MOLECULAR REORGANIZATION FOLLOWING SENSORY STIMULATION IN THE ADULT MOUSE BARREL CORTEX

Sensory information is an important factor in shaping neuronal circuits during development and adulthood. In the barrel cortex of adult rodents, cells from layer IV are able to adapt their functional state to an increased flow of sensory information from the mystacial whisker follicles. Previous studies in our group have shown that whisker stimulation induces the formation of inhibitory synapses in the corresponding barrel (Knott et al., 2002) and decreases neuronal responses toward the deflection of the stimulated whisker (Quairiaux et al., 2007). Together these observations have turned the barrel cortex into a model to study homeostatic plasticity. At the cellular level, neuronal activity triggers intracellular signaling cascades leading to a transcriptional response. To further characterize the molecular pathways involved in the synaptic changes after whisker stimulation in the adult mouse, a previous doctoral student in our group performed a microarray analysis on laser-dissected barrels in sections through layer IV. This study identified the regulation (up and down) of a series of genes in the stimulated barrels (thesis of Johnston-Wenger, 2010). We here focused on ten genes that presented the highest fold change according to the microarray analysis. Out of these genes, 7 are known as neuronal activity-dependent genes (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 and Adcyapl) whereas three have so far not been related to neuronal plasticity (Scn7a, Pcdhl5 and Cede3). The study aimed at confirming the results of the microarray analysis and localizing molecular modifications in the stimulated barrel column at the cellular level. In situ hybridization for Pcdhl5 after different periods of whisker stimulation (3, 6, 9, 15, 24 hrs) allowed us to confirm that the 1.25 fold change used for the microarray analysis is an appropriate threshold for considering a regulation significant after sensory-stimulation. Moreover, we confirmed with in situ hybridization a significant upregulation of the genes of interest in the stimulated barrels. In situ hybridization and immunohistochemistry allowed us to observe the distribution of the genes of interest and the corresponding protein products at the cellular level. Three observations were made: 1) alterations of the expression was restricted to the stimulated barrels for all genes tested; 2) within a barrel column not all cells responded to whisker stimulation with an altered gene expression; 3) in the stimulated barrels, two different patterns of mRNA and protein expression can be distinguished. We hypothesize that this segregation of the activity-induced gene expression reflects the segregation of the two principal thalamocortical pathways conveying the sensory information to the barrel cortex. Moreover, only neurons reaching the critical threshold will modify their gene expression program resulting in structural as well as physiological modifications that prevent the subsequent propagation of the excess of excitation to the postsynaptic targets. The activity-induced gene expression is therefore adapted in a cell-type-specific manner to induce a homeostatic response to the entire neuronal network involved in the integration of the sensory information. This to our knowledge the first study showing the distinct, but complementary contribution of the two thalamocortical pathways in experience-dependent plasticity in the adult mouse barrel cortex. -- L'information sensorielle nous permet de continuellement façonner nos circuits neuronaux autant durant le développement qu'à l'âge adulte. Chez le rongeur l'information sensorielle perçue par les vibrisses est intégrée au niveau du cortex somatosensoriel primaire (appelé en anglais « barrel cortex ») dont les cellules de la couche IV sont capables d'adapter leur état fonctionnel en réponse à une augmentation d'activité neuronale. Ce modèle expérimental a permis à notre groupe de recherche d'observer des changements rapides du circuit neuronal en fonction de l'activité sensorielle. En effet, la stimulation continue d'une vibrisse d'une souris adulte pendant 24 heures induit non seulement un remaniement synaptique (Knott et al., 2002), mais également des changements physiologiques au niveau des neurones du tonneau correspondant (Quairiaux et al., 2007). Ces observations nous permettent d'affirmer que le « barrel cortex » est un modèle approprié pour y étudier la plasticité synaptique. Au niveau cellulaire, l'activité neuronale déclenche des cascades de signalisation intracellulaire résultant en une réponse transcriptionnelle. Afin de caractériser les voies moléculaires impliquées dans la plasticité synaptique, une puce à ARN nous a permis de comparer l'expression de gènes entre un tonneau correspondant à une vibrisse stimulée et un tonneau d'une vibrisse non-stimulée (Nathalie). Cette analyse a révélé un certain nombre de gènes régulés de manière positive ou négative par l'augmentation de l'activité neuronale. Nous nous sommes concentrés sur 10 gènes dont l'expression est fortement régulée. L'expression de sept d'entre eux a déjà été démontrée comme dépendante de l'activité neuronale (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 otAdcyapl) alors que l'expression des trois autres (Scn7a, Pcdhl5 et Cedei) n'a pour le moment pas encore été liée à la plasticité neuronale. Le but de cette thèse est de confirmer les résultats de la puce à ARN et de déterminer dans quel type cellulaire ces gènes sont exprimés. L'hybridation in situ pour le gène Pcdhl5, après différentes périodes de stimulation des vibrisses (3, 6, 9, 15 et 24 heures), nous a permis de confirmer que le seuil de 1.25x utilisé dans l'analyse de la puce à ARN est approprié pour considérer qu'un gène est régulé de manière significative par la stimulation sensorielle. Nous avons également pu confirmer à l'aide de cette technique que la stimulation sensorielle augmente significativement l'expression de ces dix gènes. L'expression de ces gènes au niveau cellulaire a été observée à l'aide des techniques d'hybridation in situ et d'immunohistochimie. Trois observations ont été faites : 1) la régulation de ces gènes est restreinte aux tonneaux correspondants aux vibrisses stimulées ; 2) au niveau d'une colonne corticale correspondant aux vibrisses stimulées, seules certaines cellules présentent une altération de leur expression génique ; 3) au niveau des tonneaux stimulés, deux profils d'expression d'ARNm et de protéines sont observés. Notre hypothèse est que cette distribution pourrait correspondre à la terminaison ségrégée des deux voies thalamocortical qui amènent l'information sensorielle dans le cortex cérébral. De plus, seul les neurones atteignant le seuil critique d'activation modifient leur expression génique en réponse à la stimulation sensorielle. Ces changements d'expression géniques vont permettre à la cellule de modifier ses propriétés structurales et physiologiques de manière a prevenir la propagation d'un excès d'activité neuronale au niveau de ses cibles postsynaptics. L'activité neuronale agit donc spécifiquement sur certains types cellulaires de maniere a induire une réponse homéostatique au niveau du réseau neuronal impliqué dans l'integration de l'information sensorielle. Nos travaux démontrent pour une première fois que les deux voies sensorielles contribuent d'une manière distincte et complémentaire à la plasticité corticale induite par un changement de l'activité sensorielle chez la souris adulte.

