J Oral Pathol Med (2014) 43: 576–578 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
doi: 10.1111/jop.12194
wileyonlinelibrary.com/journal/jop
No evidence for Helicobacter pylori in oral lichen planus Shwetha R. Hulimavu, Leeky Mohanty, Narayan V. Tondikulam, Sadhana Shenoy, Saleha Jamadar, Abhishek Bhadranna Department of Oral and Maxillofacial Pathology, The Oxford Dental College, Bengaluru, Karnataka, India
OBJECTIVES: Oral lichen planus is a T-cell-mediated mucosal disease of unknown etiology. Numerous predisposing factors have been put forward in the etiology of this disease. This includes stress, drugs, genetic susceptibility, certain viruses, and bacterial infections. Recently, there have been studies published on possible role of Helicobacter pylori infection in pathogenesis of mucocutaneous diseases including oral lichen planus (OLP). The aim of this study was to detect immunohistochemically the presence of Helicobacter pylori in oral lichen planus. MATERIALS AND METHODS: Paraffin-embedded tissue blocks of 50 cases of OLP and 10 cases of normal buccal mucosal biopsies and 6 endoscopic biopsies of patients with peptic ulcer (control group) were sectioned and stained by hematoxylin and eosin. Serial sections of same were stained immunohistochemically using Anti-Helicobacter pylori antibody and observed under microscope for presence or absence of Helicobacter pylori. RESULTS: Except for the control group, none of the cases of OLP and normal buccal mucosal biopsies showed positivity for Helicobacter pylori. CONCLUSION: As we did not detect the presence of Helicobacter pylori in any of the OLP cases, we question the role of these organisms in the pathogenesis of OLP planus if any. J Oral Pathol Med (2014) 43: 576–578 Keywords: Helicobacter pylori; Immunohistochemistry; Oral lichen planus
Introduction Helicobacter pylori (H. pylori) are gram-negative, microaerophilic, and spiral- or coccoid-shaped bacteria considered to be the most common human pathogen colonizing the gastric mucosa, affecting nearly half of the world’s popuCorrespondence: Dr. Leeky Mohanty, Department of Oral and Maxillofacial Pathology, The Oxford Dental College, Bengaluru-560068, Karnataka, India. Tel: +919845067066, Fax: 080-2573 4656, E-mail:leekymohanty@ yahoo.com Accepted for publication March 3, 2014
lation (1). In addition to its role in causing gastritis, gastric ulcer, duodenal ulcer, gastric carcinoma, or mucosaassociated lymphoid tissue lymphoma, it has been shown to cause nongastrointestinal diseases such as rosacea, chronic urticaria, psoriasis, Behcet’s syndrome, cutaneous pruritus, and lichen planus (2). However, studies investigating the possible role of this bacterial agent in oral lichen planus (OLP) are limited, and they have generally failed to conclusively establish any causative role it may have in the pathogenesis of OLP. One of the reasons for this could be the nonspecific tests that were used in many of these studies (3–5). In this study, we have used immunohistochemistry (IHC) to look for the presence of H. pylori in OLP.
Materials and methods Fifty paraffin-embedded tissue blocks of cases diagnosed histopathologically as OLP, and 10 cases of normal buccal mucosal biopsies were retrieved from the archives of Department of Oral and Maxillofacial Pathology. Of the 50 OLP cases included, 16 cases were clinically diagnosed as reticular OLP, 17 cases were erosive OLP, 3 cases were bullous type of OLP, and in the remaining 14 cases, clinical information on the type of OLP was not available. As H.pylori has been proved unequivocally to be present in peptic ulcers, we took 6 endoscopic biopsies of patients with peptic ulcer as positive controls. The present study was reviewed and approved by our institutional ethical committee review board (Ref no: 715/10-11). Immunohistochemistry Serial sections of paraffin-embedded blocks were cut at a thickness of 4 lm for routine histological and subsequent immunohistochemical examinations. The sections taken for IHC were first mounted on poly-L-lysine-coated glass slides and incubated at 55–60°C overnight. Following this, the sections were dewaxed in xylene and hydrated through different grades of isopropyl alcohol. The sections were then quenched in methanol and 3% hydrogen peroxidase for 20 min at room temperature to block endogenous peroxidase activity. Antigen retrieval was carried out by microwave with 0.01 M sodium citrate buffer solution (pH 6.0) for three cycles of 800 W for 5 min twice and 200 W for
