Stephen P. James

Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis

BACKGROUND & AIMS Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are important regulators of mucosal inflammation and epithelial cell growth. To determine the role of iNOS and COX-2 in Helicobacter pylori-induced tissue injury, we compared their gene expression in H. pylori-induced gastritis with that in normal gastric mucosa and in non-H. pylori gastritis. METHODS In 43 patients, we assessed H. pylori infection status, histopathology, messenger RNA (mRNA) and protein expression, and cellular localization of iNOS and COX-2. RESULTS By reverse-transcription polymerase chain reaction (RT-PCR), antral iNOS and COX-2 mRNA expression was absent to low in normal mucosa (n = 10), significantly increased in H. pylori-negative gastritis (n = 13), and even more markedly increased in H. pylori-positive gastritis (n = 20). Increased iNOS and COX-2 levels were confirmed by Northern and Western blot analysis and were both greater in the gastric antrum than in the gastric body of infected patients. Immunohistochemistry also showed increased expression of both genes in H. pylori gastritis: iNOS protein was detected in epithelium, endothelium, and lamina propria inflammatory cells, and COX-2 protein localized to mononuclear and fibroblast cells in the lamina propria. CONCLUSIONS iNOS and COX-2 are induced in H. pylori-positive gastritis and thus may modulate the inflammation and alterations in epithelial cell growth that occur in this disease. Higher levels of iNOS and COX-2 in H. pylori-positive vs. -negative gastritis and in gastric antrum, where bacterial density is greatest, suggest that expression of these genes is a direct response to H. pylori infection.

Primary sclerosing cholangitis: Summary of a workshop†‡

Primary sclerosing cholangitis (PSC) is a rare but important liver disease that leads to cirrhosis and need for liver transplantation in a high proportion of cases. The disease occurs in approximately 1 per 100,000 population per year, usually presents in adulthood, and affects men more often than women. Typical serum biochemical results, autoantibodies and liver biopsy are suggestive but not diagnostic of PSC, the diagnosis requiring cholangiographic demonstration of stricturing and dilatation of the intra‐ and/or extra‐hepatic bile ducts. The natural history of PSC is variable, the average survival being 12 to 17 years. The cause of PSC is still unknown. Although considered an autoimmune disease, PSC has several atypical features and a strong genetic component. The therapy of PSC is unsatisfactory. Standard doses of ursodeoxycholic acid (UDCA) lead to improvements in biochemical abnormalities but not in histology, cholangiographic appearance or survival. Several innovative therapies have been tried in PSC, but with scant evidence of benefit. For patients with high grade strictures, endoscopic dilatation is beneficial. Liver transplantation is successful for end‐stage liver disease due to PSC and improves survival. PSC may recur after transplantation but is rarely progressive. The most dreaded complication of PSC is cholangiocarcinoma. Diagnosis of this highly malignant tumor is difficult, and there are no biomarkers for its early detection. Liver transplantation for cholangiocarcinoma has an exceedingly poor outcome, although transplantation with neoadjuvant chemoirradiation holds promise in selected patients. Thus, significant opportunities remain for basic and clinical research into the cause, natural history, and therapy of PSC. (HEPATOLOGY 2006;44:746–764.)

Primary Biliary Cirrhosis: A Model Autoimmune Disease

: Primary biliary cirrhosis is characterized by chronic inflammation and necrosis of the intrahepatic bile ducts and by chronic cholestasis. The presence of autoantibodies in serum, the association of this disease with other autoimmune diseases, the histologic appearance of typical hepatic lesions, the presence of bile duct lesions that resemble those seen in chronic graft-versus-host disease, and the possible recurrence of a chronic cholestatic syndrome in patients having hepatic transplants indicate that immune mechanisms play a role in the syndromes pathogenesis. Patients have diminished function of suppressor T cells that may be due to abnormal activation of suppressor T cells caused by interactions with autologous non-T cells. This nonspecific immunologic defect and other immune defects may cause the autoimmune manifestations of primary biliary cirrhosis. Treatments to arrest the diseases progress have included corticosteroids, azathioprine, cyclosporin, and D-penicillamine. These treatments, which affect immune functions, have not had a beneficial effect on the disease process. Better understanding of the pathogenesis of primary biliary cirrhosis is needed to develop specific immunotherapies.

A Large Toxin from Pathogenic Escherichia coli Strains That Inhibits Lymphocyte Activation

ABSTRACT The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic E. coli strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E. coli is associated with gain of this activity. Among 89 strains of E. coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.

