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One of the major virulence factors of Helicobacter pyloriis the vacuolating toxin VacA. It has been known for a long time that the toxin enters host cells by endocytosis. On the other hand there is ample evidence that VacA is able to trigger apoptosis and this effect has been attributed in part to interactions with mitochondria. However, for 10 years it was difficult to reconcile the obvious accumulation of VacA in endosomes with mitochondrial targeting. The accessibility of the mitochondria to the toxin was enigmatic. In our new study, we investigated the activities of p34, the toxic subunit of VacA, in more detail. We found that the p34 N-terminus carries a unique targeting sequence for import into mitochondria and for insertion into the mitochondrial inner membrane. By forming an anion channel in this membrane, the toxin has the ability to interfere directly with mitochondrial functions. Taking into account additional results from independent studies, we discuss the implications of our findings with respect to intracellular traffic, the remarkable possibility of a direct transfer of VacA from endosomes to mitochondria, and VacA-dependent cell death.

Les protéines RAF (A-RAF, B-RAF et C-RAF) constituent une famille de kinases jouant un rôle déterminant dans des événements cellulaires aussi importants que la différenciation, la prolifération ou la régulation de la survie. Dans cette revue, nous faisons le point sur la régulation de la survie cellulaire par ces kinases. Les mécanismes en jeu sont particulièrement intéressants dans le domaine du cancer. Des études récentes portant sur la régulation de l’apoptose par les kinases RAF permettent d’envisager des approches thérapeutiques prometteuses en oncologie.

In this paper we study natural deduction for the intuitionistic and classical (normal) modal logics obtained from the combinations of the axioms T, B, 4and 5. In this context we introduce a new multi-contextual structure, called T-sequent, that allows to design simple labelfree natural deduction systems for these logics. After proving that they are sound and complete we show that they satisfy the normalization property and consequently the subformula property in the intuitionistic case.

Tumour proteostasis and the unfolded protein response (UPR) are emerging drivers of tumour progression and important determinants of clinical efficacy of cancer therapy. Recent findings indicate that they also regulate the production of protein tumour markers. Here, we discuss how this new knowledge opens up new perspectives for cancer therapeutics.

New fluorescent tetrazines have been prepared and their electrochemistry and fluorescence efficiency evaluated. The occurrence of fluorescence as well as the wavelength were found to be strongly dependent on the substituents, which have to be electronegative heteroatoms. This has been rationalized through a computational study that showed that the crucial factor is the nature of the HOMO, which determines the existence or not of fluorescence. © WileyVCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009

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Proton pump inhibitors remain the mainstay of medical therapy in gastroesophageal reflux disease. Despite their increasing use, up to 40% of patients are not fully satisfied with their antireflux therapy. Recent data on efficacy and safety are reviewed and causes of failure are discussed.

AbstractThe first ignition experiments on the Laser Mégajoule facility will use an indirect drive scheme. Our A1040 point design target is a graded doped plastic capsule filled by permeation within a gold cylinder. The deuterium-tritium ice layer may be formed either by classical slow cooling at 1.5 K below triple point, or by rapid cooling at 2.3 K below triple point. To complete the specifications, we first studied the robustness to all technological defects with the current CEA capabilities for these two options of ice formation. The technological imperfections taken into account are regrouped into 1D errors, which keep the implosion spherical, and 3D errors, which induce a deformation of the shell. The 3D robustness is expressed in terms of deformation at peak velocity and compared to the deformation threshold obtained with 2D simulations. The 1D robustness is given by the probability of exceeding 50% of nominal yield. We have taken into account 22 1D parameters and the fusion energy is approximated by a neural network based on 2000 simulations. Although the studies are not finished yet, the first results show that the A1040 design with rapid cooling has sufficient margins with respect to technological defects.

