IgA quantification as a good predictor of the neutralizing antibodies levels after vaccination against SARS-CoV-2.

Background: Vaccination against COVID-19 was implemented very quickly, but the emergence of new variants that can evade the previous acquired immunological protection highlights the importance of understanding the mechanisms involved in the immune response generated after SARS-CoV-2 infection or vaccination. Objectives: Since most of our knowledge on the humoral immunity generated against SARS-CoV-2 has been obtained from studies with infected patients before vaccination, our goal here was to evaluate seroconversion and its correlation with the titers of neutralizing antibodies (NAbs) in individuals who received the complete initial recommended vaccination schedule with three different vaccines. Study design: We analyzed serum IgG, IgA and total NAbs against the trimeric SARS-CoV-2 Spike (S) protein or its receptor binding domain (RBD) in blood samples collected from 118 healthy individuals without known previous infection, before and after receiving the first and the second dose of CoronaVac (n = 18), ChAdOx-1 (n = 68) or BNT162b2 (n = 32) vaccines. Results: We found that although IgG titers were high in all sera collected after the two doses of these vaccines, NAbs amounts varies among the groups. In contrast, serum NAbs concentrations were much more comparable to the IgA levels, indicating that these antibodies would have a major neutralizing capacity against SARS-CoV-2. Conclusions: Altogether our data suggest that quantification of serum anti-S or anti-RBD IgA, rather than IgG, may be a valuable tool to screen NAbs and may be considered for surveillance of vaccine coverage.

Combination of E- and NS1-Derived DNA Vaccines: The Immune Response and Protection Elicited in Mice against DENV2.

The occurrence of dengue disease has increased radically in recent decades. Previously, we constructed the pE1D2 and pcTPANS1 DNA vaccines encoding the DENV2 envelope (E) and non-structural 1 (NS1) proteins, respectively. To decrease the number of plasmids in a tetravalent candidate vaccine, we constructed a bicistronic plasmid, pNS1/E/D2, encoding these two proteins simultaneously. We evaluated the protective immunity induced in mice vaccinated with the pNS1/E/D2 candidate and compared to the responses elicited by immunization with the former vaccines isolated or in combination. We transfected BHK-21 cells with the different plasmids and detected recombinant proteins by immunofluorescence and mass spectrometry assays to confirm antigen expression. BALB/c mice were inoculated with the DNA vaccines followed by a lethal DENV2 challenge. ELISA, PRNT50, and IFN-gamma ELISPOT assays were performed for the investigation of the humoral and cellular responses. We observed the concomitant expression of NS1 and E proteins in pNS1/E/D2-transfected cells. All E-based vaccines induced anti-E and neutralizing antibodies. However, anti-NS1 antibodies were only observed after immunization with the pcTPANS1 administered alone or combined with pE1D2. In contrast, splenocytes from pNS1/E/D2- or pcTPANS1 + pE1D2-vaccinated animals responded to NS1- and E-derived synthetic peptides. All the DNA vaccines conferred protection against DENV2.

Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors.

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.

T Cell Responses Induced by DNA Vaccines Based on the DENV2 E and NS1 Proteins in Mice: Importance in Protection and Immunodominant Epitope Identification.

The importance of the cellular immune response against DENV has been increasingly highlighted in the past few years, in particular for vaccine development. We have previously constructed two plasmids, pE1D2, and pcTPANS1, encoding the envelope (E) ectodomain (domains I, II, and III) and the non-structural 1 (NS1) protein of dengue virus serotype 2 (DENV2), respectively. In the present work, we analyzed the induction of the cellular response in mice immunized with these DNA vaccines and identified the immunogenic peptides. Vaccinated BALB/c mice became protected against a lethal challenge of DENV2. Depletion of CD4+ cells in vaccinated animals almost completely abolished protection elicited by both vaccines. In contrast, a significant number of pE1D2- and pcTPANS1-immunized mice survived virus challenge after depletion of CD8+ cells, although some animals presented morbidity. To identify immunogenic peptides recognized by T cells, we stimulated splenocytes with overlapping peptide libraries covering the E and NS1 proteins and evaluated the production of IFN-γ by ELISPOT. We detected two and three immunodominant epitopes in the E and NS1 proteins, respectively, and four additional NS1-derived peptides after virus challenge. Characterization by intracellular cytokine staining (ICS) revealed that both CD4+ and CD8+ T cells were involved in IFN-γ and TNF-α production. The IFN-γ ICS confirmed reaction of almost all E-derived peptides before challenge and identified other epitopes after infection. All NS1-derived peptides were able to elicit IFN-γ production in CD4+ cells, while only a few peptides induced expression of this cytokine in CD8+ T lymphocytes. Interestingly, we observed an increase in the frequency of either CD4+ or CD8+ T cells producing TNF-α after immunization with the pE1D2 and challenge with DENV2, while lymphocytes from pcTPANS1-vaccinated animals maintained ordinary TNF-α production after virus infection. We also assessed the recognition of E and NS1 immunogenic peptides in C57BL/6 mice due to the difference in MHC haplotype expression. Two NS1-derived epitopes featured prominently in the IFN-γ response with cells from both animal strains. Overall, our results emphasize the importance of the T cell response involved in protection against dengue induced by E and NS1 based DNA vaccines.

