Proteasome-Rich PaCS as an Oncofetal UPS Structure Handling Cytosolic Polyubiquitinated Proteins. In Vivo Occurrence, in Vitro Induction, and Biological Role.

In this article, we outline and discuss available information on the cellular site and mechanism of proteasome interaction with cytosolic polyubiquitinated proteins and heat-shock molecules. The particulate cytoplasmic structure (PaCS) formed by barrel-like particles, closely reproducing in vivo the high-resolution structure of 26S proteasome as isolated in vitro, has been detected in a variety of fetal and neoplastic cells, from living tissue or cultured cell lines. Specific trophic factors and interleukins were found to induce PaCS during in vitro differentiation of dendritic, natural killer (NK), or megakaryoblastic cells, apparently through activation of the MAPK-ERK pathway. Direct interaction of CagA bacterial oncoprotein with proteasome was shown inside the PaCSs of a Helicobacter pylori-infected gastric epithelium, a finding suggesting a role for PaCS in CagA-mediated gastric carcinogenesis. PaCS dissolution and autophagy were seen after withdrawal of inducing factors. PaCS-filled cell blebs and ectosomes were found in some cells and may represent a potential intercellular discharge and transport system of polyubiquitinated antigenic proteins. PaCS differs substantially from the inclusion bodies, sequestosomes, and aggresomes reported in proteinopathies like Huntington or Parkinson diseases, which usually lack PaCS. The latter seems more linked to conditions of increased cell proliferation/differentiation, implying an increased functional demand to the ubiquitin⁻proteasome system.

Polyubiquitinated proteins, proteasome, and glycogen characterize the particle-rich cytoplasmic structure (PaCS) of neoplastic and fetal cells.

A particle-rich cytoplasmic structure (PaCS) concentrating ubiquitin-proteasome system (UPS) components and barrel-like particles in clear, cytoskeleton- and organelle-free areas has recently been described in some neoplasms and in genetic or infectious diseases at risk of neoplasia. Ultrastructurally similar particulate cytoplasmic structures, interpreted as glycogen deposits, have previously been reported in clear-cell neoplasms and some fetal tissues. It remains to be investigated whether the two structures are the same, colocalize UPS components and polysaccharides, and have a role in highly proliferative cells such as fetal and neoplastic cells. We used immunogold electron microscopy and confocal immunofluorescence microscopy to examine human and mouse fetal tissues and human neoplasms. Fetal and neoplastic cells both showed colocalization of polyubiquitinated proteins, 19S and 20S proteasomes, and polysaccharides, both glycogen and chondroitin sulfate, inside cytoplasmic structures showing all distinctive features of PaCSs. Poorly demarcated and/or hybrid (ribosomes admixed) UPS- and glycogen-enriched areas, likely stages in PaCS development, were also seen in some fetal cells, with special reference to those, like primary alveolar pulmonary cells or pancreatic centroacinar cells, having a crucial role in organogenesis. UPS- and glycogen-rich PaCSs developed extensively in clear-cell neoplasms of the kidney, ovary, pancreas, and other organs, as well as, in infantile, development-related tumors replicating fetal patterns, such as choroid plexus papilloma. UPS-mediated, ATP-dependent proteolysis and its potential energy source, glycogen metabolism, may have a crucial, synergic role in embryo-/organogenesis and carcinogenesis.

Use of larvae of the wax moth Galleria mellonella as an in vivo model to study the virulence of Helicobacter pylori.

BACKGROUND: Helicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world's population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection. RESULTS: In the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD50 lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae always correlated with the induction of apoptosis in hemocytes. CONCLUSIONS: G. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients' disease status.

Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome.

BACKGROUND: Shwachman-Diamond syndrome is an autosomal recessive disorder in which severe bone marrow dysfunction causes neutropenia and an increased risk of leukemia. Recently, novel particulate cytoplasmic structures, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from patients with Helicobacter pylori gastritis and several epithelial neoplasms. DESIGN AND METHODS: Blood neutrophils from 13 cases of Shwachman-Diamond syndrome - ten with and three without SBDS gene mutation - and ten controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins. RESULTS: Many extensively disseminated particulate cytoplasmic structures, accounting for 22.78 ± 5.57% (mean ± standard deviation) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman-Diamond syndrome patients. The particulate cytoplasmic structures showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in particulate cytoplasmic structures of Helicobacter pylori-positive gastritis. Neutrophils from patients with Shwachman-Diamond syndrome frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No particulate cytoplasmic structures were observed in most control neutrophils; however, in a few cells from two cases we noted focal development of minute particulate cytoplasmic structures, accounting for 0.74 ± 0.56% of the total cytoplasm (P<0.001 versus particulate cytoplasmic structures from mutated Shwachman-Diamond syndrome patients). Neutrophils from non-mutated Shwachman-Diamond-syndrome-like patients resembled controls in two cases, and a third case showed particulate cytoplasmic structure patterns intermediate between those in controls and those in mutated Shwachman-Diamond syndrome patients. CONCLUSIONS: Particulate cytoplasmic structures are a prominent feature of neutrophils from patients with Shwachman-Diamond syndrome. They may help us to understand the mechanism of granulocyte dysfunction and the neoplastic risk of the disease.

Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.

Bidimensional Engineered Amorphous a-SnO2 Interfaces: Synthesis and Gas Sensing Response to H2S and Humidity.

Two-dimensional (2D) transition metal dichalcogenides (TMDs) and metal chalcogenides (MCs), despite their excellent gas sensing properties, are subjected to spontaneous oxidation in ambient air, negatively affecting the sensor's signal reproducibility in the long run. Taking advantage of spontaneous oxidation, we synthesized fully amorphous a-SnO2 2D flakes (≈30 nm thick) by annealing in air 2D SnSe2 for two weeks at temperatures below the crystallization temperature of SnO2 (T < 280 °C). These engineered a-SnO2 interfaces, preserving all the precursor's 2D surface-to-volume features, are stable in dry/wet air up to 250 °C, with excellent baseline and sensor's signal reproducibility to H2S (400 ppb to 1.5 ppm) and humidity (10-80% relative humidity (RH)) at 100 °C for one year. Specifically, by combined density functional theory and ab initio molecular dynamics, we demonstrated that H2S and H2O compete by dissociative chemisorption over the same a-SnO2 adsorption sites, disclosing the humidity cross-response to H2S sensing. Tests confirmed that humidity decreases the baseline resistance, hampers the H2S sensor's signal (i.e., relative response (RR) = Ra/Rg), and increases the limit of detection (LOD). At 1 ppm, the H2S sensor's signal decreases from an RR of 2.4 ± 0.1 at 0% RH to 1.9 ± 0.1 at 80% RH, while the LOD increases from 210 to 380 ppb. Utilizing a suitable thermal treatment, here, we report an amorphization procedure that can be easily extended to a large variety of TMDs and MCs, opening extraordinary applications for 2D layered amorphous metal oxide gas sensors.

Helicobacter pylori counteracts the apoptotic action of its VacA toxin by injecting the CagA protein into gastric epithelial cells.

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA(+)/VacA(+)H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.

Helicobacter pylori Hp(2-20) promotes migration and proliferation of gastric epithelial cells by interacting with formyl peptide receptors in vitro and accelerates gastric mucosal healing in vivo.

Helicobacter pylori-derived peptide RpL1 aa 2-20 (Hp(2-20)) in addition to its antimicrobial action exerts several immunomodulatory effects in eukaryotic cells by interacting with formyl peptide receptors (FPRs). It has recently been shown that activation of FPRs facilitates intestinal epithelial cell restitution. We investigated whether Hp(2-20) induces healing of injured gastric mucosa and assessed the mechanisms underlying any such effect. We investigated the expression of FPRs in two gastric epithelial cell lines (MKN-28 and AGS) at mRNA and protein level. To determine whether FPRs were functional we performed chemotaxis experiments and proliferation assays and studied the Hp(2-20)-activated downstream signaling pathway. The effect of Hp(2-20) on mucosal healing was evaluated in rats after indomethacin-induced injury. Here we show that: (1) FPRs were expressed in both cell lines; (2) Hp(2-20) stimulated migration and proliferation of gastric epithelial cells; (3) this effect was specifically mediated by formyl peptide receptor-like 1 (FPRL1) and FPRL2 and was associated with activation of FPR-related downstream signaling pathways; (4) Hp(2-20) up-regulated the expression and secretion of vascular endothelial growth factor; and (5) Hp(2-20) accelerated healing of rat gastric mucosa after injury brought about by indomethacin at both the macroscopic and microscopic levels. In conclusion, by interacting with FRPL1 and FPRL2, H. pylori-derived Hp(2-20) induces cell migration and proliferation, as well as the expression of vascular endothelial growth factor, thereby promoting gastric mucosal healing. This study provides further evidence of the complexity of the relationship between H. pylori and human gastric mucosa, and it suggests that a bacterial product may be used to heal gastric mucosal injury.

Colocalization of tracks from boron neutron capture reactions and images of isolated cells.

