Gastric Mucosa Samples Research Articles

Alu and Satα hypomethylation in Helicobacter pylori‐infected gastric mucosae

Global hypomethylation and regional hypermethylation are supposed to be hallmarks of cancer cells. During gastric carcinogenesis, in which Helicobacter pylori infection is causally involved, aberrant hypermethylation is already present in H. pylori-infected gastric mucosae. In contrast, little is known about global hypomethylation, which can be caused by hypomethylation of individual repetitive elements and other sequences. We, therefore, investigated hypomethylation of individual repetitive elements and the global 5-methylcytosine content in four groups of gastric mucosal samples that represented the time course of H. pylori infection and gastric carcinogenesis [gastric mucosae of H. pylori-negative healthy volunteers (G1, n = 34), H. pylori-positive healthy volunteers (G2, n = 42), H. pylori-positive gastric cancer patients (G3, n = 34) and H. pylori-negative gastric cancer patients (G4, n = 20)] and 52 primary gastric cancers. Major variants of Alu, LINE1 and Satα were identified, and their methylation levels were quantified by bisulfite pyrosequencing. Compared with G1, the Alu methylation level was decreased in G2, G3, G4 and cancers (89.2-97.1% of that in G1, p < 0.05). The Satα methylation level was decreased in G2 (91.6%, p < 0.05) and G3 (94.3%, p = 0.08) but not in G4 and cancers. The LINE1 methylation level was decreased only in cancers. The 5-methylcytosine content was at similar levels in G2, G3 and G4 and highly variable in cancers. These results showed that Alu and Satα hypomethylation is induced in gastric mucosae by H. pylori infection during gastric carcinogenesis, possibly in different target cells, and that global hypomethylation is not always present in human gastric cancers.

Significant increase in IgG4+ plasma cells in gastric biopsy specimens from patients with pernicious anaemia

AimTo investigate the presence of IgG4+ plasma cells in gastric mucosal biopsy samples from patients with atrophic gastritis (AG) and a history of pernicious anaemia (PA) (AG+PA+).MethodsGastric mucosal biopsy specimens...

Promoter methylation of p16, Runx3, DAPK and CHFR genes is frequent in gastric carcinoma

Transcriptional silencing induced by hypermethylation of CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including gastric cancer. A number of reports on methylation of various genes in gastric cancer have been published, but most of these studies focused on cancer tissues or only a single gene. In this study, we determined the promoter hypermethylation status and mRNA expression of 4 genes: p16, Runx3, DAPK and CHFR. Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of p16, Runx3, DAPK and CHFR gene promoters in cancer and adjacent normal gastric mucosa specimens from 70 patients with gastric cancer, as well as normal gastric biopsy samples from 30 people without cancer serving as controls. In addition, the mRNA expression of p16, Runx3, DAPK and CHFR was investigated in 34 gastric cancer patients by RT-PCR. Bisulfite DNA sequence analysis was applied to check the positive samples detected by MSP. When carcinoma specimens were compared with adjacent normal gastric mucosa samples, a significant increase in promoter methylation of p16, Runx3, DAPK and CHFR was observed, while all 30 histologically normal gastric specimens were methylation free for all 4 genes. The methylation rate of the 4 genes increased from normal stomach tissue to tumor-adjacent gastric mucosa to gastric cancer tissue. Concurrent methylation in 2 or more genes was found in 22.9% of tumor-adjacent normal gastric mucosa and 75.7% of cancer tissues. No correlation was found between hypermethylation and other clinicopathological parameters such as sex, age, and tumor location. However, the frequency of DAPK and CHFR methylation in cancer tissues was significantly associated with the extent of differentiation and lymph node metastasis (P < 0.05) and the frequency of Runx3 methylation was significantly associated with tumor size (P < 0.05). Weak expression and loss of expression of the 4 genes was observed in cancer tissues and was significantly associated with promoter hypermethylation (P < 0.05). Promoter hypermethylation of p16, Runx3, DAPK and CHFR is frequent in gastric cancer. DAPK and CHFR promoter hypermethylation may be an important help in evaluating the differentiation grade and lymph node status of gastric cancer. Weak gene expression and loss of gene expression due to promoter hypermethylation may be a cancer-specific event.

