Significance of cell-surface expression of matrix metalloproteinases and their inhibitors on gastric epithelium and infiltrating mucosal lymphocytes in progression of helicobacter pylori-associated gastritis

Background: Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) have recently been shown to be important in tissue breakdown and remodeling in gut with inflammatory bowel disease. The role of MMPs and TIMPs remains largely unexplored in Helicobacter pylori-associated gastritis (HAG). The aim of this study was to investigate the expression of these proteolytic enzymes in HAG. Methods: Cell-surface or/and intracellular expression of MMP-2, 7, 9 and MT1-MMP and TIMPs (TIMP-2 and -4) was determined in gastric epithelium and infiltrative mucosal lymphocytes (IML) in single endoscopic biopsies from H. pylori-infected (n?=?25) and uninfected (n?=?15) patients. The quantitative analysis was based on the percentage of positive cells detected by flow cytometry. Results: Secreted MMPs and TIMPs as well as membrane type 1-MMP were shown to be localized mainly on the cell surface of both epithelial cells and IML in HAG. H. pylori significantly up-regulated the cell-surface expression not only of MMPs, but also of TIMPs on IML within tissues. The expression of these molecules on IML was correlated with the grade of gastritis. Conclusions: MMPs and TIMPs expressed on gastric epithelium and H. pylori-antigen(s)-stimulated IML may be implicated in mucosal degradation and remodeling of the stomach. These might contribute to the pathogenesis and progression of atrophy and intestinal metaplasia of gastric mucosa.     

Enhanced Cell Surface Expression of Matrix Metalloproteinases and Their Inhibitors, and Tumor-Induced Host Response in Progression of Human Gastric Carcinoma

The cell surface and/or intracellular expression of the matrix metalloproteinases (MMP -2, 7, and -9 and MT1-MMP) and their inhibitors (TIMP-2 and -4) were investigated in tumor and tumor-infiltrating lymphocytes (TIL) in gastric carcinoma (n = 15) from the primary locus, metastatic gastric carcinoma (n = 20) from malignant ascites, and benign gastric mucosa (n = 20) for the control. The quantitative analysis was based on the percentage of positive cells by flow cytometry. The results clearly showed increased cell surface expression of MMP-2, -7, and -9, MT1-MMP, and TIMP-2 and -4 in both tumor cells and TIL during the development of invasion and/or metastasis of gastric carcinoma. There were equilateral correlations with cancer progression and frequency of cell surface expression of MMPs and their inhibitors, TIMPs, suggesting not only the aggressive nature of particularly metastatic gastric carcinoma, but also the presence of MMPs complexed with TIMPs on tumor cells and TIL. The enhanced cell surface expression of MMPs and TIMPs on TIL within metastatic carcinoma nests showed the result of a host response induced by tumors. These suggest that the increased cell surface expression of MMPs and TIMPs, and tumor-induced host response play a key role in gastric cancer invasion and/or metastasis.     

Coordinate cell-surface expression of matrix metalloproteinases and their inhibitors on cancer-associated myofibroblasts from malignant ascites in patients with gastric carcinoma

Purpose:The crucial role of tumor stroma in cancer cell invasion has been described in human carcinoma tissues. However, myofibroblastic invasion remains largely unexplored in malignant ascites. Purpose of this study is to investigate the spatial localization or regulation of matrix metalloproteinases (MMP-2, -7 -9, MT1-MMP) and their inhibitors (TIMP-2 and -4) on myofibroblasts from malignant ascites in 20 patients with gastric carcinoma. Methods: The quantitative flow cytometric analysis of MMPs or TIMPs on myofibroblasts was based on the percentage of double positive cells defined by anti MMPs or anti TIMPs, and anti α-smooth muscle actin (α-SMA) antibodies. Result: The results clearly showed that the coordination of the high level of cell-surface expression of secreted MMPs and TIMPs was noted on the α-SMA⁺ myofibroblasts. The finding suggests the possible formation of ternary complex, MT1-MMP/TIMPs/MMPs on the cells. The events might be a cause and result of activation processing of MMPs on the cells. Conclusion: This study provides the presence of invasive myofibroblasts with activated MMPs in close association with MMPs⁺ and TIMPs⁺ cancer cells and tumor-infiltrating lymphocytes from malignant ascites, emphasizing the importance of molecular cross-talk in tumor-host microenvironment for cancer invasion, metastasis and progression.     