H. Pylori and oral lichen planus Hulimavu et al.
14 min and later was subjected to two washes of tris buffer solution for 5 min each. Sections were then incubated with primary antibody (rabbit polyclonal to H. pylori, Novacastra, Bengaluru, India) at 1:80 dilutions for 1 hour. After washing with tris buffer solution, the sections were then incubated for 30 min with anti-mouse secondary antibody and visualized using 3, 30 -diaminobenzidine (DAB) chromogen. Counter staining was carried out with Harris hematoxylin. Sections were then viewed under the microscope; three high power fields (209) per slide were assessed for the cytotoxic associated gene A expression on the outer membrane of H. pylori.
577
A
Statistical analysis The statistical software namely SPSS 11, Bengaluru, India was used for the analysis of the data, and Microsoft Word and Excel have been used to generate tables. B
Results In our study, the age of the patients ranged from 7 to 68 years. The mean age of the patients was 42.89 15.79 years; of these, 54% were males and 46% were females. The presence or absence of H. pylori was assessed. In all the gastric mucosal biopsies, H. pylori bacteria were found in the lumen of the parietal glands (Fig. 1A–B). All cases of OLP irrespective of its clinical variant as well as the normal buccal mucosa were negative for H. pylori.
Discussion Oral lichen planus is a T-cell-mediated mucosal disease with unknown etiology. Both antigen specific and nonspecific mechanisms have been hypothesized in its pathogenesis (6) including bacterial agents like H. pylori (7). Only limited studies have been published till date investigating the possible role of H.pylori in OLP and are mainly based on nonspecific methods of identification of H. pylori which included serology, culture, and urea breath test (3–5). Immunohistochemical technique is a very sensitive (>90%) and a specific test; the H. pylori organisms can be easily identified by the pathologist even if they are few in number and hence lower interobserver variability. Perhaps most importantly, IHC can detect posttreatment coccoid forms of H. pylori which usually cannot be cultured (8). Helicobacter pylori detection in OLP has ranged from 0% to 100% in various studies (3–5, 9–11). Serological analysis found no significant association between H.pylori infection and OLP, although IgG antibodies were found in 51% of patients with OLP and 66% of control patients (3). Another study combining serology with culture found all cases to be negative (0%) for H. pylori (4). In a study carried out using urea breath test, which relies on urease activity of H. pylori, 80% of patients with OLP were found to be positive for H. pylori, but they also found H. pylori positivity in 73% of patients with skin lichen planus and 66% H. pylori positivity in control groups but without any statistical significance (5).
Figure 1 (A) Helicobacter pylori organisms seen in the lumen of parietal glands of gastric mucosa (H&E stain, 200X). (B) H. pylori organisms seen in the lumen of parietal glands of gastric mucosa (IHC using anti-H. pylori antibody, 2009).
With the advances in molecular technology, the potential difficulties encountered with other methods have been circumvented by the use of PCR. A study carried out using this technique for detection of H. pylori DNA in OLP patients found all cases of erosive OLP (100%) to be positive, whereas all the cases of nonerosive OLP (0%) were found to be negative for H. pylori DNA (9). In contrast, another PCR study failed to detect H pylori DNA in any of the 20 cases of OLP (0%) and normal mucosa (10). Possible reasons for the variations could be attributed to the sample size, geographic variation, contamination artifacts, and technique used for identification (11). Helicobacter pylori being epitheliotropic, we used another molecular technique like IHC to specifically locate these organisms in the epithelium of OLP cases. Although IHC techniques have been used in the detection of H. pylori in gastric mucosal biopsies (8, 12), to the best of our knowledge, no studies have been published in literature on OLP. In spite of using a highly sensitive and specific method, we failed to detect any H. pylori organisms, irrespective of the clinical variant of oral lichen planus.