CpG Methylation of Transcription Factor 4 in Gastric Carcinoma

Epigenetic silencing of tumor-related genes by CpG island methylation is an important mechanism for the development of many tumors, including gastric carcinoma. Deregulation of transcription factor 4 (TCF4) by promoter methylation was recently shown to play a key role in gastric carcinogenesis. The extent of methylation in the TCF4 promoter was assessed using methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PS) in 120 gastric carcinoma (GC) samples collected during gastrectomy, and in 40 normal gastric mucosa samples. The PS analysis of GCs revealed a higher frequency of TCF4 methylation (75.8%; 91/120). The methylation frequency for TCF4 by both MSP and PS techniques was significantly higher in advanced (75.0 and 91.7%, respectively) compared with early (60.0 and 60.0%, respectively, p < 0.05) GCs. There was a significant difference in TCF4 methylation between GCs and normal gastric mucosa (67.5 vs. 40.0%, respectively, by MSP and 75.8 vs. 30.0%, respectively, by PS; p < 0.05). There was significant correlation between TCF4 methylation status by PS and tumor size (p = 0.004), Lauren classification (p = 0.043), depth of invasion (p < 0.001), nodal metastasis (p = 0.021), and tumor-node-metastasis (TNM) stage (p = 0.045). These results suggest that inactivation of TCF4 by promoter methylation may play a role in the early stage of gastric carcinoma progression. Furthermore, standard polymerase chain reaction followed by PS may provide a more specific and quantitative diagnostic alternative to MSP, which may be of benefit in oncology research.

Helicobacter species detection and histopathological changes in stray cats from Niterói, Brazil.

Tightly coiled bacteria are common inhabitants of the gastric mucosae of cats and there is considerable debate whether feline helicobacters are commensal or pathogenic organisms. To determine the prevalence of Helicobacter species and gastric histopathological findings, gastric mucosa samples of 56 stray cats were collected from cardia, fundus, body, antrum and pylorus at necropsy. The samples were examined by urease test, cytology, histopathology and culture. Helicobacter species were detected in 53 cats (94.6%) by at least one detection method. Urease test results were positive in gastric samples from 50 (89.3%) cats. Cytological and histological evaluations revealed Helicobacter species in 52 (92.8%) and 50 (89.2%) cats, respectively. Helicobacter species organisms were not cultured from any cat. Regardless of the presence of gastric bacteria, most of the cats had mild to moderate gastritis. These data reveal a high prevalence of Helicobacter species in stray cats from Niterói, Brazil.

Abstract 157: Quantitative COX2 promoter methylation analysis in gastric carcinoma

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric cancinoma. DNA methylation of multiple CpG sites in promoter regions of several tumor related genes, such as hMLH1, p14, p16, and APC has reported in gastric carcinoma. Recent studies indicate that aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in a subset of gastric carcinomas (GC). To determine the occurrence of CpG island hypermethylation in GC in relation to H. pylori infection, the status and level of CpG methylation in promoter region of COX-2 was analyzed in early and advanced gastric carcinomas as opposed to normal gastric tissues. Materials and Methods: The extent of promoter methylation of COX-2 gene was assessed quantitatively using Pyrosequencing (PS) in 60 early gastric cancers (EGC) and 60 advanced gastric cancers (AGC) samples harvested upon gastrectomy, and 40 normal gastric mucosa samples from patients with benign gastric pathology. Giemsa stain was performed to detect H. pylori in neoplastic and non-neoplastic gastric mucosa. Gene product was studied by immunohistochemistry and the relationship between methylation profile of gene promoter and clinicopathological parameters was analyzed. Results: The PS analysis of GCs revealed a high frequency of COX-2 methylation in 30.0% (36/120). The methylation frequency for COX-2 gene by PS techniques was significantly higher in EGCs than in AGCs (40.0% and 20.0%, respectively, p&amp;lt;0.05) There was a significant difference of COX-2 methylation in between GCs and normal gastric tissues (30.0% vs. 10.0%, respectively by PS, p&amp;lt;0.05). The mean methylation level in 8 CpG sites of COX-2 promoter by PS was 19.9%, 33.5%, and 18.5% in normal gastric tissue, EGCs, and AGCs respectively. COX-2 gene methylation was significantly associated with H. pylori infection (p&amp;lt;0.001). However, the methylation of COX-2 promoter by PS was not correlated with reduced or absent expression of COX-2 protein (p&amp;gt;0.05). Conclusion: we found that COX-2 promoter methylation was significantly higher in the tumor tissues and was early event for gastric carcinoma development, but not in progression of GC. These results suggest that COX-2 gene methylation may be important in the initial development of gastric carcinogenesis and the H. pylori infection is associated with promoter methylation of COX-2 gene. Furthermore, standard PCR followed by PS could be a more specific and quantitative method providing a more comprehensive picture of the distribution of DNA methylation throughout the promoter regions of specific genes, which will be of benefit in oncology research. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 157.

