Molecular screening of Plasmodium sp. asymptomatic carriers among transfusion centers from Brazilian Amazon region

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The effect of CpG-ODN on antigen presenting cells of the foal

Background: Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. Methods: Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNα, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-κB p65 using a chemiluminescence assay. Results: The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNα (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells. Conclusion: CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNα cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment. © 2007 Flaminio et al; licensee BioMed Central Ltd.

Indirect cytotoxicity of a 35% hydrogen peroxide bleaching gel on cultured odontoblast-like cells

The aim of this study was to evaluate the trans-enamel and trans-dentinal effects of a 35% hydrogen peroxide (H2O2) bleaching gel on odontoblast-like cells. Enamel/dentin discs obtained from bovine incisors were mounted in artificial pulp chambers (APCs). Three groups were formed: G1- 35% H2O2; G2- 35% H2O2 + halogen light application; G3- control. The treatments were repeated 5 times and the APCs were incubated for 12 h. Then, the extract was collected and applied for 24 h on the cells. Cell metabolism, total protein dosage and cell morphology were evaluated. Cell metabolism decreased by 62.09% and 61.83% in G1 and G2, respectively. The depression of cell metabolism was statistically significant when G1 and G2 were compared to G3. Total protein dosage decreased by 93.13% and 91.80% in G1 and G2, respectively. The cells in G1 and G2 exhibited significant morphological alterations after contact with the extracts. Regardless of halogen light application, the extracts caused significantly more intense cytopathic effects compared to the control group. After 5 consecutive applications of a 35% H2O2 bleaching agent, either catalyzed or not by halogen light, products of gel degradation were capable to diffuse through enamel and dentin causing toxic effects to the cells.

Toxic effects of daily applications of 10% carbamide peroxide on odontoblast-like MDPC-23 cells

Background. Tooth bleaching has been widely studied, mainly due to the possible undesirable effects that can be caused by this esthetic procedure. The cytotoxicity of the bleaching agents and its components to pulp cells has been demonstrated in several researches. The aim of this study was to evaluate the toxic effects of successive applications of 10% carbamide peroxide (CP) gel on odontoblast-like cells. Materials and methods. Enamel-dentin discs obtained from bovine incisors were adapted to artificial pulp chambers (APCs). The groups were formed as follows: G1: Without treatment (control group); G2: 10% carbamide peroxide, CP (five applications/one per day); G3: 10% CP (one unique application); and G4: 35% hydrogen peroxide, HP (three applications of 15 min each). After treatment, cell metabolism (MTT), alkaline phosphatase (ALP) activity and plasma membrane damage (flow cytometry) were analyzed. Results. Reductions in cell metabolism and alkaline phosphatase activity along with severe damage of the cytoplasmic membrane were noted in G2. In G3, no damage was observed, compared to the control group. Intermediary values of toxicity were obtained after 35% HP application. Conclusion. It can be concluded that one application of 10% CP did not cause toxic effects in odontoblast-like cells, but the successive application of this product promoted severe cytotoxic effects. The daily application of the bleaching agents, such as used in the at-home bleaching technique, can increase the damages caused by this treatment to the dental pulp cells. © 2013 Informa Healthcare.

HPV16-associated tumors control myeloid cell homeostasis in lymphoid organs, generating a suppressor environment for T cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito de dose efetiva mínima e dose não tóxica de 5-Fluorouracil e Leucovorina sobre a atividade de células dendríticas humanas e sobre a imunogenicidade de células de câncer colorretal

