Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

From the Cover: Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes.

BACKGROUND: The purpose of this work was to characterize the expression of drug and nutrient carriers along the anterior-posterior and crypt-villus axes of the intestinal epithelium and to study the validity of utilizing whole gut tissue rather than purified epithelial cells to examine regional variations in gene expression. RESULTS: We have characterized the mRNA expression profiles of 76 % of all currently known transporters along the anterior-posterior axis of the gut. This is the first study to describe the expression profiles of the majority of all known transporters in the intestine. The expression profiles of transporters, as defined according to the Gene Ontology consortium, were measured in whole tissue of the murine duodenum, jejunum, ileum and colon using high-density microarrays. For nine transporters (Abca1, Abcc1, Abcc3, Abcg8, Slc10a2, Slc28a2, Slc2a1, Slc34a2 and Slc5a8), the mRNA profiles were further measured by RT-PCR in laser micro-dissected crypt and villus epithelial cells corresponding to the aforementioned intestinal regions. With respect to differentially regulated transporters, the colon had a distinct expression profile from small intestinal segments. The majority (59 % for p cutoff < or = 0.05) of transporter mRNA levels were constant across the intestinal sections studied. For the transporter subclass "carrier activity", which contains the majority of known carriers for biologically active compounds, a significant change (p < or = 0.05) along the anterior-posterior axis was observed. CONCLUSION: All nine transporters examined in laser-dissected material demonstrated good replication of the region-specific profiles revealed by microarray. Furthermore, we suggest that the distribution characteristics of Slc5a8 along the intestinal tract render it a suitable candidate carrier for monocarboxylate drugs in the posterior portion of the intestine. Our findings also predict that there is a significant difference in the absorption of carrier-mediated compounds in the different intestinal segments. The most pronounced differences can be expected between the adjoining segments ileum and colon, but the differences between the other adjoining segments are not negligible. Finally, for the examined genes, profiles measured in whole intestinal tissue extracts are representative of epithelial cell-only gene expression.

The high road and the low road: trafficking choices in plants.

Polar transport of the signaling molecule auxin is critical for plant development and depends on both the polar distribution of auxin efflux carriers, which pump auxin out of the cell and the alignment of these polarized cells. Two papers in this issue of Cell (Michniewicz et al., 2007; Jaillais et al., 2007) address how polar transport of these carriers occurs and describe the endosomal pathways involved.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples.

New genetic models and mesenchymal cells as tools to ameliorate anti-tumor immunity