BACKGROUND The diagnosis of dermatofibrosarcoma protuberans (DFSP) in childhood is often difficult because of the deceptive appearance of the lesions. Little is known about congenital DFSP, the frequency of which is probably underestimated because the initial lesion may pass unnoticed. OBSERVATIONS We studied 9 DFSP congenital cases (8 plaques and 1 nodule) initially suspected to be benign lesions. The first biopsies or excisions were performed after a delay of 5½ months to 15 years. All cases were CD34+. Histologic patterns were similar to the DFSP adult classic pattern in 4 cases. One case was a Bednar tumor. The histologic diagnosis of the 4 remaining cases was difficult. The collagen, type I, α 1–platelet-derived growth factor β fusion gene (COL1A1-PDGFB) was detected by means of reverse transcriptase–polymerase chain reaction or fluorescence in situ hybridization. CONCLUSIONS All cases of congenital DFSP were difficult to identify clinically. The diagnosis was suspected by means of histologic and immunohistochemical evaluation and was confirmed using molecular analyses. This study illustrates the difficulties and pitfalls of the recognition of congenital DFSP and emphasizes the value of immunohistochemical study with anti-CD34 and complementary molecular analysis for all cutaneous spindle cell tumors and plaques in neonates and infants.Arch Dermatol. 2007;143:203-210--

The adoption of a non-uniform dopant profile has substantially increased the tolerance to high mode deformations of our baseline indirect-drive design. In addition, a low deuterium-tritium (DT) gas density, obtained by 'dynamic quenching' at 2.3?K below triple point, could partly compensate for the decrease in robustness due to DT ageing. Finally, the net margin regarding all laser and target technological defects is about 2. As soon as a sufficient amount of laser beams and diagnostics is available, we will shoot pre-ignition experiments to tune the point design. We are studying new targets which need less energy for these campaigns.

Despite being clinically important and highly prevalent, several aspects of gastroesophageal reflux disease are incompletely understood. Research activity remains high, however, and we shall review some highlights from last year's publications.

A base-line capsule design for the Laser MegaJoule facility is optimized by radially grading the Ge dopant in plastic ablator. A one dimensional robustness study is performed to optimize the implosion and the entropy deposition. Then, the tolerance versus the ablator roughness is examined for modes over 10 through direct two-dimensional simulations. The stability is significantly higher than that of an uniformly doped design.

A novel class of photochromic materials based on polar azo derivatives containing functionalized biphenyl substituents has been synthesized. Once melt and cooled, these compounds formed amorphous transparent monomeric materials whose glass transition temperatures depend mainly on the bulkiness of the biphenyl substituent; by contrast, a change in polarity, evidenced by absorption spectroscopy and DFT calculations, exerts insignificant influence. Photoisomerization of the azo groups occurred both in solution and thin films; the rate constants of the thermal back-relaxation in both media increase with compound polarity. Photoconversion yields display smaller values in solid state than in solution, due to more packed surroundings; surprisingly, they increase substantially with the monomer bulkiness, despite raising Tgs. Comparative dynamic surface relief grating writings and AFM surface modulation measurements of irradiated thin films showed that the efficiency of the writing process improves dramatically for glasses consisting of more hindered and less polar compounds with a 4- and 2-fold increase in the diffraction efficiency and peak-to-trough amplitude, respectively. Such results highly contrast with those obtained in polymer matrices, where competitive polymer chain reorganization occurs. Free volume enhancement upon back-and-forth photoisomerization of bulky photochromes, facilitating the azo photoisomerization, has been put forward to explain these results.

The logic of bunched implications, BI, provides a logical analysis of a basic notion of resource that is rich enough, for example, to form the logical basis for ‘pointer logic’ and ‘separation logic’ semantics for programs that manipulate mutable data structures. We develop a theory of semantic tableaux for BI, so providing an elegant basis for efficient theorem proving tools for BI. It is based on the use of an algebra of labels for BI's tableaux to solve the resource-distribution problem, the labels being the elements of resource models. For BI with inconsistency, $\bot$, the challenge consists in dealing with BI's Grothendieck topological models within such a proof-search method, based on labels. We prove soundness and completeness theorems for a resource tableaux method TBI with respect to this semantics and provide a way to build countermodels from so-called dependency graphs. Then, from these results, we can define a new resource semantics of BI, based on partially defined monoids, and prove that this semantics is complete. Such a semantics, based on partiality, is closely related to the semantics of BI's (intuitionistic) pointer and separation logics. Returning to the tableaux calculus, we propose a new version with liberalised rules for which the countermodels are closely related to the topological Kripke semantics of BI. As consequences of the relationships between semantics of BI and resource tableaux, we prove two new strong results for propositional BI: its decidability and the finite model property with respect to topological semantics.