Socioeconomic Factors and Caries in People between 19 and 60 Years of Age: An Update of a Systematic Review and Meta-Analysis of Observational Studies.

This study is aimed to perform an update of a systematic review and meta-regression to evaluate the effect modification of the socioeconomic indicators on caries in adults. We included studies that associated social determinants with caries, with no restriction of year and language. The Newcastle-Ottawa Scale was used to evaluate the risk of bias. With regard to the meta-analysis, statistical heterogeneity was evaluated by I², and the random effect model was used when it was high. A subgroup analysis was conducted for socioeconomic indicators, and a meta-regression was performed. Publication bias was assessed through Egger's test. Sixty-one studies were included in the systematic review and 25 were included in the meta-analysis. All of the studies were published between 1975 and 2016. The most frequent socioeconomic indicators were schooling, income, and socioeconomic status (SES). In the quantitative analysis, the DMFT (decayed, missing, filled teeth) variation was attributed to the studies' heterogeneity. The increase of 10.35 units in the proportion of people with lower SES was associated with an increase of one unit in DMFT, p = 0.050. The findings provide evidence that populations with the highest proportions of people with low SES are associated with a greater severity of caries. The results suggest the need for actions to reduce the inequalities in oral health (PROSPERO [CRD42017074434]).

BALB/c mice infected with DENV-2 strain 66985 by the intravenous route display injury in the central nervous system.

Dengue is a mild flu-like arboviral illness caused by dengue virus (DENV) that occurs in tropical and subtropical countries. An increasing number of reports have been indicating that dengue is also associated to neurological manifestations, however, little is known regarding the neuropathogenesis of the disease. Here, using BALB/c mice intravenously infected with DENV-2 strain 66985, we demonstrated that the virus is capable of invading and damaging the host's central nervous system (CNS). Brain and cerebellum of infected animals revealed histological alterations such as the presence of inflammatory infiltrates, thickening of pia matter and disorganization of white matter. Additionally, it was also seen that infection lead to altered morphology of neuroglial cells and apoptotic cell death. Such observations highlighted possible alterations that DENV may promote in the host's CNS during a natural infection, hence, helping us to better understand the neuropathological component of the disease.

In Vitro Inhibition of Leishmania Attachment to Sandfly Midguts and LL-5 Cells by Divalent Metal Chelators, Anti-gp63 and Phosphoglycans.

Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous and visceral leishmaniasis, respectively. Several aspects of the vector-parasite interaction involving gp63 and phosphoglycans have been individually assayed in different studies. However, their role under the same experimental conditions was not studied yet. Here, the roles of divalent metal chelators, anti-gp63 antibodies and purified type I phosphoglycans (PGs) were evaluated during in vitro parasite attachment to the midgut of the vector. Parasites were treated with divalent metal chelators or anti-gp63 antibodies prior to the interaction with Lutzomyia longipalpis/Lutzomyia intermedia midguts or sand fly LL-5 cells. In vitro binding system was used to examine the role of PG and gp63 in parallel. Treatment with divalent metal chelators reduced Le. infantum adhesion to the Lu. longipalpis midguts. The most effective compound (Phen) inhibited the binding in both vectors. Similar results were observed in the interaction between both Leishmania species and the cell line LL-5. Finally, parallel experiments using anti-gp63-treated parasites and PG-incubated midguts demonstrated that both approaches substantially inhibited attachment in the natural parasite-vector pairs Le. infantum/Lu. longipalpis and Le. braziliensis/Lu. intermedia. Our results suggest that gp63 and/or PG are involved in parasite attachment to the midgut of these important vectors.