幽门螺杆菌感染对胃黏膜基质金属蛋白酶表达的影响及其与胃癌的关系

基质金属蛋白酶（MMPs）是一组锌依赖性内肽酶，可降解细胞外基质蛋白，有助于肿瘤细胞浸润和转移。已证实幽门螺杆菌（H．pylori）胃癌的发生有关，但其对胃癌浸润和转移的作用尚未明确。近年有研究显示H．pylori感染可使胃黏膜上皮MMPs表达增高，推测H．pylori感染可能通过MMPs而促进胃癌浸润和转移。本文就H．pylori感染对胃黏膜MMPs表达的影响及其与胃癌的关系作一综述，并对金属蛋白酶组织抑制剂（TIMPs）治疗胃癌的前景作一展望．     

Rapid elimination of Helicobacter pylori and reduction of histocompatibility leucocyte antigen-DR expression 12 h after a single dose of omeprazole, amoxycillin and metronidazole triple therapy

BACKGROUND: Studies of eradication of Helicobacter pylori and subsequent resolution of H. pylori-related gastritis, have focused mainly on medium and long-term change following eradication therapies. Results from those studies have shown that both acute and chronic inflammatory changes found in gastric mucosa eventually return to normal. However, the early events in the stomach, particularly the effects on bacterial density and acute inflammatory markers of anti-H. pylori treatment, are largely unknown. The objective of this study was therefore to examine changes in the number of H. pylori, and the severity of gastric mucosal inflammation in the gastric biopsy specimens of patients before (0 h group, n = 14) and 12 h (12 h group, n = 14) after initiating anti-H. pylori treatment. METHODS: Biopsies were assessed, either quantitatively or semi-quantitatively, for the presence of H. pylori, neutrophils, mast cells, intraepithelial lymphocytes and the expression of histocompatibility leucocyte antigen (HLA)-DR by gastric epithelium and the results were compared between groups. RESULTS: Median H. pylori scores were 5 (range 2-5) and 0 (range 0-2) in biopsies from untreated and 12 h post-treatment groups, respectively (P < 0.001). In most 12 h post-treatment biopsies, H. pylori organisms could not be identified. There was a significant reduction in HLA-DR expression by gastric epithelium (median 3.5 with range 2-4 at 0 h group vs median 2 with range 0-4, P < 0.05), but there was no significant difference in the number of intraepithelial lymphocytes, CD3+ cells, mast cells or the distribution and density of neutrophils (all P > 0.05). Furthermore, the severity of gastritis as scored with the Sydney system was similar in both untreated and treated groups. CONCLUSIONS: The results of this study indicate that elimination of H. pylori organisms and resolution of some inflammatory markers occurs as early as 12 h following a single dose of omeprazole 40 mg, amoxycillin 1.0 g and metronidazole 400 mg, which suggests that rational therapeutic strategies with shorter duration using the currently available drugs may be possible.     

Expression of cytokeratins in Helicobacter pylori -associated chronic gastritis of adult patients infected with cagA ＋ strains： An immunohistochemical study

