Poly I:C elicits broader and stronger humoral and cellular responses to a Plasmodium vivax circumsporozoite protein malaria vaccine than Alhydrogel in mice.

Malaria remains a global health challenge, necessitating the development of effective vaccines. The RTS,S vaccination prevents Plasmodium falciparum (Pf) malaria but is ineffective against Plasmodium vivax (Pv) disease. Herein, we evaluated the murine immunogenicity of a recombinant PvCSP incorporating prevalent polymorphisms, adjuvanted with Alhydrogel or Poly I:C. Both formulations induced prolonged IgG responses, with IgG1 dominance by the Alhydrogel group and high titers of all IgG isotypes by the Poly I:C counterpart. Poly I:C-adjuvanted vaccination increased splenic plasma cells, terminally-differentiated memory cells (MBCs), and precursors relative to the Alhydrogel-combined immunization. Splenic B-cells from Poly I:C-vaccinated mice revealed an antibody-secreting cell- and MBC-differentiating gene expression profile. Biological processes such as antibody folding and secretion were highlighted by the Poly I:C-adjuvanted vaccination. These findings underscore the potential of Poly I:C to strengthen immune responses against Pv malaria.

A low-carbohydrate diet with different fatty acids' sources in the treatment of obesity: Impact on insulin resistance and adipogenesis.

BACKGROUND: The search for nutritional intervention strategies against obesity has grown, highlighting the low-carbohydrate diet model. However, little is known about the impact of the quality of fatty acids consumed in this diet. Thus, we aim to investigate the influence of fatty acid quality on dietary strategy on obesity. METHODS: Male Swiss mice were diet-induced to obesity. Afterward, mice consume a low-carb diet with different types of fat: saturated, polyunsaturated ω-3, ω-6, and monounsaturated ω-9 fatty acids. Weight gain and food consumption were monitored weekly. An oral glucose tolerance test was performed and blood and tissue samples were collected for analysis of insulin resistance markers. Protein expression of insulin signaling pathway molecules, lipid metabolism, mitochondrial function, macrophage polarization, and cytokine production were analyzed. RESULTS: The high-fat diet was able to induce obesity and glucose intolerance. The switch to a low-carbohydrate dietary pattern reversed the glucose intolerance, with better results in the ω-3 and ω-9 groups. After the low-carbohydrate diet, groups ω-3 and ω-9 presented improved fasting serum glucose, insulin, and HOMA indexes. The low-carbohydrate diet also increased the activity of insulin pathway proteins such as IR, IRS1, and AKT. Furthermore, the ω-3 diet group showed greater activity of mitochondrial complexes and AMPK signaling pathway proteins. The ω-6 and ω-9 -rich diet induced M2-type macrophage polarization, as well as cytokine production modulation by the low-carbohydrate diet in the ω-3 and ω-9 groups. CONCLUSIONS: Consuming a low-carbohydrate diet pattern promotes weight loss and improves glucose intolerance in obesity. Also, the quality of lipids has a direct influence, demonstrating that the consumption of ω-3 polyunsaturated and ω-9 monounsaturated lipids can lead to more favorable outcomes for the improvement of glucose intolerance, lipid metabolism, and anti-inflammatory effects.

Adjuvant properties of IFN-γ and GM-CSF in the scFv6.C4 DNA vaccine against CEA-expressing tumors.

Tumor-associated carcinoembryonic antigen (CEA) is a natural target for vaccines against colorectal cancers. Our previous experience with a DNA vaccine with scFv6.C4, a CEA surrogate, showed a CEA-specific immune response with 40% of tumor-free mice after challenge with B16F10-CEA and 47% with MC38-CEA cells. These percentages increased to 63% after using FrC as an adjuvant. To further enhance the vaccine efficacy, we tested GM-CSF and IFNγ as adjuvants. C57BL/6J-CEA2682 mice were immunized 4 times with uP-PS/scFv6.C4, uP-PS/scFv6.C4 + uP-IFNγ, or uP-PS/scFv6.C4 + uP-GMCSF. After one week, the mice were challenged with MC38-CEA, and tumor growth was monitored over 100 days. Immunization with scFv6.C4 and scFv6.C4 + GM-CSF resulted in a gradual increase in the anti-CEA antibody titer, while scFv6.C4 + IFNγ immunization led to a rapid and sustained increase in the titer. The addition of IFNγ also induced higher CD4 + and CD8 + responses. When challenged, almost 80% of the scFv6.C4 + IFNγ-vaccinated mice did not develop tumors, while the others had a significant tumor growth delay. The probability of being tumor-free was 2700% higher using scFv6.C4 + IFNγ than scFv6.C4. The addition of GM-CSF had no additional effect on tumor protection. DNA immunization with scFv6.C4 + IFNγ, but not GM-CSF, increased the antitumor effect via readily sustained specific humoral and cytotoxic responses to CEA.