Dataset of proteins mapped on HepG2 cells and those differentially abundant after expression of the dengue non-structural 1 protein.

The data supplied in this article are related to the research article entitled "The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach" (K. Rabelo, M.R. Trugillo, S.M. Costa, B.A. Pereira, O.C. Moreira, A.T. Ferreira et al., 2016) [1]. The present article provides the inventory of peptides and proteins mapped in a hepatocyte cell line (HepG2) by mass spectrometry in the presence of the non-structural protein 1 (NS1) of Dengue 2 virus (DENV2). Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) or pMAXGFP, encoding the green fluorescent protein (GFP), a protein unrelated to dengue. Differentially abundant protein lists were obtained by comparing cells transfected with pcENS1 and controls.

The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach.

Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines. BIOLOGICAL SIGNIFICANCE: Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.

Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route.

Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy.

Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen.

Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.

Low water quality in tropical fishponds in southeastern Brazil.

Expansion of aquaculture around the world has heavily impacted the environment. Because fertilizers are needed to raise fish, one of the main impacts is eutrophication, which lowers water quality and increases the frequency of algal blooms, mostly cyanobacteria. To evaluate whether the water quality in 30 fishponds in southeastern Brazilian met the requirements of Brazilian legislation, we analyzed biotic and abiotic water conditions. We expected that the high nutrient levels due to fertilization would cause low water quality. We also analyzed cyanotoxins in seston and fish muscle in some systems where cyanobacteria were dominant. The fishponds ranged from eutrophic and hypereutrophic with high phytoplankton biomass. Although cyanobacteria were dominant in most of the systems, cyanotoxins occurred in low concentrations, possibly because only two of the 12 dominant species were potential producers of microcystins. The high phosphorus concentrations caused the low water quality by increasing cyanobacteria, chlorophyll-a, turbidity, and thermotolerant coliforms, and by depleting dissolved oxygen. We found that all the 30 systems were inappropriate for fish culture, according to Brazilian legislation, based on at least one of the parameters measured. Furthermore, there was not any single system in the water-quality thresholds, according to the Brazilian legislation, to grow fish. Our findings indicate the need for better management to minimize the impacts of eutrophication in fishponds, in addition to a rigorous control to guarantee good food.

Serviço de radiologia odontológica em municípios da região Centro-Oeste do Estado de Minas Gerais, Brasil / Dental radiologic service in cities of the midwest region of the State of Minas Gerais, Brazil

Objetivo: identificar a existência do serviço público de radiologia odontológica em municípios da região Centro-Oeste vinculados a uma Superintendência Regional de Saúde (SRS) de Minas Gerais, Brasil, e a partir daí conhecer o funcionamento do serviço ou o potencial funcionamento caso fosse implantado. Método: o estudo foi aprovado pelo Comitê de Ética em Pesquisa. Para a coleta de dados aplicou-se questionário aos coordenadores municipais de Saúde Bucal. A análise estatística descritiva foi desenvolvida no SPSS. Participaram 50 municípios de 55 vinculados a uma mesma SRS. Resultados: dos municípios participantes, 27 ofertavam o serviço radiológico em odontologia no setor público e 23 não ofertavam esse serviço. Entre os municípios que oferecem o serviço, 81,5% contam com um aparelho próprio, 11,1% utilizam todas as medidas de radioproteção, 96,3% ofertam a radiografia periapical, sem restrição para indicação (70,4%) e quantidade (96,3%). Dos municípios que não possuem o serviço radiológico público, para grande parte, apenas um aparelho próprio seria suficiente para instalação do serviço (59,1%). A maioria dos coordenadores afirmou que a radiologia ajuda muito na qualidade do serviço odontológico (87,0%), no entanto, somente uma minoria (21,7%) pretendia instalar o serviço. Conclusões: a oferta do serviço de radiologia odontológica no setor público não é uma realidade para todos os municípios avaliados. Dos que ofertam o serviço radiológico, a maioria não segue as legislações brasileiras na adoção de todas as medidas de radioproteção.