AIM:To investigate the expression of differentcytokeratins(CKs)in gastric epithelium of adult patientswith chronic gastritis infected with Helicobacter pylori(Hpylori)cagA strains.METHODS:The expression of CK 7,8,18,19 and 20was studied immunohistochemically in antral gastricbiopsies of 84 patients.All the CKs were immunostainedin cagA H pylori gastritis(57 cases),non-H pylori gastritis(17 cases)and normal gastric mucosa(10 cases).RESULTS:In cagA H pylori gastritis,CK8 wasexpressed comparably to the normal antral mucosafrom surface epithelium to deep glands.Distributionof CK18 and CK 19 was unchanged,i.e.transmucosal,but intensity of the expression was different in foveolarregion in comparison to normal gastric mucosa.Cytokeratin 18 immunoreactivity was significantly higherin the foveolar epithelium of H pylori-positive gastritiscompared to both Hpylori-negative gastritis and controls.On the contrary,decrease in CK19 immunoreactivityoccurred in foveolar epithelium of H pylori-positive gastritis.In both normal and inflamed antral mucosawithout Hpylori infection,CK20 was expressed strongly/moderately and homogenously in surface epithelium andupper foveolar region,but in H pylori-induced gastritissignificant decrease of expression in foveolar regionwas noted.Generally,in both normal antral mucosa andH pylori-negative gastritis,expression of CK7 was notobserved,while in about half cagA H pylori-infectedpatients,moderate focal CK7 immunoreactivity of theneck and coiled gland areas was registered,especially inareas with more severe inflammatory infiltrate.CONCLUSION:Alterations in expression of CK 7,18,19 and 20 together with normal expression of CK8 occurin antral mucosa of H pylori-associated chronic gastritisin adult patients infected with cagA strains.Alterationsin different cytokeratins expression might contribute toweakening of epithelial tight junctions observed in Hpylori-infected gastric mucosa.     

Expression of COX‐1, COX‐2 and inducible nitric oxide synthase in superficial gastritis,mucosal dysplasia and gastric carcinoma

OBJECTIVE : To investigate the significance of the expression of cyclooxygenase‐1 (COX‐1), cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) in superficial gastritis, gastric mucosal dysplasia and gastric carcinoma, and to study the relationship between COX‐2, iNOS, gastric carcinoma and Helicobacter pylori infection. METHODS : Polyclonal antibodies to COX‐1, COX‐2 and iNOS were used detect their expression and the status of H. pylori infection in 92 specimens of paraffin‐embedded gastric tissue. Of the 92 patients, 33 had superficial gastritis, 30 had gastric mucosal dysplasia and 29 had gastric cancer. Helicobacter pylori was detected by toluidine blue staining. RESULTS : Expression of COX‐2 and iNOS in gastric cancer (65.5%, 62.1%) was significantly higher than that in gastritis (18.2%, 18.2%; P < 0.01). Expression of COX‐2 and iNOS in gastritis with H. pylori infection was higher than that in gastric mucosal dysplasia with H. pylori infection. The expression of COX‐2 and iNOS occurred concomitantly in gastritis, dysplasia and gastric cancer. CONCLUSION : Inflammation and H. pylori infection may be able to stimulate the expression of COX‐2 and iNOS, which might be involved in gastric carcinogenesis.     

Helicobacter pylori eradication decreases the expression of glycosylphosphatidylinositol-anchored complement regulators, decay-accelerating factor and homologous restriction factor 20, in human gastric epithelium

BACKGROUND: It has previously been reported that there is a strong correlation between the expression of glycosylphosphatidylinositol (GPI)-anchored complement membrane inhibitor in gastric epithelium and the severity of inflammation of gastric mucosa. To investigate the regulation of complement activity in gastric epithelium during Helicobacter pylori (H. pylori)-associated gastritis, the expression of GPI-anchored complement membrane inhibitors, decay-accelerating factor (DAF) and 20-kDa homologous restriction factor 20 (HRF20), and membrane cofactor protein (MCP), which is a transmembrane protein, were evaluated after removal of the H. pylori stimulus. Furthermore, the expression of the complement fragment, C3c, was also investigated. METHODS: Forty-six patients with epigastric symptoms and endoscopically confirmed peptic ulcer or gastritis who had H. pylori infection of the gastric mucosa were enrolled in the present study. Biopsy specimens were obtained from the gastric antrum and corpus 1 month before and after eradication. Helicobacter pylori infection was determined by the rapid urease test, histology, and culture before eradication, and by histology, culture, and urea breath test after eradication. Gastric biopsy specimens obtained before and after eradication were evaluated for infiltration by neutrophils and mononuclear cells. The expression of complement membrane inhibitors, DAF, HRF20, and MCP and that of the main complement fragment, C3c, was immunohistochemically evaluated. RESULTS: One month after the eradication of H. pylori, the infiltration by neutrophils and mononuclear cells in the gastric mucosa decreased significantly (P < 0.0001) as compared with that before eradication. The expression of DAF, HRF20, and C3c on gastric mucosal epithelium also significantly decreased in both the antrum and the corpus (P < 0.05) 1 month after eradication. However, no change was observed in the expression of MCP. CONCLUSIONS: The decrease in the expression of GPI-anchored complement regulator and the complement after removal of a chronic microbial stimulus suggests that the gastric epithelium appears to undergo an aggressive stress of complement during H. pylori infection. Conclusively, DAF and HRF20 may play an important protective role against complement-mediated damage induced by chronic microbial stimuli in such a pathological condition.     

