Chemiluminescence assay of mucosal reactive oxygen species in gastric cancer, ulcer and antral mucosa

BACKGROUND/AIMS: Reactive oxygen species (ROS) have been implicated in inflammatory and cancerous illness, including that of the gastrointestinal tract. The oxidative damage incurred during human gastric ulcer or cancer mucosa may be related to acumination of ROS. In this study, we aimed to demonstrate oxidative stress of gastric ulcer and cancer mucosa compared to gastric antral mucosa. METHODOLOGY: Patients: Thirty-four patients with gastric ulcer and gastric cancer were enrolled in this study. Gastric mucosa specimens, taken from upper GI endoscopic biopsy, from the lesion (ulcer or cancer) and antrum were sent for the activity of O2- or H2O2 determined by chemiluminescence assay. Protein concentrations in the tissue homogenates were determined by Bio-Red protein assay. The production of O2- or H2O2 per unit of protein was calculated by dividing the tissue CL level by the protein content of a tissue. RESULTS: The oxidative stress metabolites O2- and H2O2 of mucosa were evaluated by chemiluminescence assay in gastric lesions (27 ulcers and 7 cancers) and gastric antrum. Gastric lesion showed significantly increased O2- than antral mucosa (18.77 +/- 45.18 (counts/sec x microg), 95% CI 3.01, 34.53 vs. 3.58 +/- 6.89 (counts/sec x microg), 95% CI 1.18, 5.98, p < 0.05). There was also significantly greater expression of H2O2 in gastric lesion than gastric antral mucosa (76.06 +/- 148.36 (counts/sec x microg), 95% CI 24.30, 127.83 vs. 912.41 +/- 20.22 (counts/sec x microg), 95% CI 5.35, 19.46, p = 0.008). Differences of mucosal O2- and H2O2 between gastric ulcer and cancer were not significant. There was significant correlation of O2- and H2O2 generation in gastric lesion mucosa. CONCLUSIONS: Oxidative stress is now thought to make a significant contribution to inflammatory disease and malignancy. The reason that overproduction of free radicals is a feature of such a broad spectrum of diseases derived from the fact that oxidative metabolism is a necessary part of every cell's metabolism. In this study, we demonstrated increased ROS production in gastric ulceration and cancer compared with gastric antral mucosa.     

Colonic mucosal interleukin-6 in inflammatory bowel disease.

Interleukin-6, a cytokine produced by various cell types, has a major role in inflammatory and immunological reactions. To define its potential role in inflammatory bowel disease, its concentrations in endoscopic biopsy samples from patients with ulcerative colitis and Crohn's disease were measured. The involved colonic mucosa from active disease was found to contain significantly larger amounts of interleukin-6 than that from inactive disease or normal controls. Colonic mucosal interleukin-6 levels correlated well with the grade of macroscopic inflammation, especially in patients with ulcerative colitis. The levels of interleukin-6 decreased in parallel with clinical improvement following the start of therapy in patients with both forms of inflammatory bowel disease. Mucosal interleukin-6 is thus concluded to accurately reflect the degree of colonic inflammation and may be importantly associated with inflammatory and immunological phenomena seen in inflammatory bowel disease.     

Enhanced mucosal cytokine production in inflammatory bowel disease.

Proliferation, maturation, chemotaxis, and activation of neutrophils and monocytes are mediated largely by cytokines, including colony-stimulating factors and lymphokines. Cytokines produced in the intestinal mucosa contribute to the increased migration of neutrophils and monocytes into the lesion of inflammatory bowel disease and to the activation of these inflammatory cells. Lamina propria mononuclear cells isolated from colon tissue from 14 patients with inflammatory bowel disease (IBD) and from histologically normal controls were studied. Cells from IBD-affected tissue produced significantly more colony-stimulating factor activity (1402 +/- 252 U) per 2 x 10(6) cells than those from normal mucosa (362 +/- 85 U), mainly because of the increased production of granulocyte colony-stimulating factor and interleukin 1. This was accompanied by increases in the amount of specific messenger RNA for these two cytokines in lamina propria mononuclear cells from mucosa of patients with Crohn's disease (CD) compared with normal controls. By contrast, there was a substantial reduction in interleukin 3 production in CD and in ulcerative colitis lamina propria mononuclear cells, and this was reflected in significantly reduced expression of interleukin 3 messenger RNA in CD cells. Of the agents used in the therapy of IBD, hydrocortisone and 5-aminosalicylic acid, but not cyclosporin A, markedly suppressed in vitro production of cytokines by lamina propria mononuclear cells, suggesting that their therapeutic efficacy in vivo may be due in part to down-regulation of cytokine production in the inflamed mucosa.     