In Boron Neutron Capture Therapy, the boronated drug plays a leading role in delivering a lethal dose to the tumour. The effectiveness depends on the boron macroscopic concentration and on its distribution at sub-cellular level. This work shows a way to colocalize alpha particles and lithium ions tracks with cells. A neutron autoradiography technique is used, which combines images of cells with images of tracks produced in a solid-state nuclear track detector.

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 Helicobacter 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 ZnI(2)-OsO(4 )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) 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.

CagA Effector Protein in Helicobacter pylori-Infected Human Gastric Epithelium in Vivo: From Bacterial Core and Adhesion/Injection Clusters to Host Cell Proteasome-Rich Cytosol.

A key role in the carcinogenic action of Helicobacter pylori is played by the effector protein CagA, the first identified oncoprotein of the bacterial world. However, the present knowledge in regard to the bacterial injection of CagA into epithelial cells (through a type IV secretion system) and its intracellular fate is based primarily on experimental studies in vitro. Our study was aimed to investigate, in H. pylori-infected human gastric epithelium, CagA delivery and intracellular distribution in order to identify any in vivo counterpart of the cell injection mechanism described in vitro and any intracellular cytoplasmic site of preferential CagA distribution, thus shedding light on the natural history of CagA in vivo. By transmission electron microscopy and ultrastructural immunocytochemistry (which combine precise molecule localization with detailed analysis of bacterial-host cell interaction and epithelial cell ultrastructure), we investigated endoscopic biopsies of gastric antrum from H. pylori-infected dyspeptic patients. Our findings provide support for CagA direct injection into gastric epithelial cells at bacterial adhesion sites located on the lateral plasma membrane and for its cytosolic intracellular distribution with selective concentration inside peculiar proteasome-rich areas, which might be site not only of CagA degradation but also of CagA-promoted crucial events in gastric carcinogenesis.

Classification of Helicobacter pylori Virulence Factors: Is CagA a Toxin or Not?

Since its discovery, Helicobacter pylori has been identified as the causative agent of various gastric diseases. H. pylori produces myriads of disease-associated virulence factors. These bacterial determinants can be distinguished as cell-binding factors, immunoregulatory components, survival factors, toxins, and effector proteins. For most of these factors there is consensus about their classification. However, there is a strong dispute in the literature as to whether one of the best-studied factors, CagA, represents a toxin or not. CagA displays unique functions that are clearly different from conventional toxins, and CagA counteracts the activities of an established H. pylori toxin, VacA. Canonical toxins commonly have specific (and narrow) targets, can act even in the absence of the bacterial cell, and elicit acute damage to host cells. However, there is still no agreement on the classification of CagA. Here we discuss whether CagA acts as a toxin, and propose a classification consensus for CagA.

Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes.

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.

Natural history of Helicobacter pylori VacA toxin in human gastric epithelium in vivo: vacuoles and beyond.

Uptake, intracellular trafficking and pathologic effects of VacA toxin from Helicobacter pylori have been widely investigated in vitro. However, no systematic analysis investigated VacA intracellular distribution and fate in H. pylori-infected human gastric epithelium in vivo, using ultrastructural immunocytochemistry that combines precise toxin localization with analysis of the overall cell ultrastructure and intercompartimental/interorganellar relationships. By immunogold procedure, in this study we investigated gastric biopsies taken from dyspeptic patients to characterize the overall toxin's journey inside human gastric epithelial cells in vivo. Endocytic pits were found to take up VacA at sites of bacterial adhesion, leading to a population of peripheral endosomes, which in deeper (juxtanuclear) cytoplasm enlarged and fused each other to form large VacA-containing vacuoles (VCVs). These directly opened into endoplasmic reticulum (ER) cisternae, which in turn enveloped mitochondria and contacted the Golgi apparatus. In all such organelles we found toxin molecules, often coupled with structural damage. These findings suggest direct toxin transfer from VCVs to other target organelles such as ER/Golgi and mitochondria. VacA-induced cytotoxic changes were associated with the appearance of auto(phago)lysosomes containing VacA, polyubiquitinated proteins, p62/SQSTM1 protein, cathepsin D, damaged mitochondria and bacterial remnants, thus leading to persistent cell accumulation of degradative products.

Different Polyubiquitinated Bodies in Human Dendritic Cells: IL-4 Causes PaCS During Differentiation while LPS or IFNα Induces DALIS During Maturation.

