Prevalence of anti-S100A10 antibodies in antiphospholipid syndrome patients.

INTRODUCTION: Annexin A2 (ANXA2), an endothelial cell receptor for plasminogen and tissue plasminogen activator, has been identified as a new autoantigen in antiphospholipid syndrome (APS). ANXA2 can exist as a monomer or a heterotetrameric complex with S100A10 protein. This S100A10 subunit also plays a pivotal role in the regulation of fibrinolysis. The aim of this study was to evaluate the prevalence of autoantibodies directed against S100A10 protein in patients with APS. METHODS: Patients with primary antiphospholipid syndrome (PAPS), patients with systemic lupus erythematosus (SLE) and patients with unexplained thrombosis were retrospectively included in this study. Patients were followed in the department of Internal Medicine of Amiens University Hospital, Amiens, France. IgG and IgM anti-S100A10 antibodies were detected in the serum of patients by enzyme-linked immunosorbent assay. The cut-off value for positivity was defined as 3 standard deviations above the mean optical density (OD) obtained in the sera of 116 healthy blood donors. RESULTS: The study group consisted of 116 healthy individuals and 106 patients: 42 APS patients (26 patients with PAPS and 16 patients with secondary SLE-related APS), 43 SLE patients without APS and 21 patients with unexplained thrombosis. The median age of APS patients, SLE patients without APS, patients with unexplained thrombosis and healthy individuals was 47, 38, 53 and 42 years, respectively. Anti-S100A10 antibodies were detected in 11.9% of APS patients and this prevalence was statistically higher than that observed in healthy individuals (1.7%) (p = 0.0148). Highest levels of anti-S100A10 were observed in the serum of one PAPS patient with venous thrombosis and one SLE patient with APS with a history of stroke and recurrent miscarriage. CONCLUSION: S100A10 protein, the binding partner of ANXA2, was identified as a target of autoantibodies in sera from patients with APS. Further studies involving a large cohort of APS patients are required to determine whether these antibodies could play a role in thrombogenic mechanisms of APS and to determine their diagnostic value in discriminating clinical subgroups of patients with APS, particularly those with seronegative APS.

Impact of a procalcitonin-based algorithm on the management of adhesion-related small bowel obstruction.

INTRODUCTION: Adhesion-related small bowel obstruction (ASBO) management is difficult if there are no signs of strangulation or peritonitis when intestinal transit has not been restored. The aim of the present study was to determine the impact of combining a procalcitonin (PCT)-based algorithm with clinical signs on the management of uncomplicated ASBO. METHOD: We performed a pilot, retrospective, single-center "before-after" study. During the "before" period (2007 to 2012), patients with uncomplicated ASBO (n=93, the Gastrografin® group) underwent a clinical examination and a Gastrografin® index. During the "after" period (2013 to 2016), patients with uncomplicated ASBO (n=70, the algorithm group) underwent a clinical examination and were assessed with the PCT-based algorithm. The study's primary outcome was the appropriateness of ASBO management. The secondary outcomes were the need for surgery and the time to surgery, the LOS, the morbidity and mortality rates, and the recurrence rate. RESULTS: The proportion of well-managed patients was higher in the algorithm group than in the Gastrografin® group (86% vs. 47%; P<0.001). The time to surgery (48h vs 72h; P=0.02) and the LOS (4 vs. 6days, P=0.02) were significantly lower in the algorithm group. The need for surgery was similar in both groups (31% vs. 37%, P=0.49). The morbidity (P=0.69), mortality (P=0.82) and recurrence rates (P=0.57) were similar in the two groups. CONCLUSION: The use of a PCT-based algorithm is of value in the routine clinical management of ASBO; it reduces the LOS and the time to surgery without increasing the need for surgery.

A molecular insight into the phototoxic reactions observed with vemurafenib, a first-line drug against metastatic melanoma.

The electronic properties of vemurafenib (VB) provide a rational basis for understanding its strong UVA-induced phototoxicity. Thus, solvation of hydrophobic VB by hydrogen bonding solvents controls its photophysical, photochemical and photosensitizing properties. Addition of phosphate buffered saline (PBS) to methanol (MeOH) induces a bathochromic shift of the VB absorbance spectrum and a fluorescence emission (λmax = 450 nm, quantum yield (Φ) = 0.011). Phosphorescence (λmax = 461 nm) is observed at 77 K in MeOH. 308 nm laser flash spectroscopy demonstrates that the lifetimes (τ) and quantum yields of the VB triplet state ((3)T(*)(1)) in deaerated MeOH (τMeOH = 0.41 µs, λmax ∼ 380 nm), MeOH-PBS and HSA solutions markedly depend on the microenvironment. A long-lived radical (half-life >200 µs) is also formed. The state (3)T(*)(1) is quenched by O2 and electron donors (Cys and 2'-deoxyguanosine) at a rate constant >1 × 10(9) M(-1) s(-1). UVA-irradiation of VB in air-saturated MeOH or MeOH-PBS solutions produces a UVA-absorbing photoproduct (Φ âˆ¼ 5 × 10(-4)). VB photosensitizes Trp destruction by type I (radical formation) and type II (singlet oxygen ((1)O2) formation) photodynamic reactions (Φ = 0.005). Singlet oxygen production is further demonstrated by the VB-photosensitized His oxidation (ΦMeOH = 0.006).

[Role of physical, psychological and sexual abuse in functional digestive disorders. A case-controls trial.]. / El papel del abuso físico, psicológicoy sexual en los trastornos funcionales digestivos.Un estudio de casos y controles.