Objective: To identify the existence of public dental radiology municipalities in the Midwest region linked to a Regional Superintendent of Health (SRS) of Minas Gerais, Brazil, and from there to understand the operation of the service or the potential operation if it was deployed. Method: The study was approved by the Research Ethics. For data collection questionnaire was applied to municipal coordinators of Oral Health. Descriptive statistical analysis was developed in SPSS. Participated in 50 municipalities of 55 bound to the same SRS. Results: participating municipalities, 27 ofertavam dental radiology service in the public sector and 23 not ofertavam this service. Among the municipalities offering the service, 81.5% have an apparatus, 11.1% use all measures of radioprotection, 96.3% tender the periapical without restriction indication (70.4%) and quantity (96.3%). Municipalities that do not have radiological public service, for the most part, only an apparatus would be enough to install the service (59.1%). Most coordinators said the radiology helps a lot on the quality of dental services (87.0%), however, only a minority (21.7%) planned to install the service. Conclusions: The provision of dental radiology service in the public sector is not a reality for all municipalities assessed. That offer the service of radiological, most do not follow the laws Brazilian adoption of all measures of radioprotection.

High dental caries among adults aged 35 to 44 years: case-control study of distal and proximal factors.

The aim of this study was to determine whether a high degree of dental caries severity is associated with the distal and proximal determinants of caries in a group of Brazilian adults aged 35 to 44 years. A population-based case-control study was conducted using two groups-a case group with high caries severity (DMFT ≥ 14) and a control group without high caries severity (DMFT < 14). The sample comprised adults from metropolitan Belo Horizonte, Brazil (180 cases and 180 controls matched for gender and age). The exam was performed by calibrated dentists using the DMFT index. The statistical analysis used the Mann-Whitney test and bivariate and multivariate logistic regression (the conditional backward stepwise method). The mean DMFT was 8.4 ± 3.9 in the control group and 20.1 ± 4.5 in the case group. High caries severity was associated with regular visits to the dentist, low income, use of private/supplementary dental service and not petitioning the authorities for community benefits. The results of the study underscore the importance of considering distal and proximal factors in the assessment of the severity of dental caries. Greater caries severity persists among low-income families and among groups with a low degree of social cohesion.

A systematic review of socioeconomic indicators and dental caries in adults.

Increasing evidence suggests that socioeconomic factors may be associated with an increased risk of dental caries. To provide better evidence of the association between dental caries in adults and socioeconomic indicators, we evaluated the relation between these two conditions in a thorough review of the literature. Seven databases were systematically searched: Pubmed, Cochrane, Web of Science, Bireme, Controlled Trials, Clinical Trials and the National Institute for Health and Clinical Excellence. No restrictions were placed on the language or year of publication. The search yielded 41 studies for systematic review. Two independent reviewers screened the studies for inclusion, extracted data and evaluated quality using the Newcastle-Ottawa scale. The following socioeconomic indicators were found: educational level, income, occupation, socio-economic status and the community index. These indicators were significantly associated with a greater occurrence of dental caries: the subject's education, subject's income, subject's occupation and the Gini coefficient. A high degree of heterogeneity was found among the methods. Quality varied across studies. The criteria employed for socioeconomic indicators and dental caries should be standardized in future studies. The scientific evidence reveals that educational level, income, occupation and the Gini coefficient are associated with dental caries.

The severity of dental caries in adults aged 35 to 44 years residing in the metropolitan area of a large city in Brazil: a cross-sectional study.

BACKGROUND: In recent decades, studies in the field of public health have increasingly focused on social determinants that affect the health-illness process. The epidemiological perspective considers oral health to be a reflection of socioeconomic and environmental aspects, and it is particularly influenced by the social context. The aim of the present study was to assess the association between the severity of dental caries among adults aged 35 to 44 years and characteristics on the different levels at which the determinants of caries operate (individual, social structure and social context). METHODS: A home-based, cross-sectional field study was carried out involving a sample of 1,150 adults (35 to 44 years of age) residing in metropolitan Belo Horizonte, Brazil. The DMFT (decayed, missing, filled tooth) index (≥14) was used to determine the severity of dental caries. Bivariate and multivariate analyses were carried out using the Poisson regression model with the level of significance set at 5% (p < 0.05) and 95% confidence intervals. RESULTS: The majority of the participants (68.5%) had high caries severity. The rate of high-severity caries in the group between 40 and 44 years of age was 1.15-fold (CI: 1.04-1.26) greater than that among those aged 35 to 39 years. A greater prevalence of high caries severity was found among those who frequently visited the dentist (PR = 1.18; CI: 1.07-1.30), those with a lower income (PR = 1.11; CI: 1.01-1.23), those who reported that their neighborhood did not come together in the previous year to petition political leaders for benefits (PR = 1.16; CI: 1.05-1.28) and those who are unable to make decisions (without empowerment) (PR = 1.12; CI: 1.01-1.24). CONCLUSIONS: The present study revealed high dental caries severity in adults, which was associated with individual characteristics, health-related behavior and social structure and contextual variables. These findings underscore the importance of considering social determinants involved in the health-illness process when carrying out epidemiological studies on dental caries.

Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

Differential midgut attachment of Leishmania (Viannia) braziliensis in the sand flies Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia.

The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG). An upregulation of beta-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia) braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V.) braziliensis with its natural vectors Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia) whitmani. Using two in vitro binding techniques, phosphoglycans (PGs) derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was approximately 11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL) in Brazil is discussed.

Studies on the sandfly fauna (Diptera: Psychodidae: Phlebotominae) from transmission areas of American Cutaneous Leishmaniasis in state of Acre, Brazil

Studies were undertaken on the phlebotomines in the municipalities of Bujari, Xapuri and Rio Branco in the state of Acre. The abundance of species on the ground and in the tree canopy was estimated by Standardized Index of Species Abundance. Of the 52 species identified, Lutzomyia (N.) antunesi, Lutzomyia (N.) whitmani, Lutzomyia (P.) davisi, Lutzomyia migonei, Lutzomyia (N.) umbratilis, Lutzomyia (N.) flaviscutellata, Lutzomyia (T.) ubiqui-talis, Lutzomyia (P.) hirsuta hirsuta, Lutzomyia (P.) paraensis and Lutzomyia (P.) ayrozai are known to be vectors of Leishmania, the causative agent of American cutaneous leishmaniasis. Lutzomyia (T.) auraensis, Lu. (N.) antunesi, Lu. (N.) whitmani and Lu. (P.) davisi accounted for 66.95 percent of the specimens collected. Lu. (N.) whitmani was the most abundant species, followed by Lu. (N.) antunesi and Lu. (P.) davisi. Lu. (N.) antunesi was the most abundant species in the soil as well as in the canopy. Lu. (N.) umbratilis occurred in all three municipalities and was the fifth most abundant species in the Chico Mendes Municipal Park in Rio Branco. It was collected on both the ground level as well as in the canopy; however, it was more frequently collected in the tree canopy. The present study suggests the existence of three transmission cycles of Leishmania in Acre, including the transmission of Leishmania (V.) guyanensis by Lu. (N.) umbratilis south of the Amazon River.

Effects of a saxitoxin-producer strain of Cylindrospermopsis raciborskii (cyanobacteria) on the swimming movements of cladocerans.

This study evaluated the effects of a saxitoxin-producer strain (T3) of the cyanobacteria species Cylindrospermopsis raciborskii on the swimming movements of three cladoceran species (Daphnia gessneri, D. pulex, and Moina micrura). Acute toxicity bioassays were designed to access the effects of T3 strain, of a nonsaxitoxin producer strain (NPLP-1) of the same species and of a raw water sample from Funil reservoir (Rio de Janeiro, Brazil), that contained this and other cyanobacteria. In the acute bioassays, animals were exposed to C. raciborskii filaments or Funil water for 24-48 h and then transferred to food suspensions without cyanobacterial filaments for a further 48 h. During the exposure time to T3 strain filaments there was a decrease in the number of swimming individuals, with animals showing progressive immobilization. The same effect was observed with Funil water sample. Animals stayed alive on the bottom of the test tube and recovered swimming movements when transferred to food suspensions without toxic cells. This effect was not observed with the strain NPLP-1. The cladoceran D. pulex showed to be extremely sensitive to T3 strain and to Funil water containing C. raciborskii filaments, showing complete paralysis after 24-h exposure to T3 cell densities of 10(3) and 10(4) cells mL(-1), and after 24-h exposure to only 10% of raw water. However, D. gessneri was not sensitive to both T3 and to Funil water, whereas M. micrura was intermediate in sensitivity. This is the first report on the effects of cyanobacterial saxitoxins on movements of freshwater cladocerans, showing also difference in sensitivity among closely related Daphnia species.