Drug Repurposing in Chagas Disease: Chloroquine Potentiates Benznidazole Activity against Trypanosoma cruzi In Vitro and In Vivo.

Drug combinations and drug repurposing have emerged as promising strategies to develop novel treatments for infectious diseases, including Chagas disease. In this study, we aimed to investigate whether the repurposed drugs chloroquine (CQ) and colchicine (COL), known to inhibit Trypanosoma cruzi infection in host cells, could boost the anti-T. cruzi effect of the trypanocidal drug benznidazole (BZN), increasing its therapeutic efficacy while reducing the dose needed to eradicate the parasite. The combination of BZN and COL exhibited cytotoxicity to infected cells and low antiparasitic activity. Conversely, a combination of BZN and CQ significantly reduced T. cruzi infection in vitro, with no apparent cytotoxicity. This effect seemed to be consistent across different cell lines and against both the partially BZN-resistant Y and the highly BZN-resistant Colombiana strains. In vivo experiments in an acute murine model showed that the BZN+CQ combination was eight times more effective in reducing T. cruzi infection in the acute phase than BZN monotherapy. In summary, our results demonstrate that the concomitant administration of CQ and BZN potentiates the trypanocidal activity of BZN, leading to a reduction in the dose needed to achieve an effective response. In a translational context, it could represent a higher efficacy of treatment while also mitigating the adverse effects of high doses of BZN. Our study also reinforces the relevance of drug combination and repurposing approaches in the field of Chagas disease drug discovery.

Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection.

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8+ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim+/-, Bim-/- mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim-/- mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim+/- mice. At the peak of parasitemia, peritoneal macrophages of Bim-/- mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim-/- splenocytes. Moreover, an impaired anti-T. cruzi CD8+ T-cell response was found in Bim-/- mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim-/- mice and place Bim as an important protein in the control of T. cruzi infections.

Rapamycin Improves the Response of Effector and Memory CD8+ T Cells Induced by Immunization With ASP2 of Trypanosoma cruzi.

Deficiency in memory formation and increased immunosenescence are pivotal features of Trypanosoma cruzi infection proposed to play a role in parasite persistence and disease development. The vaccination protocol that consists in a prime with plasmid DNA followed by the boost with a deficient recombinant human adenovirus type 5, both carrying the ASP2 gene of T. cruzi, is a powerful strategy to elicit effector memory CD8+ T-cells against this parasite. In virus infections, the inhibition of mTOR, a kinase involved in several biological processes, improves the response of memory CD8+ T-cells. Therefore, our aim was to assess the role of rapamycin, the pharmacological inhibitor of mTOR, in CD8+ T response against T. cruzi induced by heterologous prime-boost vaccine. For this purpose, C57BL/6 or A/Sn mice were immunized and daily treated with rapamycin for 34 days. CD8+ T-cells response was evaluated by immunophenotyping, intracellular staining, ELISpot assay and in vivo cytotoxicity. In comparison with vehicle-injection, rapamycin administration during immunization enhanced the frequency of ASP2-specific CD8+ T-cells and the percentage of the polyfunctional population, which degranulated (CD107a+) and secreted both interferon gamma (IFNγ) and tumor necrosis factor (TNF). The beneficial effects were long-lasting and could be detected 95 days after priming. Moreover, the effects were detected in mice immunized with ten-fold lower doses of plasmid/adenovirus. Additionally, the highly susceptible to T. cruzi infection A/Sn mice, when immunized with low vaccine doses, treated with rapamycin, and challenged with trypomastigote forms of the Y strain showed a survival rate of 100%, compared with 42% in vehicle-injected group. Trying to shed light on the biological mechanisms involved in these beneficial effects on CD8+ T-cells by mTOR inhibition after immunization, we showed that in vivo proliferation was higher after rapamycin treatment compared with vehicle-injected group. Taken together, our data provide a new approach to vaccine development against intracellular parasites, placing the mTOR inhibitor rapamycin as an adjuvant to improve effective CD8+ T-cell response.