Hepatitis B virus screening in contacts of blood donors with antibodies against core protein (anti-HBc), but without surface antigen (HBsAg)

To increase blood safety Brazil introduced screening for anti-HBc among blood donors in 1993. There was a decrease in the hepatitis B virus (HBV) transmission, but this measure identified a great number of HBsAg-negative, anti-HBc-positive donors. Surveillance policy determines that contacts of HBV carriers should be screened to HBV markers, but there is no recommendation about how to guide contacts of HBsAg-negative, anti-HBc-positive donors. Aiming to evaluate whether the contacts of this group are at greater risk for HBV infection, a cross-sectional study was performed to compare prevalence of HBV infection between contacts of HBsAg-positive blood donors (group I) and contacts of HBsAg-negative, anti-HBc-positive donors (group II). Contacts were submitted to a questionnaire and blood tests for HBV markers. In group I (n = 143), 53 (37.1%) were anti-HBc-positive and 11 (7.7%) were HBsAg-positive. In group II (n = 111), there were 9 and 0.9%, respectively. HBV exposure was associated with group I, sexual activity, blood transfusion, being one of the donor's parents, and living for more than ten years with the donor. Regarding the families as sample units, it was more common to find at least one member with HBV markers (p < 0.05) among the families of group I compared to group II. Contacts of HBsAg-negative, anti-HBc-positive individuals presented a much lower risk of having already been exposed to HBV and there is no need to screen them for HBV in low to moderate prevalence populations.

Constitutively active G protein-coupled receptor mutants: implications on receptor function and drug action.

Mutations of GPCRs can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis; others occur spontaneously in human diseases. The analysis of constitutively active GPCR mutants has attracted a large interest in the past decade, providing an important contribution to our understanding of the molecular mechanisms underlying receptor function and drug action.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Mutations in the SPARC-Related Modular Calcium-Binding Protein 1 Gene, SMOC1, Cause Waardenburg Anophthalmia Syndrome.

Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites.

Protein tyrosine kinases in Schistosoma mansoni

The identification and description of signal transduction molecules and mechanisms are essential to elucidate Schistosoma mansoni host-parasite interactions and parasite biology. This mini review focuses on recent advancements in the study of signalling molecules and transduction mechanisms in S. mansoni, drawing special attention to the recently identified and characterised protein tyrosine kinases of S. mansoni.

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Anchoring of an immunogenic Plasmodium falciparum circumsporozoite protein on the surface of Dictyostelium discoideum.