慢性胃炎胃黏膜细胞DNA含量和增殖活性的流式细胞仪检测

背景慢性萎缩性胃炎是一种胃癌癌前状态,研究表明正常胃黏膜、癌前病变和胃癌细胞的DNA含量随病变的进展而逐渐增高。目的应用流式细胞仪检测慢性胃炎胃黏膜细胞的DNA含量和增殖活性,探讨两者在慢性胃炎发生、发展过程中的临床意义。方法选取90例经胃镜检查诊断为慢性胃炎者的胃黏膜活检标本,制备单细胞悬液,应用流式细胞仪进行细胞DNA含量和增殖活性检测。结果所有慢性胃炎胃黏膜细胞的DNA倍体类型均为二倍体,但慢性萎缩性胃炎和慢性萎缩性胃炎伴肠化生胃黏膜细胞的增殖指数(PI)较慢性非萎缩性胃炎显著增高(P<0.05)。除慢性非萎缩性胃炎外,其余慢性胃炎组幽门螺杆菌(H.pylori)阳性患者胃黏膜细胞的PI值均较阴性患者显著增高(P<0.05)。结论慢性萎缩性胃炎和H.pylori阳性慢性胃炎胃黏膜细胞的增殖活性显著增高。应用流式细胞仪检测胃黏膜细胞的DNA含量和增殖活性,也许能成为胃癌癌前状态和癌前病变病理诊断的参考指标。     

Overexpression of MT3-MMP in hepatocellular carcinoma correlates with capsular invasion

BACKGROUND/AIMS: Extracellular matrix-degrading matrix metalloproteinases (MMPs) are invariably up-regulated in epithelial cancers and are key agonists of angiogenesis, invasion and metastasis. Recent studies have shown high levels of various MMPs, including MT1-MMP, MMP-1, MMP-2 and MMP-9, and their involvement in tumor progression in human hepatocellular carcinoma (HCC). However, the expression and role of MT3-MMP in HCC remains unclear. METHODOLOGY: We examined the immunohistochemical expression of MT3-MMP in surgically resected HCCs (n=58), hepatitis C virus (HCV) and hepatitis B virus (HBV)-related chronic hepatitis (n=34) and cirrhosis (n=24). RESULTS: MT3-MMP expression was observed in all non-cancerous liver tissues. In HCCs, 52% (30/58) of patients showed high MT3-MMP expression while the remaining 48% (28/58) of patients showed low expression. A clinicopathological survey demonstrated a significant correlation between high MT3-MMP expression and capsular invasion of carcinoma (p = 0.034) although there was no correlation between high MT3-MMP expression in HCC and overall survival or disease-free survival. CONCLUSIONS: MT3-MMP was expressed not only in chronic hepatitis and liver cirrhosis, but also in HCC, and high MT3-MMP expression correlated significantly with capsular invasion of carcinoma.     

Flow cytometric measurement of tumor necrosis factor-related apoptosis-inducing ligand and its receptors in gastric epithelium and infiltrating mucosal lymphocytes in Helicobacter pylori-associated gastritis

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors have recently been reported to be responsible for apoptotic signaling molecules. However, little is known about TRAIL-mediated apoptosis in the human glandular stomach. METHODS: Biopsies from 66 patients (28 Helicobacter pylori-negative, 38 H. pylori-positive) were investigated for phenotypic distribution of TRAIL and its receptors DR4/DR5 and DcR2 on mucosal epithelium, and infiltrating mucosal lymphocytes using flow cytometry. Apoptosis of the cells was examined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick and labeling (TUNEL). In addition, the phenotypic distribution of CD antigens on infiltrating major T- and B-lymphocytes were determined. RESULTS: Membrane-bound TRAIL and its receptors were constitutively expressed in all patients with high levels in the gastric mucosal epithelium. In particular, these protein molecules were overexpressed in mucosal lymphocytes coupled with increased proportions of CD19+ B cells, and CD3+ T cells bearing CD8+CD11b- and CD4+CD62L- surface phenotypes in H. pylori-positive gastric mucosa. The frequencies of apoptotic epithelium and infiltrating lymphocytes in H. pylori-associated gastritis were significantly greater than those of H. pylori-negative normal mucosa (P < 0.01). CONCLUSION: The present findings show that flow cytometric analysis is useful for detection of membrane-bound TRAIL and its receptors in gastric epithelium and infiltrating mucosal lymphocytes.     