INTRODUCTION: Abuse has been considered a significant factor on the development of functional gastrointestinal disorders (FGID), especially for severe and treatment-refractory patients. The aim of our study was to evaluate the presence of all FGID according to Rome II criteria, in a group of women with history of physical, psychological and/or sexual abuse. MATERIAL AND METHODS: A cross sectional study was performed in 96 women (37 +/- 12 years of age) with history of physical, psychological and/or sexual abuse (cases); and 96 open population women (36 +/- 14 years of age) (controls). The following evaluations were administered: Rome II questionnaire, a self-administered instrument to evaluate history of physical (beating), psychological(insults, public humiliation) and/or sexual abuse (rape, coercion), and HAD questionnaire. RESULTS: Among 96 women with history of abuse,91 (95%) reported to have suffered psychological abuse, 72 (75%) physical abuse, and 24 (25%)sexual abuse. Women with history of abuse had a higher prevalence of rumination (6% vs. 0%, p= 0.02), functional heartburn (26% vs. 13%, p =0.04), aerofagia (17% vs. 5%, p = 0.019), irritable bowel syndrome (38% vs. 18%, p = 0.002), fecalin continence (16% vs. 4%, p = 0.01), elevator anisyndrome (5% vs. 0%, p = 0.05), and proctalgia fugax (29% vs. 15%, p = 0.02) compared to controls. There was a positive correlation between anxiety (r = 0.5, p = 0.001) and depression scores(r = 0.45, p = 0.001), and the number of FGID. CONCLUSION: We demonstrated a high prevalence of FGID among women with history of physical,psychological, and/or sexual abuse. In this association,concomitant anxiety and depression play a significant role.

[Vocal forcing and posture: experimental studies on normal subject]. / Forçage vocal et posture : études expérimentales chez le sujet sain.

UNLABELLED: One of the well-known characteristics of vocal forcing is postural with an increase in the antero-posterior movements of the trunk and head during phonation. OBJECTIVE: we conceived different physiological experiments on normal subjects to explore in an objective way these movements. MATERIALS AND RESULTS: A series of experiments using a platform of posturography confirmed that there is an increase in the tensions in the muscles implied in the posture when the subject forces his voice because of an ambient noise. This increase is characterized by the index VCOP rms (variance of the displacement of the center of pressure in upright position) which passes from 13.19 in normal voice to 18.63 in forced voice. A complementary study was carried out with an equipment of analysis of the movements (ELITE). CONCLUSION: We could, thus, confirm the existence of the contemporary antero-posterior movements of vocal forcing. The discussion concerns the application perspectives of these experimental knowledge in the clinical field of the dysfunctional dysphonia.

Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides.

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 (PI4,5P(2)) 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 PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is 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 PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-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 PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.

A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic.

The small GTPase Rab4 is implicated in endocytosis in all cell types, but also plays a specific role in some regulated processes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for an effector(s) that specifically recognizes its GTP-bound form. We cloned a ubiquitous 69-kDa protein, Rabip4, that behaves as a Rab4 effector in the yeast two-hybrid system and in the mammalian cell. Rabip4 contains two coiled-coil domains and a FYVE-finger domain. When expressed in CHO cells, Rabip4 is present in early endosomes, because it is colocated with endogenous Early Endosome Antigen 1, although it is absent from Rab11-positive recycling endosomes and Rab-7 positive late endosomes. The coexpression of Rabip4 with active Rab4, but not with inactive Rab4, leads to an enlargement of early endosomes. It strongly increases the degree of colocalization of markers of sorting (Rab5) and recycling (Rab11) endosomes with Rab4. Furthermore, the expression of Rabip4 leads to the intracellular retention of a recycling molecule, the glucose transporter Glut 1. We propose that Rabip4, an effector of Rab4, controls early endosomal traffic possibly by activating a backward transport step from recycling to sorting endosomes.

The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release.

The pathogenic bacterium Helicobacter pylori produces the cytotoxin VacA, which is implicated in the genesis of gastric epithelial lesions. By transfect ing HEp-2 cells with DNAs encoding either the N-terminal (p34) or the C-terminal (p58) fragment of VacA, p34 was found localized specifically to mitochondria, whereas p58 was cytosolic. Incubated in vitro with purified mitochondria, VacA and p34 but not p58 translocated into the mitochondria. Microinjection of DNAs encoding VacA-GFP and p34-GFP, but not GFP-VacA or GFP-p34, induced cell death by apoptosis. Transient transfection of HeLa cells with p34-GFP or VacA-GFP induced the release of cytochrome c from mitochondria and activated the executioner caspase 3, as determined by the cleavage of poly(ADP-ribose) polymerase (PARP). PARP cleavage was antagonized specifically by co-transfection of DNA encoding Bcl-2, known to block mitochondria-dependent apoptotic signals. The relevance of these observations to the in vivo mechanism of VacA action was supported by the fact that purified activated VacA applied externally to cells induced cytochrome c release into the cytosol.

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

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

Effect of Helicobacter pylori on polymorphonuclear leukocyte migration across polarized T84 epithelial cell monolayers: role of vacuolating toxin VacA and cag pathogenicity island.

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.

The p21 Rho-activating toxin cytotoxic necrotizing factor 1 is endocytosed by a clathrin-independent mechanism and enters the cytosol by an acidic-dependent membrane translocation step.

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