A quimioterapia antitumoral convencional, baseada na aplicação da dose máxima tolerável de drogas antineoplásicas, está comumente associada com mielossupressão e causa diminuição da viabilidade e número de células dendríticas (DCs), principais células apresentadoras de antígenos (APCs) do sistema imune. Uma alternativa para limitar os efeitos tóxicos desses agentes é a quimioterapia metronômica, baseada na administração das drogas em doses baixas e frequentes que, entre outros aspectos, melhora a eficácia antitumoral pela inibição da angiogênese no sítio do tumor. Estudos prévios do grupo indicam que doses ultrabaixas de determinados agentes antineoplásicos modulam positivamente o estado de ativação e as funções das células dendríticas, sugerindo que a associação entre vacinas de DCs e quimioterapia metronômica possibilitaria a estimulação da resposta imune, ao mesmo tempo em que as células tumorais sofrem o efeito dos agentes antineoplásicos em doses ultrabaixas. Assim, hipotetizamos que o uso de 5-Fluorouracil e Leucovorina, drogas de escolha para o tratamento do câncer colorretal, em dose efetiva mínima ou dose ultrabaixa não tóxica, poderia modular positivamente as DCs humanas e potencializar a imunogenicidade de células tumorais, além de torná-las mais sensíveis à ação de linfócitos T citotóxicos. Desse modo, o presente estudo tem o objetivo de avaliar o efeito in vitro da dose efetiva mínima e dose não tóxica de 5-Fluorouracil e Leucovorina sobre as funções de células dendríticas humanas e sobre a imunogenicidade de células de câncer colorretal humano da linhagem HT-29. Com esse propósito as DCs humanas foram sensibilizadas com lisatos de células tumorais pré tratadas com as doses previamente determinadas das drogas e avaliadas quanto à capacidade de apresentação de antígenos e indução de linfócitos T citotóxicos... (Resumo completo, clicar acesso eletrônico abaixo)

Periodontal pathogens directly promote autoimmune experimental arthritis by inducing a TLR2- and IL-1-Driven Th17 response

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cytocompatibility of HEMA-free resin-based luting cements according to application protocols on dentine surfaces

To evaluate the transdentinal cytotoxicity of resin-based luting cements (RBLCs), with no HEMA in their composition, to odontoblast-like cells. Human dentine discs 0.3 mm thick were adapted to artificial pulp chambers (APCs) and placed in wells of 24-well plates containing 1 mL of culture medium (DMEM). Two categories of HEMA-free RBLCs were evaluated: group 1, self-adhesive Rely X Unicem (RU; 3M ESPE), applied directly to the dentine substrate; and group 2, Rely X ARC (RARC; 3M ESPE), applied to dentine previously acid-etched and treated with a bonding agent. In group 3 (control), considered as representing 100% cell metabolic activity, no treatment was performed on dentine. The APC/disc sets were incubated for 24 h or 7 days at 37 °C and 5% CO2 . Then, the extracts (DMEM + dental materials components that diffused through dentine) were applied to cultured odontoblast-like MDPC-23 cells for 24 h. After that, the cell viability (MTT assay), cell morphology (SEM), total protein production (TP) and alkaline phosphatase (ALP) activity were assessed. Data from MTT assay and TP production were analysed by Kruskal-Wallis and Mann-Whitney tests (α = 5%). Data from ALP activity were analysed by one-way anova and Tukey's test (α = 5%). In group 1, a slight reduction in cell viability (11.6% and 16.8% for 24-h and 7-day periods, respectively) and ALP activity (13.5% and 17.9% for 24-h and 7-day periods, respectively) was observed, with no significant difference from group 3 (control) (P > 0.05). In group 2, a significant reduction in cell viability, TP production and ALP activity compared with group 3 (control) occurred (P < 0.05), regardless of incubation time. Alteration in MDPC-23 cell morphology was observed only in group 2. HEMA-free Rely X ARC cement caused greater toxicity to odontoblast-like MDPC-23 cells than did Rely X Unicem cement when both resin-based luting materials were applied to dentine as recommended by the manufacturer.

Transdentinal cytotoxicity of resin-based luting cements to pulp cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific.

Temporal Trends of Asthma Mortality Rates in Brazil from 1980 to 2010

Objective. Mortality from asthma has varied among countries during the last several decades. This study aimed to identify temporal trends of asthma mortality in Brazil from 1980 to 2010. Method. We analyzed 6840 deaths of patients aged 5-34 years that occurred in Brazil with the underlying cause of asthma. We applied a log-linear model using Poisson regression to verify peaks and trends. We also calculated the point estimation and 95% confidence interval (CI 95%) of the annual percent change (APC) of the mortality rates, and the average annual percent change (AAPC) for 2001-2010. Results. A decline was observed from 1980 to 1992 [APC = -3.4 (-5.0 to -1.8)], followed by a nonsignificant rise until 1996 [APC = 6.8 (-1.4 to 15.6)], and a new downward trend from 1997 to 2010 [APC = -2.7 (-3.9 to -1.6)]. The APCs varied according to age strata: 5-14 years from 1980 to 2010 [-0.3 (-1.1 to 0.5)]; 15-24 years from 1980 to 1991 [-2.1 (-5.0 to 0.9)], from 1992 to 1996 [6.8 (-6.7 to 22.2)], and from 1997 to 2010 [-3.9 (-5.7 to -2.0)]; 24-25 years from 1980 to 1992 [-2.5 (-4.6 to -0.3)], from 1993 to 1995 [12.0 (-21.1 to 59.1)], and from 1996-2010 [-1.7 (-3.0 to -0.4)]. AAPC from 2001 to 2010 was -1.7 (-3.0 to -0.4); the decline for this period was significant for patients over 15 years old, women, and those living in the Southeast region. Conclusion. Asthma mortality rates in Brazil have been declining since the late 1990s.

