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ObjectivesPersistent infection with specific genotypes of human papillomaviruses (HPVs) is the main cause of invasive cervical cancer (ICC). Only a few of the various HPV types account for most of the cases worldwide, and geographical differences in their distribution are evident. Data from locally prevalent genotypes are essential in view of introduction of HPV type-specific prophylactic vaccines.MethodsIn this work, we have investigated HPV type distribution in samples of ICC cases that occurred in Uruguayan women. DNA extracted from ICC treated in Centro Hospitalario Pereira Rossell of Montevideo between 1999 and 2007 were analyzed. Search and typing were performed by polymerase chain reaction using generic GP5+/GP6+ primers and specific primers for HPV types 16, 18, 33, and 45. Positive GP5+/GP6+ samples, which were negative for all 4 high-risk HPV-specific types screened were further analyzed by sequencing.ResultsHuman papillomavirus DNA sequences were found in 163 (92.6%) of 176 cases. The most prevalent genotypes were HPV16 (67.6%) and HPV18 (8.5%) followed by HPV45 (6.8%) and HPV33 (3.4%), as single or mixed infection. Other less frequent genotypes were HPV31, HPV35, HPV39, HPV51, HPV52, HPV58, HPV66, and HPV73. The viral type could not be determined (HPV X) in 1 case (0.6%) of the HPV DNA–positive cervical cancers and double infections were found in 1.7% of the cases. The higher percentage of most aggressive HPV (16/18/45) genotypes was detected in cases diagnosed at younger than 60 years old, whereas these genotypes were less frequent in older patients.ConclusionWe conclude that HPV types 16, 18, and 45 have a very high prevalence in ICC of Uruguayan women. Results provide evidence that 16 of 18 infections are more aggressive, but most cancers could be vaccine preventable.

International audience; The surface of Trypanosoma cruzi is covered by a dense glycocalix which is characteristic of each stage of the life cycle. Its composition and complexity depend mainly on mucin-like proteins. A remarkable feature of O-glycan biosynthesis in trypanosomes is that it initiates with the addition of a GlcNAc instead of the GalNAc residue that is commonly used in vertebrate mucins. The fact that the interplay between trans-sialidase and mucin is crucial for pathogenesis, and both families have stage-specific members is also remarkable. Recently the enzyme that transfers the first GlcNAc from UDP-GlcNAc to a serine or threonine residue was kinetically characterized. The relevance of this enzyme is evidenced by its role as catalyzer of the first step in O-glycosylation. In this paper we describe how this gene is expressed differentially along the life cycle with a pattern that is very similar to that of trans-sialidases. Its localization was determined, showing that the protein predicted to be in the Golgi apparatus is also present in reservosomes. Finally our results indicate that this enzyme, when overexpressed, enhances T. cruzi infectivity.

International audience; Primary cilia are conserved organelles that play crucial roles as mechano- and chemosensors, as well as transducing signaling cascades. Consequently, ciliary dysfunction results in a broad range of phenotypes: the ciliopathies. Bardet-Biedl syndrome (BBS), a model ciliopathy, is caused by mutations in 16 known genes. However, the biochemical functions of the BBS proteins are not fully understood. Here we show that the BBS7 protein (localized in the centrosomes, basal bodies and cilia) probably has a nuclear role by virtue of the presence of a biologically confirmed nuclear export signal. Consistent with this observation, we show that BBS7 interacts physically with the polycomb group (PcG) member RNF2 and regulate its protein levels, probably through a proteasome-mediated mechanism. In addition, our data supports a similar role for other BBS proteins. Importantly, the interaction with this PcG member is biologically relevant because loss of BBS proteins leads to the aberrant expression of endogenous RNF2 targets in vivo, including several genes that are crucial for development and for cellular and tissue homeostasis. Our data indicate a hitherto unappreciated, direct role for the BBS proteins in transcriptional regulation and potentially expand the mechanistic spectrum that underpins the development of ciliary phenotypes in patients.

International audience; The fast reaction of (*)NO and O(2)(*-) to give ONOO(-) has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 ≤ k(f) ≤ 20 × 10(9) M(-1) s(-1)) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form (*)NO + O(2)(*-) = ONOO(-) with respect to its dependence on temperature, viscosity, and [ONOO(-)](eq) by determining [ONOO(-)](eq) and [(*)NO](eq). The equilibrium forward reaction rate constant (k(f)(eq)) has negative activation energy, in contrast to that found under irreversible conditions. In contradiction to the law of mass action, we demonstrate that the equilibrium constant depends on ONOO(-) concentration. Therefore, a wide range of k(f)(eq) values could be derived (7.5-21 × 10(9) M(-1) s(-1)). Of general interest, the variations in k(f) can thus be explained by its dependence on the distance between ONOO(-) particles (sites of generation of (*)NO and O(2)(*-)).

International audience; The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

It is widely accepted that the majority of cancers result from multiple cellular events leading to malignancy after a prolonged period of clinical latency, and that the immune system plays a critical role in the control of cancer progression. Bovine leukemia virus (BLV) is an oncogenic member of the Retroviridae family. Complete genomic sequences of BLV strains isolated from peripheral blood mononuclear cells (PBMC) from cattle have been previously reported. However, a detailed characterization of the complete genome of BLV strains directly isolated from bovine tumors is much needed in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV in cattle. In this study, we performed a molecular characterization of BLV complete genomes from bovine B-cell lymphosarcoma isolates. A nucleotide substitution was found in the glucocorticoid response element (GRE) site of the 5' long terminal repeat (5'LTR) of the BLV isolates. All amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5'LTR were not found in these studies. Amino acid substitutions were found in the nucleocapsid, gp51 and G4 proteins. Premature stop-codons in R3 were observed. Few mutations or amino acid substitutions may be needed to allow BLV provirus to achieve silencing. Substitutions that favor suppression of viral expression in malignant B cells might be a strategy to circumvent effective immune attack.