ABSTRACTRecent studies have shown that the human fecal microbiota is composed of a consortium of species specific to the host and resistant to modifications over time. Antibiotics are known to affect the intestinal microflora, and ensuing changes may result in antibiotic-associated diarrhea. It is therefore important to characterize the nature and amplitude of these modifications and the ability of this ecosystem to return to its original profile—i.e., its resilience. Six healthy volunteers received oral amoxicillin (1.5 g/day) for 5 days. Fecal samples were collected at day 0 (D0) before antibiotic treatment and at set intervals until 60 days thereafter. Fecal DNA was isolated, and V6-to-V8 regions of the 16S rRNA genes were amplified by PCR with general primers and analyzed by temporal temperature gradient gel electrophoresis. Dominant species profiles were compared on the basis of similarity (Pearson correlation coefficient). Dominant species profiles at D0 were used as a reference. The fecal microbiota showed a major shift in dominant species upon antibiotic treatment, starting 24 h after treatment initiation and reaching an average similarity of only 74% after 4 days. Within 30 days following antibiotic treatment, the fecal microbiota tended to reach an average similarity of 88% to the D0 value; within 60 days, the average similarity to the D0 value was 89%. However, in one subject, important modifications persisted for at least 2 months, with similarity to the D0 value remaining below 70%. We demonstrated the resilience of the dominant human fecal microbiota upon short-course antibiotic challenge. Yet the persistence of long-term alterations in some subjects may explain susceptibilities to antibiotic-associated diarrhea. Furthermore, these findings suggest that strategies reinforcing the ability of the fecal microbiota to resist modifications would be of clinical relevance.

ABSTRACTThe mucosa-associated microbiota lining the gut epithelium might play a central role in the activation and/or perpetuation of mucosal inflammation in Crohn's disease (CD). We sought for localized dysbiosis by comparing the biodiversity and composition of the microbiotas in ulcerated and nonulcerated mucosal samples from patients with CD. Biopsy samples (n= 75) of ulcerated and adjacent nonulcerated mucosa were collected during colonoscopy from 15 patients, from the ileum, right colon, left colon, and rectum. Temporal temperature gradient gel electrophoresis (TTGE) of bacterial 16S rRNAs was used to evaluate the dominant bacterial species. TTGE profiles were compared using software that calculates similarity percentages. For a given patient, average similarity indexes between ulcerated and nonulcerated mucosal TTGE profiles ranged from 95.2% ± 4.2% to 97.9% ± 1.7% (means ± standard deviations) for the different segments. The mean values did not differ significantly. Average interindividual similarity indexes for a given segment among the different patients ranged from 33.6% ± 15.5% to 42.0% ± 25.6%. In CD, the dominant microbiotas do not differ qualitatively between ulcerated and nonulcerated mucosae. Biodiversity remains high in ulcerated mucosa. This argues against a pathogenic role of localized qualitative dysbiosis in CD-associated ulceration.

Abstract: In this abstract we emphasize the role of a semantic structure called resource graph in order to study the provability in some resource-sensitive logics, like the Bunched Implications Logic (BI) or the Non-commutative Logic (NL). Such a semantic structure is appropriate for capturing the particular interactions between different kinds of connectives (additives and multiplicatives in BI, commutatives and non-commutatives in NL) that occur during proof-search and is also well-suited for providing countermodels in case of non-provability. We illustrate the key points with a tableau method with labels and constraints for BI and then present tools, namely BILL and CheckBI, which are respectively dedicated to countermodel generation and verification in this logic. [Copyright &y& Elsevier]

A novel general synthetic strategy, based on a convergent approach, allowed us to prepare a series of conjugated 6,6‘-distyryl-3,3‘-bipyridine derivatives via the Suzuki reaction. First, the key electron-donor and electron-acceptor 6-(arylvinylene)-3-bromopyridine building blocks were synthesized by Knoevenagel- or Siegrist-type reactions. Second, some of them were transformed to the corresponding pyridylboronic esters. Finally, for the first time, we successfully demonstrated that such blocks can be homo- and cross-coupled in high yields and multigram scales, leading to noncentrosymmetic or symmetric chromophores. Their mesogenic, electrochemical, and optical properties have been investigated depending on the electronic structure. In this series, push−pull compounds are liquid crystals and promising for NLO applications. Whatever the structure, all of these compounds exhibit a high electron affinity and are strongly fluorescent. As an application, lasing properties of one push−pull and one symmetrical compound are reported. In addition, a blue-emitting LED was fabricated whose performances at 10 mA/cm² are a luminous efficiency of 3.9 cd/A, a power efficiency of 1.4 lm/W, and an external quantum efficiency of 2.9%. Thus, this versatile synthetic route is of particular interest due to the potential applications of the chromophores in several optoelectronic fields.

