Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12198
Immunohistochemical Expressions of MUC2, MUC5AC, and MUC6 in Normal, Helicobacter pylori Infected and Metaplastic Gastric Mucosa of Children and Adolescents Ji Sook Park,* Jung-Sook Yeom,* Ji-Hyun Seo,* Jae-Young Lim,* Chan-Hoo Park,* Hyang-Ok Woo,* HeeShang Youn,* Jin-Su Jun,* Ji-Hoe Park,* Gyung-Hyuck Ko,† Seung-Chul Baik,‡ Woo-Kon Lee,‡ Myung-Je Cho‡ and Kwang-Ho Rhee‡ *Department of Pediatrics, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, Jinju, Gyeongsangnam-do, Korea, †Department of Pathology, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, Jinju, Gyeongsangnam-do, Korea, ‡Department of Microbiology, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, Jinju, Gyeongsangnam-do, Korea
Keywords Immunohistochemistry, Helicobacter pylori, gastric mucin. Reprint requests to: Hee-Shang Youn, Department of Pediatrics, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, 90, Chilam dong, Jinju si, Gyeongsangnam-do 660-702, Korea. E-mail: [email protected]
Abstract Objectives: The aim of this study was to investigate expression of gastric mucins in children and adolescents and to assess their relations with age and Helicobacter pylori (H. pylori) infection. Methods: Gastric biopsies were collected from 259 pediatric and adulthood patients with gastrointestinal symptoms among all of patients undergone gastroduodenoscopy from 1990 to 2004 at Gyeongsang National University hospital and assorted based on H. pylori infection, age, and intestinal metaplasia as follows; H. pylori infection before 5 years of age or not, H. pylori infection between 5 and 9 years of age or not, H. pylori infection between 10 and 14 years of age or not, H. pylori infection between 20 and 29 years of age or not and intestinal metaplasia between 21 and 35 years of age. Total 810 tissue slides from the subjects were examined regarding expressions of Mucin2 (MUC2), Mucin5AC (MUC5AC), and Mucin6 (MUC6) in nine groups using immunohistochemical stains. A semiquantitative approach was used to score the staining extent of tissue slide. Results: Increased expressions of MUC2, MUC5AC, and MUC6 were noted in intestinal metaplasia compared with subjects infected with H. pylori between 20 and 29 years. Gastric expressions of MUC5AC were decreased in older than 5 years with H. pylori compared with in older than 5 years without H. pylori (p < .001). Expressions of MUC2 and MUC6 did not change significantly by H. pylori status. Some nuclear expressions of MUC2 and MUC6 were noted in children without intestinal metaplasia. Conclusions: MUC5AC might be affected by chronic H. pylori infection. In addition to biomarkers for intestinal metaplasia or prognostic factors for gastric cancer in adults, MUC2 and MUC6 in children might have an another role, based on ectopic gastric nuclear expressions of MUC2 and MUC6 in children without intestinal metaplasia.
Helicobacter pylori (H. pylori) is one of the most common pathogens in the human gastrointestinal tract. H. pylori infection seems to start from infancy and the infection rate is up to 70–80% during childhood at around 7 years of age in Korea, although there are diverse rates of H. pylori infection according to living areas [1].
© 2015 John Wiley & Sons Ltd, Helicobacter
Although the infection rate has been decreasing recently [2], chronic H. pylori infection is usually associated with diverse gastrointestinal diseases including ulcers, intestinal metaplasia, and gastric cancer during adulthood [3]. Chronic gastric inflammation is usually caused by chronic H. pylori infection and dietary,
1
Gastric Expressions of MUC2, MUC5AC and MUC6
environmental or genetic factors and chronic gastric inflammation could play a role in developing gastric cancer [4]. H. pylori can adhere to the gastric mucosa and affect the gastric microenvironment. The gastric mucin consisting of apomucin and O-linked carbohydrate side chain can protect the gastric epithelium from diverse chemical or physical stimuli. Twelve genes encoding apomucins have been reported. Of them, MUC1, MUC5AC, and MUC6 apomucins are usually expressed in normal human gastric mucosa, MUC2 apomucin is focally expressed in the part of normal human gastric mucosa, but MUC2 is generally expressed in the duodenum [5,6]. The expressions of gastric mucins have been studied as biomarkers for intestinal metaplasia or prognostic factors for gastric cancer in adults [7–10]. But to the best of our knowledge, there has been few reports about gastric mucin in children only [11]. Chronic H. pylori infection can play a pivotal role in the development of intestinal metaplasia [12]. Even though there have been several controversies [13,14], intestinal metaplasia has been considered as a precancerous state and could progress to gastric cancer through several steps [3]. However, H. pylori usually disappears in the human stomach after intestinal metaplasia and the association between gastric epithelial changes and chronic H. pylori infection remains unclear yet [15]. We hypothesized that chronic H. pylori infection might affect expressions of gastric mucins before intestinal metaplasia and tried to investigate the expressions of gastric mucins according to H. pylori infection and ages using immunohistochemical stains.
