Helicobacter pylori HP(2-20) induces eosinophil activation and accumulation in superficial gastric mucosa and stimulates VEGF-alpha and TGF-beta release by interacting with formyl-peptide receptors.

Eosinophils participate in the immune response against Helicobacter pylori, but little is known about their role in the gastritis associated to the infection. We recently demonstrated that the Hp(2-20) peptide derived from H. pylori accelerates wound healing of gastric mucosa by interacting with N-formyl peptide receptors (FPRs) expressed on gastric epithelial cells. The aim of the present study was to investigate whether eosinophils play a role in the repair of gastric mucosa tissue during H. pylori infection. Immuno-histochemistry and transmission electron microscopy were used to detect eosinophils in gastric mucosal biopsies. Eosinophil re-distribution occurred in the gastric mucosa of H. pylori-infected patients: their density did not change in the deep mucosal layer, whereas it increased in the superficial lamina propria just below the foveolar epithelium; eosinophils entered the epithelium itself as well as the lumen of foveolae located close to the area harboring bacteria, which in turn were also engulfed by eosinophils. The H. pylori-derived peptide Hp(2-20) stimulated eosinophil migration through the engagement of FPR2 and FPR3, and also induced production of VEGF-A and TGF-beta, two key mediators of tissue remodelling. We also demonstrate that Hp(2-20) in vivo induced eosinophil infiltration in rat gastric mucosa after injury brought about by indomethacin. This study suggests that eosinophil infiltrate could modulate the capacity of gastric mucosa to maintain or recover its integrity thereby shedding light on the role of eosinophils in H. pylori infection.

Prevalence of double pituitary adenomas in a surgical series: Clinical, histological and genetic features.

BACKGROUND: The term double pituitary adenomas (DPA) is usually referred to those rare lesions showing two distinct cellular components. Genetic background may sustain the proliferation of more than one cell at the same time but no information is available on the presence of aip mutations in these patients. AIM: We report the prevalence and the endocrinological, neuroradiological, histopathological and genetic features of DPA detected in a large surgical series. The contribution of pituitary transcription factor immunostains in DPA was also evaluated. SUBJECTS AND METHODS: One-hundred-forty-four patients undergoing surgery for tumors of the sellar region were evaluated. Histopathology, immunohistochemistry and the mutational analysis for the entire coding region of the AIP and MEN1 genes were performed. RESULTS: One-hundred-seventeen patients out of 144 had a pituitary adenoma. DPA was found in 3 (2.6%) out of 117 patients with pituitary adenoma. Immunohistochemistry and transcription factors analysis demonstrated two not yet described histotype associations in DPA. The coexistence of somatotroph-lactotroph and silent mammosomatotroph histotype in 1 case and the coexistence of sparsely granulated lactotroph and null cell adenomas in the remaining two cases were first identified. Sequencing data for the coding region of the aip and the menin gene resulted in wild type sequences in all patients with DPA. CONCLUSIONS: The prevalence of DPA observed in our unselected surgical series is not negligible (2.6%). Furthermore, the evaluation of the treatment outcome would suggest that the clinical management of DPAs requires a careful diagnostic approach and follow- up.

High cell sensitivity to Helicobacter pylori VacA toxin depends on a GPI-anchored protein and is not blocked by inhibition of the clathrin-mediated pathway of endocytosis.

Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains ("lipid rafts"), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.

Mammary tumors induced in rats by adriamycin and daunomycin.

The two anthracycline antitumor antibiotics, Adriamycin and daunomycin (DM), induced a high incidence of mammary tumors, both fibroadenomas and adenocarcinomas, in female rats that received a single i.v. dose, thus confirming previous results. The incidence of DM-induced adenocarcinomas increased with the dose of the drug, whereas the incidence of Adriamycin-induced adenocarcinomas showed a plateau at 5 mg/kg and above. Adriamycin- and DM-induced fibroadenomas showed a peak at lower doses (about 5 to 6 mg/kg). With the highest DM dose (12.5 mg/kg) used, there was a slight prevalence of adenocarcinomas over fibroadenomas.

