Advances on the early cellular events occurring upon exposure of human macrophages to aluminum oxyhydroxide adjuvant.

Aluminum compounds are the most widely used adjuvants in veterinary and human vaccines. Despite almost a century of use and substantial advances made in recent decades about their fate and biological effects, the exact mechanism of their action has been continuously debated, from the initial "depot-theory" to the direct immune system stimulation, and remains elusive. Here we investigated the early in vitro response of primary human PBMCs obtained from healthy individuals to aluminum oxyhydroxide (the most commonly used adjuvant) and a whole vaccine, in terms of internalization, conventional and non-conventional autophagy pathways, inflammation, ROS production, and mitochondrial metabolism. During the first four hours of contact, aluminum oxyhydroxide particles, with or without adsorbed vaccine antigen, (1) were quickly recognized and internalized by immune cells; (2) increased and balanced two cellular clearance mechanisms, i.e. canonical autophagy and LC3-associated phagocytosis; (3) induced an inflammatory response with TNF-α production as an early event; (4) and altered mitochondrial metabolism as assessed by both decreased maximal oxygen consumption and reduced mitochondrial reserve, thus potentially limiting further adaptation to other energetic requests. Further studies should consider a multisystemic approach of the cellular adjuvant mechanism involving interconnections between clearance mechanism, inflammatory response and mitochondrial respiration.

High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer.

BACKGROUND: Carbonic anhydrase IX (CAIX) is an enzyme upregulated by hypoxia during tumour development and progression. This study was conducted to assess if the expression of CAIX in tumour tissue and/or plasma can be a prognostic factor in patients with non-small cell lung cancer (NSCLC). METHODS: Tissue microarrays containing 555 NSCLC tissue samples were generated for quantification of CAIX expression. The plasma level of CAIX was determined by ELISA in 209 of these NSCLC patients and in 58 healthy individuals. The CAIX tissue immunostaining and plasma levels were correlated with clinicopathological factors and patient outcome. RESULTS: CAIX tissue overexpression correlated with shorter overall survival (OS) (P=0.05) and disease-specific survival (DSS) of patients (P=0.002). The CAIX plasma level was significantly higher in patients with NSCLC than in healthy individuals (P<0.001). A high level of CAIX in the plasma of patients was associated with shorter OS (P<0.001) and DSS (P<0.001), mostly in early stage I+II NSCLC. Multivariate Cox analyses revealed that high CAIX tissue expression (P=0.002) was a factor of poor prognosis in patients with resectable NSCLC. In addition, a high CAIX plasma level was an independent variable predicting poor OS (P<0.001) in patients with NSCLC. CONCLUSION: High expression of CAIX in tumour tissue is a predictor of worse survival, and a high CAIX plasma level is an independent prognostic biomarker in patients with NSCLC, in particular in early-stage I+II carcinomas.

RET gene mutations are not involved in the origin of human testicular seminoma.

Testicular germ cell cancers are the most common solid malignancies in young men, but their pathogenesis remains undetermined although some epidemiological data have implicated both environmental and genetic factors. Glial cell-derived neurotrophic factor (GDNF) is secreted by Sertoli cells, and promotes germ stem cell proliferation by activating RET, a tyrosine kinase receptor. Over-expression of GDNF in adult transgenic mice induces the development of testicular tumours that mimic human seminoma, the most frequent testicular germ cell tumour. Activating mutations of RET were previously reported in several types of cancer, including thyroid, pituitary, adrenal and melanoma cancer. Both mouse experimental model and clinical studies suggested that mutations or selective polymorphisms of RET might be associated with human seminoma. To verify this hypothesis, we conducted this study in a French University Hospital and carried out an association study using tissue samples from 66 paraffin-embedded seminoma tumours. The most frequently mutated exons of the RET proto-oncogene were sequenced to identify mutations or selective polymorphisms. No somatic mutations were identified. The polymorphic variants frequencies did not differ from those in a control Caucasian population. Human classical seminoma that occurs in young men does not appear to be linked with mutations or relevant polymorphisms of RET.

The sensitivity of activated Cys Ret mutants to glial cell line-derived neurotrophic factor is mandatory to rescue neuroectodermic cells from apoptosis.