Rectal mucosal plasma cells in inflammatory bowel disease.

To achieve optimum staining and reproducible counts of plasma cells in paraffin embedded tissue with the immunoperoxidase technique we have found it essential to obtain a plateau count by titration of antisera for each specimen. This modification was used to study IgA, IgM, IgE, and IgG plasma cells in rectal biopsies from 20 controls, 20 patients with ulcerative proctocolitis, 20 with Crohn's colitis, 20 with non-specific proctitis, 15 with bacterial colitis, and seven with Crohn's disease but no apparent large bowel involvement. Counts were correlated with the characteristic histological features of inflammatory bowel disease. In controls the ratio of the mean counts for IgA, IgM, IgE, and IgG plasma cells was 8:3:3:1. All types of plasma cells were very significantly increased in the patients with ulcerative proctocolitis, Crohn's colitis, and non-specific proctitis and counts correlated with the severity of inflammation. There was no significant difference between the counts in these three groups. All counts tended to be higher in bacterial colitis than in controls, the difference being significant for IgA and IgE. When matched for severity of inflammation there was no significant difference between the counts in bacterial colitis and inflammatory bowel disease. The counts in patients with Crohn's disease but no large bowel involvement were not significantly different from controls. These results suggest that changes in plasma cell counts in inflammatory bowel disease are a non-specific response to mucosal damage, possible by a luminal irritant, and do not differentiate the type of inflammatory bowel disease.     

Gut mucosal lymphocytes in inflammatory bowel disease

We have developed an enzymatic technique for isolating human intestinal mucosal lymphoid cells. This method was found to be superior to mechanical methods in regard to cell yield and survival. It is based on treating mucosa with serum-free solutions containing collagenase and deoxyribonuclease, followed by isolating the lymphoid cells through centrifugation steps involving fetal calf serum and ficoll-hypaque. Exposure of peripheral blood lymphocytes to the components of the enzymatic solution did not appreciably alter their uptake of tritiated thymidine in the presence or absence of mitogens. Application of the method to derive lymphoid cells from Crohn's disease, ulcerative colitis, and normal intestinal mucosa has shown that gut mucosal lymphocytes from inflammatory bowel disease (1) exceed the number of those from normal mucosa by a factor of 3 to 5; (2) show different degrees of tritiated thymidine uptake, spontaneously and in response to mitogens, depending upon the time they are harvested during the dissociation process; (3) are better stimulators than responders in the allogeneic mixed lymphocyte reaction; (4) generate suppressor cell activity comparable to that of peripheral blood lymphocytes; (5) cannot, in contrast to peripheral blood lymphocytes, generate antibody-dependent cell mediated cytotoxicity; and (6) produce an average of 5 times more IgM than equal numbers of peripheral blood lymphocytes.     

肠黏膜屏障与炎症性肠病

炎症性肠病（Innammatory bowel disease，IBD）是一组病因不明的慢性肠道炎症性疾病，主要包含两个独立的疾病，溃疡性结肠炎（Ulcerative colitis，UC）和克罗恩病（Crohn’s disease，CD）。近年研究发现，肠黏膜屏障功能异常在IBD发病机制中发挥重要作用。更好地了解正常及疾病状态下肠黏膜屏障的结构和功能可以为IBD的治疗提供新的思路。     

炎症性肠病患者肠黏膜组织细胞凋亡

炎症性肠病(inflammatory bowel disease,IBD)是发生在胃肠道慢性非特异性炎症性疾病,其病因及发病机制尚未十分明确,可能与环境因素、遗传因素和免疫因素等有关.研究表明细胞凋亡在IBD的发病中起着重要的作用,表现为炎症肠黏膜组织内存在细胞凋亡紊乱,肠上皮细胞凋亡过度、黏膜固有层组织内淋巴细胞凋亡抵抗,以及PMN凋亡迟滞.这是造成IBD肠道炎症发生和持续的重要原因.研究发现发生细胞凋亡的主要机制在于激活了Fas/FasL信号传导途径、Bcl-2和Bax调节途径而实施的.研究细胞凋亡机制对揭示IBD的发病机制.靶向性阻断细胞凋亡通路治疗IBD发生有重要意义.     

Pathology of inflammatory bowel disease in colorectal mucosal biopsies

The importance of colorectal biopsies in the diagnosis and management of inflammatory bowel disease (IBD) is reviewed and the histologic criteria for identifying the different types of colitis are outlined. While most of the histopathologic features of the various forms of colitis are characteristic, none is pathognomonic. Accordingly, emphasis is placed on considering all clinical and investigational data in evaluating histopathologic changes found in colorectal mucosal biopsies.     

