DOI 10.1007/s10517-015-2913-x Cell Technologies in Biology and Medicine, No. 4, February, 2015
157
Immune Homeostasis of Human Gastric Mucosa in Helicobacter pylori Infection I. V. Reva1, T. Yamamoto3, S. S. Vershinina2, and G. V. Reva1,2 Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 44-51, January, 2015 Original article submitted March 3, 2014 We present the results of electron microscopic, microbiological, immunohistochemical, and molecular genetic studies of gastric biopsy specimens taken for diagnostic purposes according by clinical indications during examination of patients with gastrointestinal pathology. Immune homeostasis of the gastric mucosa against the background of infection with various pathogen strains of Helicobacter pylori was studied in patients of different age groups with peptic ulcer, gastritis, metaplasia, and cancer. Some peculiarities of Helicobacter pylori contamination in the gastric mucosa were demonstrated. Immune homeostasis of the gastric mucosa in different pathologies was analyzed depending on the Helicobacter pylori genotype. Key Words: Helicobacter pylori; immunocytes; metaplasia; peptic ulcer; carcinogenesis
According to WHO data, gastrointestinal (GIT) diseases account for more than 60% morbidities in the world. In Russia, more than 120,000 surgeries are annually performed for pathologies requiring stomach resection, such as cancer or ulcers of the stomach and duodenum. The prevalence of peptic ulcers and stomach cancers steadily increases in most counties [4,12]. In 70-90% cases, stomach cancer is associated with Helicobacter pylori (HbP) infection. The presence of HbP increases the risk of stomach cancer by about 6 times; HbP is detected in about half cases of stomach cancer [9]. It was hypothesized that chronic gastritis leads to intestinal metaplasia (atrophic gastritis) followed by malignant transformation [15]. At the final stages, biopsy can be HbP-negative, but immunological tests show previous infection [5]. In 1994, International Agency for Research on Cancer has recognized HbP as class I carcinogen as the main cause of stomach cancer in humans [3,10,14], but no exhaustive proofs for causative role of HbP were presented until now [8]. The role of HbP (in Russian patients, HbP genome is poorly studied) in the pathogenesis of the ulcer process, metaplasia, and cancer development, as well as its in1 Engineering School, 2School of Biomedicine, Far Eastern Federal University, Vladivostok; Russia; 3International Medical Research Center (IMERC), Niigata, Japan. Address for correspondence: [email protected]. G. V. Reva
fluence on the local immune homeostasis in the gastric mucosa remain unclear. In oncology, the efficiency of the treatment and prevention of mortality depends on early diagnosis, etiological, and pathogenetic factors of this disease. Therefore, diagnostic monitoring should include not only clinical symptoms at different stages of the disease, but also molecular genetic features of HbP. The pathogenesis of peptic ulcer and carcinogenesis against the background HbP infection is now considered depending on the HbP strain, presence of cagA, vacA, and babA genes associated with gastric tumors, and other factors [15]. Inefficiency of antibiotic therapy of HbP infections related to antibiotic resistance, the existence of GIT areas shielding the bacteria from antibiotics, and the appearance of multidrug-resistant HbP isolates (including clarithromycin- and quinolone-resistant strains) require more comprehensive analysis of the ulcerative process in GIT and the role HbP infections in the carcinogenesis. Here we studied the influence of HbP on changes in the gastric mucosa for optimization of the diagnostic methods in gastric ulcer. We addressed the peculiarities of the immune homeostasis of the gastric mucosa under normal and pathological conditions, peculiarities of HbP genome in patients of the Primorsky Territory, and
0007-4888/15/15910157 © 2015 Springer Science+Business Media New York
158 peculiarities of immune homeostasis in case of gastric mucosa infection with various HbP strains.
