Camila Peres-Buzalaf

Prostaglandin E-2 and the Suppression of Phagocyte Innate Immune Responses in Different Organs

The local and systemic production of prostaglandin E2 (PGE2) and its actions in phagocytes lead to immunosuppressive conditions. PGE2 is produced at high levels during inflammation, and its suppressive effects are caused by the ligation of the E prostanoid receptors EP2 and EP4, which results in the production of cyclic AMP. However, PGE2 also exhibits immunostimulatory properties due to binding to EP3, which results in decreased cAMP levels. The various guanine nucleotide-binding proteins (G proteins) that are coupled to the different EP receptors account for the pleiotropic roles of PGE2 in different disease states. Here, we discuss the production of PGE2 and the actions of this prostanoid in phagocytes from different tissues, the relative contribution of PGE2 to the modulation of innate immune responses, and the novel therapeutic opportunities that can be used to control inflammatory responses.

Proteomic Analysis of Liver in Rats Chronically Exposed to Fluoride

Fluoride (F) is a potent anti-cariogenic element, but when ingestion is excessive, systemic toxicity may be observed. This can occur as acute or chronic responses, depending on both the amount of F and the time of exposure. The present study identified the profile of protein expression possibly associated with F-induced chronic hepatotoxicity. Weanling male Wistar rats (three-weeks old) were divided into three groups and treated with drinking water containing 0, 5 or 50 mg/L F for 60 days (n=6/group). At this time point, serum and livers were collected for F analysis, which was done using the ion-sensitive electrode, after hexamethyldisiloxane-facilitated diffusion. Livers were also submitted to histological and proteomic analyses (2D-PAGE followed by LC-MS/MS). Western blotting was done for confirmation of the proteomic data A dose-response was observed in serum F levels. In the livers, F levels were significantly increased in the 50 mg/L F group compared to groups treated with 0 and 5 mg/L F. Liver morphometric analysis did not reveal alterations in the cellular structures and lipid droplets were present in all groups. Proteomic quantitative intensity analysis detected 33, 44, and 29 spots differentially expressed in the comparisons between control vs. 5 mg/L F, control vs. 50 mg/L F, and 5 mg/L vs. 50 mg/L F, respectively. From these, 92 proteins were successfully identified. In addition, 18, 1, and 5 protein spots were shown to be exclusive in control, 5, and 50 mg/L F, respectively. Most of proteins were related to metabolic process and pronounced alterations were seen for the high-F level group. In F-treated rats, changes in the apolipoprotein E (ApoE) and GRP-78 expression may account for the F-induced toxicity in the liver. This can contribute to understanding the molecular mechanisms underlying hepatoxicity induced by F, by indicating key-proteins that should be better addressed in future studies.

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

Background Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-γ production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-β and IL-10 production, which could be associated with long-term protection. Conclusions/Significance We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections.

Low intensity lasers differently induce primary human osteoblast proliferation and differentiation.

Among various compounds used in research and clinic for degenerative bone diseases, low level laser therapy (LLLT), comprising low level lasers (LLL) and light emitting diodes (LEDs), has been investigated regarding its effects on bone metabolism. They have specific wavelengths but in general act as a cellular biomodulator, and as a therapeutic agent, rebalancing and normalizing their activity. However, they are not standardized yet, since their parameters of use are relevant for the effects and mechanisms of action. Therefore, the aim of this study was to compare the influence of two spectrums of LLL and LED phototherapy, at the same energy densities (10 and 50J/cm(2)), on human osteoblasts proliferation and differentiation. The involvement of ERK signaling on proliferation was also investigated by evaluating its activation during proliferation under different phototherapies by western blotting and CFSE-based osteoblast proliferation was measured in a presence or absence of the ERK-specific inhibitor. Osteogenic differentiation was evaluated through in vitro mineralization and gene expression of type I collagen (COL1A1) and osteonectin (SPARC) by Real Time- PCR. Increases in viable cells and proliferation were obtained after irradiation, regardless of LLLT type. However, only red at 10J/cm(2) and infrared at both doses, but not LED, induced ERK1/2 activation. In the presence of ERK inhibitor, the LLL-induced proliferation was prevented. In addition, while COL1A1 gene expression was upregulated by red laser, SPARC does so by infrared stimulation. However, LED, at both doses, increased both COL1A1 and SPARC expression. All LLLT increased mineralization, dependent on the dose and time. Thus, LLL and LED differently modulated the metabolism of human osteoblasts, increasing proliferation by mechanism dependent or not of ERK signaling activation and osteogenic differentiation markers.