Abstract 4940: E-cadherin methylation is preceded in normal mucosa of diffuse type gastric carcinoma

Abstract Background: The importance of loss of E-cadherin (encoded by CDH1) to cancer progression is well established. CDH1 is mutationally inactivated in nearly half of diffuse type gastric cancer (DGC). Although CDH1 promoter methylation has been suggested to be a major second hit in hereditary diffuse gastric cancer, its role in sporadic DGCs is not clarified. Searching for the second hit methylation in potential precursor lesions in sporadic DGC, we investigated the CDH1 methylation frequency in a series of DGCs and matching normal mucosas and then compared the result with the CDH1 methylation frequency in normal mucosas from benign gastric pathology. Materials and Methods: The extent of promoter methylation of CDH1 gene was assessed quantitatively using Pyrosequencing in 72 DGCs with two matching normal mucosas from adjacent and remote foci, and 24 normal gastric mucosa samples from patients with benign gastric pathology. Gene product was studied by immunohistochemistry and the relationship between methylation profile of gene promoter and clinicopathological parameters was analyzed. Results: The Pyrosequencing analysis of DGCs revealed a high frequency of CDH1 methylation in 77.8% (56/72). The methylation frequency for CDH1 gene in matching normal mucosas from adjacent and remote foci was 70.8% (51/72) and 72.2% (52/72), respectively. According to the distance from the tumor location the result did not show significant difference. Moreover, total of 65 out of 72 cases (90.3%) showed at least one focus with CDH1 methylation. Frequency of CDH1 methylation in normal gastric mucosa samples with benign gastric pathology was 50.0% (12/24). There was a significant difference of CDH1 methylation in between normal mucosas from DGCs and normal mucosas with benign gastric pathology (90.3% vs. 50.0%, respectively, p&amp;lt;0.001). CDH1 methylation in matching normal gastric mucosas was counted as a statistically significant risk factor when adjusted for age and sex using a binary logistic regression model (p&amp;lt;0.001). Additionally loss or diminished expression of E-cadherin was confirmed by immunohistochemistry in 69 of 72 tumor samples (96.9%) and its down expression was not uncommonly observed in adjacent normal gastric mucosas. Conclusion: We found that CDH1 promoter methylation in the normal gastric mucosas from DGC samples was significantly higher than in the normal gastric mucosas with benign pathology but had a similar frequency with tumor tissues. These results suggest that CDH1 gene methylation may be important in the initial development of sporadic DGC tumorigenesis and the presence of DGC progenitor cells may be traced by the E-cadherin down-expression in normal gastric mucosa from different foci. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4940.

OC-039 Altered gene expression levels of MAD2, BUB1, aurora kinase A and B in early gastric cancers