Hirschsprung's disease (HSCR), a frequent developmental defect of the enteric nervous system is due to loss-of-function mutations of RET, a receptor tyrosine kinase essential for the mediation of glial cell-derived neurotrophic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutations (e.g., Cys(609), Cys(620), and Cys(634)) of the same gene are responsible for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by causing a covalent Ret dimerization, leading to ligand-independent activation of its tyrosine kinase. In this context, the association of Cys(609)- or Cys(620)-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys(634) Ret variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here that despite their constitutively activated kinase, the mere expression of these three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys(634) Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys(609) or Cys(620) mutations, which impair the terminal Ret glycosylation required for its insertion at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together, these data support the idea that sensitivity to GDNF is the mandatory condition, even for constitutively activated Ret mutants, to rescue neuroectodermic cells from apoptosis. These findings may help clarify how a gain-of-function mutation can be associated with a developmental defect.

Immunotherapy in Non-Small Cell Lung Cancer: Biological Principles and Future Opportunities.

Immunotherapy aims to amplify the anticancer immune response through reactivation of the lymphocytic response raised against several tumor neo-antigens. To obtain an effective immune response, this therapeutic approach requires that a number of immunological checkpoints be passed, such as the activation of excitatory costimulatory signals or the avoidance of coinhibitory molecules. Among the immune checkpoints, the interaction of the membrane-bound ligand PD-1 and its receptor PD-L1 has received much attention because of remarkable efficacy in numerous clinical trials for various cancer types, including non-small cell lung cancer (NSCLC). However, several limitations exist with these therapeutic agents when used as monotherapy, with objective responses observed in only 30-40% of patients, with the majority of patients demonstrating innate resistance, and approximately 25% of responders later demonstrating disease progression. Recent developments in the understanding of cancer immunology have the potential to identify mechanisms of innate and acquired resistance to immune checkpoint inhibitors through translational research in human samples. This review focuses on the biological basic principles for immunological checkpoint blockade, and highlights the current status and the perspectives of this therapeutic approach in NSCLC patients.

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2.

Recently c-Cbl has been reported to be phosphorylated upon CSF-1 stimulation. The product of the c-cbl proto-oncogene (c-Cbl) is a 120 kDa protein harboring several docking sites for Src homology 2 (SH2) domain containing proteins and proline-rich regions that have been shown to allow its constitutive association with the SH3 domains of Grb2. We demonstrate here that CSF-1 exposure of stable transfectant CHO cells expressing the CSF-1 receptor induced the sustained tyrosine phosphorylation of c-Cbl and its subsequent association with Crk-II and the p85 kDa subunit of the PI 3-kinase, while it constitutively associates with Grb2. We demonstrate by in vitro experiments that these associations require the SH2 domain of Crk-II and both the C- and N-terminal SH2 domains of the p85 subunit of the PI 3-kinase. cCbl is the major PI 3-kinase-containing protein in c-Fms expressing CHO cells upon CSF-1 stimulation. Thus c-Cbl behaves as a core protein, allowing the formation of a quaternary complex including, Crk-II, PI 3-kinase and Grb2. We provide evidence that this multiprotein complex can interact with the tyrosine phosphorylated CSF-1 receptor through the unoccupied SH2 domain of Grb2.

The multiple endocrine neoplasia type 2B point mutation switches the specificity of the Ret tyrosine kinase towards cellular substrates that are susceptible to interact with Crk and Nck.

The RET proto-oncogene encodes a Tyrosine Kinase Receptor (RTK) which plays an important function in the proliferation and/or differentiation of neuroectodermic cells. Germline mutation of a methionine to a threonine within the RET TK domain predisposes to the Multiple Endocrine Neoplasia type 2B (MEN 2B). It has been demonstrated that, unlike c-Ret, the MEN 2B mutated Ret displays constitutive TK activity, tyrosine autophosphorylation and transforms fibroblasts. However, this oncoprotein is more than a fully activated wild-type (WT) Ret TK since it also displays modified substrate specificity. Change in substrate specificity leads to the tyrosine autophosphorylation of MEN 2B Ret on new sites as well as the phosphorylation of several novel downstream targets. But, none of these substrates have been identified and the ability of MEN 2B Ret phosphoprotein to interact with Src Homology 2 (SH2) domain containing molecules has been poorly investigated. In this report, using a constitutively activated Ret TK form, Ret-ptc 2, we demonstrate that the MEN 2B as the activated WT Ret TK binds to several SH2 signalling proteins such as Shc, Grb-2, Phospholipase Cgamma, Crk and Nck. However, in contrast to the activated WT form, expression of the MEN 2B mutated Ret-ptc 2 results in the tyrosine phosphorylation of a panel of proteins which interestingly interact with Crk and Nck. We identified Paxillin, a cytoskeletal protein as one of the Crk associated proteins that is dramatically phosphorylated in MEN 2B but not in WT Ret expressing cells. These data suggest that MEN 2B mutated Ret triggers distinct signalling pathways that might be related to its transforming power.