Necrotizing enterocolitis (NEC) is among the most severe conditions that can affect preterm infants. Although the etiology of NEC remains unknown, initial bacterial colonization could play a pivotal role in the development of NEC. To further explore the putative relationship between pathogen microorganisms and NEC, we conducted a prospective case-control study in 12 preterm infants with a new approach based on molecular techniques. Over an inclusion period of 24 mo, 12 neonates of <34 wk gestational age admitted to the neonatal unit were enrolled. The group included three cases of NEC, and nine control infants without evidence of NEC who were matched for gestational age and birth weight. Stool samples were collected at weekly intervals from all infants. PCR and temporal temperature gradient gel electrophoresis of 16S ribosomal DNA were used to detect the establishment of bacterial communities in the digestive tract. A salient feature of the bacteriological pattern was observed only in the three infants who later developed NEC A band corresponding to the Clostridium perfringenssubgroup could be detected in early samples, before diagnosis. There was no evidence for this specific band in any of the nine controls. To our knowledge, the current report is the first to demonstrate that the use of molecular techniques based on the study of bacterial 16S rRNA genes allowed the recognition of C. perfringensspecies in the first 2 wk of life of three infants who later displayed symptoms of NEC. A significant temporal relationship was thus established between early colonization by Clostridiumand the later development of NEC. Compared with conventional bacteriological culturing methods, the use of this new molecular approach to analyze the gastrointestinal ecosystem should therefore allow a more complete and rapid assessment of intestinal flora. Although the current data do not constitute definitive proof that the identified bacterial species was a causative agent in the development of NEC, they outline the promise of this new technique based on molecular biology, and suggest that large-scale studies on a much wider population at high risk for NEC may be warranted.

The simplified perturbation method of Vandenboomgaerde et al. (2002) is applied to both the Richtmyer–Meshkov and the Rayleigh–Taylor instabilities. This theory is devoted to the calculus of the growth rate of the perturbation of the interface in the weakly nonlinear stage. In the standard approach, expansions appear to be series in time. We build accurate approximations by retaining only the terms with the highest power in time. This simplifies and accelerates the solution. High order expressions are then easily reachable. For the Richtmyer–Meshkov instability, multimode configurations become tractable and the selection mode process can be studied. Inferences for the intermediate nonlinear regime are also proposed. In particular, a class of homothetic configurations is inferred; its validity is verified with numerical simulations even as vortex structures appear at the interface. This kind of method can also be used for the Rayleigh–Taylor instability. Some examples are presented.

The initial evolution of the momentum and buoyancy fluxes in a freely decaying, stably stratified homogeneous turbulent flow with r.m.s. velocity u'0 and integral lengthscale l0 is calculated using a weakly inhomogeneous and unsteady form of the rapid distortion theory (RDT) in order to study the growth of small temporal and spatial perturbations in the large-scale mean stratification N(z, t) and mean velocity profile ū(z, t) (here N is the local Brunt–Väisälä frequency and ū is the local velocity of the horizontal mean flow) when the ratio of buoyancy forces to inertial forces is large, i.e. Nl0/u'0≫1. The lengthscale L of the perturbations in the mean profiles of stratification and shear is assumed to be large compared to l0 and the presence of a uniform background mean shear can be taken into account in the model provided that the inertial shear forces are still weaker than the buoyancy forces, i.e. when the Richardson number Ri = (N/∂zū)2≫1 at each height. When a mean shear perturbation is introduced initially with no uniform background mean shear and uniform stratification, the analysis shows that the perturbations in the mean flow profile grow on a timescale of order N-1. When the mean density profile is perturbed initially in the absence of a background mean shear, layers with significant density gradient fluctuations grow on a timescale of order N−10 (where N0 is the order of magnitude of the initial Brunt–Väisälä frequency) without any associated mean velocity gradients in the layers. These results are in good agreement with the direct numerical simulations performed by Galmiche et al. (2002) and are consistent with the earlier physically based conjectures made by Phillips (1972) and Posmentier (1977). The model also shows that when there is a background mean shear in combination with perturbations in the mean stratification, negative shear stresses develop which cause the mean velocity gradient to grow in the density layers. The linear analysis for short times indicates that the scale on which the mean perturbations grow fastest is of order u'0/N0, which is consistent with the experiments of Park et al. (1994). We conclude that linear mechanisms are widely involved in the formation of shear and density layers in stratified flows as is observed in some laboratory experiments and geophysical flows, but note that the layers are also significantly influenced by nonlinear and dissipative processes at large times.