Introduction Losses and/or gains of whole chromosomes (aneuploidy) are extremely common in most cancers. Indeed aneuploidy is thought to occur early in carcinogenesis and the genetic instability induced is thought to provide a pool of selectable clones for tumour evolution. We have been interested in aneuploidy as a biomarker for GI tract cancer for some time. It is believed that alterations in the expression of key components of the mitotic checkpoint drive aneuploidy, by impairing the ability of this checkpoint to prevent chromosome mis-segregation. Here we aimed to correlate gene expression levels of the mitotic checkpoints MAD2, Bub1, aurora kinase A and B with aneuploidy levels and histology in premalignant gastric tissues. Methods We collected over 50 cytology brush samples of gastric mucosa during routine endoscopic examination. We examined aneuploidy levels in these exfoliated cells using fluorescent in situ hybridisation, specifically looking at copy numbers of chromosome 1. Concurrently, during endoscopy, biopsy samples were obtained from the same gastric epithelium for histological assessment and for CLO testing and further biopsies were obtained and used to extract RNA to determine mitotic checkpoint gene expression levels using the real-time PCR. Specifically the expression levels Mad2, Bub1, aurora kinase A and B were assessed. Results Aneuploidy levels mirrored histological progression in the stomach as previously described (from an average of 2.6% in normal gastric epithelium to over 6% in intestinal metaplasia/atrophic gastritis). RNA was extracted from 46 samples for mitotic checkpoint gene expression analysis. Mad2 and Bub1 expression levels were found to be decreased significantly in the small number of samples with intestinal metaplasia. We also demonstrated that the levels of Mad2 and Bub1 were also diminished in the 28 patients with gastritis. Conversely, Aurora kinase A and B were upregulated in the H pylori infected tissues in particular, compared to histologically normal tissue. Comprehensive data analysis is now underway comparing mitotic checkpoint expression to histology, aneuploidy level and to key clinical parameters. Conclusion Mitotic checkpoint dysregulation occurs in pre-malignant gastric tissue concurrently with rising levels of aneuploidy. These mitotic checkpoint abnormalities may underlie the induction of aneuploidy and may represent the mechanism by which chromosomal instability is induced in early neoplasia.

Insulin-like growth factor binding protein-3 gene methylation and protein expression in gastric adenocarcinoma

Objective The aim of this study was to evaluate IGFBP-3 protein expression, its correlation with gene promoter methylation pattern in gastric carcinogenesis and with clinicopathological characteristics. Design Forty-three normal gastric mucosa and 94 adenocarcinoma samples were investigated through methylation specific PCR, after bisulfite modification. Immunohistochemistry was analyzed using peroxidase in 54 gastric cancer and 20 normal gastric mucosa samples. Results IGFBP-3 expression was higher in tumor samples than in normal mucosa ( p < 0.0001). Intestinal type presented a higher frequency of protein expression than diffuse type ( p = 0.0412). Methylation frequency of IGFBP-3 promoter in gastric samples revealed, respectively, 95.7% and 97.7% in neoplastic and non-neoplastic samples. The frequency of IGFBP-3 methylation did not differ between tumor and normal samples (95.7% versus 97.7%, p = 0.7810). We did not observe a significant correlation between IGFBP-3 promoter methylation and protein expression. Conclusion In summary, our study did not observe any influence of IGFBP-3 promoter methylation on protein expression. Moreover we propose that IGFBP-3 immunostaining in gastric tissue may be a useful marker for malignancy.

Increased Number of CpG Island Hypermethylation in Tumor Suppressor Genes of Non-Neoplastic Gastric Mucosa Correlates with Higher Risk of Gastric Cancer