Helicobacter pylori stimulates inducible nitric oxide synthase expression and activity in a murine macrophage cell line

BACKGROUND & AIMS Helicobacter pylori uniquely colonizes the human stomach and produces gastric mucosal inflammation. High-output nitric oxide production by inducible nitric oxide synthase (iNOS) is associated with immune activation and tissue injury. Because mononuclear cells comprise a major part of the cellular inflammatory response to H. pylori infection, the ability of H. pylori to induce iNOS in macrophages was assessed. METHODS H. pylori preparations were added to RAW 264.7 murine macrophages, and iNOS expression was assessed by Northern blot analysis, enzyme activity assay, and NO2- release. RESULTS Both whole H. pylori and French press lysates induced concentration-dependent NO2- production, with peak levels 20-fold above control. These findings were paralleled by marked increases in iNOS messenger RNA and enzyme activity levels. iNOS expression was synergistically increased with interferon gamma, indicating that the H. pylori effect can be amplified by other macrophage-activating factors. Studies of lipopolysaccharide (LPS) content and polymyxin B inhibition of LPS suggested that the H. pylori effect was attributable to both LPS-dependent and -independent mechanisms. CONCLUSIONS iNOS expression in macrophages is activated by highly stable H. pylori products and may play an important role in the pathogenesis of H. pylori-associated gastric mucosal disease.

Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia

The adenomatous polyposis coli (APC) tumor suppressor gene is mutationally inactivated in both familial and sporadic forms of colorectal cancers. In addition, hypermethylation of CpG islands in the upstream portion of APC, a potential alternative mechanism of tumor suppressor gene inactivation, has been described in colorectal cancer. Because a subset of both gastric and colorectal cancers display the CpG island methylator phenotype, we hypothesized that epigenetic inactivation of APC was likely to occur in at least some gastric cancers. APC exhibits two forms of transcripts from exons 1A and 1B in the stomach. Therefore, we investigated CpG island methylation in the sequences upstream of exons 1A and 1B, i.e., promoters 1A and 1B, respectively. We evaluated DNAs from 10 gastric cancer cell lines, 40 primary gastric cancers, and 40 matching non-cancerous gastric mucosae. Methylated alleles of promoter 1A were present in 10 (100%) of 10 gastric cancer cell lines, 33 (82.5%) of 40 primary gastric cancers, and 39 (97.5%) of 40 non-cancerous gastric mucosae. In contrast, promoter 1B was unmethylated in all of these same samples. APC transcripts from exon 1A were not expressed in nine of the 10 methylated gastric cancer cell lines, whereas APC transcripts were expressed from exon 1B. Thus, expression from a given promoter correlated well with its methylation status. We conclude that in contrast to the colon, methylation of promoter 1A is a normal event in the stomach; moreover, promoter 1B is protected from methylation in the stomach and thus probably does not participate in this form of epigenetic APC inactivation.

Lymphocytes isolated from the intestinal lamina propria of normal nonhuman primates have increased expression of genes associated with T-cell activation.

Synthesis of interleukin-2 (IL-2) and expression of interleukin-2 receptors play central roles in T-cell activation, proliferation, and differentiation. The state of activation of T lymphocytes in the intestinal lamina propria was compared with that of circulating lymphocytes and lymphocytes isolated from the spleen or mesenteric lymph nodes of normal nonhuman primates. Lamina propria lymphocytes (LPL) had significantly higher proliferation in response to recombinant IL-2 compared with the other populations. In agreement with this finding, LPL had a significantly higher percentage of interleukin-2 receptor-positive (IL-2R+) cells as determined by staining with fluoresceinated monoclonal anti-IL-2R antibody. Two-color immunofluorescence staining showed that both CD4+ and CD8+ lamina propria T cells were IL-2R+. It was also found that the percentage of major histocompatibility complex class II-positive T cells was higher in the lamina propria. Northern blot analysis with a cDNA specific for the IL-2R showed that unstimulated LPL had easily detectable IL-2R mRNA, whereas no IL-2R mRNA was found in unstimulated lymphocyte populations from other sites. The activation of the IL-2R gene in LPL was not associated with the activation of other cellular genes (actin, major histocompatibility complex class I). Although no IL-2 bioactivity was measured in culture supernatants of unstimulated lymphocytes, concanavalin A-stimulated LPL produced significantly more IL-2 than other lymphocytes. This finding was confirmed at the molecular level as IL-2 mRNA was not detected in unstimulated LPL but was found in concanavalin A-stimulated LPL. Thus, normal lamina propria T lymphocytes have selective expression of genes associated with cell activation.