Estrogen-induced growth inhibition of human seminoma cells expressing estrogen receptor beta and aromatase.

It is now well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. Studying a human testicular seminoma cell line, JKT-1, we show here that 17beta-estradiol (10(-12) to 10(-6) M) induced in vitro a significant dose-dependent decrease of cell growth. This antiproliferative effect was maximum after 4 days of exposure at a physiologically intratesticular concentration of 10(-9) M, close to the K(d) of ER, and reversed by ICI 182780, an ER antagonist, suggesting an ER-mediated pathway. By RT-PCR and Western blot we were able to confirm that JKT-1, like tumoral seminoma cells and normal human testicular basal germ cells, expresses estrogen receptor beta (ERbeta), including ERbeta1 and ERbeta2, a dominant negative variant, but not ERalpha. Using immunofluorescence and confocal microscopy, ERbeta was observed as perinuclear intracytoplasmic spots in JKT-1 and tumoral seminoma cells without significant translocation of ERbeta into the nucleus, under 17beta-estradiol exposure. Double staining observed by confocal microscopy revealed that ERbeta colocalized in JKT-1 cells with cytochrome C, a mitochondrial marker. We report for the first time the expression of a functional aromatase complex in seminoma cells as assessed by RT-PCR, Western blot and enzymatic assay. Seminoma cells are able to respond to estrogens through a possible autocrine or paracrine loop. These preliminary results support estrogen-dependency of human testicular seminoma, the most frequent tumor of young men, and suggest potential pharmacological use. Whether this estrogen control, however, involves an ERbeta-mediated stimulation of cell apoptosis and/or an ERbeta-mediated inhibition of cell proliferation, remains to be further determined.

Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) increases the adherence to epithelia and the oxidative burst of human polymorphonuclear leukocytes but decreases bacteria phagocytosis.

Recruitment of polymorphonuclear leukocytes (PMNL) is a hallmark of both urinary and digestive infections caused by Escherichia coli. Cytotoxic necrotizing factor 1 (CNF-1) is a toxin produced by uropathogenic E. coli strains that mediates its effects via the activation of small GTP-binding proteins. However, the role and the consequences of CNF-1 on PMNL physiology remain largely unknown. In this study, we provide evidence that CNF-1 dramatically affects the PMNL cytoskeleton architecture by inducing an increased content of F-actin. Furthermore, we demonstrate that CNF-1 increases functional features of PMNL, such as superoxide generation and adherence on epithelial T84 monolayers, but significantly decreases their phagocytic function. Our results suggest that CNF-1 may behave as a virulence factor in urinary or digestive infection by stimulating PMNL cytotoxicity as a result of its enhancing effect on their adherence to epithelial cells as well as the production of radical oxygen products. Moreover, the decreased phagocytosis of PMNL induced by CNF-1 likely facilitates growth of bacteria. In these conditions, CNF-1 would intervene in the initiation and in the perpetuation of the inflammatory process.

KRAS Mutations in Lung Adenocarcinoma: Molecular and Epidemiological Characteristics, Methods for Detection, and Therapeutic Strategy Perspectives.

KRAS mutations are detected in over one third of lung adenocarcinomas, most frequently in Caucasian and smoker patients. The impact of KRAS mutations on lung adenocarcinoma prognosis is currently subject to debate, as is their impact on the response to chemotherapy and EGFR tyrosine kinase inhibitors. The different methods for KRAS status assessment, based on histological and cytological samples or biological fluids, offer varying sensitivities. Since no treatments are available in clinical routine for KRAS-mutated lung cancer patients, one of the current major challenges in thoracic oncology is developing new dedicated strategic therapies. Different molecules can be developed that act on a post-transcriptional KRAS protein level, blocking its cytoplasmic membrane recruitment. The efficacy of these molecules' targeting of the different signaling pathways activated by the KRAS mutation (such as the MEK and BRAF pathways) is related to the particular KRAS mutation subtype. New therapeutic strategies are currently focused on certain genes linked with KRAS inducing a synthetic lethal interaction. The purpose of this work is to provide an overview of i) the recent epidemiological and molecular findings concerning KRASmutated lung adenocarcinoma, ii) the prognostic impact of KRAS mutations, in particular during response to treatment, iii) the available methods for detecting this mutation, and iv) the current molecules under development for new therapeutic strategies and the clinical trials targeting this genomic alteration.