Early skin immunological disturbance after Plasmodium-infected mosquito bites

Although the role of regulatory T cells (Tregs) during malaria infection has been studied extensively, such studies have focused exclusively on the role of Treg during the blood stage of infection; little is known about the detailed mechanisms of Tregs and sporozoite deposition in the dermis by mosquito bites. In this paper we show that sporozoites introduced into the skin by mosquito bites increase the mobility of skin Tregs and dendritic cells (DCs). We also show differences in MHC class II and/or C086 expression on skin-resident dendritic cell subtypes and macrophages. From the observed decrease of the number of APCs into draining lymph nodes, suppression of CD28 expression in conventional CD4 T cells, and a low homeostatic proliferation of skin-migrated CD4 T found in nude mice indicate that Tregs may play a fundamental role during the initial phase of malaria parasite inoculation into the mammalian host. (C) 2012 Elsevier Inc. All rights reserved.

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon Basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Marcatura di molecole biologiche a funzione antigenica per lo studio e la caratterizzazione di protocolli di vaccinoterapia in oncologia medica

Dendritic Cells (DCs) derived from human blood monocytes that have been nurtured in GM-CSF and IL-4, followed by maturation in a monocyte-conditioned medium, are the most potent APCs known. These DCs have many features of primary DCs, including the expression of molecules that enhance antigen capture and selective receptors that guide DCs to and from several sites in the body, where they elicit the T cell mediated immune response. For these features, immature DCs (iDC) loaded with tumor antigen and matured (mDC) with a standard cytokine cocktail, are used for therapeutic vaccination in clinical trials of different cancers. However, the efficacy of DCs in the development of immunocompetence is critically influenced by the type (whole lysate, proteins, peptides, mRNA), the amount and the time of exposure of the tumor antigens used for loading in the presentation phase. The aim of the present study was to create instruments to acquire more information about DC antigen uptake and presentation mechanisms to improve the clinical efficacy of DCbased vaccine. In particular, two different tumor antigen were studied: the monoclonal immunoglobulin (IgG or IgA) produced in Myeloma Multiple, and the whole lysate obtained from melanoma tissues. These proteins were conjugated with fluorescent probe (FITC) to evaluate the kinetic of tumor antigen capturing process and its localization into DCs, by cytofluorimetric and fluorescence microscopy analysis, respectively. iDC pulsed with 100μg of IgG-FITC/106 cells were monitored from 2 to 22 hours after loading. By the cytofluorimetric analysis it was observed that the monoclonal antibody was completely captured after 2 hours from pulsing, and was decreased into mDC in 5 hours after maturation stimulus. To monitor the lysate uptake, iDC were pulsed with 80μg of tumor lysate/106 cells, then were monitored in the 2h to 22 hours interval time after loading. Then, to reveal difference between increasing lysate concentration, iDC were loaded with 20-40-80-100-200-400μg of tumor lysate/106 cells and monitored at 2-4-8-13h from pulsing. By the cytofluorimetric analysis, it was observed that, the 20-40-80-100μg uptake, after 8 hours loading was completed reaching a plateau phase. For 200 and 400μg the mean fluorescence of cells increased until 13h from pulsing. The lysate localization into iDC was evaluated with conventional and confocal fluorescence microscopy analysis. In the 2h to 8h time interval from loading an intensive and diffuse fluorescence was observed within the cytoplasmic compartment. Moreover, after 8h, the lysate fluorescence appeared to be organized in a restricted cloudy-shaded area with a typical polarized aspect. In addition, small fluorescent spots clearly appeared with an increment in the number and fluorescence intensity. The nature of these spot-like formations and cloudy area is now being investigated detecting the colocalization of the fluorescence lysate and specific markers for lysosomes, autophagosomes, endoplasmic reticulum and MHCII positive vesicles.