K-ras and p53 gene mutations in pancreatic cancer: ductal and nonductal tumors progress through different genetic lesions.

We studied K-ras and p53 gene mutations in a panel of 57 primary pancreatic cancers including ductal and nonductal tumors. DNAs were obtained from formalin-fixed, paraffin-embedded material. Target sequences were amplified by polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis and sequencing. Both K-ras and p53 genes were frequently mutated in ductal cancers (25 of 35, 71.4%; 18 of 35, 51.4%, respectively). K-ras mutations were confined to the second position of codon 12 where base transitions and transversions were equally observed. p53 changes were mainly missense mutations. Transitions and transversions were found equally with a prevalence of G:C-->A:T changes among transitions. No gene alterations were present in the 6 exocrine nonductal tumors and (with one exception) in the 12 endocrine tumors analyzed. Our results indicate that mutated K-ras and p53 genes can cooperate in the establishment of ductal pancreatic cancers, whereas other genetic events have to be present in nonductal tumors. Moreover, K-ras alterations may represent an early event in ductal tumorigenesis, as suggested both by the high gene mutation frequency and by the presence of mutations in low-grade tumors. On the contrary, p53 gene changes seem to represent an event required for the malignancy progression of ductal tumors from lower to higher grades.

Cell biology, clinicopathological profile, and classification of gastro-enteropancreatic endocrine tumors.

Recent developments in the field of endocrine cell biology and pathology at both morphological and molecular levels are briefly outlined and discussed as a basis for endocrine tumor characterization. The main tools available for identifying the endocrine nature of the tumors, their pathogenetic interpretation. and experimental reproduction with special emphasis on tumor antecedents are reported. Based on this, classifications of endocrine tumors of the pancreas and gastrointestinal tract are developed, covering most clinical (hyperfunctional syndromes included), pathological, and biological patterns, with special emphasis on tumor prognosis.

Na+,K(+)-ATPase of gastric cells. A target of Helicobacter pylori cytotoxic activity.

The present study shows a direct impairing action of a cytotoxin-producing Helicobacter pylori strain on the Na+,K(+)-ATPase (evaluated as K(+)-dependent phosphatase activity) of human gastric epithelial cells in culture. The toxin itself is likely involved in this action which may also account for the cell edema found in vivo in Helicobacter pylori-colonized stomach.

p53 gene mutations and protein nuclear accumulation are early events in intestinal type gastric cancer but late events in diffuse type.

We screened for p53 alterations in 71 early gastric cancers of differing histological types and growth patterns, 18 advanced cancers of diffuse type, 19 dysplastic lesions, and 12 extensive intestinal metaplasia cases. Tumors were investigated for gene mutations (exons 5-8) with PCR-based denaturing gradient gel electrophoresis and sequencing techniques, and for protein accumulation with immunohistochemical methods. Nontumor samples were studied with immunohistochemistry alone. Of the early cancers, intestinal tumors showed a much higher p53 mutation frequency (41%) than did diffuse cancers (4%). When comparing early and advanced tumors of the same type, we observed a similarity in mutation frequency (41 versus about 50%) for intestinal tumors, and a significant increase for diffuse tumors (from 4 to 33%). Immunopositive case distribution between tumor types and stages paralleled that of mutated cases. Immunohistochemical and genetic analysis gave concordant results for all samples with gene mutations. Eighteen of the 65 (28%) nonmutated tumors displayed significant immunoreactivity. Early tumors that massively penetrated the submucosa, i.e., the early tumors for which prognosis is worst, showed the highest frequency both of p53 gene mutation and of nonmutated protein accumulation. Twelve of 19 dysplastic lesions showed significant immunoreactivity, whereas intestinal metaplasias proved unreactive in all but a few cells. Our results yield two implications: that p53 alterations have a crucial and early role in gastric carcinogenesis of intestinal type, likely acting at the transition step between metaplasia and dysplasia; and that the alterations are mainly associated with tumor progression in cancer of diffuse type.

Gastric argyrophil carcinoidosis in patients with Zollinger-Ellison syndrome due to type 1 multiple endocrine neoplasia. A newly recognized association.