Adhesion of human monocytic THP-1 cells to endothelial cell adhesion molecules or extracellular matrix proteins via beta1 integrins regulates heparin binding epidermal growth factor-like growth factor (HB-EGF) expression.

Adherence to endothelium and then to the extracellular matrix is a prerequisite for extravasation of monocytes into injured tissues. There, monocytes differentiate into macrophages and express heparin binding epidermal growth factor-like growth factor (HB-EGF), a key growth factor involved in normal wound healing. We investigated whether the interaction of human monocytic THP-1 cells with the endothelial cell adhesion molecules (vascular CAM-1, VCAM-1; intercellular adhesion molecule-1 ICAM-1 and endothelial-selectin, E-selectin), or the extracellular matrix (ECM) proteins (fibronectin, FN; laminin, LN and fibrinogen, FG) regulate HB-EGF expression. We have shown that adherence of THP-1 cells via VCAM-1, E-selectin or FN, which are all overexpressed at sites of inflammation, potentiates HB-EGF mRNA expression. In contrast, adhesion of THP-1 cells via ICAM-1 or FG, has no significant effect. Since THP-1 cells interact with ICAM-1 and FG through beta2 integrins, and with VCAM-1 and FN via beta1 integrins, regulation of HB-EGF expression appears to be specific to beta1 integrin ligation. In addition, we demonstrate that THP-1 binding to LN, through the beta1 integrin VLA-6, down regulates HB-EGF expression. Thus physiologically, transient destruction of LN and expression of VCAM-1, E-selectin and fibronectin at sites of inflammation, may locally induce HB-EGF overexpression.

Human monocytes express amphiregulin and heregulin growth factors upon activation.

In the last few years, three new heparin binding growth factors that interact with the Epidermal Growth Factor receptor (EGFR) and/or the related p185erbB-2 tyrosine kinase have been identified. Amphiregulin (AR) and Heparin-Binding EGF-like growth factor (HB-EGF) bind and activate the EGFR while Heregulin (HRG) acts through the p185erbB-2 and p180erbB-4 tyrosine kinases. Recently, activated macrophages were reported to secrete a p185erbB-2- and a heparin binding EGFR-stimulatory activities. We show here that activated monocytes secrete AR, HRG and HB-EGF-like molecules. Indeed, upon activation with Phorbol12, 13-dibutyrate (PDBu), the human monocytic-like THP-1 cells expressed high levels of AR, HRG and HB-EGF transcripts and released heparin binding factors that induced tyrosine phosphorylation of the EGFR in A431 cells and a protein of 185 kDa in MDA MB 453 cells. Similarly, activation of peripheral blood monocytes induces a dramatic increase of these three genes. Since EGFR, cerbB-2, c-erbB-4 transcripts are not or hardly detected upon activation, the occurrence of autocrine loops in these cells is unlikely. Therefore, secretion of these factors by activated monocytes may be implicated in the paracrine activation of the erb receptors thereby contributing to the epithelial and connective tissue proliferation.

Detection of circulating tumor cells from lung cancer patients in the era of targeted therapy: promises, drawbacks and pitfalls.

Interest in biomarkers in the field of thoracic oncology is focused on the search for new robust tests for diagnosis (in particular for screening), prognosis and theragnosis. These biomarkers can be detected in tissues and/or cells, but also in biological fluids, mainly the blood. In this context, there is growing interest in the detection of circulating tumor cells (CTCs) in the blood of lung cancer patients since CTC identification, enumeration and characterization may have a direct impact on diagnosis, prognosis and theragnosis in the daily clinical practice. Many direct and indirect methods have been developed to detect and characterize CTCs in lung cancer patients. However, these different approaches still hold limitations and many of them have demonstrated unequal sensitivity and specificity. Indeed, these methods hold advantages but also certain disadvantages. Therefore, despite the promises, it is currently difficult and premature to apply this methodology to the routine care of lung cancer patients. This situation is the consequence of the analysis of the methodological approaches for the detection and characterization of CTCs and of the results published to date. Finally, the advent of targeted cancer therapies in thoracic oncology has stimulated considerable interest in non-invasive detection of genomic alterations in tumors over time through the analysis of CTCs, an approach that may help clinicians to optimize therapeutic strategies for lung cancer patients. We describe here the main methods for CTC detection, the advantages and limitations of these different approaches and the potential usefulness and value of CTC characterization in the field of thoracic oncology.