Cell Technologies in Biology and Medicine, No. 4, February, 2015
TABLE 1. Age-Distribution of the Examinees Age group
MATERIALS AND METHODS The study was conducted in accordance with the provisions of the Declaration of Helsinki (2000) and with the approval of the Ethics Committee of the Far Eastern Federal University. Biological specimens and biopsy samples of the gastric mucosa were obtained from 244 patients aged from 6.5 months to 78 years (Table 1) during fiberoptic gastroduodenoscopy performed at the Russian-Japanese endoscopic center of the Far Eastern Regional Medical Center (2004-2011) and Center for Maternal and Child Welfare (2008-2011). We obtained and analyzed 799 biopsy specimens from different compartments of the gastric mucosa from 244 patients. The mucus obtained from GIT was analyzed by phase-contrast microscopy. Esophagogastrojejunoscopy with gastrobiopsy was performed in all patients with clinical manifestations according to recommendations [2]. For endoscopic examination of the upper GIT, Pentax FG24V and FG29V forward-view fiberoptic instruments were used; biopsy specimens were taken using standard forceps with cup-shaped jaws (KW-2415S, KW-1815S). For examination of the stomach and small intestine below the Treitz ligament in children, a standard pediatric esophagogastroscope (7.8 mm insertion tube diameter) was used. The specimens were taken from the cardia, antrum, and fundus of the stomach according to the Sydney system and WHO gold standard. The results were interpreted according to the criteria of the morphological section of Sydney classification, updates to the international classification of gastritis, and visual analog scale with the standards of semiquantitative evaluation of morphological changes. Sections (3-5 μ) sliced from paraffin blocks were stained with hematoxylin and eosin. In additions, cytological samples (impression smears) were analyzed. The morphological study was based on immunohistochemical phenotyping by differentiation cluster (CD) markers. Immunohistochemical analysis was used for detection of HbP, activity of Ki-67 gene, CD4, CD8, CD68, and CD163 (Dako) for illustration and subsequent comparative quantitative analysis in patients of different age groups and in the dynamics of the disease. The intensity of proliferative processes in the epithelium was evaluated by the mitotic index (Ki-67 marker) by counting mitoses per 100 cells. Macrophages were identified by CD68 marker (highly glycosylated transmembrane glycoprotein located in lysosomes) with monoclonal antibodies (clone KR1, code No. M0814). Unmasking of antigenic determinants was performed in a glass container filled with reducing solution in a
Number of examined patients
From 10 days to 1 year
7
1-2 years
11
3-7 years
5
8-12 years
19
13-16 years
13
17-21 years
24
22-35 years
29
36-55 years
47
56-65 years
34
66-75 years
39
76-80 years
11
>80 years
5
water bath for 1 h. Some preparations were exposed to microwaves that ensure the best unmasking effect. For antigen unmasking, 10 mM citrate buffer (pH 6.0) or Dako TRS (Target Retrieval Solution, code No. S1700) was used. Cold preparations were washed in distilled water. The antibodies were used in dilutions 1:50 and 1:100. Modern highly sensitive immunohistochemical analysis was performed using EPOS and EnVision automated systems. Immunocompetent cells were identified by the same scheme irrespective antigen localization in cell structures (membranes, lysosomes, nucleus, Golgi complex). The presence of HbP was detected by inoculation into growth medium and molecular genetic analysis of the isolated HbP strains followed by PCR analysis for pathogenic genes. Additionally, HbP infection was verified by immunohistochemical method with antibodies to HbP. The images were obtained using an Olympus АХ80 microscope and DP75 digital camera with original morphometry software; Hitachi SU8000 scanning electron microscope was also used. Statistical processing of the data, evaluation of significance of differences between the groups by the distribution parameters, and comparison of the group of samples were carried out using methods of variation statistics by confidence intervals for the difference between the means and parametric Student’s t test at p80 years (N=5)
14/1356
1%
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161
Fig. 2. Proliferative activity in the epithelial layer of the normal gastric mucosa in children (a) and adults (b) and in malignization (c). Immunohistochemical detection of Ki-67, ×400.
cells exclusively in the wall mucus. In healthy individuals and in patients with acute and chronic gastritis and ulcers, no bacteria inside epitheliocytes were detected (Fig. 1). In acute gastritis, high content of HbP was found in the lumens of gastric glands. Macrophages with villous surface surrounded by HbP were seen at the bottom of gastric glands (Fig. 1, a). The relief and microvilli on the surface of the mucosa were preserved. In chronic gastritis, the number of bacteria was lower, microvilli were atrophic, probably due to inflammation, and the surface looked wavy with larger folds (Fig. 1, b). The epithelium was preserved. Signs of damage to the apical surface of the epithelium and destruction of some microvilli were noted. Epithelial cells without microvilli and with hypertrophied microvilli were found. Some folds of hypertrophies mucosa were covered with epithelial cells with smooth surface, on the surface of some epithelial cells, smooth areas and relief areas with villous processes were seen. The following morphological types of microorganisms were distinguished: 1) with long and short, sometimes with clublike flagella located
on one pole of the bacterial cell and 2) with wide short and long flagella located on the lateral surface of HbP. These morphological differences can reflect different functional states of HbP or different species (molecular genetic analysis has identified ~100 HbP species). In ulcerative process, the mucosa was partially destroyed (Fig. 1, c), the epithelium was somewhere absent and the lamina propria was exposed; preserved epithelium with microvilli was seen along the ulcer edges. Immunohistochemical analysis of biopsy specimens from the cardia, fundus, and antrum showed the presence of HbP only on the surface of epithelial cells without contamination (Fig. 1, d-f). Our findings do not confirm the data that HbP invades the lamina propria of the gastric mucosa involved in specific inflammation (gastritis, duodenitis) with participation of local and humoral immune mechanisms associated with phagocytosis, blast transformation of lymphocytes, and production of immunoglobulins. Evaluation of the regeneration potential of surface epithelium in the gastric wall in healthy and HbPpositive individuals of different age showed that pro-
Fig. 3. Normal human gastric mucosa (a-c), chronic gastritis (d), ulcer (e), and malignization (f). Immunohistochemical analysis, ×400.