Role of mucosal dendritic cells in inflammatory bowel disease

The gastrointestinal innate and adaptive immune system continuously faces the challenge of potent stimuli from the commensal microflora and food constituents. These local immune responses require a tight control, the outcome of which is in most cases the induction of tolerance. Local T cell immunity is an important compartment of the specif ic intestinal immune system. T cell reactivity is programmed during the initial stage of its activation by professional presenting cells. Mucosal dendritic cells （DCs） are assumed to play key roles in regulating immune responses in the antigenrich gastrointestinal environment. Mucosal DCs are a heterogeneous population that can either initiate （innate and adaptive） immune responses, or control intestinal inflammation and maintain tolerance. Defects in this regulation are supposed to lead to the two major forms of inflammatory bowel disease （IBD）, Crohn＇s disease （CD） and ulcerative colitis （UC）. This review will discuss the emerging role of mucosal DCs in regulating intestinal inflammation and immune responses.     

The role of the mucosal immune system in inflammatory bowel disease.

Continued delineation of the major factors that lead to intestinal inflammation will provide critical insights into many of the pathophysiologic events leading to tissue destruction in IBD. The exploration of exciting and important new areas, such as the role of adhesion molecules, proinflammatory cytokines, and the activation of lymphocytes and phagocytes, will contribute significantly to a better understanding of the mechanisms that sustain the intestinal inflammatory process. Determining the mechanisms of amplification and perpetuation of intestinal inflammation as well as learning more about the natural suppression of intestinal inflammation by the normal cellular and cytokine networks of the mucosal immune system will open exciting new therapeutic approaches. It is encouraging to see realistic and testable working models emerge from the combined efforts of many committed investigators who have been engaged in studying the role of the mucosal immune system in the pathophysiology of IBD. A great deal more remains to be learned in this rapidly advancing area, and we can look forward with confidence to continued advances in the study of IBD.     

Characteristics of isolated intestinal mucosal lymphoid cells in inflammatory bowel disease.

Immunologic characteristics of intestinal mucosal lymphoid cells from patients with inflammatory bowel disease and controls have been compared. Mononuclear cells isolated by enzymatic means from intestinal tissues involved with inflammatory bowel disease were present in greater numbers, with increased proportions of macrophages and B-lymphocytes, particularly cells bearing intrinsic membrane immunoglobulin G. Synthesis of immunoglobulin G, measured by radioimmunoassay, was increased tenfold in inflammatory bowel disease, while immunoglobulin A synthesis per 10(6) cells was unchanged. "Null" or K-lymphocytes were absent from all populations, and antibody-dependent cellular cytotoxicity (a K-cell-mediated function) was not demonstrable. Taken together, the results fail to support a role for antibody-dependent cellular cytotoxicity or a defect in secretory immunoglobulin A, but rather focus attention upon possible forms of immunoglobulin G-mediated tissue damage in the pathogenesis or perpetuation of inflammatory bowel disease.     

Homocysteine triggers mucosal microvascular activation in inflammatory bowel disease

OBJECTIVES: Increased homocysteine contributes to the pathophysiology of several chronic inflammatory diseases. Whether homocysteine could participate in mucosal inflammation in inflammatory bowel disease (IBD) has not been explored yet. Our aims were to study the levels of plasma and mucosal homocysteine in IBD patients and to assess whether homocysteine can trigger an inflammatory reaction on human intestinal microvascular endothelial cells (HIMECs). METHODS: Homocysteine was measured in the plasma, mucosal biopsy, and lamina propria mononuclear cell (LPMC) supernatants from normal and IBD subjects. HIMEC were cultured in presence of homocysteine, TNF-alpha, or folic acid, alone or in combination. Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular cell adhesion molecule 1 was measured by flow cytometry and monocyte chemoattractant protein-1 (MCP-1) production by ELISA. Phosphorylation of p38 and p42/44 was assessed by immunoblot in HIMEC extracts. T-cell- and monocyte-HIMEC adhesion assays were used to evaluate the impact of homocysteine on leukocyte adhesion to intestinal endothelial cells. RESULTS: Patients with IBD displayed significantly higher homocysteine plasma and mucosal levels than control subjects. IBD-derived LPMC released higher homocysteine than control-derived LPMC. Treatment of HIMEC with homocysteine, and synergistically with the combination of TNF-alpha and homocysteine, triggered HIMEC inflammation, resulting in VCAM-1 up-regulation, MCP-1 production, and p38 phosphorylation. These events lead to an increased capacity of HIMEC to adhere T- and monocyte cells and were blocked by folic acid treatment. CONCLUSIONS: Homocysteine is increased in both the mucosa and plasma of patients with Crohn's disease and ulcerative colitis and contributes to the inflammatory state of the mucosal IBD endothelium. Therefore, homocysteine could play a proinflammatory role in IBD, which can be efficiently targeted by folic acid supplementation.     