Freely decaying turbulent flows in a stably stratified fluid are simulated with a pseudo-spectral numerical code solving the fully nonlinear Navier–Stokes equations under the Boussinesq approximation with periodic boundary conditions. The flow is decomposed into a turbulent field and a horizontal mean flow ū(z, t) defined as the average of the horizontal velocity component in a horizontal plane at height z and time t. Similarly, the density field is decomposed into a turbulent field and a (stable) mean density profile ρ(z, t) defined as the average of the density field in a horizontal plane at height z and time t. Attention is paid to the effect of the turbulent velocity field on an initial z-periodic horizontal mean flow (Simulation A) or an initial z-periodic perturbation of the mean density profile (Simulation B). Both A and B are performed under conditions of moderate and strong stratification and are compared to the non-stratified simulations. Simulation A shows that the turbulence–mean flow interaction is strongly affected by the buoyancy forces. In the absence of a stratification, the mean flow perturbation decays rapidly due to the turbulent diffusion of momentum. When a moderate stratification is applied, the mean flow perturbation decays much more slowly whereas it oscillates and grows with time when the stratification is strong. These results are interpreted by defining a time-dependent eddy viscosity. Whereas the eddy viscosity coefficient has positive values in the non-stratified simulation, it is affected by the buoyancy forces and decreases after a period of order N−1. For large times, the eddy diffusivity oscillates and its time-averaged value over a few turnover timescales is positive but small when the stratification is moderate, and roughly zero when the stratification is strong. These results are interpreted by defining a time-dependent eddy viscosity. Whereas the eddy viscosity coefficient has positive values in the non-stratified simulation, it is affected by the buoyancy forces and decreases after a period of order N−1 in the stratified simulations (where N is the Brunt–Väisälä frequency associated with the background linear stratification). At large time, we find that the eddy viscosity remains roughly zero when the stratification is moderate, whereas it oscillates but remains persistently negative in the strongly stratified case, which causes the horizontal mean flow to accelerate. We conclude that the presence of a stable stratification strongly affects the temporal behaviour of the mean quantities ū and ρ in turbulent flows and partly explains the formation of horizontal layers in stratified geofluids such as oceans and atmospheres.

Donor–acceptor complex formation has been shown to occur between carbazole, an electron donor, and lutetium bisphthalocyanine, an acceptor, through π–π interactions. New transitions due to complexation between the two molecules are observed in the near IR, at about 1500 nm, in the solid state (KBr matrix); from the evolution of the absorption bands with the experimental conditions, it appears that more than one charge transfer complex exists. The nature of the molecular orbitals of the donor and acceptor molecules could explain why these interactions are weak.

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P2) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P2. This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P2-induced talin activation. Finally, sequestration of PI4,5P2by a specific pleckstrin homology domain confirms that PI4,5P2is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P2exposes the integrin-binding site on talin. We propose that PI4,5P2-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P2to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.

Helicobacter pylorivacuolating 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.

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.

AbstractIn this letter, we present a study of surface-enhanced Raman spectra of thin layers of phthalocyanine (Pc) complexes deposited on Ag and Au rough surfaces. the materials of this work are MgPc (monophthalocyanine) and the asymmetrical lutetium bisphthalocyanine (tBu)4PcLuPcCl8(Pc'LuPc') under three oxidation states. Enhancement of specific bands is observed depending on the surface and the oxidation state. SERS signatures of neutral, oxidised and reduced Pc'LuPc' are identified.

Helicobacter pylori infection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis. The mechanisms underlying this process are poorly documented. The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced by H. pylori. In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains of H. pylori. We have evaluated the role of the vacuolating cytotoxin VacA and of the cag pathogenicity island (PAI) of H. pylori in PMNL migration via their effects on T84 epithelial cells. We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positive H. pylori strains. In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells with cag PAI-positive and cagE-positive H. pylori strains. Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL. These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that the cag PAI, with a pivotal role for the cagE gene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response.

