Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application

Proteasome Particle-Rich Structures Are Widely Present in Human Epithelial Neoplasms: Correlative Light, Confocal and Electron Microscopy Study

A novel cytoplasmic structure has been recently characterized by confocal and electron microscopy in H. pylori-infected human gastric epithelium, as an accumulation of barrel-like proteasome reactive particles colocalized with polyubiquitinated proteins, H. pylori toxins and the NOD1 receptor. This proteasome particle-rich cytoplasmic structure (PaCS), a sort of focal proteasome hyperplasia, was also detected in dysplastic cells and was found to be enriched in SHP2 and ERK proteins, known to play a role in H. pylori-mediated gastric carcinogenesis. However, no information is available on its occurrence in neoplastic growths. In this study, surgical specimens of gastric cancer and various other human epithelial neoplasms have been investigated for PaCSs by light, confocal and electron microscopy including correlative confocal and electron microscopy (CCEM). PaCSs were detected in gastric cohesive, pulmonary large cell and bronchioloalveolar, thyroid papillary, parotid gland, hepatocellular, ovarian serous papillary, uterine cervix and colon adenocarcinomas, as well as in pancreatic serous microcystic adenoma. H. pylori bodies, their virulence factors (VacA, CagA, urease, and outer membrane proteins) and the NOD1 bacterial proteoglycan receptor were selectively concentrated inside gastric cancer PaCSs, but not in PaCSs from other neoplasms which did, however, retain proteasome and polyubiquitinated proteins reactivity. No evidence of actual microbial infection was obtained in most PaCS-positive neoplasms, except for H. pylori in gastric cancer and capsulated bacteria in a colon cancer case. Particle lysis and loss of proteasome distinctive immunoreactivities were seen in some tumour cell PaCSs, possibly ending in sequestosomes or autophagic bodies. It is concluded that PaCSs are widely represented in human neoplasms and that both non-infectious and infectious factors activating the ubiquitin-proteasome system are likely to be involved in their origin. PaCS detection might help clarify the role of the ubiquitin-proteasome system in carcinogenesis

Evidence for transepithelial dendritic cells in human H. pylori active gastritis.

Background: Despite extensive experimental investigation stressing the importance of bacterial interaction with dendritic cells (DCs), evidence regarding direct interaction of H. pylori or its virulence products with DCs in the human gastric mucosa is lacking. Methods: Human gastric mucosa biopsies, with or without H. pylori infection and active inflammation, were investigated at light and electron microscopy level with immunocytochemical tests for bacterial products (VacA, urease, outer membrane proteins) and DC markers (DC-SIGN, CD11c, CD83) or with the DC-labeling ZnI2-OsO4 technique. Parallel tests with cultured DCs were carried out. Results: Cells reproducing ultrastructural and cytochemical patterns of DCs were detected in the lamina propria and epithelium of heavily infected and inflamed (but not of normal or moderately inflamed) mucosa, where DC luminal endings directly contact H. pylori and take up their virulence products. Cytotoxic changes (mitochondrial swelling, cytoplasmic vacuolation, autophagy) were observed in intraepithelial DCs and reproduced in cultured DCs incubated with H. pylori broth culture filtrates to obtain intracellular accumulation of VacA and urease. Granulocytes were also seen to contact and heavily phagocytose luminal H. pylori, while macrophages remained confined to basal epithelium, though taking up bacteria and bacterial products. Conclusion: Human DCs can enter H. pylori-infected gastric epithelium, in association with other innate immunity cells, to take up bacteria and their virulence products. This process is likely to be important for bacterial sensing and pertinent immune response, however it may also generate DC cytotoxic changes potentially hampering their function

Chaperone molecules concentrate together with the ubiquitin–proteasome system inside particulate cytoplasmic structures: possible role in metabolism of misfolded proteins