Correction: CXCR3 chemokine receptor contributes to specific CD8+ T cell activation by pDC during infection with intracellular pathogens.

[This corrects the article DOI: 10.1371/journal.pntd.0008414.].

STING Signaling Drives Production of Innate Cytokines, Generation of CD8+ T Cells and Enhanced Protection Against Trypanosoma cruzi Infection.

A variety of signaling pathways are involved in the induction of innate cytokines and CD8+ T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-ß production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-ß, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-ß, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-ß, IL-12, CXCL9, IFN-γ, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-γ, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-ß and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-γ and IFN-γ/perforin-producing CD8+ T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.

CXCR3 chemokine receptor contributes to specific CD8+ T cell activation by pDC during infection with intracellular pathogens.

Chemokine receptor type 3 (CXCR3) plays an important role in CD8+ T cells migration during intracellular infections, such as Trypanosoma cruzi. In addition to chemotaxis, CXCR3 receptor has been described as important to the interaction between antigen-presenting cells and effector cells. We hypothesized that CXCR3 is fundamental to T. cruzi-specific CD8+ T cell activation, migration and effector function. Anti-CXCR3 neutralizing antibody administration to acutely T. cruzi-infected mice decreased the number of specific CD8+ T cells in the spleen, and those cells had impaired in activation and cytokine production but unaltered proliferative response. In addition, anti-CXCR3-treated mice showed decreased frequency of CD8+ T cells in the heart and numbers of plasmacytoid dendritic cells in spleen and lymph node. As CD8+ T cells interacted with plasmacytoid dendritic cells during infection by T. cruzi, we suggest that anti-CXCR3 treatment lowers the quantity of plasmacytoid dendritic cells, which may contribute to impair the prime of CD8+ T cells. Understanding which molecules and mechanisms guide CD8+ T cell activation and migration might be a key to vaccine development against Chagas disease as those cells play an important role in T. cruzi infection control.

Obesity-related inflammatory modulation by juçara berry (Euterpe edulis Mart.) supplementation in Brazilian adults: a double-blind randomized controlled trial.

PURPOSE: Obesity is an inflammatory-related disease, which recruits immune system cells triggering to imbalanced production of cytokines. Obesity management and treatment using foods bioactive compounds have gained clinical and scientific relevance. Juçara (Euterpe edulis Mart.) fruit is rich in fibers, unsaturated lipids and, anthocyanins showing potential health benefits. Thus, we investigated the effect of juçara pulp intake on inflammatory status of monocytes from obese individuals. METHODS: It is a placebo-controlled, randomized double-blind trial. Twenty-seven obese participants (BMI between 30.0 and 39.9 kg/m2) of both genders from 31 to 59-year-old, divided into two groups: 5 g juçara freeze-dried pulp or 5 g of placebo for 6 weeks. Before and after supplementation, blood samples were collected and monocytes obtained and stimulated with lipopolysaccharides. After 24 h of incubation, the cells and supernatants were analyzed. RESULTS: Post-treatment, juçara reduced TLR4, and IL-6 mRNA compared to placebo. Juçara also increased IL-10 mRNA in post-treatment. The protein expression of TLR4 pathway post-treatment, MYD88 expression reduced in juçara group compared to placebo. The juçara post-treatment reduced pIKKα/ß compared to the placebo. Ob-R protein levels were higher in the juçara group post-treatment compared to pre-treatment. IL-6, TNF-α, and MCP-1 production by monocytes were reduced by juçara in post-treatment compared to pre-treatment levels. The supplementation increased IL-10 in juçara group with LPS compared to pre-treatment and versus juçara group without LPS. CONCLUSION: These results demonstrated a proinflammatory state at the beginning, which was improved by juçara pulp consumption. Our results suggest juçara pulp as a potential tool against the proinflammatory status of obesity.