International audience; Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC(50) 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, K(D) 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC(50) = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC(50) attained with other antihapten VHHs reported thus far. Despite the modest overall antihapten sdAbs response in llamas, a small subpopulation of high-affinity VHHs is generated that can be isolated by careful design of the selection process.

Antibody-dependent cell cytotoxicity (ADCC) plays a critical role in monoclonal antibody (mAb)-mediated cancer therapy. ADCC, however, has not been directly shown in vivo but inferred from the requirement for IgG Fc receptors (FcγR) in tumor rejection in mice. Here, we investigated the mechanism of action of a Tn antigen-specific chimeric mAb (Chi-Tn), which binds selectively to a wide variety of carcinomas, but not to normal tissues, in both humans and mice. Chi-Tn mAb showed no direct toxicity against carcinomas cell lines in vitro but induced the rejection of a murine breast tumor in 80% to 100% of immunocompetent mice, when associated with cyclophosphamide. Tumor rejection was abolished in Fc receptors–associated γ chain (FcR-γ)–deficient mice, suggesting a role for ADCC. Indeed, tumor cells formed stable conjugates in vivo with FcR-γ chain-expressing macrophages and neutrophils in Chi-Tn mAb-treated but not in control mAb-treated mice. The contact zone between tumor cells and ADCC effectors accumulated actin, FcγR and phospho-tyrosines. The in vivo formed ADCC synapses were organized in multifocal supra-molecular activation clusters. These results show that in vivo ADCC mediated by macrophages and neutrophils during tumor rejection by Chi-Tn mAb involves a novel type of multifocal immune synapse between effectors of innate immunity and tumor cells. Cancer Res; 71(15); 5134–43. ©2011 AACR.

Trypanosoma cruzi tryparedoxin 1 (TcTXN1) is an oxidoreductase belonging to the thioredoxin superfamily, which mediates electron transfer between trypanothione and peroxiredoxins. In trypanosomes TXNs, and not thioredoxins, constitute the oxido-reductases of peroxiredoxins. Since, to date, there is no information concerning TcTXN1 substrates in T. cruzi, the aim of this work was to characterize TcTXN1 in two aspects: expression throughout T. cruzi life cycle and subcellular localization; and the study of TcTXN1 interacting-proteins. We demonstrate that TcTXN1 is a cytosolic and constitutively expressed protein in T. cruzi. In order to start to unravel the redox interactome of T. cruzi we designed an active site mutant protein lacking the resolving cysteine, and validated the complex formation in vitro between the mutated TcTXN1 and a known partner, the cytosolic peroxiredoxin. Through the expression of this mutant protein in parasites with an additional 6xHis-tag, heterodisulfide complexes were isolated by affinity chromatography and identified by 2-DE/MS. This allowed us to identify fifteen TcTXN1 proteins which are involved in two main processes: oxidative metabolism and protein synthesis and degradation. Our approach led us to the discovery of several putatively TcTXN1-interacting proteins thereby contributing to our understanding of the redox interactome of T. cruzi. Fil: Piñeyro, María Dolores. Universidad de la Republica Facultad de Medicina; Uruguay. Instituto Pasteur de Montevideo; Uruguay Fil: Parodi Talice, Adriana. Universidad de la Republica; Uruguay. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Uruguay Fil: Portela, Magdalena. Universidad de la República; Uruguay. Universidad de la Republica; Uruguay Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Robello, Carlos. Instituto Pasteur de Montevideo; Uruguay. Universidad de la Republica Facultad de Medicina; Uruguay

International audience; Trans-sialidases are surface-located proteins in Trypanosoma cruzi that participate in key parasite-host interactions and parasite virulence. These proteins are encoded by a large multigenic family, with tandem-repeated and individual genes dispersed throughout the genome. While a large number of genes encode for catalytically active enzyme isoforms, many others display mutations that involve catalytic residues. The latter ultimately code for catalytically inactive proteins with very high similarity to their active paralogs. These inactive members have been shown to be lectins, able to bind sialic acid and galactose in vitro, although their cellular functions are yet to be fully established. We now report structural and biochemical evidence extending the current molecular understanding of these lectins. We have solved the crystal structure of one such catalytically inactive trans-sialidase-like protein, after soaking with a specific carbohydrate ligand, sialyl-α2,3-lactose. Instead of the expected trisaccharide, the binding pocket was observed occupied by α-lactose, strongly suggesting that the protein retains residual hydrolytic activity. This hypothesis was validated by enzyme kinetics assays, in comparison to fully active wild-type trans-sialidase. Surface plasmon resonance also confirmed that these trans-sialidase-like lectins are not only able to bind small oligosaccharides, but also sialylated glycoproteins, which is relevant in the physiologic scenario of parasite infection. Inactive trans-sialidase proteins appear thus to be β-methyl-galactosyl-specific lectins, evolved within an exo-sialidase scaffold, thus explaining why their lectin activity is triggered by the presence of terminal sialic acid.

Background During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes. Methods We evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation. Results Repeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation. Conclusions Here we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.