori and whether the genes of H pylori change in the gallbladder[22]. We conclude that H pylori are present in the gallbladder and may relate to the incidence of cholecystitis, which requires further research.
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S- Editor Liu Y L- Editor Zhu LH E- Editor Che YB
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