Cell Technologies in Biology and Medicine, No. 4, February, 2015
Note. “–” – no data. Significant difference from the norm (p80 years
13.54±0.09
4.83±0.15 3.46±0.18
2.94±0.12 1.48±0.12
1.50±0.30 3.90±0.11
3.00±0.19 3.17±0.06
3.33±0.15 1.81±0.15
1.67±0.18 3.96±0.18
4.71±0.15
2.95±0.13 76-80 years
14.18±0.08
3.24±0.11 66-75 years
24.55±0.11
6.23±0.12
5.47±0.11 3.91±0.10 2.00±0.07 3.40±0.14 4.16±0.10 2.09±0.14 4.43±0.12 3.77±0.06 56-65 years
24.90±0.04
5.90±0.13 5.17±0.07
3.78±0.16 2.32±0.15
1.87±0.08 3.30±0.18
3.20±0.03 4.77±0.16
6.52±0.09 1.97±0.21
2.14±0.23 5.07±0.09
5.16±0.12
3.91±0.14 36-55 years
26.41±0.11
3.63±0.12 22-35 years
28.59±0.14
4.84±0.18
5.15±0.17 3.14±0.14 1.85±0.05 3.40±0.05 3.83±0.16 1.95±0.15 4.93±0.16 3.15±0.14 17-21 years
25.16±0.17
4.41±0.05 2.86±0.09
2.93±0.07 1.88±0.16
1.62±0.10 3.20±0.06
3.50±0.16 3.75±0.11
3.48±0.15 1.32±0.12
1.52±0.17 4.72±0.10
4.14±0.14
2.81±0.08 13-16 years
23.94±0.16
2.29±0.07 8-12 years
23.73±0.04
4.15±0.17 2.81±0.12 1.36±0.09 2.80±0.04 2.64±0.07 1.14±0.13 3.90±0.11 1.94±0.05 3-7 years
23.6±0.08
–
– 2.30±0.17
2.64±0.12 1.25±0.09
1.50±0.04 –
– 2.18±0.08
1.60±0.09 0.90±0.04
1.11±0.11 –
– 3.22±0.04
1-2 years
3.41±0.14
1.32±0.07
1.51±0.06
From 10 days to 1 year
gastritis norm ulcer gastritis norm
gastritis
ulcer
norm
CD68 CD4+/CD8+ Age
Number of cells per field of view on section of the biopsy specimen (M±m)
TABLE 6. Parameters of Immune Homeostasis of the Gastric Mucosa in Norm and under Pathological Conditions
CD163
ulcer
162
liferative activity was maximum in children aged from 9 months to 10 years, while in adults, only single cells that entered irreversible mitosis were observed (Fig. 2, a, b; Table 5). Metaplasia and carcinogenesis were also associated with high mitotic activity of cells in the damaged sites of the gastric mucosa (Fig. 2, c). This explains, why the ulcerative process in adults often develops as a result of mismatch of extensive alteration and limited reparative capacities of cambial cells. The immune homeostasis of the epithelium, both under normal conditions and against the background of HbP infection without clinical manifestations, is maintained by solitary CD68 (Fig. 3, a), CD163 (Fig. 3, b), and CD4 (Fig. 3, c) immunocytes; the number of effector CD4 (Fig. 3, d), CD8 (Fig. 3, e) immunocytes increase in acute and chronic gastritis and gastric ulcer; they were abundant in the lamina propria of the gastric mucosa in gastritis (Table 6). Malignization was associated with the absence of effector immunocytes in the pathological focus (Fig. 3, f). Despite the fact that HbP is considered the key pathogenetic factor in triggering the cascade oncogenetic changes in the GIT mucosa, our findings suggest that pathogenic and opportunistic microorganisms and opportunistic saprophytic microflora are only one of the causes determining alteration and dysfunction in GIT. This is confirmed by the fact that despite high rate of HbP infection in population of the Primorsky Territory, ulcerative processes developed in not all infected individuals and had seasonal variations; carcinogenesis also developed in not all HbP carriers. We believe that barrier properties of GIT mucosa epithelium depending on various factors including surface and secretory epithelium and numerous differons of immune cells located among them are one of the key morphological elements in prevention of the pathological process in GIT. Generalized or local shifts in immune homeostasis in the mucosa serve as a trigger of the pathological processes in GIT. The observed age dependence of the pathology and infection of the gastric mucosa (>60% cancers occur in people above 65) indicates that HbP can be a cause of carcinogenesis induction [13]. It is known that cancer can involve the cardia (upper portion of the stomach), fundus, or pylorus. The middle and lower portions of the stomach are more susceptible for carcinogenesis induction. Therefore, equal content of microorganisms in different portions of the stomach suggests that the degree of colonization of the gastric mucosa with HbP does not play a role in carcinogenesis. We believe that hyperplasia of microvilli on epithelial cells occurs first and prevents HbP adhesion to the cell membranes. Then, enzymes (phospholipase, protease) and cytokines produced by HbP destroy microvilli, damage the gastric mucosa, and impair the resistance of