J Oral Pathol Med
H. Pylori and oral lichen planus Hulimavu et al.
578
Studies investigating the presence of H. pylori in normal oral mucosa, saliva, supragingival and subgingival plaque have shown varying detection rates of H. pylori from 0 to 100% (11). So this raises the doubt whether oral cavity acts as a real H.pylori reservoir or whether it is transiently stored in the mouth as a result of gastroesophageal reflux or when passing to the stomach (13). In our study, when the normal buccal mucosal biopsies were assessed for presence of H. pylori, none of the biopsies showed positivity. To conclude, in our study, despite using a specific technique like IHC with a substantial sample size, we did not detect H. pylori organisms in any of our OLP cases or normal buccal mucosal tissues. Thus, we question the very existence of these organisms in the oral cavity and its role in the pathogenesis of oral lichen planus if any.
References 1. Zou QH, Li RQ. Helicobacter pylori in the oral cavity and gastric mucosa: a meta analysis. J Oral Pathol Med 2011; 40: 317–24. 2. Hernando Harder AC, Booken N, Goerdit S, Singer MV, Herman H. Helicobacter pylori infection and dermatologic diseases. Euro J Dermatol 2009; 19: 431–44. 3. Poushahidi S, Farnaz F, Ebrahimi H, et al. Lack of association between Helicobacter pylori infection and oral lichen planus. Asian Pac J Cancer Prev 2012; 13: 1745–7. 4. Shimoyama T, Horie N, Kato T, Kaneko T, Komiyama K. Helicobacter Pylori in oral ulcerations. J Oral Sci 2000; 42: 225–9. 5. Zenouz TA, Mehdipour M, Heydarlou MJ, Gholizadeh N. Relationship between lichen planus and Helicobacter pylori infection. J Dent Res Dent Clin Dent Prospect 2010; 4: 17–20.
J Oral Pathol Med
6. Sugerman PB, Savage NW, Wallsh LJ, Zhao ZZ, Zhou XJ, Khan A. The pathogenesis of oral lichen planus. Crit Rev Oral Biol Med 2002; 13: 350–65. 7. Moravvej H, Hoseini H, Barikbin B, Molekzdeh R, Razavi GM. Association of Helicobacter pylori with lichen planus. Indian J Dermatol 2008; 52: 138–40. 8. Jonkers D, Stobberingh E, De Bruine A, Arends JW, Stockbugger R. Evaluation of immunohistochemistry for the detection of Helicobacter pylori in gastric mucosal biopsies. J Infect 2007; 35: 149–54. 9. Attia E, Abdel Fattah NS, Abdella HM. Upper gastrointestinal findings and detection of Helicobacter pylori in patients with oral lichen planus. Clin Exp Dermatol 2010; 35: 355–60. 10. Riggio MP, Lennon A, Wray D. Detection of Helicobacter pylori DNA in recurrent aphthous stomatitis tissue by PCR. J Oral Pathol Med 1999; 29: 507–13. 11. Burgers R, Schneider BW, Reischl U, et al. Helicobacter pylori in human oral cavity and stomach. Eur J Oral Sci 2008; 116: 297–304. 12. Semino-Mora C, Doi SQ, Marty A, Simko V, Carlstedt I, Dubois A. Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma. J Infect Dis 2003; 187: 1165–77. 13. McCarthy PL, Shklar G. Lichen Planus. Diseases of the oral mucosa, 2nd edn. Philadelphia: Lea and Febriger, 1980; 203–4.
Acknowledgement We thank Dr. Aparna, Senior consultant pathologist, Narayana Hrudayalaya for providing tissue sections of gastric mucosal biopsies for our study.