Rectal mucosal nitric oxide in differentiation of inflammatory bowel disease and irritable bowel syndrome.

BACKGROUND & AIMS: Differentiating patients with functional bowel disorders from those with inflammatory bowel disease (IBD) can be difficult. Rectal luminal levels of nitric oxide (NO) are greatly increased in IBD. To further evaluate this disease marker, we compared NO in patients with irritable bowel syndrome (IBS) with those found in patients with active IBD and in healthy control subjects. METHODS: Rectal NO was measured with chemiluminescence technique by using a tonometric balloon method in 28 healthy volunteers, 39 patients with IBS, 86 with IBD (Crohn's disease and ulcerative colitis), and 12 patients with collagenous colitis. In addition, NO was measured before and after a 4-week treatment period in patients with active ulcerative colitis and repeatedly during 2 weeks in healthy volunteers. RESULTS: NO was low in healthy control subjects (median, 45; 25th-75th percentile, 34-64 parts per billion [ppb]), and variations over time were small. In IBS patients NO was slightly increased (150, 53-200 ppb; P < .001), whereas patients with active IBD or collagenous colitis had greatly increased NO levels (3475, 575-8850 ppb, and 9950, 4475-19,750 ppb, respectively; P < .001). With a cutoff level of 250 ppb, NO had a sensitivity of 95% and a specificity of 91% in discriminating between active bowel inflammation and IBS. Rectal NO correlated with disease activity in IBD and collagenous colitis and decreased markedly in IBD patients responding to anti-inflammatory treatment. CONCLUSIONS: Rectal NO is a minimally invasive and rapid tool for discriminating between active bowel inflammation and IBS and a possibly useful add-on for monitoring patients with IBD.     

Limitations in assessment of mucosal healing in inflammatory bowel disease

An emerging parameter to define the effectiveness of new therapeutic agents in clinical trials,and by extension,for use in day-to-day clinical practice has been labeled mucosal healing.It has been hypothesized that complete healing of the intestinal mucosa in inflammatory bowel diseases should result in reduced disease complications,reduced hospitalization and reduced surgical treatment.By implication,the natural history of inflammatory bowel disease might then be altered. Measurement of mucosal healing,how...     

Limitations in assessment of mucosal healing in inflammatory bowel disease

An emerging parameter to define the effectiveness of new therapeutic agents in clinical trials,and by extension,for use in day-to-day clinical practice has been labeled mucosal healing.It has been hypothesized that complete healing of the intestinal mucosa in inflammatory bowel diseases should result in reduced disease complications,reduced hospitalization and reduced surgical treatment.By implication,the natural history of inflammatory bowel disease might then be altered. Measurement of mucosal healing,how...     

Alterations of the mucosal immune system in inflammatory bowel disease

The normal intestinal immune system is under a balance in which proinflammatory and anti-inflammatory cells and molecules are carefully regulated to promote a normal host mucosal defense capability without destruction of intestinal tissue. Once this careful regulatory balance is disturbed, nonspecific stimulation and activation can lead to increased amounts of potent destructive immunologic and inflammatory molecules being produced and released. The concept of balance and regulation of normal mucosal immune and inflammatory events is indicative of how close the intestine is to developing severe inflammation. The normal intestinal mucosal immune system is constantly stimulated by lumenal contents and bacteria. The stimulatory molecules present in the intestinal lumen that activate and induce subsequent mucosal immunologic and inflammatory events include bacterial cell wall products, such as peptidoglycans and lipopolysaccharides, as well as other chemotactic and toxic bacterial products that are produced by the many different types of bacteria within the gastrointestinal tract. These highly stimulatory bacterial cell wall products are capable of activating macrophages and T lymphocytes to release potent proinflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). IL-1, IL-6, and TNF-α increase the presence of human leukocyte antigen (HLA) class II antigen-presenting molecules on the surfaces of epithelial cells, endothelial cells, macrophages, and B cells, thus increasing their ability to present lumenal antigens and bacterial products. The proinflammatory cytokines IL-1 and TNF-α also increase the ability of epithelial cells, endothelial cells, macrophages, and fibroblasts to secrete potent chemotactic cytokines, such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), which serve to increase the movement of macrophages and granulocytes from the circulation into the inflamed mucosa. Thus, through lumenal exposure to potent, nonspecific stimulatory bacterial products, the state of activation of the intestinal immune system and mucosal inflammatory pathways are markedly up-regulated. This raises the question of whether there is a deficiency in effective down-regulation through the absence of normally suppressive cytokines such as interleukin-10 (IL-10), transforming growth factor-β (TGF-β), interleukin-4 (IL-4), and IL-1 receptor antagonist. Normally, the turning off of the active and destructive immunologic and inflammatory events should occur following the resolution of a bacterial or viral infection that has been appropriately defended against and controlled by the mucosal immune system. In inflammatory bowel disease (IBD), however, the down-regulatory events and processes that should turn off the immunologic and inflammatory protective processes, once the pathogenic agent has been cleared, appear to be deficient or only partially effective. We may find that we ultimately are dealing with disease processes that have more than one genetic or cellular basis. The improved understanding of the immunopathophysiology of IBD will allow exploration of novel immunologic and genetic approaches, such as gene replacement therapy, administration of a suppressor cytokine or an altered cell surface antigen, the administration ofhumanized monoclonal antibodies directed against proinflammatory cytokines, or the development of newer strategies against fundamenial cell biologic mechanisms such as adhesion molecules.     