CXCR3 chemokine receptor guides Trypanosoma cruzi-specific T-cells triggered by DNA/adenovirus ASP2 vaccine to heart tissue after challenge.

CD8+ T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8+ T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi, the causative agent of Chagas disease. After T. cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8+ T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8+ T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8+ T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T. cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2KK-restricted TEWETGQI-specific CD8+ T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8+ T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T. cruzi-specific effector CD8+ T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies.

scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor.

The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

Therapeutical effects of vaccine from Trypanosoma cruzi amastigote surface protein 2 by simultaneous inoculation with live parasites.

The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 - Control Group (CTRL); Group 2 - Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 - Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 8 plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 8 pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection.

Therapeutic effects of vaccine derived from amastigote surface protein-2 (ASP-2) against Chagas disease in mouse liver.

This study investigated the efficacy of the vaccine in liver of mice infected with the Trypanosoma cruzi (T. cruzi) and immunized with AdASP-2. For this purpose, histopathological analysis and gene expression of COX-2, TNF-alpha, TNFR, iNOS, cytochrome C, caspase-3, TLR4, IL-6 and IL10 were evaluated. The following groups were used in this study: Group 1 - Control Group (CTRL) animals received AdßGal vehicle; Group 2 - Infected Group (TC) animals were infected with T. cruzi; Group 3 - Immunized Group (AdASP-2): animals were immunized by AdASP-2 vaccine; Group 4 - Immunized and Infected Group (AdASP-2+TC) animals were infected with T. cruzi and immunized by AdSP-2 vaccine. A significant decrease of amastigote nests was noticed in the group of animals that were immunized with AdASP-2 and infected on the same day. COX-2 and TNF-alpha gene expressions increased in TC group, whereas TNF-alpha decreased in the TC+AdASP-2 group. TNFR expression was high in AdASP-2+TC group. iNOS expression was high for all experimental groups whereas cytochrome C decreased for all experimental groups. Caspase 3 increased in TC and TC+AdASP-2 groups. The gene expression of TLR4 and IL-10 showed an increase in AdASP-2+TC group. Finally, hepatic fibrosis was noticed to TC and AdASP-2 + TC groups. Taken together, our results demonstrated that vaccination with AdASP-2 was effective against the acute phase of experimental Chagas disease as a result of a more powerful and rapid immune response closely related to expression of some inflammatory genes, such as iNOS, TNF-alpha, TLR 4, and IL-10.

Supplementation of Juçara Berry (Euterpe edulis Mart.) Modulates Epigenetic Markers in Monocytes from Obese Adults: A Double-Blind Randomized Trial.

Nutrigenomics is an emerging field in obesity since epigenetic markers can be modified by environmental factors including diet. Considering juçara composition-rich in anthocyanins, monounsaturated fatty acids (MUFAs) and fibers-it has the potential for epigenetic modulation. We evaluated the juçara supplementation modulating the serum fatty acids profile and epigenetic markers in monocytes of adult obese humans. It was a randomized double-blind, controlled trial with 27 obese (Body mass index between 30.0 and 39.9 kg/m²) participants of both genders aged from 31 to 59 years, divided into juçara group (5 g juçara freeze-dried pulp) or placebo group (5 g of maltodextrin) for 6 weeks. Before and after supplementation, blood samples were collected. The serum and monocytes cells obtained were cultured and stimulated with lipopolysaccharides as proinflammatory stimulus. After 24 h of incubation, the cells and supernatants were collected and analyzed. Juçara improved the serum fatty acids profile on unsaturated fatty acids levels. The epigenetic markers evaluated were improved post-treatment. Also, the methylated DNA level was increased after treatment. We find that juçara supplementation is a predictor of methyl CpG binding proteins 2 (MeCP2) in monocytes. Concluding, juçara supplementation improved the serum fatty acids profile, modulating the epigenetic markers in monocytes from obese individuals.