Materials and Methods Tissue Samples Endoscopic gastric biopsies were obtained from 259 pediatric patients and adulthood volunteers to examine their gastrointestinal symptoms for the purposes of their diagnoses. All study subjects were diagnosed between 1990 and 2004 at the department of Pediatrics, Gyeonsang National University Hospital. The biopsies were kept using paraffin blocks at the National Biobank of Korea after pathologic diagnoses. This study was carried out after obtaining clearance from the ethical board of the hospital (GNUHIRB-5413). Gastric specimens in this study were obtained from the National Biobank of Korea. We studied 115 normal gastric specimens, 111 H. pylori-infected specimens of subjects aged under 30 years and 33 gastric specimens with intestinal metaplasia of subjects between 20 and 35 years of age. Normal, H. pylori-infected gastric mucosa or intestinal
2
Park et al.
metaplasia was proved pathologically by experienced pathologist. Gastric specimens were assorted and divided in nine groups based on H. pylori infection and age as follows; under 5 years of age with H. pylori infection or not, between 5 and 9 years with H. pylori infection or not, between 10 and 14 years with H. pylori infection or not, between 20 and 29 years with H. pylori infection or not, and between 21 and 35 years with intestinal metaplasia (Table 1).
Monoclonal Antibodies Commercial three monoclonal antibodies (Thermoscientific LabVision, Waltham, MA, USA) were used to determine the expression of gastric mucins (MUC2, MUC5AC, and MUC6). Dilutions of MUC2, MUC5AC, and MUC6 were 1 : 100, 1 : 30, and 1 : 100, respectively.
Immunohistochemistry Paraffin sections were cut with 2–3 lm thickness, dewaxed, and rehydrated. MUC2, MUC5AC, and MUC6 immunohistochemistry (IHC) were performed on 810 tissue slides from 259 subjects according to standard procedures as previously described using LP polymer kit (Lab VisionTM UltraVisonTM LP Detection System: HRP Polymer (Ready to Use), Thermoscientific, Waltham, MA, USA) [16]. The biopsy specimens were heated in citrate buffer (pH 6.0) for 25 minutes and washes 4 times. To reduce nonspecific background staining caused by endogenous peroxidase, tissue slides were incubated in hydrogen peroxide block for 10 minutes. We also applied ultra V block and incubated the tissue slides for 5 minutes at room temperature (RT). MUC2, MUC5AC, and MUC6 (primary antibodies) were applied and incubated for 30 minutes at RT and wash for 3 minutes using 0.05 mol/L TRIS–HCl buffer. Primary antibody enhancer (TL-125PB; Thermo Scientific, UK) was applied and washed using buffer solution. Horse-Radish Peroxidase (HRP polymer) was applied at RT in the dark and washed. Diaminobenzidine (DAB) reaction was applied using DAB Plus substrate System (TA-125-HDX; Thermo Scientific) for 3–5 minutes and was stopped after darkening under light microscope. After washing, counter stain and coverslip using an aqueous mounting media were performed. Stains were performed on 272 gastric tissue slides for MUC2, 270 slides for MUC5AC and 268 for MUC6. To check technical errors of IHC, stains of gastric and duodenal tissues from the patients and volunteers were performed at the same time because MUC2 has been known to be expressed in small intestinal mucosa, and MUC5AC and MUC6 generally expressed in gastric
© 2015 John Wiley & Sons Ltd, Helicobacter
Gastric Expressions of MUC2, MUC5AC and MUC6
Park et al.