Background/Aim: We investigated the relationship of gastric cancer (GC) and CpG island hypermethylation (CIHM) in tumor suppressor genes of non-neoplastic gastric mucosa. Methods: Gastric mucosa samples were obtained from 125 GC and 180 non-GC subjects. CIHM of p14, p16, DAP-kinase and CDH1 genes were determined by methylation-specific polymerase chain reaction. High CIHM was defined as three or all methylated CpG islands. Results: Rates of CIHM of p14, CDH1, DAP-kinase, and high CIHM were significantly higher in all GC samples than non-GC samples (p14: 32.2 vs. 50.4%; OR = 1.70, 95% CI = 1.03–2.80, p = 0.04, CDH1: 36.1 vs. 84.0%; OR = 8.65, 95% CI = 14.74–15.77, p < 0.0001, DAP-kinase: 42.2 vs. 83.2%; OR = 5.98, 95% CI = 3.37–10.62, p < 0.0001, and high CIHM: 44.4 vs. 80.8%; OR = 4.40, 95% CI = 2.51–7.72, p < 0.0001). CIHM in CDH1 and DAP-kinase were associated with a greater risk of GC including all of its different subtypes. An increased number of CIHM was associated with an increased risk of all GC (1 CIHM; OR = 2.33, 95% CI = 0.82–6.64, p = 0.11, 2 CIHM; OR = 4.89, 95% CI = 1.79–13.37, p = 0.002, 3 CIHM; OR = 9.43, 95% CI = 3.20–27.78, p < 0.0001, and all CIHM OR = 24.71, 95% CI = 6.70–91.18, p < 0.0001). Three and all CIHM were closely associated with a higher risk of intestinal-type GC, Helicobacter pylori-positive infection status, male gender, and middle and older GC while 3 CIHM was closely associated with a higher risk of diffuse-type GC, H. pylori-negative infection status and younger GC. Conclusions: CIHM of CDH1 and DAP-kinase in non-neoplastic gastric mucosa corresponded to a risk of GC regardless of histological subtype, H. pylori infection status, gender and generation. An increased number of CIHM correlates with a higher GC risk including its various clinico-pathological subtypes.

Analysis of DNA Methylation Status of the Promoter of Human Telomerase Reverse Transcriptase in Gastric Carcinogenesis

Telomerase is expressed in normal somatic cells and reactivated in majority of tumor cells. Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is a rate-limiting factor of telomerase activity. Evidence has shown that gastric cancer is the result of genetics and epignomics. DNA methylation is one of the most important research fields in epigenomics. It is one of the mechanisms resulting in gene silencing in carcinogenesis. Genomic DNAs were extracted from normal gastric mucosa, precancerous lesions and gastric cancer samples and were modified by sodium bisulfite. The modified genomic DNAs were amplified by PCR with primers that did not contain CpG sites. Each PCR product was sequenced. By matching the sequencing results and the original sequence, the status of each sample was obtained. PCR was carried out to identify hTERT expression. The promoter of hTERT in gastric cancer was more methylated than in the precancerous lesions and normal gastric mucosa (p<0.05). hTERT was absent in normal gastric mucosa and its positive rate was higher in gastric cancer than in precancerous lesions (p<0.05). hTERT promoter in gastric cancer was more methylated than in the precancerous lesions and normal gastric mucosa. This may suggest that the degree of methylation of the hTERT promoter was increased during gastric carcinogenesis and may be a potential biological maker in early diagnosis of gastric cancer. During gastric carcinogenesis, expression of hTERT was increased. This may suggest that methylation of hTERT may influence expression of hTERT.

Methylation ofGATA-4andGATA-5and development of sporadic gastric carcinomas

To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis. Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed. GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides). Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.

Helicobacter pyloriin dental plaque and stomach of patients from Northern Brazil

To establish whether virulence factor genes vacA and cagA are present in Helicobacter pylori (H. pylori) retrieved from gastric mucosa and dental plaque in patients with dyspepsia. Cumulative dental plaque specimens and gastric biopsies were submitted to histological examination, rapid urease test and polymerase chain reaction (PCR) assays to detect the presence of cagA and vacA polymorphisms. Detection of H. pylori from dental plaque and gastric biopsy samples was greater by PCR compared to histological examination and the rapid urease test. DNA from H. pylori was detected in 96% of gastric mucosa samples and in 72% of dental plaque samples. Sixty-three (89%) of 71 dental plaque samples that were H. pylori-positive also exhibited identical vacA and cagA genotypes in gastric mucosa. The most common genotype was vacAs1bm1 and cagA positive, either in dental plaque or gastric mucosa. These virulent H. pylori isolates were involved in the severity of clinical outcome. These pathogenic strains were found simultaneously in dental plaque and gastric mucosa, which suggests that gastric infection is correlated with the presence of H. pylori in the mouth.