Renal Proteome in Mice with Different Susceptibilities to Fluorosis

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice (susceptible to dental fluorosis, n = 18) and 129P3/J mice (resistant, n = 18) were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies.

Celecoxib improves host defense through prostaglandin inhibition during Histoplasma capsulatum infection.

Prostaglandins act as mediators of inflammation and, similar to cytokines, function as immune modulators during innate and adaptive immune responses. Therefore, using a pharmacological inhibitor, celecoxib, we investigated the role of prostaglandins in host defense against Histoplasma capsulatum infection in C57BL/6 mice. Our results showed that treatment with celecoxib inhibited cyclooxygenase 2, reduced the total fungal burden, and reduced the concentration of PGE2, cytokines, lymphocytes, neutrophils, and mononuclear cells in the bronchoalveolar space and lung parenchyma. In addition, celecoxib treatment increased the synthesis of nitric oxide, IFN-γ, LTB4, and the phagocytic capacity of alveolar macrophages. Moreover, celecoxib treatment increased the survival of mice after infection with a lethal inoculum of H. capsulatum. These results suggest that prostaglandins alter the host immune response and play an important role in the pathogenesis of histoplasmosis. Thus, the inhibition of prostaglandins could be a valuable immunomodulatory strategy and antifungal therapy for histoplasmosis treatment.

Bone Response to Fluoride Exposure Is Influenced by Genetics

Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality. In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group (ratio ≥1.5 or ≤0.5; p<0.05). Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue.

Low-Level Fluoride Exposure Increases Insulin Sensitivity in Experimental Diabetes

The effect of chronic fluoride (F) exposure from the drinking water on parameters related to glucose homeostasis was investigated. Wistar rats were randomly distributed into 2 groups (diabetic [D] and nondiabetic [ND]; n = 54 each). In D, diabetes was induced with streptozotocin. Each group was further divided into 3 subgroups (0, 10, or 50 mgF/L in drinking water). After 22 days of treatment, plasma and liver samples were collected. No alterations in glycemia, insulinemia, KITT, and HOMA2-IR (homeostasis model assessment 2 of insulin resistance) were seen for ND. F-exposure of D rats led to significantly lower insulinemia, without alterations in glycemia (increased %S). Proteomic analysis detected 19, 39, and 16 proteins differentially expressed for the comparisons D0 vs. D10, D0 vs. D50, and D10 vs. D50, respectively. Gene Ontology with the most significant terms in the comparisons D0 vs. D10, D0 vs. D50, and D50 vs. D10 were organic acid metabolic process and carboxylic acid metabolic process, organic acid metabolic process, and cellular ketone metabolic process. Analysis of subnetworks revealed that proteins with fold changes interacted with GLUT4 in comparison D0 vs. D10. Among these proteins, ERj3p was present in D10. Upregulation of this protein in the presence of F might help to explain the higher %S found in these animals. These data suggest that fluoride might enhance glucose homeostasis in diabetes and identify specific biological mechanisms that merit future studies.

Experimental Calcium Silicate-Based Cement with and without Zirconium Oxide Modulates Fibroblasts Viability

The aim of this study was to verify whether the use of zirconium oxide as a radiopacifier of an experimental calcium silicate-based cement (WPCZO) leads to cytotoxicity. Fibroblasts were treated with different concentrations (10 mg/mL, 1 mg/mL, and 0.1 mg/mL) of the cements diluted in Dulbeccos modified Eagles medium (DMEM) for periods of 12, 24, and 48 h. Groups tested were white Portland cement (WPC), white Portland cement with zirconium oxide (WPCZO), and white mineral trioxide aggregate Angelus (MTA). Control group cells were not treated. The cytotoxicity was evaluated through mitochondrial-activity (MTT) and cell-density (crystal violet) assays. All cements showed low cytotoxicity. In general, at the concentration of 10 mg/mL there was an increase in viability of those groups treated with WPC and WPCZO when compared to the control group (p<0.05). A similar profile for the absorbance values was noted among the groups: 10 mg/mL presented an increase in viability compared to the control group. On the other hand, smaller concentrations presented a similar or lower viability compared to the control group, in general. A new dental material composed of calcium silicate-based cement with 20% zirconium oxide as the radiopacifier showed low cytotoxicity as a promising material to be exploited for root-end filling.

Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts

Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm2 or 3.8J/cm2, respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm2 promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.