Table 1 Characteristics of study subjects and their specimens of gastric antrum Group of patients H. pylori
Age (years)
Sex
Number of patients or volunteers
Median age (range in years)
Negative
Immunohistochemical Expressions of MUC2, MUC5AC, and MUC6 in Normal, Helicobacter pylori Infected and Metaplastic Gastric Mucosa of Children and Adolescents Ji Sook Park,* Jung-Sook Yeom,* Ji-Hyun Seo,* Jae-Young Lim,* Chan-Hoo Park,* Hyang-Ok Woo,* HeeShang Youn,* Jin-Su Jun,* Ji-Hoe Park,* Gyung-Hyuck Ko,† Seung-Chul Baik,‡ Woo-Kon Lee,‡ Myung-Je Cho‡ and Kwang-Ho Rhee‡ *Department of Pediatrics, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, Jinju, Gyeongsangnam-do, Korea, †Department of Pathology, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, Jinju, Gyeongsangnam-do, Korea, ‡Department of Microbiology, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, Jinju, Gyeongsangnam-do, Korea
Keywords Immunohistochemistry, Helicobacter pylori, gastric mucin. Reprint requests to: Hee-Shang Youn, Department of Pediatrics, Gyeonsang National University School of Medicine, Institute of Health Science, Gyeongsang National University Hospital, 90, Chilam dong, Jinju si, Gyeongsangnam-do 660-702, Korea. E-mail: [email protected]
Abstract Objectives: The aim of this study was to investigate expression of gastric mucins in children and adolescents and to assess their relations with age and Helicobacter pylori (H. pylori) infection. Methods: Gastric biopsies were collected from 259 pediatric and adulthood patients with gastrointestinal symptoms among all of patients undergone gastroduodenoscopy from 1990 to 2004 at Gyeongsang National University hospital and assorted based on H. pylori infection, age, and intestinal metaplasia as follows; H. pylori infection before 5 years of age or not, H. pylori infection between 5 and 9 years of age or not, H. pylori infection between 10 and 14 years of age or not, H. pylori infection between 20 and 29 years of age or not and intestinal metaplasia between 21 and 35 years of age. Total 810 tissue slides from the subjects were examined regarding expressions of Mucin2 (MUC2), Mucin5AC (MUC5AC), and Mucin6 (MUC6) in nine groups using immunohistochemical stains. A semiquantitative approach was used to score the staining extent of tissue slide. Results: Increased expressions of MUC2, MUC5AC, and MUC6 were noted in intestinal metaplasia compared with subjects infected with H. pylori between 20 and 29 years. Gastric expressions of MUC5AC were decreased in older than 5 years with H. pylori compared with in older than 5 years without H. pylori (p < .001). Expressions of MUC2 and MUC6 did not change significantly by H. pylori status. Some nuclear expressions of MUC2 and MUC6 were noted in children without intestinal metaplasia. Conclusions: MUC5AC might be affected by chronic H. pylori infection. In addition to biomarkers for intestinal metaplasia or prognostic factors for gastric cancer in adults, MUC2 and MUC6 in children might have an another role, based on ectopic gastric nuclear expressions of MUC2 and MUC6 in children without intestinal metaplasia.
Helicobacter pylori (H. pylori) is one of the most common pathogens in the human gastrointestinal tract. H. pylori infection seems to start from infancy and the infection rate is up to 70–80% during childhood at around 7 years of age in Korea, although there are diverse rates of H. pylori infection according to living areas [1].
© 2015 John Wiley & Sons Ltd, Helicobacter
Although the infection rate has been decreasing recently [2], chronic H. pylori infection is usually associated with diverse gastrointestinal diseases including ulcers, intestinal metaplasia, and gastric cancer during adulthood [3]. Chronic gastric inflammation is usually caused by chronic H. pylori infection and dietary,
1
Gastric Expressions of MUC2, MUC5AC and MUC6
environmental or genetic factors and chronic gastric inflammation could play a role in developing gastric cancer [4]. H. pylori can adhere to the gastric mucosa and affect the gastric microenvironment. The gastric mucin consisting of apomucin and O-linked carbohydrate side chain can protect the gastric epithelium from diverse chemical or physical stimuli. Twelve genes encoding apomucins have been reported. Of them, MUC1, MUC5AC, and MUC6 apomucins are usually expressed in normal human gastric mucosa, MUC2 apomucin is focally expressed in the part of normal human gastric mucosa, but MUC2 is generally expressed in the duodenum [5,6]. The expressions of gastric mucins have been studied as biomarkers for intestinal metaplasia or prognostic factors for gastric cancer in adults [7–10]. But to the best of our knowledge, there has been few reports about gastric mucin in children only [11]. Chronic H. pylori infection can play a pivotal role in the development of intestinal metaplasia [12]. Even though there have been several controversies [13,14], intestinal metaplasia has been considered as a precancerous state and could progress to gastric cancer through several steps [3]. However, H. pylori usually disappears in the human stomach after intestinal metaplasia and the association between gastric epithelial changes and chronic H. pylori infection remains unclear yet [15]. We hypothesized that chronic H. pylori infection might affect expressions of gastric mucins before intestinal metaplasia and tried to investigate the expressions of gastric mucins according to H. pylori infection and ages using immunohistochemical stains.