Quantification of cyclooxygenase-2 methylation in gastric mucosa by denaturing high performance liquid chromatography assay

To setup a quantitative assay for detection of cyclooxygenase-2 (COX-2) methylation in human gastric mucosa samples. A standard analysis system was established by denaturing high performance liquid chromatography (DHPLC) under the condition of 55 degrees C oven temperature and a linear acetonitrile gradient (4.0/min). While, a total of 10 cases of gastric biopsy samples were detected for methylation status of COX-2. The complete methylated human promyelocytic leukemia cells (HL-60) and unmethylated gastric cancer cell line (MGC803) were used as positive and negative control. The proportion of the methylated copies of COX-2 was calculated according to the peak heights of methylated (M) and unmethylated (U) COX-2 in same PCR amplicon. The formula was Y = 1.0608 x M/(M + U), R(2) = 0.9894. Among 10 biopsy samples, the proportions of methylated copies of COX-2 in 2 cases of dysplasia were higher than superficial gastritis and chronic atrophy gastritis (24.5%, 18.4% vs 7.6%, 9.6%). The methylation of COX-2 promoter CpG islands can be detected in human gastric mucosa samples by quantitative DHPLC assay, which could be used in the population-based study of precancerous gastric lesions.

HTERT methylation and expression in gastric cancer

Gastric cancer is the second most prevalent cause of cancer death worldwide. DNA methylation is a common event in gastric carcinogenesis. hTERT seems to be the rate-limiting determinant of telomerase activation, which is responsible for stability and life span. hTERT hypermethylation has been associated with telomerase expression. In the present study, we investigated the promoter methylation status and hTERT protein expression in gastric cancer and normal mucosa samples. One hundred and nine gastric cancer and 53 normal mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analysed using peroxidase in 55 gastric cancer and 18 normal gastric mucosa samples. This is the first study evaluating hTERT methylation status in gastric carcinogenesis. We did not observe hTERT protein expression in normal gastric mucosa. Moreover, hTERT expression was observed in 80% of tumours and was associated with gastric cancer (p < 0.0001). Partial methylation was the most frequent pattern in gastric samples, even in normal mucosa. The frequency of specimens presenting hypermethylation was significantly higher in tumours than in normal mucosa samples (p = 0.0002), although the presence of hypermethylated promoter was not associated with a higher frequency of hTERT expression. A low correlation between hTERT protein expression and methylation was verified in gastric cancer samples. There was a clear difference in the frequency of hTERT expression and methylation within tumoral and non-tumoral tissues. Methylation status and telomerase expression may be useful for the diagnosis of gastric cancer and may have an impact on the anti-telomerase strategy for cancer therapy.

Helicobacter pyloriStimulates Dendritic Cells To Induce Interleukin-17 Expression from CD4+T Lymphocytes

Helicobacter pylori is a human gastroduodenal pathogen that leads to active chronic inflammation characterized by T-cell responses biased toward a Th1 phenotype. It has been accepted that H. pylori infection induces a Th17 response. At mucosal sites, dendritic cells (DCs) have the capacity to induce effector T cells. Here, we evaluate the role of DCs in the H. pylori-induced interleukin-17 (IL-17) response. Immunohistochemistry and immunofluorescence were performed on human gastric mucosal biopsy samples and showed that myeloid DCs in H. pylori-infected patients colocalized with IL-23- and that IL-17-producing lymphocytes were present in H. pylori-infected antral biopsy samples. In parallel, human monocyte-derived DCs stimulated in vitro with live H. pylori cells produced significant levels of IL-23 in the absence of IL-12 release. The subsequent incubation of H. pylori-infected DCs with autologous CD4(+) T cells led to gamma interferon (IFN-gamma) and IL-17 expression. The inhibition of IL-1 and, to a lesser extent, IL-23 inhibited IL-17 production by T cells. Finally, isogenic H. pylori mutant strains not expressing major virulence factors were less effective in inducing IL-1 and IL-23 release by DCs and IL-17 release by T cells than parental strains. Altogether, we can conclude that DCs are potent inducers of IL-23/IL-17 expression following H. pylori stimulation. IL-1/IL-23 as well as H. pylori virulence factors seem to play an important role in mediating this response.