Lactoferrin sequestration and its contribution to iron-deficiency anemia in Helicobacter pylori-infected gastric mucosa

BACKGROUND AND AIM: It is known that lactoferrin serves as a source of iron for Helicobacter pylori in gastric mucosa. The present study was undertaken to investigate the relationship between lactoferrin and H. pylori infection coexistent with iron-deficiency anemia by determining the lactoferrin levels in gastric biopsy specimens, and by locating the major sites of lactoferrin expression, according to the presence or absence of iron-deficiency anemia. METHODS: One hundred and one adolescents who underwent gastroduodenoscopy were divided into four groups: controls without H. pylori infection (NL; n =43); patients with H. pylori infection (HP; n = 26); patients with iron-deficiency anemia (IDA; n = 6); and patients with H. pylori gastritis and coexisting iron-deficiency anemia (HPIDA; n = 26). The gastric mucosal levels of lactoferrin were measured by immunoassay. Immunohistochemical technique was used to allow identification of the location and quantification of the lactoferrin expression. RESULTS: The mucosal level of lactoferrin was highest (3.93 +/- 2.73 ng/microg protein) in HPIDA, followed by 2.67 +/- 1.79 ng/microg protein in HP, 0.59 +/- 0.57 ng/microg protein in NL and 0.14 +/- 0.10 ng/microg protein in IDA. Their multiple comparisons were statistically significant at the 0.05 level. After the eradication of H. pylori in 12 HPIDA patients who underwent follow-up endoscopy, the mean mucosal level of lactoferrin decreased significantly, while the blood hemoglobin level correspondingly increased. The major sites of lactoferrin expression by immunohistochemistry were in glands and neutrophils within epithelium. Lactoferrin was stained weakly in NL and IDA, and strongly in HP and HPIDA. CONCLUSION: The lactoferrin sequestration in the gastric mucosa of HPIDA was remarkable, and this finding seems to give a clue that leads to the clarification of the mechanism by which H. pylori infection contributes to iron-deficiency anemia.     

吸烟对冠状动脉搭桥术前大隐静脉桥血管中MMPs/TIMPs基因表达的影响

目的探讨吸烟对冠状动脉搭桥术前大隐静脉桥血管中基质金属蛋白酶及其组织型抑制剂基因表达的影响。方法按入选标准选择110例冠状动脉搭桥患者进行研究,按术前吸烟状态分为吸烟组组45例,对照组36例,戒烟组29例。收集整理所有入选病人详细术前临床资料。术中收集大隐静脉标本,以real-time RT-PCR、免疫组化和western-blot方法阐明吸烟及戒烟对CABG术前大隐静脉桥血管中MMP-2、MMP-9、TIMP-2、TIMP-3的基因表达情况。结果三组术前临床资料无统计学差异。与对照组相比,吸烟组和戒烟组大隐静脉桥血管中MMP-2和MMP-9基因表达明显增强,P〈0.05,并以吸烟组更甚。戒烟6个月以上,与吸烟组相比,MMP-2和MMP-9基因表有所降低,P〈0.05。与MMPs基因表达情况相反,TIMP-2、TIMP-3mRNA的表达在吸烟组和戒烟组中均有降低,分别P〈0.05,也同样以吸烟组更甚,P〈0.05。结论吸烟作为心血管病明确的独立危险因素,严重影响冠状动脉搭桥术前大隐静脉桥血管中MMPs/TIMPs基因表达平衡,并且这种平衡的打破,将对大隐静脉血管中细胞外基质的构成产生重要影响。