The immunologic basis of inflammatory bowel disease

The idiopathic inflammatory bowel diseases (IBD) comprise a spectrum of disorders that are marked by the presence of chronic inflammation of the gastrointestinal tract which cannot be ascribed to a specific pathogen (1, 2). At one end of the spectrum is ulcerative colitis, a disease that affects the large bowel exclusively and which is characterized by mucosal ulceration, superficial inflammatory-cell infiltration of the bowel wall, and, in extensive long-standing cases, neoplastic transformation. Prominent symptoms in this form of IBD include severe bloody diarrhea, pain on defecation, weight loss, and, in children, growth failure. At the other end of the spectrum is Crohns disease (regional enteritis or regional ileitis), a disease that in contrast to ulcerative colitis can affect any part of the alimentary canal, from the mouth to the rectum, but which most commonly involves the terminal ileum and the ascending colon. In this condition, the major pathology consists of a focal, transmural inflammatory,cell infiltration that is sometimes granulomatous in character and which may be complicated by intestinal strictures, fistula formation, and perforation, Symptoms that commonly occur in Crohns disease include abdominal pain of a cramping nature and systemic complaints such as fever

Mucosal T cells provide helper function but do not proliferate when stimulated by specific antigen in lymphogranuloma venereum proctitis in nonhuman primates

To study antigen-specific immune responses of gut-associated T lymphocytes after gastrointestinal infection, Cynomolgus monkeys were inoculated rectally with Chlamydia trachomatis of the L2 [lymphogranuloma venereum (LGV)] strain. Infected monkeys developed a chronic proctitis with the appearance of LGV-specific immunoglobulin G-antibodies in the serum. Lymphocytes isolated from the peripheral blood, the spleen, and draining lymph nodes had a vigorous antigen-specific proliferative response to LGV in vitro. Both T and B cells proliferated in response to stimulation with LGV, but B-cell proliferation was T-cell-dependent, as shown by cell separation techniques and cell-cycle analysis with dual-laser flow cytometry. Lymphocytes isolated from both involved and uninvolved lamina propria did not proliferate in response to LGV stimulation, whereas mitogen-induced proliferation was not different in lamina propria lymphocytes and the other lymphocyte populations. This lack of antigen-specific proliferation was not caused by a suppressor effect of mucosal T cells or monocytes or the absence of antigen-presenting cells. In contrast, lamina propria T lymphocytes from infected animals were able to provide antigen-specific help for polyclonal immunoglobulin synthesis by immune B lymphocytes after stimulation with LGV. Thus, in LGV proctitis in monkeys, mucosal antigen-reactive T cells differ from lymphocytes in other sites in that they can provide helper function, but are not able to proliferate in response to LGV antigens.

Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia

A significant portion of gastric cancers exhibit defective DNA mismatch repair, manifested as microsatellite instability (MSI). High-frequency MSI (MSI-H) is associated with hypermethylation of the human mut-L homologue 1 (hMLH1) mismatch repair gene promoter and diminished hMLH1 expression in advanced gastric cancers. However, the relationship between MSI and hMLH1 hypermethylation has not been studied in early gastric neoplasms. We therefore investigated hMLH1 hypermethylation, hMLH1 expression and MSI in a group of early gastric cancers and gastric adenomas. Sixty-four early gastric neoplasms were evaluated, comprising 28 adenomas, 18 mucosal carcinomas, and 18 carcinomas with superficial submucosal invasion but clear margins. MSI was evaluated using multiplex fluorescent PCR to amplify loci D2S123, D5S346, D17S250, BAT 25 and BAT 26. Methylation-specific PCR was performed to determine the methylation status of hMLH1. In two hypermethylated MSI-H cancers, hMLH1 protein expression was also evaluated by immunohistochemistry. Six of sixty-four early gastric lesions were MSI-H, comprising 1 adenoma, 4 mucosal carcinomas, and 1 carcinoma with superficial submucosal invasion. Two lesions (one adenoma and one mucosal carcinoma) demonstrated low-frequency MSI (MSI-L). The remaining 56 neoplasms were MSI-stable (MSI-S). Six of six MSI-H, one of two MSI-L, and none of thirty MSI-S lesions showed hMLH1 hypermethylation (P<0.001). Diminished hMLH1 protein expression was demonstrated by immunohistochemistry in two of two MSI-H hypermethylated lesions. hMLH1 promoter hypermethylation is significantly associated with MSI and diminished hMLH1 expression in early gastric neoplasms. MSI and hypermethylation-associated inactivation of hMLH1 are more prevalent in early gastric cancers than in gastric adenomas. Thus, hypermethylation-associated inactivation of the hMLH1 gene can occur early in gastric carcinogenesis.