Résumé Le but final de ce projet est d'utiliser des cellules T ou des cellules souches mésenchymateuses modifiées génétiquement afin de surexprimer localement les deux chémokines CXCL13 et CCL2 ensemble ou chacune séparément à l'intérieur d'une tumeur solide. CXCL13 est supposé induire des structures lymphoïdes ectopiques. Un niveau élevé de CCL2 est présumé initier une inflammation aiguë. La combinaison des deux effets amène à un nouveau modèle d'étude des mécanismes régulateur de la tolérance périphérique et de l'immunité tumorale. Les connaissances acquises grâce à ce modèle pourraient permettre le développement ou l'amélioration des thérapies immunes du cancer. Le but premier de ce travail a été l'établissement d'un modèle génétique de la souris permettant d'exprimer spécifiquement dans la tumeur les deux chémokines d'intérêt à des niveaux élevés. Pour accomplir cette tâche, qui est en fait une thérapie génétique de tumeurs solides, deux types de cellules porteuses potentielles ont été évaluées. Des cellules CD8+ T et des cellules mésenchymateuses de la moelle osseuse transférées dans des receveurs portant une tumeur. Si on pouvait répondre aux besoins de la thérapie génétique, indépendamment de la thérapie immune envisagée, on posséderait là un outil précieux pour bien d'autres approches thérapeutiques. Plusieurs lignées de souris transgéniques ont été générées comme source de cellules CD8+ T modifiées afin d'exprimer les chémokines d'intérêt. Dans une approche doublement transgénique les propriétés de deux promoteurs spécifiques de cellules T ont été combinées en utilisant la technologie Cre-loxP. Le promoteur de granzyme B confère une dépendance d'activation et le promoteur distal de lck assure une forte expression constitutive dès que les cellules CD8+ T ont été activées. Les transgènes construits ont montré une bonne performance in vivo et des souris qui expriment CCL2 dans des cellules CD8+ T activées ont été obtenues. Ces cellules peuvent maintenant être utilisées avec différents protocoles pour transférer des cellules T cytotoxiques (CTL) dans des receveurs porteur d'une tumeur, permettant ainsi d'évaluer leur capacité en tant que porteuse de chémokine d'infiltrer la tumeur. L'établissement de souris transgéniques, qui expriment pareillement CXCL13 est prévu dans un avenir proche. L'évaluation de cellules mésenchymateuses de la moelle osseuse a démontré que ces cellules se greffent efficacement dans le stroma tumoral suite à la co-injection avec des cellules tumorales. Cela représente un outil précieux pour la recherche, vu qu'il permet d'introduire des cellules manipulées dans un modèle tumoral. Les résultats confirment partiellement d'autres résultats rapportés dans un modèle amélioré. Cependant, l'efficacité et la spécificité suggérées de la migration systémique de cellules mésenchymateuses de la moelle osseuse dans une tumeur n'ont pas été observées dans notre modèle, ce qui indique, que ces cellules ne se prêtent pas à une utilisation thérapeutique. Un autre résultat majeur de ce travail est l'établissement de cultures de cellules mésenchymateuses de la moelle osseuse in vitro conditionnées par des tumeurs, ce qui a permis à ces cellules de s'étendre plus rapidement en gardant leur capacité de migration et de greffe. Cela offre un autre outil précieux, vu que la culture in vitro est un pas nécessaire pour une manipulation thérapeutique. Abstract The ultimate aim of the presented project is to use genetically modified T cells or mesenchymal stem cells to locally overexpress the two chemokines CXCL13 and CCL2 together or each one alone inside a solid tumor. CXCL13 is supposed to induce ectopic lymphoid structures and a high level of CCL2 is intended to trigger acute inflamation. The combination of these two effects represents a new model for studying mechanisms that regulate peripheral tolerance and tumor immunity. Gained insights may help developing or improving immunotherapy of cancer. The primary goal of the executed work was the establishment of a genetic mouse model that allows tumor-specific expression of high levels of the two chemokines of interest. For accomplishing this task, which represents gene therapy of solid tumors, two types of potentially useful carrier cells were evaluated. CD8+ T cells and mesenchymal bone marrow cells to be used in adoptive cell transfers into tumor-bearing mice. Irrespectively of the envisaged immunotherapy, satisfaction of so far unmet needs of gene therapy would be a highly valuable tool that may be employed by many other therapeutic approaches, too. Several transgenic mouse lines were generated as a source of CD8+ T cells modified to express the chemokines of interest. In a double transgenic approach the properties of two T cell-specific promoters were combined using Cre-loxP technology. The granzyme B promoter confers activation-dependency and the lck distal promoter assures strong constitutive expression once the CD8+ T cell has been activated. The constructed transgenes showed a good performance in vivo and mice expressing CCL2 in activated CD8+ T cells were obtained. These cells can now be used with different protocols for adoptively transferring cytotoxic T cells (CTL) into tumor-bearing recipients, thus allowing to study their capacity as tumor-infiltrating chemokine carrier. The establishment of transgenic mice likewisely expressing CXCL13 is expected in the near future. In addition, T cells from generated single transgenic mice that have high expression of an EGFP reporter in both CD4+ and CD8+ cells can be easily traced in vivo when setting up adoptive transfer conditions. The evaluation of mesenchymal bone marrow cells demonstrated that these cells can efficiently engraft into tumor stroma upon local coinjection with tumor cells. This represents a valuable tool for research purposes as it allows to introduce manipulated stromal cells into a tumor model. Therefore, the established engraftment model is suited for studying the envisaged immunotherapy. These results confirm to some extend previously reported results in an improved model, however, the suggested systemic tumor homing efficiency and specificity of mesenchymal bone marrow cells was not observed in our model indicating that these cells may not be suited for therapeutic use. Another major result of the presented work is the establishment oftumor-conditioned in vitro culture of mesenchymal bone marrow cells, which allowed to more rapidly expand these cells while maintaining their tumor homing and engrafting capacities. This offers another valuable tool as in vitro culture is a necessary step for therapeutic manipulations.

Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1·8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Comportamiento del orificio de implantación del catéter en pacientes en diálisis peritoneal en relación a los cuidados

Infections of the catheter wound in peritoneal dialysis are the most frequent cause of morbility in patients who undergo this technique. There are a number of procedures for the care of the wound and it is not easy to define a single method that will guarantee good condition of the wound. In order to evaluate the behaviour of the wound related to the procedure used in their care, we studied 306 patients over 24 months, compiling socio-demographic and clinical variables. We found a high incidence of infections caused by gram-positive skin and mucous germs, with a strong correlation with the fact that the patient/family carer is a nasal carrier of staphylococcus aureus and that they appear more frequently in patients who do not remove the wound dressing in the shower. We also detected an increase in pseudomonas infections when the patient does not dry the wound with a hair-dryer

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells.

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.

Pneumocystis jirovecii transmission from immunocompetent carriers to infant.

We report a case of Pneumocystis jirovecii transmission from colonized grandparents to their infant granddaughter. Genotyping of P. jirovecii showed the same genotypes in samples from the infant and her grandparents. These findings support P. jirovecii transmission from immunocompetent carrier adults to a susceptible child.