Population biology and diet of the puffer fish Lagocephalus laevigatus (Tetraodontiformes: Tetraodontidae) in Caraguatatuba Bay, south-eastern Brazil

This study describes the spatio-temporal distribution, population biology, and diet of the puffer fish Lagocephalus laevigatus in Caraguatatuba Bay, south-eastern Brazil. Monthly samples were taken between August 2003 and October 2004 by trawls in two areas, south and north, at depths of 1 to 4 m. The fish were measured and their sex and reproductive stage determined. The abundance of this species was compared between areas and among months, and the items in the diet were identified and quantified. Lagocephalus laevigatus was rare in Caraguatatuba Bay, where only 199 small individuals (4.8 to 15.4 cm) were obtained in the entire study period, suggesting that this species uses the estuary as a nursery. None of the specimens of L. laevigatus captured in Caraguatatuba Bay were sexually mature. Higher densities of L. laevigatus in the bay were recorded in the south area and between October and December 2003, i.e. in the spring, suggesting that spawning may occur from late winter to spring (August through to November). The diet items consumed by L. laevigatus in Caraguatatuba Bay were, as expected from the current literature, crustaceans, mainly amphipods, and fish. However, the most-consumed item was the sea whip Leptogorgia setacea (Cnidaria). This feeding habit may be related to the presence of toxins (tetrodotoxin and saxitoxin) that are frequently found in the skin and viscera of L. laevigatus, which may be sequestered from the sea whip, which possibility still needs to be specifically evaluated.

Emtricitabine-Tenofovir Concentrations and Pre-Exposure Prophylaxis Efficacy in Men Who Have Sex with Men

Drug concentrations associated with protection from HIV-1 acquisition have not been determined. We evaluated drug concentrations among men who have sex with men in a substudy of the iPrEx trial (1). In this randomized placebo-controlled trial, daily oral doses of emtricitabine/tenofovir disoproxil fumarate were used as pre-exposure prophylaxis (PrEP) in men who have sex with men. Drug was detected less frequently in blood plasma and in viable cryopreserved peripheral blood mononuclear cells (PBMCs) in HIV-infected cases at the visit when HIV was first discovered compared with controls at the matched time point of the study (8% versus 44%; P < 0.001) and in the 90 days before that visit (11% versus 51%; P < 0.001). An intracellular concentration of the active form of tenofovir, tenofovir-diphosphate (TFV-DP), of 16 fmol per million PBMCs was associated with a 90% reduction in HIV acquisition relative to the placebo arm. Directly observed dosing in a separate study, the STRAND trial, yielded TFV-DP concentrations that, when analyzed according to the iPrEx model, corresponded to an HIV-1 risk reduction of 76% for two doses per week, 96% for four doses per week, and 99% for seven doses per week. Pro-phylactic benefits were observed over a range of doses and drug concentrations, suggesting ways to optimize PrEP regimens for this population.

Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Background: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in Sao Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naive individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5 x less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Frequency of subtype B and F1 dual infection in HIV-1 positive, Brazilian men who have sex with men

Background: Because various HIV vaccination studies are in progress, it is important to understand how often inter- and intra-subtype co/superinfection occurs in different HIV-infected high-risk groups. This knowledge would aid in the development of future prevention programs. In this cross-sectional study, we report the frequency of subtype B and F1 co-infection in a clinical group of 41 recently HIV-1 infected men who have sex with men (MSM) in Sao Paulo, Brazil. Methodology: Proviral HIV-1 DNA was isolated from subject's peripheral blood polymorphonuclear leukocytes that were obtained at the time of enrollment. Each subject was known to be infected with a subtype B virus as determined in a previous study. A small fragment of the integrase gene (nucleotide 4255-4478 of HXB2) was amplified by nested polymerase chain reaction (PCR) using subclade F1 specific primers. The PCR results were further confirmed by phylogenetic analysis. Viral load (VL) data were extrapolated from the medical records of each patient. Results: For the 41 samples from MSM who were recently infected with subtype B virus, it was possible to detect subclade F1 proviral DNA in five patients, which represents a co-infection rate of 12.2%. In subjects with dual infection, the median VL was 5.3 x 10(4) copies/ML, whereas in MSM that were infected with only subtype B virus the median VL was 3.8 x 10(4) copies/ML (p > 0.8). Conclusions: This study indicated that subtype B and F1 co-infection occurs frequently within the HIV-positive MSM population as suggested by large number of BF1 recombinant viruses reported in Brazil. This finding will help us track the epidemic and provide support for the development of immunization strategies against the HIV.