Qualidade higienicossanitária de hortaliças cruas servidas em restaurante institucional da Baixada Santista, SP / Hygienicosanitary quality of raw vegetables served in institutional restaurant of Baixada Santista, SP

A qualidade higienicossanitária de verduras consumidas cruas constitui fator importante para a prevenção das doenças de origem alimentar. O objetivo dessa pesquisa foi analisar a qualidade higienicossanitária de hortaliças cruas servidas em um restaurante da Baixada Santista. Para isso, foram realizadas 30 coletas de amostras, num período de 4 meses e analisada a qualidade microbiológica e a presença de parasitas. Para avaliação de Boas Práticas em serviço de alimentação, foi aplicado um checklist e realizado o treinamento de colaboradores. Nas análises microbiológicas constatou-se que 53,3% das amostras foram consideradas impróprias para consumo para o parâmetro de coliformes a 35ºC e termotolerantes e também foi constatada ausência total de Salmonella segundo a RDC nº 12 de 02/01/2001. Quanto às análises parasitológicas, 10% das amostras apresentaram resultados positivos. A partir da aplicação do checklist, os dados coletados demonstraram falta de qualidade sanitária no preparo das saladas, inadequação quanto à higiene pessoal e ambiental, e inadequação das edificações e instalações em 51,83% do checklist aplicado. O treinamento em Boas Práticas de Manipulação deve ser executado periodicamente com todos os colaboradores. Conclui-se que a falta de qualidade higienicossanitária no preparo das saladas e a inadequação das boas práticas observadas, podem comprometer a qualidade da refeição e a saúde do consumidor.

The hygienic-sanitary quality of ready-to-eat foods is important to the prevention of food-borne diseases. The objective of this research was to evaluate the hygienic-sanitary quality of raw vegetables served at Baixada Santista. For this, 30 samples of raw salads were sampled, in a period of four months and were submitted to microbiological and parasitologycal analysis. For the evaluation of Good Practices in food service, a checklist was applied and training of handlers. In the microbiological analysis, 53,3% of the samples were considered improper for consumption for the coliform parameter at 35ºC and thermotolerant and was also found absence of Salmonella according to RDC nº12 of 01/02/2001. Regarding the parasitological analysis, ten percent of samples had parasites. From the application of the checklist, the data show lack of sanitary quality without preparation of salads, inadequate personal and environmental hygiene, and inadequate buildings and facilities in 51,83% of the checklists applied. Training in Good Handling Practices should be performed periodically with all handlers. It is concluded that the lack of hygienic-sanitary quality without preparation of the salads and an inadequacy of the observed good practices can compromise a quality of the meal and a health of the consumer.

TLR3 Is a Negative Regulator of Immune Responses Against Paracoccidioides brasiliensis.

Toll-like receptors (TLRs) comprise the best-characterized pattern-recognition receptor (PRR) family able to activate distinct immune responses depending on the receptor/adaptor set assembled. TLRs, such as TLR2, TLR4 and TLR9, and their signaling were shown to be important in Paracoccidioides brasiliensis infections. However, the role of the endosomal TLR3 in experimental paracoccidioidomycosys remains obscure. In vitro assays, macrophages of the bone marrow of WT or TLR3-/- mice were differentiated for evaluation of their microbicidal activity. In vivo assays, WT or TLR3-/- mice were infected intratracheally with Paracoccidioides brasiliensis yeasts for investigation of the lung response type induced. The cytotoxic activity of CD8+ T cells was assessed by cytotoxicity assay. To confirm the importance of CD8+ T cells in the control of infection in the absence of tlr3, a depletion assay of these cells was performed. Here, we show for the first time that TLR3 modulate the infection against Paracoccidioides brasiliensis by dampening pro-inflammatory response, NO production, IFN+CD8+T, and IL-17+CD8+T cell activation and cytotoxic function, associated with granzyme B and perforin down regulation. As conclusion, we suggest that TLR3 could be used as an escape mechanism of the fungus in an experimental paracoccidioidomycosis.