I. V. Reva, T. Yamamoto, et al.
epithelial cells to hydrochloric acid. Adaptation of the gastric epithelium to changing environment against the background HbP-infection in the first stages of the pathological process can manifest in enhanced proliferative activity aimed at the maintenance of the barrier properties of the surface epithelium after exhaustion of the regenerative potential and development of intestinal metaplasia. Gastric cancer is known to develop typically against the background of atrophic gastritis and intestinal metaplasia. Studies performed over the last 50 years have demonstrated that gastritis against the background of HbP infection was the main cause of intestinal metaplasia. This suggests that timely cure or prevention of gastritis can prevent gastric cancer. The established role of immune effector cells of the gastric mucosa epithelium suggests the necessity of taking this parameter of the immune homeostasis into account not only as an important prognostic marker of malignancy, but also as a morphological substrate that can influence the pathogenesis of the developing carcinogenesis. Analysis on the large statistical material has proven that complete eradication of HbP almost 2-fold increased the risk of developing reflux esophagitis, Barrett’s esophagus, and cardioesophageal adenocarcinoma and that the presence of HbP in the stomach reduces the risk of gastric cancer [7], these data suggest that disturbances in local immune homeostasis play an important role in carcinogenesis. HbP, as well as other microorganisms present in the mucosa of different visceral systems act as promoters of the oncological process against the background of impaired barrier properties of the mucosa epithelium. The study was supported by Scientific Foundation of the Far Eastern Federal University, within the
163 framework of the State Task 2014/36 (February 3, 2014) and the International Grant of the Far Eastern Federal University (Agreement No. 13-09-0602-m_a, November 6, 2013).
REFERENCES 1. M. Aragona, C. A. Buda, S. Panetta, et al., Oncol. Rep., 7, No. 5, 987-990 (2000). 2. P. R. Bishop, M. J. Nowicki, W. L. May, et al., Gastrointest. Endosc., 55, No. 6, 624-630 (2002). 3. L. Chiariotti, T. Angrisano, S. Keller, et al., Med. Microbiol. Immunol., 202, No. 5, 327-337 (2013). 4. E. M. Co and N. L. Schiller, Antimicrob. Agents Chemother., 50, No. 12, 4174-4176 (2006). 5. M. R. Couturier, E. Tasca, C. Montecucco, and M. Stein, Infect. Immunol., 74, No. 1, 273-281 (2006). 6. A. Dlugosz, G. Adler, A. Ciechanowicz, et al., Eur. J. Gastroenterol. Hepatol., 14, No. 11, 1231-1235 (2002). 7. A. Dubois, Gastroenterology, 132, No. 3, 1177-1180 (2007). 8. Y. Han, W. W. Tu, Y. G. Wen, et al., Med. Oncol., 30, No. 2, 564 (2013). 9. F. Hensel, C. Knörr, R. Hermann, et al., Int. J. Cancer, 81, No. 2, 229-235 (1999). 10. P. C. Konturek, S. J. Konturek, and T. Brzozowski, J. Physiol. Pharmacol., 60, No. 3, 3-21 (2009). 11. Y. López-Vidal, S. Ponce-de-León, G. Castillo-Rojas, et al., PLoS One, 3, No. 12, e3849, doi: 10.1371/journal.pone.0003849 (2008). 12. M. Micev and M. Cosić-Micev, Acta Chir. Iugosl., 58, No. 1, 39-52 (2011). 13. K. Rembiasz, P. C. Konturek, D. Karcz, et al., Dig. Dis. Sci., 50, No. 3, 474-482 (2005). 14. C. Semino-Mora, S. Q. Doi, A. Marty, et al., J. Infect. Dis., 187, No. 8, 1165-1177 (2003). 15. C. F. Zambon, F. Navaglia, D. Basso, et al., J. Clin. Pathol., 56, No. 4, 287-291 (2003).