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an "immune exhaustion'', with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28(-)CD57(+)CD8(+) T cells between the groups. However, the frequency of Tim-3(+)CD8(+) and Tim-3(+)CD4(+) exhausted T cells, but not PD-1(+) T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1(+)CD8(+) T cells were directly associated with T cell immune activation in children. The frequency of Tim-3(+)CD8(+) T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.

Broad and Cross-Clade CD4(+) T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-gamma secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-gamma and TNF-alpha, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background: The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods: Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings: Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0.04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0.06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0.04; Pol p = 0.13; Gag p = 0.89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p. 0.50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4.7 vs 5.1) but the difference was not significant (p = 0.27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0.30). Interpretation: Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.

Expansion of a subset of CD14(high)CD16(neg)CCR2(low/neg) monocytes functionally similar to myeloid-derived suppressor cells during SIV and HIV infection

Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes express the CD14(++)CD16(-) CCR2(+) phenotype and migrate to inflammatory sites by quickly responding to CCL2 signaling. Here, we identified and characterized the expansion of a novel monocyte subset during HIV and SIV infection, which were undistinguishable from classical monocytes, based on CD14 and CD16 expression, but expressed significantly lower surface CCR2. Transcriptome analysis of sorted cells demonstrated that the CCR2(low/neg) cells are a distinct subpopulation and express lower levels of inflammatory cytokines and activation markers than their CCR2(high) counterparts. They exhibited impaired phagocytosis and greatly diminished chemotaxis in response to CCL2 and CCL7. In addition, these monocytes are refractory to SIV infection and suppress CD8(+) T cell proliferation in vitro. These cells express higher levels of STAT3 and NOS2, suggesting a phenotype similar to monocytic myeloid-derived cells, which suppress expansion of CD8(+) T cells in vivo. They may reflect an antiproliferative response against the extreme immune activation observed during HIV and SIV infections. In addition, they may suppress antiviral responses and thus, have a role in AIDS pathogenesis. Antiretroviral therapy in infected macaque and human subjects caused this population to decline, suggesting that this atypical phenotype is linked to viral replication. J. Leukoc. Biol. 91: 803-816; 2012.

Lack of evidence to support the association of a single IL28B genotype SNP rs12979860 with the HTLV-1 clinical outcomes and proviral load

Background: The Interleukin 28B (IL28B) rs12979860 polymorphisms was recently reported to be associated with the human T-cell leukemia virus type 1 (HTLV-1) proviral load (PvL) and the development of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Methods: In an attempt to examine this hypothesis, we assessed the association of the rs12979860 genotypes with HTLV-1 PvL levels and clinical status in 112 unrelated Brazilian subjects (81 HTLV-1 asymptomatic carriers, 24 individuals with HAM/TSP and 7 with Adult T cell Leukemia/Lymphoma (ATLL)). Results: All 112 samples were successfully genotyped and their PvLs compared. Neither the homozygote TT nor the heterozygote CT mutations nor the combination genotypes (TT/CT) were associated with a greater PvL. We also observed no significant difference in allele distribution between asymptomatic carriers and patients with HTLV-1 associated HAM/TSP. Conclusions: Our study failed to support the previously reported positive association between the IL28B rs12979860 polymorphisms and an increased risk of developing HAM/TSP in the Brazilian population.