We report four cases of gastric argyrophil carcinoidosis arising in the oxyntic mucosa of patients with Zollinger-Ellison syndrome as part of type 1 multiple endocrine neoplasia (MEN) syndrome. Multiple mucosal and submucosal carcinoids were seen in combination with innumerable hyperplastic and dysplastic growths of argyrophil endocrine cells disseminated in the entire acidopeptic mucosa. Histochemical and ultrastructural investigation indicated that the argyrophil enterochromaffin-like (ECL) cell, a type of endocrine cell normally restricted to the oxyntic mucosa and very sensitive to gastrin stimulation, was a major component of the carcinoidosis. Five similar cases were found in the literature. Because argyrophil ECL cell carcinoid or carcinoidosis is unusual in patients who have the Zollinger-Ellison syndrome but do not have MEN, we believe that the genetic trait of the MEN syndrome has an important permissive role in the promotion of gastric carcinoids through the hypergastrinemia inherent to Zollinger-Ellison syndrome.

Hyperplastic, dysplastic, and neoplastic enterochromaffin-like-cell proliferations of the gastric mucosa. Classification and histogenesis.

Endocrine cells of the gastric oxyntic mucosa, and especially the enterochromaffin-like (ECL) cells, are the progenitors of gastrin-promoted proliferative lesions whose tumorigenic potential largely depends on the background condition in which they arise. Hypertrophic gastropathy due to the familial multiple endocrine neoplasia (MEN-1)-associated or sporadic Zollinger-Ellison syndrome (ZES), diffuse chronic atrophic gastritis restricted to the corpus-fundus (type A CAG), with or without associated pernicious anemia, and Helicobacter pylori-related multifocal chronic atrophic gastritis are the usual background for such growths. The endocrine cell lesions have been classified as pseudohyperplasia (cell clustering unassociated with cell proliferation), hyperplasia (diffuse, linear, micronodular, adenomatoid), dysplasia (enlarged, adenomatous or fused micronodules, microinfiltration, nodular growth), and neoplasia (intramucosal or invasive carcinoids). The entire spectrum of endocrine cell proliferation, from hyperplasia to dysplasia and neoplasia, has been observed in MEN-ZES and diffuse type A CAG. Both hyperplastic and pseudohyperplastic changes occur with some frequency in the H. pylori-related chronic gastritis associated with ulcer disease or dyspepsia. However, because no progression to dysplastic or neoplastic lesions has thus far been documented in these latter conditions, their role in gastric endocrine cell tumorigenesis appears negligible.

Intestinal and diffuse gastric cancers arise in a different background of Helicobacter pylori gastritis through different gene involvement.

Investigation of extensively sampled nontumor gastric mucosa from 205 early gastric cancers showed Helicobacter pylori colonization in 85% of cases, including 100% of diffuse and 78% (83% in 97 cases with Swiss rolls) of glandular or mixed cancers. Intestinal metaplasia, including its type III variant, was prominent in the mucosa associated with glandular and mixed (but not diffuse) early cancers. Both glandular (usually called "intestinal") and diffuse-type cancers showed admixtures of intestinal and gastric tumor cell phenotypes. Both p53 gene mutations and p53 protein immunostaining were essentially restricted to glandular or mixed cancers and associated dysplastic lesions. Their appearance in the advanced stage of diffuse cancer was partly due to a change of the histologic pattern from glandular to diffuse during progression of some tumors. Loss of laminin, beta I integrin, or zonula adherens junctions was a common finding in both early and advanced diffuse cancer. It is concluded that two main pathways operate in gastric carcinogenesis, both starting from H. pylori gastritis and both leading to phenotypically variable, often mixed gastric/intestinal tumor growth. However, only one of the two pathways involves intestinal metaplasia, its type III variant, p53 gene alteration, and dysplasia to end in glandular cancer. In the other pathway, diffuse cancer apparently arises directly from hyperplastic, sometimes atypical necks of mostly nonmetaplastic gastric glands, through primary involvement of genes affecting cell-cell and cell-matrix junctional proteins.