Conflict of interest None.
doi: 10.1111/jop.12194
wileyonlinelibrary.com/journal/jop
No evidence for Helicobacter pylori in oral lichen planus Shwetha R. Hulimavu, Leeky Mohanty, Narayan V. Tondikulam, Sadhana Shenoy, Saleha Jamadar, Abhishek Bhadranna Department of Oral and Maxillofacial Pathology, The Oxford Dental College, Bengaluru, Karnataka, India
OBJECTIVES: Oral lichen planus is a T-cell-mediated mucosal disease of unknown etiology. Numerous predisposing factors have been put forward in the etiology of this disease. This includes stress, drugs, genetic susceptibility, certain viruses, and bacterial infections. Recently, there have been studies published on possible role of Helicobacter pylori infection in pathogenesis of mucocutaneous diseases including oral lichen planus (OLP). The aim of this study was to detect immunohistochemically the presence of Helicobacter pylori in oral lichen planus. MATERIALS AND METHODS: Paraffin-embedded tissue blocks of 50 cases of OLP and 10 cases of normal buccal mucosal biopsies and 6 endoscopic biopsies of patients with peptic ulcer (control group) were sectioned and stained by hematoxylin and eosin. Serial sections of same were stained immunohistochemically using Anti-Helicobacter pylori antibody and observed under microscope for presence or absence of Helicobacter pylori. RESULTS: Except for the control group, none of the cases of OLP and normal buccal mucosal biopsies showed positivity for Helicobacter pylori. CONCLUSION: As we did not detect the presence of Helicobacter pylori in any of the OLP cases, we question the role of these organisms in the pathogenesis of OLP planus if any. J Oral Pathol Med (2014) 43: 576–578 Keywords: Helicobacter pylori; Immunohistochemistry; Oral lichen planus
Introduction Helicobacter pylori (H. pylori) are gram-negative, microaerophilic, and spiral- or coccoid-shaped bacteria considered to be the most common human pathogen colonizing the gastric mucosa, affecting nearly half of the world’s popuCorrespondence: Dr. Leeky Mohanty, Department of Oral and Maxillofacial Pathology, The Oxford Dental College, Bengaluru-560068, Karnataka, India. Tel: +919845067066, Fax: 080-2573 4656, E-mail:leekymohanty@ yahoo.com Accepted for publication March 3, 2014
lation (1). In addition to its role in causing gastritis, gastric ulcer, duodenal ulcer, gastric carcinoma, or mucosaassociated lymphoid tissue lymphoma, it has been shown to cause nongastrointestinal diseases such as rosacea, chronic urticaria, psoriasis, Behcet’s syndrome, cutaneous pruritus, and lichen planus (2). However, studies investigating the possible role of this bacterial agent in oral lichen planus (OLP) are limited, and they have generally failed to conclusively establish any causative role it may have in the pathogenesis of OLP. One of the reasons for this could be the nonspecific tests that were used in many of these studies (3–5). In this study, we have used immunohistochemistry (IHC) to look for the presence of H. pylori in OLP.
Materials and methods Fifty paraffin-embedded tissue blocks of cases diagnosed histopathologically as OLP, and 10 cases of normal buccal mucosal biopsies were retrieved from the archives of Department of Oral and Maxillofacial Pathology. Of the 50 OLP cases included, 16 cases were clinically diagnosed as reticular OLP, 17 cases were erosive OLP, 3 cases were bullous type of OLP, and in the remaining 14 cases, clinical information on the type of OLP was not available. As H.pylori has been proved unequivocally to be present in peptic ulcers, we took 6 endoscopic biopsies of patients with peptic ulcer as positive controls. The present study was reviewed and approved by our institutional ethical committee review board (Ref no: 715/10-11). Immunohistochemistry Serial sections of paraffin-embedded blocks were cut at a thickness of 4 lm for routine histological and subsequent immunohistochemical examinations. The sections taken for IHC were first mounted on poly-L-lysine-coated glass slides and incubated at 55–60°C overnight. Following this, the sections were dewaxed in xylene and hydrated through different grades of isopropyl alcohol. The sections were then quenched in methanol and 3% hydrogen peroxidase for 20 min at room temperature to block endogenous peroxidase activity. Antigen retrieval was carried out by microwave with 0.01 M sodium citrate buffer solution (pH 6.0) for three cycles of 800 W for 5 min twice and 200 W for