The circumsporozoite protein (CSP), a major antigen of Plasmodium falciparum, was expressed in the slime mold Dictyostelium discoideum. Fusion of the parasite protein to a leader peptide derived from Dictyostelium contact site A was essential for expression. The natural parasite surface antigen, however, was not detected at the slime mold cell surface as expected but retained intracellularly. Removal of the last 23 amino acids resulted in secretion of CSP, suggesting that the C-terminal segment of the CSP, rather than an ectoplasmic domain, was responsible for retention. Cell surface expression was obtained when the CSP C-terminal segment was replaced by the D. discoideum contact site A glycosyl phosphatidylinositol anchor signal sequence. Mice were immunized with Dictyostelium cells harboring CSP at their surface. The raised antibodies recognized two different regions of the CSP. Anti-sporozoite titers of these sera were equivalent to anti-peptide titers detected by enzyme-linked immunosorbent assay. Thus, cell surface targeting of antigens can be obtained in Dictyostelium, generating sporozoite-like cells having potentials for vaccination, diagnostic tests, or basic studies involving parasite cell surface proteins.

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.

Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91%, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5% when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.

Effects of dietary protein type on oxidized cholesterol-induced alteration in age-related modulation of lipid metabolism and indices of immune function in rats.

Exogenous oxidized cholesterol disturbs both lipid metabolism and immune functions. Therefore, it may perturb these modulations with ageing. Effects of the dietary protein type on oxidized cholesterol-induced modulations of age-related changes in lipid metabolism and immune function was examined using differently aged (4 weeks versus 8 months) male Sprague-Dawley rats when casein, soybean protein or milk whey protein isolate (WPI) was the dietary protein source, respectively. The rats were given one of the three proteins in diet containing 0.2% oxidized cholesterols mixture. Soybean protein, as compared with the other two proteins, significantly lowered both the serum thiobarbituric acid reactive substances value and cholesterol, whereas it elevated the ratio of high density lipoprotein-cholesterol/cholesterol in young rats, but not in adult. Moreover, soybean protein, but not casein and WPI, suppressed the elevation of Delta6 desaturation indices of phospholipids in both liver and spleen, particularly in young. On the other hand, WPI, compared to the other two proteins, inhibited the leukotriene B4 production of spleen, irrespective of age. Soybean protein reduced the ratio of CD4(+)/CD8(+) T-cells in splenic lymphocytes. Therefore, the levels of immunoglobulin (Ig)A, IgE and IgG in serum were lowered in rats given soybean protein in both age groups except for IgA in adult, although these observations were not shown in rats given other proteins. Thus, various perturbations of lipid metabolism and immune function caused by oxidized cholesterol were modified depending on the type of dietary protein. The moderation by soybean protein on the change of lipid metabolism seems to be susceptible in young rats whose homeostatic ability is immature. These observations may be exerted through both the promotion of oxidized cholesterol excretion to feces and the change of hormonal release, while WPI may suppress the disturbance of immune function by oxidized cholesterol in both ages. This alleviation may be associated with a large amount of lactoglobulin in WPI. These results thus showed a possibility that oxidized cholesterol-induced perturbations of age-related changes of lipid metabolism and immune function can be moderated by both the selection and combination of dietary protein.

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

TRADD protein is an essential component of the RIG-like helicase antiviral pathway

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

Prevalence and level of antibodies to the circumsporozoite protein of human malaria parasites in five states of the Amazon region of Brazil

The aim of this study was to determine the prevalence of malaria infection and antibodies against the repetitive epitopes of the circumsporozoite (CS) proteins of Plasmodium falciparum, P. malariae, P. vivax VK210, P. vivax VK247, and P. vivax-like in individuals living in the states of Rondônia, Pará, Mato Grosso, Amazonas, and Acre. Active malaria transmission was occurring in all studied sites, except in Acre. P. falciparum was the predominant species in Pará and Rondônia and P. vivax in Mato Grosso. Infection by P. malariae was low but this Plasmodium species was detected in Rondônia (3.5%), Mato Grosso (2.5%), and Pará (0.8%). High prevalence and levels of serological reactivity against the CS repeat peptides of P. falciparum were detected in Rondônia (93%) and Pará (85%). Sera containing antibodies against the CS repeat of P. malariae occurred more frequently in Rondônia (79%), Pará (76%), and Amazonas (68%). Antibodies against the repeat epitope of the standard CS protein of P. vivax VK210, P. vivax VK247, and P. vivax-like were more frequent in Rondônia, Pará, and Mato Grosso. The high frequency of reactions to P. malariae in most of the areas suggests that the infection by this Plasmodium species has been underestimated in Brazil.