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Immune Homeostasis of Human Gastric Mucosa in Helicobacter pylori Infection I. V. Reva1, T. Yamamoto3, S. S. Vershinina2, and G. V. Reva1,2 Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 44-51, January, 2015 Original article submitted March 3, 2014 We present the results of electron microscopic, microbiological, immunohistochemical, and molecular genetic studies of gastric biopsy specimens taken for diagnostic purposes according by clinical indications during examination of patients with gastrointestinal pathology. Immune homeostasis of the gastric mucosa against the background of infection with various pathogen strains of Helicobacter pylori was studied in patients of different age groups with peptic ulcer, gastritis, metaplasia, and cancer. Some peculiarities of Helicobacter pylori contamination in the gastric mucosa were demonstrated. Immune homeostasis of the gastric mucosa in different pathologies was analyzed depending on the Helicobacter pylori genotype. Key Words: Helicobacter pylori; immunocytes; metaplasia; peptic ulcer; carcinogenesis
According to WHO data, gastrointestinal (GIT) diseases account for more than 60% morbidities in the world. In Russia, more than 120,000 surgeries are annually performed for pathologies requiring stomach resection, such as cancer or ulcers of the stomach and duodenum. The prevalence of peptic ulcers and stomach cancers steadily increases in most counties [4,12]. In 70-90% cases, stomach cancer is associated with Helicobacter pylori (HbP) infection. The presence of HbP increases the risk of stomach cancer by about 6 times; HbP is detected in about half cases of stomach cancer [9]. It was hypothesized that chronic gastritis leads to intestinal metaplasia (atrophic gastritis) followed by malignant transformation [15]. At the final stages, biopsy can be HbP-negative, but immunological tests show previous infection [5]. In 1994, International Agency for Research on Cancer has recognized HbP as class I carcinogen as the main cause of stomach cancer in humans [3,10,14], but no exhaustive proofs for causative role of HbP were presented until now [8]. The role of HbP (in Russian patients, HbP genome is poorly studied) in the pathogenesis of the ulcer process, metaplasia, and cancer development, as well as its in1 Engineering School, 2School of Biomedicine, Far Eastern Federal University, Vladivostok; Russia; 3International Medical Research Center (IMERC), Niigata, Japan. Address for correspondence: [email protected]. G. V. Reva
fluence on the local immune homeostasis in the gastric mucosa remain unclear. In oncology, the efficiency of the treatment and prevention of mortality depends on early diagnosis, etiological, and pathogenetic factors of this disease. Therefore, diagnostic monitoring should include not only clinical symptoms at different stages of the disease, but also molecular genetic features of HbP. The pathogenesis of peptic ulcer and carcinogenesis against the background HbP infection is now considered depending on the HbP strain, presence of cagA, vacA, and babA genes associated with gastric tumors, and other factors [15]. Inefficiency of antibiotic therapy of HbP infections related to antibiotic resistance, the existence of GIT areas shielding the bacteria from antibiotics, and the appearance of multidrug-resistant HbP isolates (including clarithromycin- and quinolone-resistant strains) require more comprehensive analysis of the ulcerative process in GIT and the role HbP infections in the carcinogenesis. Here we studied the influence of HbP on changes in the gastric mucosa for optimization of the diagnostic methods in gastric ulcer. We addressed the peculiarities of the immune homeostasis of the gastric mucosa under normal and pathological conditions, peculiarities of HbP genome in patients of the Primorsky Territory, and
0007-4888/15/15910157 © 2015 Springer Science+Business Media New York
158 peculiarities of immune homeostasis in case of gastric mucosa infection with various HbP strains.