H. Pylori and oral lichen planus Hulimavu et al.
14 min and later was subjected to two washes of tris buffer solution for 5 min each. Sections were then incubated with primary antibody (rabbit polyclonal to H. pylori, Novacastra, Bengaluru, India) at 1:80 dilutions for 1 hour. After washing with tris buffer solution, the sections were then incubated for 30 min with anti-mouse secondary antibody and visualized using 3, 30 -diaminobenzidine (DAB) chromogen. Counter staining was carried out with Harris hematoxylin. Sections were then viewed under the microscope; three high power fields (209) per slide were assessed for the cytotoxic associated gene A expression on the outer membrane of H. pylori.
577
A
Statistical analysis The statistical software namely SPSS 11, Bengaluru, India was used for the analysis of the data, and Microsoft Word and Excel have been used to generate tables. B
Results In our study, the age of the patients ranged from 7 to 68 years. The mean age of the patients was 42.89 15.79 years; of these, 54% were males and 46% were females. The presence or absence of H. pylori was assessed. In all the gastric mucosal biopsies, H. pylori bacteria were found in the lumen of the parietal glands (Fig. 1A–B). All cases of OLP irrespective of its clinical variant as well as the normal buccal mucosa were negative for H. pylori.
Discussion Oral lichen planus is a T-cell-mediated mucosal disease with unknown etiology. Both antigen specific and nonspecific mechanisms have been hypothesized in its pathogenesis (6) including bacterial agents like H. pylori (7). Only limited studies have been published till date investigating the possible role of H.pylori in OLP and are mainly based on nonspecific methods of identification of H. pylori which included serology, culture, and urea breath test (3–5). Immunohistochemical technique is a very sensitive (>90%) and a specific test; the H. pylori organisms can be easily identified by the pathologist even if they are few in number and hence lower interobserver variability. Perhaps most importantly, IHC can detect posttreatment coccoid forms of H. pylori which usually cannot be cultured (8). Helicobacter pylori detection in OLP has ranged from 0% to 100% in various studies (3–5, 9–11). Serological analysis found no significant association between H.pylori infection and OLP, although IgG antibodies were found in 51% of patients with OLP and 66% of control patients (3). Another study combining serology with culture found all cases to be negative (0%) for H. pylori (4). In a study carried out using urea breath test, which relies on urease activity of H. pylori, 80% of patients with OLP were found to be positive for H. pylori, but they also found H. pylori positivity in 73% of patients with skin lichen planus and 66% H. pylori positivity in control groups but without any statistical significance (5).
Figure 1 (A) Helicobacter pylori organisms seen in the lumen of parietal glands of gastric mucosa (H&E stain, 200X). (B) H. pylori organisms seen in the lumen of parietal glands of gastric mucosa (IHC using anti-H. pylori antibody, 2009).
With the advances in molecular technology, the potential difficulties encountered with other methods have been circumvented by the use of PCR. A study carried out using this technique for detection of H. pylori DNA in OLP patients found all cases of erosive OLP (100%) to be positive, whereas all the cases of nonerosive OLP (0%) were found to be negative for H. pylori DNA (9). In contrast, another PCR study failed to detect H pylori DNA in any of the 20 cases of OLP (0%) and normal mucosa (10). Possible reasons for the variations could be attributed to the sample size, geographic variation, contamination artifacts, and technique used for identification (11). Helicobacter pylori being epitheliotropic, we used another molecular technique like IHC to specifically locate these organisms in the epithelium of OLP cases. Although IHC techniques have been used in the detection of H. pylori in gastric mucosal biopsies (8, 12), to the best of our knowledge, no studies have been published in literature on OLP. In spite of using a highly sensitive and specific method, we failed to detect any H. pylori organisms, irrespective of the clinical variant of oral lichen planus.