Two types of polyubiquitin-reactive cytoplasmic bodies, particulate cytoplasmic structures (PaCS) and dendritic cell (DC) aggresome-like induced structures (DALIS), were analyzed by electron microscopy, immunocytochemistry, immunoblotting, and flow cytometry in DC obtained from human blood monocytes incubated with GM-CSF plus IL-4 (IL4-DC), GM-CSF plus IFNα (IFN-DC), or GM-CSF alone (GM-DC), with or without LPS maturation. PaCS developed as monomorphic aggregates of proteasome-reactive barrel-like particles only in ribosomes-rich cytoplasmic areas of differentiating IL4-DC. In contrast, DALIS formed as vesicular bodies storing K63-linked ubiquitinated proteins by coalescence of increased endosomal structures, in IFN-DC or after LPS maturation of GM-DC. DALIS-forming cells showed incomplete morphological and functional DC-type differentiation when compared to PaCS-forming IL4-DC. PaCS and DALIS may have different function as well as different origin and cytochemistry. DALIS may be a transient accumulation site of potentially antigenic polyubiquitinated proteins during their processing and presentation. PaCS are found under physiologic or pathologic conditions associated with increased/deranged protein synthesis and increased ubiquitin-proteasome activity. Given its high heat-shock protein content PaCS may work as a quality control structure for newly synthesized, cytosolic proteins. This comparative analysis suggests that PaCS and DALIS have distinctive roles in DC.

Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells.

CeO2 nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(iii)/Ce(iv) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(iii). By coupling on individual cells synchrotron micro-X-ray fluorescence (µXRF) with micro-XANES (µXANES) we demonstrated that the Ce(iii)/Ce(iv) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(iv) oxidation state, while a higher Ce(iii) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

Correction: Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells.

Correction for 'Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells' by Daniela Ferraro, et al., Nanoscale, 2017, 9, 1527-1538.

Helicobacter pylori Employs a Unique Basolateral Type IV Secretion Mechanism for CagA Delivery.

The Helicobacter pylori (Hp) type IV secretion system (T4SS) forms needle-like pili, whose binding to the integrin-ß1 receptor results in injection of the CagA oncoprotein. However, the apical surface of epithelial cells is exposed to Hp, whereas integrins are basolateral receptors. Hence, the mechanism of CagA delivery into polarized gastric epithelial cells remains enigmatic. Here, we demonstrate that T4SS pilus formation during infection of polarized cells occurs predominantly at basolateral membranes, and not at apical sites. Hp accomplishes this by secreting another bacterial protein, the serine protease HtrA, which opens cell-to-cell junctions through cleaving epithelial junctional proteins including occludin, claudin-8, and E-cadherin. Using a genetic system expressing a peptide inhibitor, we demonstrate that HtrA activity is necessary for paracellular transmigration of Hp across polarized cell monolayers to reach basolateral membranes and inject CagA. The contribution of this unique signaling cascade to Hp pathogenesis is discussed.

Relationship between VacA Toxin and Host Cell Autophagy in Helicobacter pylori Infection of the Human Stomach: A Few Answers, Many Questions.

Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach of about half the global population and represents the greatest risk factor for gastric malignancy. The relevance of H. pylori for gastric cancer development is equivalent to that of tobacco smoking for lung cancer. VacA toxin seems to play a pivotal role in the overall strategy of H. pylori towards achieving persistent gastric colonization. This strategy appears to involve the modulation of host cell autophagy. After an overview of autophagy and its role in infection and carcinogenesis, I critically review current knowledge about the action of VacA on host cell autophagy during H. pylori infection of the human stomach. Although VacA is a key player in modulation of H. pylori-induced autophagy, a few discrepancies in the data are also evident and many questions remain to be answered. We are thus still far from a definitive understanding of the molecular mechanisms through which VacA affects autophagy and the consequences of this toxin action on the overall pathogenic activity of H. pylori.

Overestimation of nanoparticles-induced DNA damage determined by the comet assay.

The increasing use of engineered nanoparticles (NPs) in a wide range of commercial products raises concern about the possible risks that NPs pose to human health. Many aspects of the interaction between living cells and NPs are still unclear, and a reliable assessment of NP genotoxicity would be important. One of the most common tests used for genotoxicity is the comet assay, a sensitive method measuring DNA damage in individual cells. The assay was originally developed for soluble molecules, but it is also used in the assessment of genotoxicity of NPs. However, concerns have been raised recently about the reliability of this test in the case of NPs, but no conclusive results have been presented. Using nuclei isolated from human epithelial cells incubated with NPs, we obtained clear evidence of overestimation of NP genotoxicity by the comet assay in the case of CeO2, TiO2, SiO2, and polystyrene NPs. Removal of the NPs in the cytoplasm was effective in eliminating this genotoxicity overestimation (ex post damage) and determining the actual damage produced by the NPs during incubation with the cells (ex ante damage). This method could improve significantly the determination of NP genotoxicity in eukaryotic cells.