Thymopoiesis and regulatory T cells in healthy children and adolescents

OBJECTIVES: The purpose of this study was to investigate the association between T cell receptor excision circle levels in peripheral blood mononuclear cells and regulatory T cells that co-express CD25 and Foxp3 in healthy children and adolescents of different ages. MATERIALS AND METHODS: The quantification of signal-joint T-cell receptor excision circle levels in the genomic DNA of peripheral blood mononuclear cells was performed using real-time quantitative PCR. The analysis of CD4, CD8, CD25, and Foxp3 expression was performed using flow cytometry. RESULTS: Ninety-five healthy controls (46 females and 49 males) ranging in age from 1 to 18 years were analyzed. The mean T-cell receptor excision circle count in all individuals was 89.095 +/- 36.790 T-cell receptor excision circles per microgram of DNA. There was an inverse correlation between T-cell receptor excision circles counts and age (r = -0.846; p < 0.001) as well as between the proportion of CD4(+)CD25(+)Foxp3(+) T cells and age (r = -0.467; p = 0.04). In addition, we observed a positive correlation between the amount of CD4(+)CD25(+)Foxp3(+) T cells and the amount of T-cell receptor excision circles per microgram of DNA in individuals of all ages (r = -0.529; p = 0.02). CONCLUSIONS: In this study, we observed a decrease in the thymic function with age based on the fact that the level of T-cell receptor excision circles in the peripheral blood positively correlated with the proportion of regulatory T cells in healthy children and adolescents. These findings indicate that although T-cell receptor excision circles and regulatory T cells levels decrease with age, homeostasis of the immune system and relative regulatory T cells population levels are maintained in the peripheral blood.

Estudo histológico e comparativo das papilas linguais dos cervídeos Mazama americana e Mazama gouzoubira por microscopia de luz e eletrônica de varredura

O tipo de alimentação depende do ambiente em que o animal se encontra, sendo este um fator responsável pela alteração da morfologia, como a estratificação e o nível de queratinização da língua, e a funcionalidade da mesma. Dentre as estruturas morfológicas funcionais da língua, as papilas vêm merecendo destaque devido a sua estreita relação com a dieta. Foram utilizadas duas espécies de cervídeos: cinco Mazama gouazoubira e duas Mazama americana, dividindo-se a língua em três partes: ápice, corpo e raiz. Analisou comparativamente a língua de duas espécies por meio de microscopia de luz e eletrônica de varredura. As papilas filiforme, fungiforme e valada apresentaram na língua das duas espécies estudadas, e com a mesma distribuição, mudando apenas a quantidade e formato de papilas filiformes no ápice lingual e a quantidade e disposição das papilas valadas na raiz da língua, fator este que pode ser ligado à dieta dos animais. . Além disto, sua distribuição é semelhante à de outras espécies de herbívoros.

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

Background: Leishmania (Viannia) shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from L. (V.) shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained. Methods: F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 mu g of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated. Results: The F1 fraction induced a high degree of protection associated with an increase in IFN-gamma, a decrease in IL-4, increased cell proliferation and activation of CD8(+)T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4(+) central memory T lymphocytes and activation of both CD4+ and CD8(+) T cells. In addition, F1-immunized groups showed an increase in IgG2a levels. Conclusions: The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.

Faster HIV-1 Disease Progression among Brazilian Individuals Recently Infected with CXCR4-Utilizing Strains

Introduction: Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus's co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression. Methods: A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/mu L. Results: Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4,350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4.350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4.350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype. Conclusions: Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.

Do pets reduce the likelihood of sudden unexplained death in epilepsy?

Purpose: To assess the relationship between the presence of pets in homes of epilepsy patients and the occurrence of sudden unexpected death in epilepsy (SUDEP). Methods: Parents or relatives of SUDEP patients collected over a ten-year period (2000-2009) in a large epilepsy unit were asked if the patient lived together with any domestic pet at the time of death or not. Patients who did not experience SUDEP served as controls. Results and conclusions: Eleven out of the 1092 included patients (1%) experienced SUDEP, all with refractory symptomatic epilepsy, but none of them had pets in their homes at the time of death. In contrast, the frequency of pet-ownership in the control group (n = 1081) was 61%. According to previous studies there are some indications that human health is directly related to companionship with animals in a way that domestic animals prevent illness and facilitate recovery of patients. Companion animals can buffer reactivity against acute stress, diminish stress perception and improve physical health. These factors may reduce cardiac arrhythmias and seizure frequency, factors related to SUDEP. Companion animals may have a positive effect on well-being, thus irnproving epilepsy outcome. (c) 2012 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.