Ubiquitin-proteasome system (UPS) proteins and proteolytic activity are localized in a recently identified cytoplasmic structure characterized by accumulation of barrel-like particles, which is known as the particulate cytoplasmic structure (PaCS). PaCSs have been detected in neoplastic, preneoplastic, chronically infected, and fetal cells, which produce high amounts of misfolded proteins to be degraded by the UPS. Chaperone molecules are crucial in the early stages of handling misfolded proteins; therefore, we searched for these molecules in PaCSs. Heat shock proteins (Hsp), Hsp90, Hsp70, Hsp40, and Bcl-2-associated athanogene (Bag)3 chaperones, although not Bag6, were selectively concentrated into PaCSs of several cell lines and ex vivo fetal or neoplastic cells. Present findings point to PaCSs as an integrated, active UPS center well equipped for metabolism of misfolded proteins, especially in cells under physiological (fetal development) or pathological (neoplasia or inflammation) stress

Particle-Rich Cytoplasmic Structure (PaCS): Identification, Natural History, Role in Cell Biology and Pathology

Cytoplasmic structures showing a selective concentration of both polyubiquitinated proteins and proteasome have been described in various epithelial, hematopoietic, mesenchymal and neural cells in vitro or in fetal tissues, as well as in chronically-infected, mutated preneoplastic and neoplastic tissues. These cytoplasmic structures differ from other ubiquitin-reactive cytoplasmic bodies, like sequestosomes, aggresome-like-induced structures in dendritic cells (DALIS)/non-dendritic cells (ALIS) and aggresomes in showing distinctive ultrastructural organization (particle-rich cytoplasmic structure or PaCS), a cytochemical pattern and a functional profile. Their formation can be induced in vitro in dendritic or natural killer cells by trophic factors and interleukin treatment. They originate in close connection with ribosomes, while, as a result of their growth, the cytoskeleton and other surrounding organelles are usually dislocated outside their core. Interestingly, these particulate cytoplasmic structures are often found to fill cytoplasmic blebs forming proteasome- and polyubiquitinated protein-discharging vesicles, called ectosomes, which are found to detach from the cell and freely float in the extracellular space. To clearly point out the importance of the polyubiquitinated proteins and proteasome containing cytoplasmic structures, their role in cell biology and pathology has been carefully analyzed

The contribution of cell phenotype to the behavior of gastric cancer.

none8noSeveral histochemical studies suggest a role of tumor cell phenotype and related differentiation markers in the prognostic assessment of gastric cancer. Unfortunately, most studies have dealt with single or a few markers and have paid limited attention to their interplay with tumor histological types, which are potentially informative of prognosis. In this study, 292 invasive (T1b to T4) gastric cancers with prolonged follow-up and carefully analyzed histotype, inclusive of histotype-based grade, were investigated histochemically with a panel of 14 phenotypic markers known to be expressed in normal gut tissues and gastric cancer. Three of seven intestinal type markers investigated showed a trend for improved prognosis, one of which, CDX2, was stage independent. Three among gastric and pancreatobiliary duct markers (MUC1, MUC6, and pepsinogen II), predicted more severe prognosis stage independently, as did a combination of eight potentially informative (p < 0.1 at univariable Cox analysis) markers. Cancers with predominantly intestinal phenotype had significantly better prognosis than those with predominantly gastric, mixed, or poorly defined phenotypes; among the latter, those with high lymphocyte response, with favorable outcome, were separated from anaplastic cancers, with ominous prognosis. At multivariable analysis, CDX2 and the eight marker combination proved to be stage- and grade-independent predictors. When individually considered, and with the exception of CDX2, the biomarkers investigated gave an appreciable, although moderate, contribution to the prognostic evaluation of gastric cancer. Combined analysis of all potentially informative markers gave more important information, highly additive to both stage and histotype-based grade.noneSolcia E;Klersy C;Vanoli A;Grillo F;Manca R;Tava F;Luinetti O;Fiocca RSolcia, E; Klersy, C; Vanoli, A; Grillo, Federica; Manca, R; Tava, F; Luinetti, O; Fiocca, Robert

Mesenchymal stromal cell infusions as rescue therapy for corticosteroid-refractory adult autoimmune enteropathy