ABSTRACTHelicobacter pyloriinfection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis. The mechanisms underlying this process are poorly documented. The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced by H. pylori. In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains of H. pylori. We have evaluated the role of the vacuolating cytotoxin VacA and of the cagpathogenicity island (PAI) of H. pyloriin PMNL migration via their effects on T84 epithelial cells. We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positive H. pyloristrains. In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells with cagPAI-positive and cagE-positiveH. pyloristrains. Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL. These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that the cagPAI, with a pivotal role for the cagEgene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response.

Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic strains of Escherichia coli, activates the p21 Rho-GTP-binding protein, inducing a profound reorganization of the actin cytoskeleton. CNF1 binds to its cell surface receptor on HEp-2 cells with high affinity (Kd= 20 pM). In HEp-2 cells the action of CNF1 is not blocked in the presence of filipin, a drug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and internalization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is similar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cross the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that required by diphtheria toxin. As shown for diphtheria toxin, the pH-dependent membrane translocation step of CNF1 could be mimicked at the level of the plasma membrane by a brief exposure to a pH of ≤5.2. CNF1 is the first bacterial toxin described that uses both a clathrin-independent endocytic mechanism and an acidic-dependent membrane translocation step in its delivery of the catalytic domain to the cell cytosol.

Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic strains of Escherichia coli, activates the p21 Rho-GTP-binding protein, inducing a profound reorganization of the actin cytoskeleton. CNF1 binds to its cell surface receptor on HEp-2 cells with high affinity (K(d) = 20 pM). In HEp-2 cells the action of CNF1 is not blocked in the presence of filipin, a drug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and internalization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is similar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cross the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that required by diphtheria toxin. As shown for diphtheria toxin, the pH-dependent membrane translocation step of CNF1 could be mimicked at the level of the plasma membrane by a brief exposure to a pH of &lt;/=5.2. CNF1 is the first bacterial toxin described that uses both a clathrin-independent endocytic mechanism and an acidic-dependent membrane translocation step in its delivery of the catalytic domain to the cell cytosol.

BACKGROUND: Sodium butyrate, a product of colonic bacterial fermentation, is able to inhibit cell proliferation and to stimulate cell differentiation of colonic epithelial cell lines. It has been proposed that these cellular effects could be linked to its ability to cause hyperacetylation of histone through the inhibition of histone deacetylase. AIM: To analyse the molecular mechanisms of butyrate action on cell proliferation/differentiation and to compare them with those of trichostatin A, a well known inhibitor of histone deacetylase. METHODS: HT-29 cells were grown in the absence or presence of butyrate or trichostatin A. Cell proliferation and cell cycle distribution were studied after DNA staining by crystal violet and propidium iodide respectively. Cell cycle regulatory proteins were studied by western blot and reverse transcription-polymerase chain reaction. Cell differentiation was followed by measuring brush border enzyme activities. Histone acetylation was studied by acid/urea/Triton acrylamide gel electrophoresis. RESULTS: Butyrate blocked cells mainly in the G(1) phase of the cell cycle, whereas trichostatin A was inhibitory in both G(1) and G(2) phases. Butyrate inhibited the mRNA expression of cyclin D1 without affecting its protein expression and stimulated the protein expression of cyclin D3 without affecting its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3. Butyrate and trichostatin A stimulated p21 expression both at the mRNA and protein levels, whereas their effects on the expression of cyclin dependent kinases were slightly different. Moreover, butyrate strongly stimulated the activity of alkaline phosphatase and dipeptidyl peptidase IV, whereas trichostatin A had no effect. Finally, a six hour exposure to butyrate or trichostatin A induced histone H4 hyperacetylation. At 15 and 24 hours, histone H4 remained hyperacetylated in the presence of butyrate, whereas it returned to control levels in the presence of trichostatin A. CONCLUSIONS: The data may explain how butyrate acts on cell proliferation/differentiation, and they show that trichostatin A does not reproduce every effect of butyrate, mainly because of its shorter half life.