Mucinous cystic tumors of the pancreas: clinicopathological features, prognosis, and relationship to other mucinous cystic tumors.

The clinicopathological features of 56 patients with mucinous cystic tumors (MCTs) of the pancreas were studied. Particular attention was paid to the prognosis of MCTs and the relationship to their ovarian, hepatic, and retroperitoneal counterparts. To distinguish MCTs from pancreatic intraductal papillary-mucinous tumors, MCTs were defined as tumors lacking communication with the duct system and containing mucin-producing epithelium, usually supported by ovarian-like stroma. All 56 tumors occurred in women (mean age 48.2 years) and were preferentially (93%) located in the body and tail of the pancreas. In accordance with the WHO classification, MCTs were divided into adenomas (n = 22), borderline tumors (n= 12), and noninvasive and invasive carcinomas (n = 22). Survival analysis revealed the extent of invasion to be the most significant prognostic factor (p<0.0001). Malignancy correlated with multilocularity and presence of papillary projections or mural nodules, loss of ovarian-like stroma, and p53 immunoreactivity. Stromal luteinization with expression of tyrosine hydroxylase, calretinin, or alpha inhibin was found in 66% of the cases. We conclude that the biologic behavior of MCTs is predictable on the basis of the extent of invasion. The similarities (i.e. gender, morphology, stromal luteinization) between pancreatic MCT and its ovarian, hepatobiliary, and retroperitoneal counterparts suggest a common pathway for their development.

Human cytomegalovirus early infection, acute rejection, and major histocompatibility class II expression in transplanted lung. Molecular, immunocytochemical, and histopathologic investigations.

The present study aimed to investigate the relationship between acute rejection and human cytomegalovirus (HCMV) infection, as well as the coexpression of HLA-DR and immediate-early (IE) viral antigens, in 143 transbronchial biopsies and bronchoalveolar lavage fluids of 32 lung transplant recipients. We investigated the occurrence of morphologically overt viral infection with conventional histopathology, the expression of IE antigens with single labeling immunohistochemistry, the coexpression of IE antigens and HLA-DR molecules with double labeling techniques, and the presence of viral IE genes with polymerase chain reaction. Histopathologic study showed overt viral infections (12.6%) in 18 of the 143 biopsies; 8 were in a context of pneumonia and 10 were localizations without surrounding inflammatory cells; immunohistochemistry showed IE viral antigen expression in 31 (21.67%); PCR detected viral IE genes in 73/143 lavage fluids and biopsies (51%). The double labeling immunohistochemical technique showed that most IE antigen-expressing, noncytopathic cells were either HLA-DR negative in areas without infiltrates, or HLA-DR positive in those areas where inflammatory infiltrates were consistent, in the absence of viral cytopathy, with acute rejection. The results indicate that, in transplanted lung, the frequency of morphologically occult HCMV infection (as detected by immunohistochemically and/or PCR) is much higher than that of morphologically overt viral infection. The occurrence of inflammatory infiltrates (consistent with acute rejection) around morphologically occult infected cells and the possible lack of inflammation around both early- and late-infected cells suggest that in biopsies with occult infection the infiltrates should be attributed to allograft reaction. This conclusion would be in keeping with the coexpression of HLA-DR and HCMV IE in infiltrate-rich biopsies that are consistent with acute rejection, as well as with the absence of HLA-DR expression in IE antigen-positive cells in infiltrate-free-areas.

The pathology of the gastrointestinal endocrine system.