Adult autoimmune enteropathy (AIE) is a rare cause of malabsorption syndrome unresponsive to dietary restriction. Its diagnostic hallmarks are small-bowel villous atrophy and antienterocyte autoantibodies. Therapy is based mainly on nutritional support and immunosuppression. We treated a 61-year-old woman with corticosteroid-refractory AIE and life-threatening malabsorption syndrome with systemic infusions of autologous, bone marrow-derived, mesenchymal stromal cells (MSCs) as rescue therapy. The MSCs were expanded ex vivo following a previously used Good Manufacturing Practice procedure, and 2 intravenous infusions of 1.8 7 10(6) MSCs/kg body weight were administered 2 weeks apart. Analysis of circulating and mucosal regulatory T-and B-cell numbers, and of serum and secretory immunoglobulin levels, was performed before and after treatment. The MSC infusions were safe and effective, leading to disappearance of disease hallmarks and recovery from the life-threatening condition. Increases in mucosal regulatory T-cell numbers and secretory immunoglobulin levels were also observed. The benefit, however, was transient, and a further MSC infusion resulted in the same short efficacy. This case encourages the use of MSCs to treat patients with life-threatening, corticosteroid-refractory AIE and suggests that MSC infusion can attenuate, albeit transiently, the autoimmune attack

Ovarian serous papillary, cervical endometrioid, and thyroid papillary carcinomas.

<p>Juxtanuclear PaCSs in ovarian serous papillary (<b>A</b>, 10,000x), cervical endometrioid (<b>D</b>, 10,000x), and thyroid papillary carcinoma (<b>G</b>, 10,000x), enlarged in <b>B</b>, <b>E</b>, and <b>H</b> (all 25,000x) and even further in <b>C</b>, <b>F</b>, and <b>I</b> (all, 50,000x), respectively, to show their particulate ultrastructure (partially lost in <b>I</b> and, focally, in <b>F</b>) and 20S proteasome immunogold reactivity.</p

<i>H. pylori</i>-colonized non-neoplastic human gastric epithelium (A–C) and well differentiated, gland-forming, deeply invasive, gastric cancer (D–M).

<p>(<b>A</b>) Foveolar epithelium showing 20S proteasome immunofluorescence (green) of infranuclear PaCSs. Overlapping of confocal fluorescence microscopy and toluidine blue images of a semithin resin section from an aldehyde-osmium-fixed block (500x). (<b>B</b>) Juxtanuclear PaCS (asterisk) in a foveolar cell (5,000x) from an adjacent section to <b>A</b>. (<b>C</b>) High resolution (X 65,000) TEM of a boxed PaCS area in <b><i>B</i></b> to show its particulate ultrastructure and 20S proteasome immunogold reactivity. (<b>D</b>) Toluidine blue stained semithin resin section (500x); note pink stained PaCSs (arrowheads), many of which subluminal. (<b>E</b>) Correlative confocal and electron microscopy (CCEM), of a consecutive thin section of the boxed area in <i>D,</i> shows 20S proteasome immunofluorescence (green spots) on a TEM background (5,000x). The juxtaluminal PaCS (arrowhead) is enlarged in <b>F</b> (20,000x) whose boxed area is further enlarged in <b>G</b> (50,000x) to illustrate its particulate ultrastructure with 20S proteasome immunogold reactivity. The same proteasome antibody was used on each face of the gilder finder grid used for CCEM. Note several basally located PaCSs in <b>E</b>. (<b>H</b>) Another thin section (5,000x) from the same tumour block with several supranuclear PaCSs, one of which (arrowhead) is enlarged in the top link inset (18,000x) to show an inner bacterial body (for comparison, another intracellular bacterium positive for H. pylori OMPs immunogold (21,000x) is shown in the bottom right inset), while an juxtaluminal PaCS (boxed area) is enlarged in <b><i>I</i></b> (25,000x) to illustrate NOD1 immunogold reactivity. N, nucleus. (<b>L</b>) TEM (20.000x) of another PaCS in another section from the same block immunostained with <i>H. pylori</i> OMP antibodies; boxed area enlarged in M (50,000x) to better magnify the immunogold particles and barrel-like putative proteasome particles, one of which (boxed in <b>M</b>) is further enlarged in the <b>L</b> inset (200,000x) to illustrate the characteristic punctuate-aligned structure.</p