Cell Technologies in Biology and Medicine, No. 4, February, 2015
TABLE 1. Age-Distribution of the Examinees Age group
MATERIALS AND METHODS The study was conducted in accordance with the provisions of the Declaration of Helsinki (2000) and with the approval of the Ethics Committee of the Far Eastern Federal University. Biological specimens and biopsy samples of the gastric mucosa were obtained from 244 patients aged from 6.5 months to 78 years (Table 1) during fiberoptic gastroduodenoscopy performed at the Russian-Japanese endoscopic center of the Far Eastern Regional Medical Center (2004-2011) and Center for Maternal and Child Welfare (2008-2011). We obtained and analyzed 799 biopsy specimens from different compartments of the gastric mucosa from 244 patients. The mucus obtained from GIT was analyzed by phase-contrast microscopy. Esophagogastrojejunoscopy with gastrobiopsy was performed in all patients with clinical manifestations according to recommendations [2]. For endoscopic examination of the upper GIT, Pentax FG24V and FG29V forward-view fiberoptic instruments were used; biopsy specimens were taken using standard forceps with cup-shaped jaws (KW-2415S, KW-1815S). For examination of the stomach and small intestine below the Treitz ligament in children, a standard pediatric esophagogastroscope (7.8 mm insertion tube diameter) was used. The specimens were taken from the cardia, antrum, and fundus of the stomach according to the Sydney system and WHO gold standard. The results were interpreted according to the criteria of the morphological section of Sydney classification, updates to the international classification of gastritis, and visual analog scale with the standards of semiquantitative evaluation of morphological changes. Sections (3-5 μ) sliced from paraffin blocks were stained with hematoxylin and eosin. In additions, cytological samples (impression smears) were analyzed. The morphological study was based on immunohistochemical phenotyping by differentiation cluster (CD) markers. Immunohistochemical analysis was used for detection of HbP, activity of Ki-67 gene, CD4, CD8, CD68, and CD163 (Dako) for illustration and subsequent comparative quantitative analysis in patients of different age groups and in the dynamics of the disease. The intensity of proliferative processes in the epithelium was evaluated by the mitotic index (Ki-67 marker) by counting mitoses per 100 cells. Macrophages were identified by CD68 marker (highly glycosylated transmembrane glycoprotein located in lysosomes) with monoclonal antibodies (clone KR1, code No. M0814). Unmasking of antigenic determinants was performed in a glass container filled with reducing solution in a
Number of examined patients
From 10 days to 1 year
7
1-2 years
11
3-7 years
5
8-12 years
19
13-16 years
13
17-21 years
24
22-35 years
29
36-55 years
47
56-65 years
34
66-75 years
39
76-80 years
11
>80 years
5
water bath for 1 h. Some preparations were exposed to microwaves that ensure the best unmasking effect. For antigen unmasking, 10 mM citrate buffer (pH 6.0) or Dako TRS (Target Retrieval Solution, code No. S1700) was used. Cold preparations were washed in distilled water. The antibodies were used in dilutions 1:50 and 1:100. Modern highly sensitive immunohistochemical analysis was performed using EPOS and EnVision automated systems. Immunocompetent cells were identified by the same scheme irrespective antigen localization in cell structures (membranes, lysosomes, nucleus, Golgi complex). The presence of HbP was detected by inoculation into growth medium and molecular genetic analysis of the isolated HbP strains followed by PCR analysis for pathogenic genes. Additionally, HbP infection was verified by immunohistochemical method with antibodies to HbP. The images were obtained using an Olympus АХ80 microscope and DP75 digital camera with original morphometry software; Hitachi SU8000 scanning electron microscope was also used. Statistical processing of the data, evaluation of significance of differences between the groups by the distribution parameters, and comparison of the group of samples were carried out using methods of variation statistics by confidence intervals for the difference between the means and parametric Student’s t test at p80 years (N=5)
14/1356
1%
I. V. Reva, T. Yamamoto, et al.
161
Fig. 2. Proliferative activity in the epithelial layer of the normal gastric mucosa in children (a) and adults (b) and in malignization (c). Immunohistochemical detection of Ki-67, ×400.
cells exclusively in the wall mucus. In healthy individuals and in patients with acute and chronic gastritis and ulcers, no bacteria inside epitheliocytes were detected (Fig. 1). In acute gastritis, high content of HbP was found in the lumens of gastric glands. Macrophages with villous surface surrounded by HbP were seen at the bottom of gastric glands (Fig. 1, a). The relief and microvilli on the surface of the mucosa were preserved. In chronic gastritis, the number of bacteria was lower, microvilli were atrophic, probably due to inflammation, and the surface looked wavy with larger folds (Fig. 1, b). The epithelium was preserved. Signs of damage to the apical surface of the epithelium and destruction of some microvilli were noted. Epithelial cells without microvilli and with hypertrophied microvilli were found. Some folds of hypertrophies mucosa were covered with epithelial cells with smooth surface, on the surface of some epithelial cells, smooth areas and relief areas with villous processes were seen. The following morphological types of microorganisms were distinguished: 1) with long and short, sometimes with clublike flagella located
on one pole of the bacterial cell and 2) with wide short and long flagella located on the lateral surface of HbP. These morphological differences can reflect different functional states of HbP or different species (molecular genetic analysis has identified ~100 HbP species). In ulcerative process, the mucosa was partially destroyed (Fig. 1, c), the epithelium was somewhere absent and the lamina propria was exposed; preserved epithelium with microvilli was seen along the ulcer edges. Immunohistochemical analysis of biopsy specimens from the cardia, fundus, and antrum showed the presence of HbP only on the surface of epithelial cells without contamination (Fig. 1, d-f). Our findings do not confirm the data that HbP invades the lamina propria of the gastric mucosa involved in specific inflammation (gastritis, duodenitis) with participation of local and humoral immune mechanisms associated with phagocytosis, blast transformation of lymphocytes, and production of immunoglobulins. Evaluation of the regeneration potential of surface epithelium in the gastric wall in healthy and HbPpositive individuals of different age showed that pro-
Fig. 3. Normal human gastric mucosa (a-c), chronic gastritis (d), ulcer (e), and malignization (f). Immunohistochemical analysis, ×400.