Value of mucosal assessment and biomarkers in inflammatory bowel disease

Clinical remission at a single point in time provides surprisingly little predictive value for future inflammatory bowel disease (IBD) activity owing to the waxing and waning nature of the disease course. Furthermore, patients often present with complications of IBD despite apparent clinical remission, suggesting that undetected subclinical inflammation is driving these complications. This has led to research on a variety of surrogate markers of biologically significant asymptomatic inflammatory disease activity, including endoscopic healing, histologic normalization and biomarkers of inflammation in the blood and stool. If these have strong predictive value, they could be used to risk-stratify patients and justify the early use of immunomodulators and anti-TNF agents. Mucosal healing has been associated with positive outcomes in IBD, but the supporting data are largely retrospective and subject to channeling bias, and it is not clear whether complete mucosal healing produces better outcomes than partial healing. Stool and blood biomarkers correlate well with mucosal inflammation, but are imperfect surrogates for mucosal healing. Before using surrogate markers of intestinal inflammation to justify long-term, potentially toxic and costly therapy, prospective longitudinal studies are needed to identify surrogate end points with cut points that justify changes in therapy, and which therapies provide cost-effective benefits for mild, moderate or severe inflammation.     

Studies on isolated gut mucosal lymphocytes in inflammatory bowel disease

Digestive Diseases and Sciences - To determine whether a defective proliferation of gut mucosal lymphocytes is a contributory factor to the pathogenesis of inflammatory bowel disease, we assessed...     

Interleukin 4 in inflammatory bowel disease and mucosal immune reactivity

BACKGROUND & AIMS: Interleukin (IL) 4 has immunoregulatory and anti- inflammatory activities, but little is known about IL-4 in the human gut. We investigated production of IL-4 by isolated lamina propria mononuclear cells (LPMCs) from normal and inflamed intestine and its capacity to modulate local immune responses. METHODS: IL-4 levels were measured by enzyme-linked immunosorbent assay in cultures of control and inflammatory bowel disease LPMCs, and the effect of IL-4 on LPMC proliferation and interaction with IL-2, IL-1 beta, lipopolysaccharide, bacterial antigens, superantigen, and antibodies to various T-cell receptors was investigated. RESULTS: Various stimuli induced LPMCs to produce IL-4, but inflammatory bowel disease cells expressed IL-4 messenger RNA and secreted protein in significantly lower amounts than control cells. IL-4 failed to stimulate proliferation by fresh LPMCs, but a vigorous dose-dependent response was observed after preactivation by phytohemagglutinin, IL-2, or IL-4. When added to fresh LPMCs, IL-4 inhibited IL-2-induced proliferation. IL-4 amplified proliferation to IL-1 beta, lipopolysaccharide, peptidoglycan-polysaccharide complexes, staphylococcus enterotoxin A, and antibodies to the CD3 and CD28 receptors but not to tetanus toxoid. CONCLUSIONS: Decreased production of IL-4 in inflammatory bowel disease may cause defective immunosuppressive and anti-inflammatory mechanisms and may contribute to disease pathogenesis. The ability of IL-4 to differentially modulate LPMC reactivity probably influences mucosal immune homeostasis. (Gastroenterology 1996 Jun;110(6):1683-95)