Role of CFTR in oxidative stress and suicidal death of renal cells during cisplatin-induced nephrotoxicity.

The clinical use of the antineoplastic drug cisplatin is limited by its deleterious nephrotoxic side effect. Cisplatin-induced nephrotoxicity is associated with an increase in oxidative stress, leading ultimately to renal cell death and irreversible kidney dysfunction. Oxidative stress could be modified by the cystic fibrosis transmembrane conductance regulator protein (CFTR), a Cl(-) channel not only involved in chloride secretion but as well in glutathione (GSH) transport. Thus, we tested whether the inhibition of CFTR could protect against cisplatin-induced nephrotoxicity. Using a renal proximal cell line, we show that the specific inhibitor of CFTR, CFTR(inh)-172, prevents cisplatin-induced cell death and apoptosis by modulating the intracellular reactive oxygen species balance and the intracellular GSH concentration. This CFTR(inh)-172-mediated protective effect occurs without affecting cellular cisplatin uptake or the formation of platinum-DNA adducts. The protective effect of CFTR(inh)-172 in cisplatin-induced nephrotoxicity was also investigated in a rat model. Five days after receiving a single cisplatin injection (5 mg/kg), rats exhibited renal failure, as evidenced by the alteration of biochemical and functional parameters. Pretreatment of rats with CFTR(inh)-172 (1 mg/kg) prior to cisplatin injection significantly prevented these deleterious cisplatin-induced nephrotoxic effects. Finally, we demonstrate that CFTR(inh)-172 does not impair cisplatin-induced cell death in the cisplatin-sensitive A549 cancer cell line. In conclusion, the use of a specific inhibitor of CFTR may represent a novel therapeutic approach in the prevention of nephrotoxic side effects during cisplatin treatment without affecting its antitumor efficacy.

Drug delivery by polymeric nanoparticles induces autophagy in macrophages.

Drug delivery nanosystems are currently used in human therapy. In preliminary studies we have observed that Eudragit RS nanoparticles, prepared by nanoprecipitation or double emulsion techniques, are cytotoxic for NR8383 rat macrophages. In this study, we expand our previous analysis and suggest that unloaded Eudragit RS nanoparticles prepared by nanoprecipitation (NP/ERS) may induce important morphological and biochemical cellular modifications leading to cellular death. In NR8383 rat macrophages cell line exposed to doses varying from 15 to 100 µg/mL, NP/ERS nanoparticles are internalized inside the cells, reach the mitochondria and alter the structure of these organelles. In addition, the exposure to nanoparticles induces cellular autophagy as demonstrated by electron microscopy analysis, microchip array, qRT-PCR and Western blot assays. Although toxicity of nanoparticles has already been evidenced, it is the first time that results show clearly that the toxicity of polymeric nanovectors may be related to an activation of autophagy.

Autophagy and Crohn's disease: at the crossroads of infection, inflammation, immunity, and cancer.

Inflammatory bowel diseases (IBD) are common inflammatory disorders of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease (CD). The incidences of IBD are high in North America and Europe, affecting as many as one in 500 people. These diseases are associated with high morbidity and mortality. Colorectal cancer risk is also increased in IBD, correlating with inflammation severity and duration. IBD are now recognized as complex multigenetic disorders involving at least 32 different risk loci. In 2007, two different autophagy-related genes, ATG16L1 (autophagy-related gene 16-like 1) and IRGM (immunity-related GTPase M) were shown to be specifically involved in CD susceptibility by three independent genome-wide association studies. Soon afterwards, more than forty studies confirmed the involvement of ATG16L1 and IRGM variants in CD susceptibility and gave new information on the importance of macroautophagy (hereafter referred to as autophagy) in the control of infection, inflammation, immunity and cancer. In this review, we discuss how such findings have undoubtedly changed our understanding of CD pathogenesis. A unifying autophagy model then emerges that may help in understanding the development of CD from bacterial infection, to inflammation and finally cancer. The Pandora's box is now open, releasing a wave of hope for new therapeutic strategies in treating Crohn's disease.

Epithelial intestinal cell apoptosis induced by Helicobacter pylori depends on expression of the cag pathogenicity island phenotype.