Cell Technologies in Biology and Medicine, No. 4, February, 2015
Note. “–” – no data. Significant difference from the norm (p80 years
13.54±0.09
4.83±0.15 3.46±0.18
2.94±0.12 1.48±0.12
1.50±0.30 3.90±0.11
3.00±0.19 3.17±0.06
3.33±0.15 1.81±0.15
1.67±0.18 3.96±0.18
4.71±0.15
2.95±0.13 76-80 years
14.18±0.08
3.24±0.11 66-75 years
24.55±0.11
6.23±0.12
5.47±0.11 3.91±0.10 2.00±0.07 3.40±0.14 4.16±0.10 2.09±0.14 4.43±0.12 3.77±0.06 56-65 years
24.90±0.04
5.90±0.13 5.17±0.07
3.78±0.16 2.32±0.15
1.87±0.08 3.30±0.18
3.20±0.03 4.77±0.16
6.52±0.09 1.97±0.21
2.14±0.23 5.07±0.09
5.16±0.12
3.91±0.14 36-55 years
26.41±0.11
3.63±0.12 22-35 years
28.59±0.14
4.84±0.18
5.15±0.17 3.14±0.14 1.85±0.05 3.40±0.05 3.83±0.16 1.95±0.15 4.93±0.16 3.15±0.14 17-21 years
25.16±0.17
4.41±0.05 2.86±0.09
2.93±0.07 1.88±0.16
1.62±0.10 3.20±0.06
3.50±0.16 3.75±0.11
3.48±0.15 1.32±0.12
1.52±0.17 4.72±0.10
4.14±0.14
2.81±0.08 13-16 years
23.94±0.16
2.29±0.07 8-12 years
23.73±0.04
4.15±0.17 2.81±0.12 1.36±0.09 2.80±0.04 2.64±0.07 1.14±0.13 3.90±0.11 1.94±0.05 3-7 years
23.6±0.08
–
– 2.30±0.17
2.64±0.12 1.25±0.09
1.50±0.04 –
– 2.18±0.08
1.60±0.09 0.90±0.04
1.11±0.11 –
– 3.22±0.04
1-2 years
3.41±0.14
1.32±0.07
1.51±0.06
From 10 days to 1 year
gastritis norm ulcer gastritis norm
gastritis
ulcer
norm
CD68 CD4+/CD8+ Age
Number of cells per field of view on section of the biopsy specimen (M±m)
TABLE 6. Parameters of Immune Homeostasis of the Gastric Mucosa in Norm and under Pathological Conditions
CD163
ulcer
162
liferative activity was maximum in children aged from 9 months to 10 years, while in adults, only single cells that entered irreversible mitosis were observed (Fig. 2, a, b; Table 5). Metaplasia and carcinogenesis were also associated with high mitotic activity of cells in the damaged sites of the gastric mucosa (Fig. 2, c). This explains, why the ulcerative process in adults often develops as a result of mismatch of extensive alteration and limited reparative capacities of cambial cells. The immune homeostasis of the epithelium, both under normal conditions and against the background of HbP infection without clinical manifestations, is maintained by solitary CD68 (Fig. 3, a), CD163 (Fig. 3, b), and CD4 (Fig. 3, c) immunocytes; the number of effector CD4 (Fig. 3, d), CD8 (Fig. 3, e) immunocytes increase in acute and chronic gastritis and gastric ulcer; they were abundant in the lamina propria of the gastric mucosa in gastritis (Table 6). Malignization was associated with the absence of effector immunocytes in the pathological focus (Fig. 3, f). Despite the fact that HbP is considered the key pathogenetic factor in triggering the cascade oncogenetic changes in the GIT mucosa, our findings suggest that pathogenic and opportunistic microorganisms and opportunistic saprophytic microflora are only one of the causes determining alteration and dysfunction in GIT. This is confirmed by the fact that despite high rate of HbP infection in population of the Primorsky Territory, ulcerative processes developed in not all infected individuals and had seasonal variations; carcinogenesis also developed in not all HbP carriers. We believe that barrier properties of GIT mucosa epithelium depending on various factors including surface and secretory epithelium and numerous differons of immune cells located among them are one of the key morphological elements in prevention of the pathological process in GIT. Generalized or local shifts in immune homeostasis in the mucosa serve as a trigger of the pathological processes in GIT. The observed age dependence of the pathology and infection of the gastric mucosa (>60% cancers occur in people above 65) indicates that HbP can be a cause of carcinogenesis induction [13]. It is known that cancer can involve the cardia (upper portion of the stomach), fundus, or pylorus. The middle and lower portions of the stomach are more susceptible for carcinogenesis induction. Therefore, equal content of microorganisms in different portions of the stomach suggests that the degree of colonization of the gastric mucosa with HbP does not play a role in carcinogenesis. We believe that hyperplasia of microvilli on epithelial cells occurs first and prevents HbP adhesion to the cell membranes. Then, enzymes (phospholipase, protease) and cytokines produced by HbP destroy microvilli, damage the gastric mucosa, and impair the resistance of