Materials and Methods Tissue Samples Endoscopic gastric biopsies were obtained from 259 pediatric patients and adulthood volunteers to examine their gastrointestinal symptoms for the purposes of their diagnoses. All study subjects were diagnosed between 1990 and 2004 at the department of Pediatrics, Gyeonsang National University Hospital. The biopsies were kept using paraffin blocks at the National Biobank of Korea after pathologic diagnoses. This study was carried out after obtaining clearance from the ethical board of the hospital (GNUHIRB-5413). Gastric specimens in this study were obtained from the National Biobank of Korea. We studied 115 normal gastric specimens, 111 H. pylori-infected specimens of subjects aged under 30 years and 33 gastric specimens with intestinal metaplasia of subjects between 20 and 35 years of age. Normal, H. pylori-infected gastric mucosa or intestinal
2
Park et al.
metaplasia was proved pathologically by experienced pathologist. Gastric specimens were assorted and divided in nine groups based on H. pylori infection and age as follows; under 5 years of age with H. pylori infection or not, between 5 and 9 years with H. pylori infection or not, between 10 and 14 years with H. pylori infection or not, between 20 and 29 years with H. pylori infection or not, and between 21 and 35 years with intestinal metaplasia (Table 1).
Monoclonal Antibodies Commercial three monoclonal antibodies (Thermoscientific LabVision, Waltham, MA, USA) were used to determine the expression of gastric mucins (MUC2, MUC5AC, and MUC6). Dilutions of MUC2, MUC5AC, and MUC6 were 1 : 100, 1 : 30, and 1 : 100, respectively.
Immunohistochemistry Paraffin sections were cut with 2–3 lm thickness, dewaxed, and rehydrated. MUC2, MUC5AC, and MUC6 immunohistochemistry (IHC) were performed on 810 tissue slides from 259 subjects according to standard procedures as previously described using LP polymer kit (Lab VisionTM UltraVisonTM LP Detection System: HRP Polymer (Ready to Use), Thermoscientific, Waltham, MA, USA) [16]. The biopsy specimens were heated in citrate buffer (pH 6.0) for 25 minutes and washes 4 times. To reduce nonspecific background staining caused by endogenous peroxidase, tissue slides were incubated in hydrogen peroxide block for 10 minutes. We also applied ultra V block and incubated the tissue slides for 5 minutes at room temperature (RT). MUC2, MUC5AC, and MUC6 (primary antibodies) were applied and incubated for 30 minutes at RT and wash for 3 minutes using 0.05 mol/L TRIS–HCl buffer. Primary antibody enhancer (TL-125PB; Thermo Scientific, UK) was applied and washed using buffer solution. Horse-Radish Peroxidase (HRP polymer) was applied at RT in the dark and washed. Diaminobenzidine (DAB) reaction was applied using DAB Plus substrate System (TA-125-HDX; Thermo Scientific) for 3–5 minutes and was stopped after darkening under light microscope. After washing, counter stain and coverslip using an aqueous mounting media were performed. Stains were performed on 272 gastric tissue slides for MUC2, 270 slides for MUC5AC and 268 for MUC6. To check technical errors of IHC, stains of gastric and duodenal tissues from the patients and volunteers were performed at the same time because MUC2 has been known to be expressed in small intestinal mucosa, and MUC5AC and MUC6 generally expressed in gastric
© 2015 John Wiley & Sons Ltd, Helicobacter
Gastric Expressions of MUC2, MUC5AC and MUC6
Park et al.
Table 1 Characteristics of study subjects and their specimens of gastric antrum Group of patients H. pylori
Age (years)
Sex
Number of patients or volunteers
Median age (range in years)
Negative