J Oral Pathol Med
H. Pylori and oral lichen planus Hulimavu et al.
578
Studies investigating the presence of H. pylori in normal oral mucosa, saliva, supragingival and subgingival plaque have shown varying detection rates of H. pylori from 0 to 100% (11). So this raises the doubt whether oral cavity acts as a real H.pylori reservoir or whether it is transiently stored in the mouth as a result of gastroesophageal reflux or when passing to the stomach (13). In our study, when the normal buccal mucosal biopsies were assessed for presence of H. pylori, none of the biopsies showed positivity. To conclude, in our study, despite using a specific technique like IHC with a substantial sample size, we did not detect H. pylori organisms in any of our OLP cases or normal buccal mucosal tissues. Thus, we question the very existence of these organisms in the oral cavity and its role in the pathogenesis of oral lichen planus if any.
References 1. Zou QH, Li RQ. Helicobacter pylori in the oral cavity and gastric mucosa: a meta analysis. J Oral Pathol Med 2011; 40: 317–24. 2. Hernando Harder AC, Booken N, Goerdit S, Singer MV, Herman H. Helicobacter pylori infection and dermatologic diseases. Euro J Dermatol 2009; 19: 431–44. 3. Poushahidi S, Farnaz F, Ebrahimi H, et al. Lack of association between Helicobacter pylori infection and oral lichen planus. Asian Pac J Cancer Prev 2012; 13: 1745–7. 4. Shimoyama T, Horie N, Kato T, Kaneko T, Komiyama K. Helicobacter Pylori in oral ulcerations. J Oral Sci 2000; 42: 225–9. 5. Zenouz TA, Mehdipour M, Heydarlou MJ, Gholizadeh N. Relationship between lichen planus and Helicobacter pylori infection. J Dent Res Dent Clin Dent Prospect 2010; 4: 17–20.
J Oral Pathol Med
6. Sugerman PB, Savage NW, Wallsh LJ, Zhao ZZ, Zhou XJ, Khan A. The pathogenesis of oral lichen planus. Crit Rev Oral Biol Med 2002; 13: 350–65. 7. Moravvej H, Hoseini H, Barikbin B, Molekzdeh R, Razavi GM. Association of Helicobacter pylori with lichen planus. Indian J Dermatol 2008; 52: 138–40. 8. Jonkers D, Stobberingh E, De Bruine A, Arends JW, Stockbugger R. Evaluation of immunohistochemistry for the detection of Helicobacter pylori in gastric mucosal biopsies. J Infect 2007; 35: 149–54. 9. Attia E, Abdel Fattah NS, Abdella HM. Upper gastrointestinal findings and detection of Helicobacter pylori in patients with oral lichen planus. Clin Exp Dermatol 2010; 35: 355–60. 10. Riggio MP, Lennon A, Wray D. Detection of Helicobacter pylori DNA in recurrent aphthous stomatitis tissue by PCR. J Oral Pathol Med 1999; 29: 507–13. 11. Burgers R, Schneider BW, Reischl U, et al. Helicobacter pylori in human oral cavity and stomach. Eur J Oral Sci 2008; 116: 297–304. 12. Semino-Mora C, Doi SQ, Marty A, Simko V, Carlstedt I, Dubois A. Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma. J Infect Dis 2003; 187: 1165–77. 13. McCarthy PL, Shklar G. Lichen Planus. Diseases of the oral mucosa, 2nd edn. Philadelphia: Lea and Febriger, 1980; 203–4.
Acknowledgement We thank Dr. Aparna, Senior consultant pathologist, Narayana Hrudayalaya for providing tissue sections of gastric mucosal biopsies for our study.
Conflict of interest None.
