Expression and function of Angiopoietins and their tie receptors in human basophils and mast cells.

The Angiopoietin/Tie system is a key regulator of vascular remodeling, maturation, angiogenesis and lymphangiogenesis. In humans there are three angiopoietins: Angiopoietin-1 (Ang1), Angiopoietin-2 (Ang2), and Angiopoietin-4 (Ang4). Ang1 and Ang2 are the best characterized angiopoietins. The angiopoietin receptor system consists of two type I tyrosine kinase receptors (Tie1 and Tie2). Tie2 binds all known angiopoietins. We sought to characterize Ang1, Ang2, Tie1 and Tie2 expression and functions in human basophils and mast cells. Basophils, LAD-2 cells and Human Lung Mast Cells (HLMCs) constitutively express Ang1 and Ang2 mRNA. Intracellular staining for Ang1 and Ang2 was stronger in basophils than in mast cells. Immunoelectron microscopy demonstrated Ang1 in cytoplasmic vesicles of basophils. The protein kinase C activators phorbol diester (PMA) and bryostatin 1 (Bryo1) stimulated basophils to rapidly release a large amount of Ang1. PMA-induced Ang1 release was inhibited by brefeldin A. Tie1 and Tie2 mRNAs were expressed in basophils, LAD-2 and HLMCs. Basophils, LAD-2 and HLMCs expressed Tie1 on the cell surface. HLMCs and LAD-2 expressed Tie2 on the cell surface, whereas basophils did not. Ang1, but not Ang2, induced migration of mast cells through the engagement of Tie2. Neither Ang1 nor Ang2 induced basophil chemotaxis. We have identified a novel mechanism of cross-talk between human basophils and mast cells mediated by the Ang1/Tie2 system that might be relevant in the orchestration of inflammatory and neoplastic angiogenesis.

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.

Silent familial isolated pituitary adenomas: histopathological and clinical case report.

Familial isolated pituitary adenoma (FIPA) is a rare condition independent of Carney Complex or MEN1. An international multicenter study recently described 28 nonfunctioning pituitary adenomas in 26 families with only two homogeneous nonsecreting phenotype families consistent of silent GH and silent gonadotroph adenomas, respectively. We present the clinical, genetic, and morphological analysis of two silent pituitary adenomas occurring in a man and his daughter, and discuss the differential diagnosis associated with their histological, immunohistochemical, and ultrastructural features. The patients developed invasive nonsecreting macroadenomas manifesting only with compressive symptoms. Genetic analysis in the father showed no MEN-1 germ-line mutation. Tissue samples obtained after paraseptal trans-sphenoidal surgery were studied by immunohistochemistry for adenohypophyseal hormones, low molecular weight cytokeratins (CAM 5.2), proliferation markers, and anterior pituitary transcription factors (Pit-1 and SF-1) and by electron microscopy for secretory granules. The clinical, histological, and immunohistochemical features of the lesions posed a differential diagnosis between a null cell adenoma and a silent corticotroph adenoma (Type II); on the basis of immunohistochemical stains for cytokeratin and adenohypophysis cell lineage markers, tumor behavior and ultrastructural studies we concluded for the second. The reported cases represent an as yet undescribed example of homogeneous family with silent corticotroph adenomas (Type II). Our observations support the trend for more aggressive behavior in nonsecreting FIPAs as compared with sporadic adenomas.

Lymph node reticulum cell neoplasm with progression into cytokeratin-positive interstitial reticulum cell (CIRC) sarcoma: a case study.

AIMS: To detail on sequential biopsies the morphological and immunohistochemical features of a case of primary lymph nodal fibroblastic reticulum cell (FBRC) tumour which progressed into a clinically aggressive cytokeratin-positive interstitial reticulum cell (CIRC) sarcoma. METHODS AND RESULTS: A 70-year-old female underwent surgical excision of an enlarged submandibular lymph node. The nodal architecture was effaced by a neoplastic proliferation of medium to large cells, round to oval to spindle in shape, growing in a storiform pattern. The tumour stained for vimentin, CD68, factor XIIIa, alpha1-antitrypsin, fascin and actin. Dendritic and endothelial cell markers were negative. A diagnosis of FBRC tumour was made by combining pathological and clinical data. The patient received no therapy but 5 months later the tumour relapsed, exhibiting a deceptively pleomorphic cytology, phenotypic changes (strong cytokeratin positivity), intense p53 expression and aggressive clinical course with fatal outcome. In-situ hybridization for Epstein-Barr virus was negative. CONCLUSIONS: We speculate that the morphological changes and p53 expression of the relapsing neoplasm might reflect tumour cell dedifferentiation, in keeping with the aggressive clinical course. The intense p53 expression suggests that this oncoprotein might also play a role in reticulum cell tumorigenesis.

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.

Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium.

The mechanisms by which Helicobacter pylori releases its virulence factors are poorly known. Active secretion has been proposed for some products, including a vacuolating toxin (VacA). Outer membrane vesicles represent another mechanism by which some Gram-negative bacteria may release virulence factors. This study sought to localize VacA by immunocytochemistry in H. pylori cells, to determine whether H. pylori produces outer membrane vesicles, and to investigate whether such vesicles might constitute a vehicle for the delivery of bacterial virulence factors to the gastric mucosa. Small (50-300 nm) membrane vesicles were found in H. pylori culture media from both H. pylori strain 60190 and strain CCUG 17874. These vesicles appeared to originate from blebs arising on the bacterial outer membrane. VacA was immunolocalized in the periplasm and outer membrane of intact bacteria and also in outer membrane blebs and vesicles. Both soluble secreted VacA and VacA-containing vesicles bound to, and were internalized by, MKN28 cells and were detectable in the gastric mucosa from H. pylori-infected humans. The release of outer membrane vesicles by H. pylori may represent a mechanism, additional to secretory pathways, for the delivery of bacterial toxins and antigens to the gastric mucosa.

Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.

The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than approximately 90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.