Among endocrine tumors occurring in the gastrointestinal tract, midgut argentaffin EC cell carcinoids, gastric argyrophil ECL cell carcinoids, duodenal gastrin cell tumors, and rectal trabecular L cell carcinoids (in order of decreasing frequency) are those occurring more frequently. Together, they account for more than 80% of such tumors. Duodenal somatostatin cell tumors, gangliocytic paragangliomas, and differentiated neuroendocrine carcinomas are also well-defined tumor entities. The carcinoid syndrome, either classical, with intermittent flushing, hypotension, and diarrhea, or atypical, with persistent histamine-type red flushing, bronchospasm, and no diarrhea, and Zollinger-Ellison syndrome, with severe peptide ulcer disease, are the only hyperfunctional syndromes consistently found in association with these tumors. The carcinoid syndrome occurs in about 10% of gastrointestinal carcinoids, usually in their advanced, metastatic stage. The Zollinger-Ellison syndrome occurs in association with about 40% of intestinal gastrin cell tumors, including small intramural growths. Tumor prognosis depends on the mode and site of presentation, histology, cell type(s), size, level of invasion, metastases (especially distant metastases), and associated clinical syndrome or background disease. Hormones, trophic factors, inherited genetic traits, somatic mutations, and some chronic inflammatory processes are pathogenetically important in a large proportion of cases.

Complement-mediated binding of immunoglobulins to some endocrine cells of the pancreas and gut.

Unspecific binding of immunoglobulins to gastrin, somatostain and glucagon cells of the gastrointestinal mucosa or pancreas has been found to occur through a nonantigen-antibody mechanism mediated by the C14 fraction of complement. The phenomenon represents an important drawback in hormone immunohistochemistry, which can be overcome by using complement deprived, highly dilute anti-hormone sera.

On the immunocytochemical localization of the vasoactive intestinal polypeptide.

The distribution of vasoactive intestinal polypeptide (VIP) immunoreactive nerves and endocrine cells in the gastrointestinal tract and pancreas of a number of mammalian and submammalian species has been examined in order to throw light on the exact localization of this peptide. Seven out of 8 VIP antisera demonstrated numerous nerve fibers in the gut, whereas one antiserum (TR2) revealed only scattered, few nerve fibers. The distribution of endocrine cells demonstrated by the different VIP antisera varied considerably. Thus, some antisera demonstrated only endocrine cells in the feline antrum, others only colonic endocrine cells and still others only endocrine cells of the upper gut and pancreas. The variability in staining pattern of endocrine cells as well as recent radioimmunological data makes it opportune to suggest that true VIP is a neuronal peptide and that endocrine cells store peptides resembling, but not being identical with, VIP (VIPoids).

Proton pump inhibitors, enterochromaffin-like cell growth and Helicobacter pylori gastritis.

In both rodents and humans the development of gastrin-promoted gastric argyrophil enterochromaffin-like cell carcinoids requires the involvement of a genetic factor inherent to multiple endocrine neoplasia syndrome or of type A autoimmune chronic atrophic gastritis. Prolonged severe hypergastrinaemia acting on non-gastritic mucosa, as in Zollinger-Ellison syndrome patients, results in diffuse argyrophil enterochromaffin-like cell hyperplasia but, as a rule, does not produce tumours. Combination of chronic atrophic gastritis (mostly related to Helicobacter pylori infection) with hypergastrinaemia frequently causes linear and micronodular hyperplasia of argyrophil cells, whereas carcinoids are exceptional. No tumours or pre-neoplastic lesions have been observed in patients treated long-term with proton pump inhibitors, apart from rare cases in patients with combined Zollinger-Ellison and multiple endocrine neoplasia syndromes. A moderate increase in the incidence of argyrophil cell clustering, with or without hyperplasia, probably results from the parallel evolution of ulcer-associated Helicobacter gastritis into chronic atrophic gastritis. Eradication of H. pylori with a combination of proton pump inhibitors and antibiotics suppresses gastritis and prevents ulcer recurrence.

Review article: potential gastrointestinal effects of long-term acid suppression with proton pump inhibitors.