BACKGROUND: Peptide YY (PYY) is involved in the regulation of several gut functions, including secretion and motility. It exerts its effects through a family of six receptors, commonly named the Y receptor family. AIMS: To characterise the effects of PYY on strips of rat proximal colon in vitro, and to determine the pathways and receptors involved. METHODS: Contractions of strips removed from the muscle layer of rat proximal colon were recorded under isometric conditions, using PYY, Y receptor agonists and antagonists, and nerve blockers. Reverse transcription-polymerase chain reaction was also performed to detect the presence of mRNA coding for Y receptors. Finally, smooth muscle cells were isolated to estimate the cell length and intracellular Ca(2+) concentration in the presence and absence of PYY. RESULTS: PYY, neuropeptide Y (NPY), pancreatic polypeptide (PP) and [Leu31,Pro34]NPY induced a dose dependent contraction of strips from proximal colon. Tetrodotoxin partially inhibited the PYY and NPY induced contractions, and strongly inhibited the PP induced contraction. Specific antagonists showed the involvement of cholinergic nicotinic receptors and NK1 receptor. BIBP 3226, a specific Y1 antagonist, did not modify the colonic smooth muscle response to PYY, whereas blocking L-type Ca(2+) channels with D-600 abolished its effects. Moreover, PYY induced an increase in intracellular Ca(2+) concentration, associated with a reduction in cell length. mRNA encoding Y1 and Y4 receptors were detected in the muscle strips. CONCLUSIONS: These findings suggest that PYY stimulates colonic contractile activity in vitro through (a) a nervous Y4 dependent pathway and (b) a pathway involving a potential new receptor on myocytes.

BACKGROUND: Interleukin (IL) 1beta converting enzyme (now known as caspase 1) is able to process pro-IL-1beta into its active form and is involved in proapoptotic signalling. AIMS: To characterise IL-1 and caspase 1 expression in human colonic epithelial cells. METHODS: Intracellular IL-1 content (IL-1alpha and IL-1beta) was measured by ELISA in freshly isolated human normal colonocytes. Caspase 1 expression was determined both at the mRNA level using in situ hybridisation and reverse transcription polymerase chain reaction, and at the protein level by immunoblotting experiments using antibodies specific for the proform of caspase 1 and for its cleavage forms. RESULTS: Low amounts of IL-1beta were found in nearly all preparations (92%), and IL-1alpha was detected in only 45% of human colonocyte preparations. The normal colonic epithelium strongly expressed caspase 1, both at the mRNA level and at the protein level in its latent form. In contrast, caspase 1 was not expressed in colon cancer (primary colonic adenocarcinomas and cancer cell lines). CONCLUSIONS: The demonstration that the human colonic epithelial barrier is able to express caspase 1 and its substrate IL-1beta reinforces the concept that, under certain conditions, the epithelium could trigger an inflammatory reaction. In addition, the finding that caspase 1 was downregulated in colonic adenocarcinomas supports the concept that disrupted apoptosis pathways may be involved in tumour formation and/or may confer resistance to treatment.

BACKGROUND: Endoscopic oesophageal changes are diagnostically helpful and identify patients exposed to the risk of disease chronicity. However, there is a serious lack of agreement about how to describe and classify the appearance of reflux oesophagitis AIMS: To examine the reliability of criteria that describe the circumferential extent of mucosal breaks and to evaluate the functional and clinical correlates of patients with reflux disease whose oesophagitis was graded according to the Los Angeles system. METHODS: Forty six endoscopists from different countries used a detailed worksheet to evaluate endoscopic video recordings from 22 patients with the full range of severity of reflux oesophagitis. In separate studies, Los Angeles system gradings were correlated with 24 hour oesophageal pH monitoring (178 patients), and with clinical trials of omeprazole treatment (277 patients). RESULTS: Evaluation of circumferential extent of oesophagitis by the criterion of whether mucosal breaks extended between the tops of mucosal folds, gave acceptable agreement (mean kappa value 0.4) among observers. This approach is used in the Los Angeles system. An alternative approach of grouping the circumferential extent of mucosal breaks as occupying 0-25%, 26-50%, 51-75%, 76-99%, or 100% of the oesophageal circumference, gave unacceptably high interobserver variation (mean kappa values 0-0.15) for all but the lowest category of extent (mean kappa value 0.4). Severity of oesophageal acid exposure was significantly (p&lt;0.001) related to the severity grade of oesophagitis. Preteatment oesophagitis grades A-C were related to heartburn severity (p&lt;0.01), outcomes of omeprazole (10 mg daily) treatment (p&lt;0.01), and the risk for symptom relapse off therapy over six months (p&lt;0.05). CONCLUSIONS: Results add further support to previous studies for the clinical utility of the Los Angeles system for endoscopic grading of oesophagitis.