Helicobacter pylori has been shown to induce chronic active gastritis and peptic ulcer and may contribute to the development of duodenal ulcer. Previous studies have shown that H. pylori mediates apoptosis of gastric epithelial cells via a Fas-dependent pathway. However, evidence for the induction of such a mechanism in intestinal epithelial cells (IEC) by H. pylori infection has not been demonstrated yet. This study was performed (i) to ascertain that H. pylori can induce IEC apoptosis; (ii) to delineate the role of the cag pathogenicity island (PAI), cagE, and vacA gene products in this process; and (iii) to verify whether the Fas-dependent pathway is involved in this phenomenon. When T84 cells were exposed to VacA(+)/cag PAI(+) H. pylori strains (CCUG 17874 and 60190), they exhibited apoptosis hallmarks as assessed by morphological studies, as well as annexin V and 3,3'-dihexyloxacarbocyanine iodide staining. In contrast, few or no apoptotic features could be detected after incubation with an isogenic mutant of strain 60190 in which the cagE gene was disrupted (60190:C(-) strain) or with a VacA(-)/cag PAI(-) H. pylori strain (G21). In addition, activation of caspase-3 during infection with VacA(+)/cag PAI(+) H. pylori strains was inhibited by pretreatment of IEC with an antagonistic anti-Fas antibody (ZB4). Taken together, these findings indicate that H. pylori triggers apoptosis in IEC via a Fas-dependent pathway following a process that depends on the expression of the cag PAI.

Implication of mitogen-activated protein kinases in T84 cell responses to enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) infection of T84 cells induces a decrease in transepithelial resistance, the formation of attaching and effacing (A/E) lesions, and cytokine production. The purpose of this study was to investigate the ability of EPEC to activate mitogen-activated protein (MAP) kinases in T84 cells and to correlate these signaling pathways with EPEC-induced cell responses. T84 cells were infected with either the wild-type (WT) EPEC strain E2348/69 or two mutants, intimin deletion strain CVD206 (deltaeaeA) and type III secretion apparatus mutant strain CVD452 (deltaescN::aphA). Infection of T84 cells with WT but not mutant EPEC strains induced tyrosine phosphorylation of several proteins in T84 cells, including the p46 and p52 Shc isoforms. Kinetics studies revealed that ERK1/2, p38, and c-Jun N-terminal kinase (JNK) MAP kinases were activated in cells infected with strain E2348/69 but not with the mutant strains. Inhibition of MAP kinases with PD98059 or SB203580 did not affect the EPEC-induced decrease in transepithelial resistance or actin accumulation beneath the WT bacteria, but these two inhibitors significantly decreased interleukin-8 (IL-8) synthesis. We demonstrate that EPEC induces activation of ERK1/2, p38, and JNK cascades, which all depend on bacterial adhesion and expression of the bacterial type III secretion system. ERK1/2 and p38 MAP kinases were equally implicated in IL-8 expression but did not participate in A/E lesion formation or transepithelial resistance modification, indicating that the signaling pathways involved in these events are distinct.

Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Escherichia coli-infected T84 cells.

Use of the nonpathogenic yeast Saccharomyces boulardii in the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenic Escherichia coli (EPEC)-associated disease demonstrates that S. boulardii abrogated the alterations induced by an EPEC strain on transepithelial resistance, [(3)H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence of S. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence of S. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence of S. boulardii. Altogether, this study demonstrated that S. boulardii exerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.

Disrupted traffic of connexin 43 in human testicular seminoma cells: overexpression of Cx43 induces membrane location and cell proliferation decrease.

Connexins, the constitutive proteins of gap junctions, are considered to be tumour suppressive agents and are often impaired in the tumourigenic processes. In the present study, the expression of connexin 43 (Cx43), which is involved in the control of spermatogenesis through Sertoli/germ cell coupling, has been investigated in human testicular seminoma cells (tumours and the JKT-1 cell line). Cx43 was immunolocalized in the Golgi apparatus without membrane expression and was detected by immunoblotting in JKT-1 as exclusive 70 kD bands. No mutation could be found by sequencing the transcript obtained by RT-PCR. Transfection with a Cx43-V5 vector reproduced the same gel shift, identifying these 70 kD bands as Cx43. The Cx43-70 kD bands were also expressed in normal testicular tissue, associated with the classical 43 kD isoforms. Stable transfection of JKT-1 with a Cx43-GFP vector allowed restoration of Cx43 membrane expression, functional cell coupling, and inhibition of the cell proliferation rate. Storage of Cx43 in the Golgi apparatus may correspond during spermatogenesis to an intermittent physiological process that becomes permanent in malignant seminoma cells as a result of the tumourigenic process. By preventing Cx43 membrane expression, this disrupted traffic may itself participate in tumour promotion.