This review examines the evidence for the development of adverse effects due to prolonged gastric acid suppression with proton pump inhibitors. Potential areas of concern regarding long-term proton pump inhibitor use have included: carcinoid formation; development of gastric adenocarcinoma (especially in patients with Helicobacter pylori infection); bacterial overgrowth; enteric infections; and malabsorption of fat, minerals, and vitamins. Prolonged proton pump inhibitor use may lead to enterochromaffin-like cell hyperplasia, but has not been demonstrated to increase the risk of carcinoid formation. Long-term proton pump inhibitor treatment has not been documented to hasten the development or the progression of atrophic gastritis to intestinal metaplasia and gastric cancer, although long-term studies are required to allow definitive conclusions. At present, we do not recommend that patients be tested routinely for H. pylori infection when using proton pump inhibitors for prolonged periods. Gastric bacterial overgrowth does increase with acid suppression, but important clinical sequelae, such a higher rate of gastric adenocarcinoma, have not been seen. The risk of enteric infection may increase with acid suppression, although this does not seem to be a common clinical problem with prolonged proton pump inhibitor use. The absorption of fats and minerals does not appear to be significantly impaired with chronic acid suppression. However, vitamin B12 concentration may be decreased when gastric acid is markedly suppressed for prolonged periods (e.g. Zolllinger-Ellison syndrome), and vitamin B12 levels should probably be assessed in patients taking high-dose proton pump inhibitors for many years. Thus, current evidence suggests that prolonged gastric acid suppression with proton pump inhibitors rarely, if ever, produces adverse events. Nevertheless, continued follow-up of patients taking proton pump inhibitors for extended periods will provide greater experience regarding the potential gastrointestinal adverse effects of long-term acid suppression.

Antral gastrin cell hyperfunction and Helicobacter pylori infection.

BACKGROUND: Antral gastrin cell hyperfunction (AGCH), is a rare cause of duodenal ulcer associated with non-tumour hypergastrinaemia and acid hypersecretion. AIM: To investigate the role of Helicobacter pylori in AGCH. PATIENTS: Twelve AGCH patients and eight H. pyloripositive non-hypergastrinaemic duodenal ulcer patients were compared. METHODS: Basal and peak acid outputs, gastrin-stimulation (meal and bombesin) tests, and immunohistochemistry for antral G and D cells were performed. One year after H. pylori eradication, six AGCH patients were again investigated with the same tests. RESULTS: Significantly more basal, and stimulated gastrin and acid secretion, were found in AGCH compared to the H. pylori-positive duodenal ulcer patients (P < 0.01). G cell counts were significantly higher in AGCH than in duodenal ulcer patients (118.8, range 58-192.4, vs. 86.1, range 49-184; P < 0.05), and the resulting G/D cell ratio was also higher in AGCH patients (4.2, range 2.6-5.6, vs. 3.3, range 1.9-4.3; P < 0.05). H. pylori was present in the gastric mucosa of all 12 AGCH patients. Cure of infection in six AGCH individuals resulted in marked a decrease of gastrin levels associated with a significant (23.7%: P < 0.05) decrease of G cell count and an increase (12%; P < 0.05) of D cell count. CONCLUSIONS: The results indicate that AGCH may result from H. pylori overstimulation of gastrin cell function in patients with some presently undefined, familial predisposition and that an imbalance of the G/D cell ratio may have a role in the genesis of hypergastrinaemia.

Gastric mucosal atrophy: interobserver consistency using new criteria for classification and grading.

BACKGROUND AND AIMS: Considerable difficulties persist amongst pathologists in agreeing on the presence and severity of gastric atrophy. An international group of pathologists pursued the following aims: (i) to generate an acceptable definition and a simple reproducible classification of gastric atrophy; and (ii) to develop guidelines for the recognition of atrophy useful for increasing agreement among observers. METHODS: After redefining atrophy as the 'loss of appropriate glands' and examining histological samples from different gastric compartments, three categories were identified: (i) negative; (ii) indefinite; (iii) atrophy, with and without intestinalization. Atrophy was graded on a three-level scale. Interobserver reproducibility of the classification was tested by kappa statistics (general and weighted) in a series of 48 cases. RESULTS: The medians of the general agreement and weighted kappa values were 0.78 and 0.73, respectively. The weighted kappa coefficients, obtained by cross-tabulating the evaluation of each pathologist against all others, were, with only one exception, > 0.4 (moderate to excellent agreement). CONCLUSIONS: By using the definition of atrophy as the loss of appropriate glands and distinguishing the two main morphological entities of metaplastic and non-metaplastic types, a high level of agreement was achieved by a group of gastrointestinal pathologists trained in different cultural contexts.