I. V. Reva, T. Yamamoto, et al.
epithelial cells to hydrochloric acid. Adaptation of the gastric epithelium to changing environment against the background HbP-infection in the first stages of the pathological process can manifest in enhanced proliferative activity aimed at the maintenance of the barrier properties of the surface epithelium after exhaustion of the regenerative potential and development of intestinal metaplasia. Gastric cancer is known to develop typically against the background of atrophic gastritis and intestinal metaplasia. Studies performed over the last 50 years have demonstrated that gastritis against the background of HbP infection was the main cause of intestinal metaplasia. This suggests that timely cure or prevention of gastritis can prevent gastric cancer. The established role of immune effector cells of the gastric mucosa epithelium suggests the necessity of taking this parameter of the immune homeostasis into account not only as an important prognostic marker of malignancy, but also as a morphological substrate that can influence the pathogenesis of the developing carcinogenesis. Analysis on the large statistical material has proven that complete eradication of HbP almost 2-fold increased the risk of developing reflux esophagitis, Barrett’s esophagus, and cardioesophageal adenocarcinoma and that the presence of HbP in the stomach reduces the risk of gastric cancer [7], these data suggest that disturbances in local immune homeostasis play an important role in carcinogenesis. HbP, as well as other microorganisms present in the mucosa of different visceral systems act as promoters of the oncological process against the background of impaired barrier properties of the mucosa epithelium. The study was supported by Scientific Foundation of the Far Eastern Federal University, within the
163 framework of the State Task 2014/36 (February 3, 2014) and the International Grant of the Far Eastern Federal University (Agreement No. 13-09-0602-m_a, November 6, 2013).
REFERENCES 1. M. Aragona, C. A. Buda, S. Panetta, et al., Oncol. Rep., 7, No. 5, 987-990 (2000). 2. P. R. Bishop, M. J. Nowicki, W. L. May, et al., Gastrointest. Endosc., 55, No. 6, 624-630 (2002). 3. L. Chiariotti, T. Angrisano, S. Keller, et al., Med. Microbiol. Immunol., 202, No. 5, 327-337 (2013). 4. E. M. Co and N. L. Schiller, Antimicrob. Agents Chemother., 50, No. 12, 4174-4176 (2006). 5. M. R. Couturier, E. Tasca, C. Montecucco, and M. Stein, Infect. Immunol., 74, No. 1, 273-281 (2006). 6. A. Dlugosz, G. Adler, A. Ciechanowicz, et al., Eur. J. Gastroenterol. Hepatol., 14, No. 11, 1231-1235 (2002). 7. A. Dubois, Gastroenterology, 132, No. 3, 1177-1180 (2007). 8. Y. Han, W. W. Tu, Y. G. Wen, et al., Med. Oncol., 30, No. 2, 564 (2013). 9. F. Hensel, C. Knörr, R. Hermann, et al., Int. J. Cancer, 81, No. 2, 229-235 (1999). 10. P. C. Konturek, S. J. Konturek, and T. Brzozowski, J. Physiol. Pharmacol., 60, No. 3, 3-21 (2009). 11. Y. López-Vidal, S. Ponce-de-León, G. Castillo-Rojas, et al., PLoS One, 3, No. 12, e3849, doi: 10.1371/journal.pone.0003849 (2008). 12. M. Micev and M. Cosić-Micev, Acta Chir. Iugosl., 58, No. 1, 39-52 (2011). 13. K. Rembiasz, P. C. Konturek, D. Karcz, et al., Dig. Dis. Sci., 50, No. 3, 474-482 (2005). 14. C. Semino-Mora, S. Q. Doi, A. Marty, et al., J. Infect. Dis., 187, No. 8, 1165-1177 (2003). 15. C. F. Zambon, F. Navaglia, D. Basso, et al., J. Clin. Pathol., 56, No. 4, 287-291 (2003).