Validation of a transendoscopic glandular and nonglandular gastric biopsy technique in horses

In order to study the evolution of histopathological and immunohistochemical changes in the gastric mucosa of horses with EGUS (equine gastric ulcer syndrome), a feasible, useful, valid and safe in vivo gastric biopsy technique is required. To determine the average gastric mucosal healing time following endoscopic gastric biopsy sampling, and evaluate the feasibility, safety and usefulness of samples obtained by this method for histopathological analysis. Six mature mares from the Faculté de Médecine Vétérinaire research herd were used. Transendoscopic gastric biopsy was performed on Days 0 and 9 using a flexible forceps with oval and fenestrated jaws to obtain gastric mucosal samples from 4 different sites: cardia (C), fundus (F), margo plicatus (MP) and glandular mucosa (GL). A maximum of 4 samples per site was taken and processed routinely for histopathology, evaluated by a pathologist. On Days 1-4 and 9-11 the lesions created by the biopsies were evaluated by gastroscopy. Lesions were evaluated over time based on a score from 0-4, where 4 was the most severe. Biopsy samples could be obtained from all targeted sites except C. No abnormal clinical signs were observed up to 7 days post biopsy. The average biopsy lesion scores decreased significantly with time for all sites. The average lesion score was significantly higher for the MP compared to the other sites at Days 1 and 2. Samples taken from the nonglandular portion of the stomach were considered inadequate for histopathology, while those taken from the glandular mucosa were adequate. The transendoscopic gastric biopsy technique described here is a feasible, safe and useful technique for obtaining samples from the equine gastric glandular mucosa. Although biopsy samples could be obtained from several areas in the nonglandular mucosa, these were very small, took longer to heal and were not considered adequate for histopathological evaluation, and another technique should therefore be validated.

Effect of polymorphisms of IL-17A, -17F and MIF genes on CpG island hyper-methylation (CIHM) in the human gastric mucosa

CpG island hyper-methylation (CIHM) is one of the major events in the gastric carcinogenesis and also occurs in non-neoplastic gastric mucosa. IL-17A, -17F and MIF have a crucial role in the gastric inflammation and carcinogenesis. The CIHM status in the non-cancerous gastric mucosa, in relation to IL-17A (-197G>A, rs2275913), -17F (7488T>C, rs763780) and MIF (-173G>C and -794 tetranucleotide repeats) polymorphisms was investigated. Gastric mucosa samples were obtained from 121 cancer free subjects. CIHM of p14, p16, DAP-kinase and CDH1 genes were determined by methylation-specific polymerase chain reaction (MSP). CIHM high was defined as three or all CpG islands methylated. We employed the PCR-SSCP (multiplex PCR for IL-17A and -17F) method to detect the gene polymorphisms. No association were found between CIHM status and L-17A (-197G>A), IL-17F (7488T>C) and MIF (-173G>C) polymorphisms. MIF 5-CATT repeat carrier (5/5+5/6+5/7) held a significantly higher risk of CIHM of DAP-kinase (OR=2.33, 95% CI=1.07-5.09, p=0.03) and CIHM high (OR=3.63, 95% CI=1.31-10.08, p=0.01). Weak association was also found between the same genotype and increased risk of CIHM of p16 (OR=2.45, 95% CI=0.90-6.68, p=0.08) and CDH1 (OR=2.23, 95% CI=0.94-5.32, p=0.07). 6-CATT repeat carrier (5/6+6/6+6/7) was significantly associated with reduced risk of CIHM of p16 (OR=0.31, 95% CI=0.11-0.90, p=0.03), CDH1 (OR=0.40, 95% CI=0.17-0.98, p=0.045), DAP-kinase (OR=0.37, 95% CI=0.17-0.83, p=0.02) and CIHM high (OR=0.25, 95% CI=0.09-0.74, p=0.01). -7-CATT repeat carrier (6/7+7/7) was weakly associated with reduced risk of CIHM of p16 (OR=0.34, 95% CI=0.10-1.13, p=0.08), DAP-kinase (OR=0.43, 95% CI=0.17-1.06, p=0.07) and CIHM high (OR=0.38, 95%CI=0.12-1.20, p=0.098). The present results provided the first evidence that the genetic polymorphisms MIF polymorphism is associated with CIHM status in the human gastric mucosa. Genetic polymorphisms of MIF-794-CATT repeat may be involved in methylation related carcinogenesis in the stomach.