The infection mechanism of vaccinia virus is largely unknown. Neither the attachment protein of extracellular enveloped virus (EEV), the biologically relevant infectious form of the virus, nor its cellular receptor has been identified. Surprisingly, all former attempts using antibodies to block EEV infection of cellsin vitrohad failed. Here, we report the production of an anti-envelope hyperimmune serum with EEV neutralizing activity and show that a polyclonal antiserum against the extraviral domain of protein B5R also inhibited EEV infection.In vivo,mice vaccinated with B5R protein were protected against a lethal vaccinia virus challenge. This protectivity is likely to be mediated by neutralizing antibodies. Protein A33R, but not A34R and A36R, also proved to be protective in active and passive vaccination experiments. However, in contrast to B5R, A33R protectivity did not correlate with antibody titers. Because anti-A33R antibodies did not neutralize EEVin vitro,the protectivity mediated by A33R protein probably involves a mechanism different from simple antibody binding. Taken together, our results suggest that antibodies to a specific protective epitope or epitopes on protein B5R are able to prevent EEV infection. The protein encoded by the B5R gene is therefore likely to play a crucial role in the initial steps of vaccinia virus infection—binding to a host cell and entry into its cytoplasm.

This paper focuses on the use of linear logic as a specification language for the operational semantics of advanced concepts of programming such as concurrency and object-orientation. Our approach is based on a refinement of linear logic sequent calculi based on the proof-theoretic characterization of logic programming. A well-founded combination of higher-order logic programming and linear logic will be used to give an accurate encoding of the traditional features of concurrent object-oriented programming languages, whose corner-stone is the notion of encapsulation.

Background/Aims:Immunotherapy using interleukin 2 has had disappointing results in the treatment of colon cancer. Overcoming escape mechanisms, such as lack of antigen presentation and absence of accessory adhesion molecules on cancer cells, may increase its efficiency. We tried to do so by modifying the phenotype of the weakly immunogenic rat colon cancer PROb cells with sodium butyrate. Methods:After in vitro treatment with butyrate, PROb cells were tested for lymphokineactivated killer cell sensitivity and, using cytofluorometry, expression of adhesion molecules. We then treated established PROb peritoneal carcinomatoses with intraperitoneal injections of interleukin 2 and butyrate. Tumors were studied histologically and immunohistochemically. We tested the specificity of the immune protection by subsequent subcutaneous challenges with either PROb or glioma cells and by Winn's assay. Results:Butyrate increased lymphokine-activated killer cell sensitivity and expression of major histocompatibility complex class I and intercellular adhesion molecule 1 in vitro. Interleukin 2/butyrate combination resulted in cases of complete cure of carcinomatosis with specific protection against PROb cells. We noticed a complex stroma reaction with numerous functional antigen presenting cells close to PROb cells. Conclusions:The complete regression of tumor masses may be attributed, at least in part, to a butyrate-induced increase in immunogenicity of the cancer cells. This new combined immunotherapy may be of interest in the treatment of colon cancer.

BACKGROUND & AIMS:It is uncertain whether peptide YY (PYY) inhibits human intestinal secretion directly through enterocyte receptors or via indirect neural mechanisms. Thus, the effect of PYY on prostaglandin E2 (PGE2)-induced jejunal secretion in normal volunteers was measured, and it was determined whether a dopamine and sigma antagonist affected PYY effect. METHODS:Jejunal absorption of water and electrolytes was measured by a perfusion method in 6 volunteers. A double-blind crossover study was performed, involving intraluminal infusion of PGE2, intravenous infusion of human PYY, and intramuscular injection of haloperidol or placebo. RESULTS:PGE2 induced net secretion of water and electrolytes (P < 0.01 vs. basal). The effect of PGE2 was reduced by about half with 30 pmol x kg(-1) x h(-1) of PYY (plasma PYY, 96 +/- 12 pg/mL) and suppressed by 90 pmol x kg(-1) x h(-1) of PYY (P < 0.01; plasma PYY, 268 +/- 22 pg/mL). Plasma PYY was correlated negatively (P < 0.01) with net fluxes of water, Cl-, Na+, and K+. Haloperidol suppressed the effect of PYY on PGE2-induced secretion (P < 0.05). CONCLUSIONS:PYY administered in doses producing slightly supraphysiological plasma levels inhibits PGE2-induced secretion in normal humans. Sigma or dopamine receptors (probably neuronal ones) are involved in this effect. (Gastroenterology 1997 May;112(5):1520-8)