Chronic nonsteroidal anti‐inflammatory drug (NSAID) use suppresses multiple CpG islands hyper methylation (CIHM) of tumor suppressor genes in the human gastric mucosa

There have been reports showing a protective role of nonsteroidal anti-inflammatory drugs (NSAIDs) against gastrointestinal cancers. CpG island hyper methylation (CIHM) of tumor suppressor genes is a major event in carcinogenesis. We investigated the CIHM status of non-cancerous gastric mucosa in chronic NSAID users and non-users and assessed the effect of NSAIDs on CIHM. Gastric mucosa samples were obtained from 51 chronic NSAID users and 180 non-users. CIHM of p14(ARF), p16(INK4a), death-associated protein kinase (DAP-kinase), and E-cadherin (CDH1) genes were determined by methylation-specific PCR. CIHM high was defined as two or more CpG islands methylated. CIHM of p14, p16, CDH1, and CIHM high were lower in chronic NSAID users than in non-users (p14: non-users vs users = 32.2%vs 9.8%, P = 0.003; p16: non-users vs users = 35.0%vs 15.7%, P = 0.02; CDH1: non-users vs users = 36.1%vs 9.8%, P = 0.0009; CIHM high: non-users vs users = 44.4%vs 17.6%, P = 0.0009). NSAID use was also associated with decreased number of CIHM by anova (R = -0.32, P < 0.0001). Multivariate logistic regression analysis with adjustment for sex, age, Helicobacter pylori infection, and NSAID use revealed that NSAID use was inversely correlated with all four CIHM and CIHM high as an independent factor (p14: odds ratio [OR] = 0.17, 95% confidence interval [CI] = 0.06-0.48; p16: OR = 0.32, 95% CI = 0.14-0.75; DAP-kinase: OR = 0.45, 95% CI = 0.22-0.92; CDH1: OR = 0.18, 95% CI = 0.06-0.48; CIHM high: OR = 0.21, 95% CI = 0.09-0.49). No association was found between CIHM status and the duration or dose of NSAIDs. Chronic NSAID use suppresses CIHM in human gastric mucosa. NSAIDs may have a suppressive role against methylation-related gastric carcinogenesis.

Increased number of methylated CpG islands correlates with Helicobacter pylori infection, histological and serological severity of chronic gastritis

Promoter hypermethylation of tumor suppressor genes is one of the major events in gastric carcinogenesis. Promoter hypermethylation is also present in non-neoplastic gastric epithelium as age-related phenomenon and some reports suggest the potential association between promoter hypermethylation and Helicobacter pylori infection. Here, we examined whether methylation of multiple promoter CpG islands would occur by H. pylori infection and correlate with histological or serological severity of chronic gastritis. One hundred and ninety-one gastric mucosa samples were obtained by endoscopy. The promoter methylation status of the p14, p16, DAP-kinase and CDH1 genes were determined by methylation-specific-polymerase chain reaction. The degree of gastritis in the antrum was assessed according to the updated Sydney system in 150 participants. The pepsinogen (PG) I/II ratio was calculated based on the data of serum PG I and PG II levels measured by radioimmunoassay in 54 selected cases. CpG island methylation was found in 32.5% for p14, 35.1% for p16, 43.5% for DAP-kinase and 36.1% for CDH1, whereas non, 1, 2, 3, and all methylation of four promoter CpG sites were present in 46 (24.1%), 59 (30.9%), 46 (24.1%), 30 (15.7%), and 10 (5.2%) participants, respectively. A strong association between the increased number of methylated CpG islands and H. pylori infection was observed (P<0.0001). An increased number of methylated CpG islands was also associated with severity of neutrophil infiltration (P<0.0001), mononuclear cell infiltration (P<0.0001) and atrophy (P=0.0021) in all, and severity of neutrophil infiltration (P=0.0177) and mononuclear cell infiltration (P=0.0004) in H. pylori-positive participants. An increased number of methylated CpG islands correlated with lower PG I/II ratio in all (P=0.0105) and H. pylori-infected participants (P=0.074). Multiple promoter CpG islands would be methylated by H. pylori infection, and an increased number of methylated CpG sites correlate with histological and serological severity of chronic gastritis.