Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS(-/-)) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS(-/-) mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS(-/-) mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS(-/-) mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS(-/-) mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-gamma and TNF-alpha) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis. (c) 2008 Elsevier Masson SAS. All rights reserved.

Heat-killed Enterococcus faecalis Alters Nitric Oxide and CXCL12 Production but not CXCL8 and CCL3 Production by Cultured Human Dental Pulp Fibroblasts

Introduction: Fibroblasts are the most abundant cells in dental pulp. To investigate their capacity to produce the chemokines CCL3, CXCL8, and CXCL12 as well as nitric oxide (NO), we evaluated the production of these mediators in supernatants of cultured human dental pulp fibroblasts (HDPF) stimulated by heat-killed Enterococcus faecalis (HKEF). Methods: Primary cultures of HDPF were stimulated with medium alone or HKEF (1:1, 10:1, or 100:1 bacteria:fibroblast) for 1, 6, and 24 hours. Chemokines and NO were assessed through enzyme-linked immunosorbent assay and Griess reaction, respectively. Statistical analysis was performed by using analysis of variance and Tukey post test. Results: CCL3 was not detected, whereas constitutive CXCL8 was not affected. Production of CXCL12 was increased at 1 and 6 hours, and NO was increased at the concentration of 1:1 bacteria:fibroblast at 24 hours. Viability and proliferation assays did not reveal cell number differences. Conclusions: These findings demonstrate that heat-killed E. faecalis is able to increase production of CXCL12 and NO by HDPF. (J Endod 2010;36:91-94)

Influence of curing rate on softening in ethanol, degree of conversion, and wear of resin composite

Purpose: To investigate the effect of curing rate on softening in ethanol, degree of conversion, and wear of resin composites. Methods: With a given energy density and for each of two different light-curing units (QTH or LED), the curing rate was reduced by modulating the curing mode. Thus, the irradiation of resin composite specimens (Filtek Z250, Tetric Ceram, Esthet-X) was performed in a continuous curing mode and in a pulse-delay curing mode. Wallace hardness was used to determine the softening of resin composite after storage in ethanol. Degree of conversion was determined by infrared spectroscopy (FTIR). Wear was assessed by a three-body test. Data were submitted to Levene`s test, one and three-way ANOVA, and Tukey HSD test (alpha= 0.05). Results: Immersion in ethanol, curing mode, and material all had significant effects on Wallace hardness. After ethanol storage, resin composites exposed to the pulse-delay curing mode were softer than resin composites exposed to continuous cure (P< 0.0001). Tetric Ceram was the softest material followed by Esthet-X and Filtek Z250 (P< 0.001). Only the restorative material had a significant effect on degree of conversion (P< 0.001): Esthet-X had the lowest degree of conversion followed by Filtek Z250 and Tetric Ceram. Curing mode (P= 0.007) and material (P< 0.001) had significant effect on wear. Higher wear resulted from the pulse-delay curing mode when compared to continuous curing, and Filtek Z250 showed the lowest wear followed by Esthet-X and Tetric Ceram. (Am J Dent 2011;24:115-118).

Differential Production of Macrophage Inflammatory Protein-1 alpha, Stromal-Derived Factor-1, and IL-6 by Human Cultured Periodontal Ligament and Gingival Fibroblasts Challenged With Lipopolysaccharide From P. gingivalis

Background: Fibroblasts are considered important cells in periodontitis. When challenged by different agents, they respond through the release of cytokines that participate in the inflammatory process. The aim of this study is to evaluate and compare the expression and production of macrophage inflammatory protein (MIP)-1 alpha, stromal-derived factor (SDF)-1, and interleukin (IL)-6 by human cultured periodontal ligament and gingival fibroblasts challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Methods: Fibroblasts were cultured from biopsies of gingival tissue and periodontal ligament of the same donors and used on the fourth passage. After confluence in 24-well plates, the culture medium alone (control) or with 0.1 to 10 mu g/ml of LPS from P. gingivalis was added to the wells, and after 1, 6, and 24 hours, the supernatant and the cells were collected and analyzed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results: MIP-1 alpha, SDF-1, and IL-6 protein production was significantly greater in gingival fibroblasts compared to periodontal ligament fibroblasts. IL-6 was upregulated in a time-dependent manner, mainly in gingival fibroblasts (P<0.05), which secreted more MIP-1 alpha in the lowest concentration of LPS used (0.1 mu g/ml). In contrast, a basal production of SDF-1 that was inhibited with the increase of LPS concentration was detected, especially after 24 hours (P<0.05). Conclusion: The distinct ability of the gingival and periodontal ligament fibroblasts to secrete MIP-1 alpha, SDF-1, and IL-6 emphasizes that these cells may differently contribute to the balance of cytokines in the LPS-challenged periodontium. J Periodontol 2010;81:310-317.

Chronic ethanol intake inhibits in vitro osteogenesis induced by osteoblasts differentiated from stem cells

The study investigated whether chronic ethanol (ETH) intake and subsequent ETH exposure of cell cultures affects osteoblast differentiation by evaluating key parameters of in vitro osteogenesis. Rats were treated with 5-20% (0.85-3.43 mM) ETH, increasing by 5% per week for a period of 4 weeks (habituation), after which the 20% level was maintained for 15 days (chronic intake). Bone-marrow stem cells from control (CONT) or ETH-treated rats were cultured in osteogenic medium which was either supplemented (ETH) or not supplemented (CONT) with 1.3 mm ethanol. Thus, four groups relating to rat treatment/culture supplementation were evaluated: (1) CONT/CONT, (2) ETH/CONT, (3) CONT/ETH and (4) ETH/ETH Cell morphology, proliferation and viability, total protein content, alkaline phosphatase (ALP) activity and bone-like nodule formation were evaluated. Chronic ethanol intake significantly reduced both food and liquid consumption and body weight gain. No difference was seen in cell morphology among treatments. Cell number was affected at 7 and 10 days as follows: CONT/CONT = CONT/ETH < ETH/CONT = ETH/ETH. Doubling time between 3 and 10 days was greater in groups of CONT animals: ETH/ETH = ETH/CONT < CONT/ETH = CONT/CONT. Cell viability and ALP activity were not affected by either animal treatment or culture exposure to ethanol. At day 21, the total protein content was affected as follows: ETH/ETH = CONT/ETH < ETH/CONT = CONT/CONT. Bone-like nodule formation was affected as follows: ETH/ETH < CONT/ETH < ETH/CONT < CONT/CONT. These results show that chronic ethanol intake, followed by the exposure of osteoblasts to ethanol, inhibited the differentiation of osteoblasts, as indicated by an increased proliferation rate and reduced bone-like nodule formation. Copyright (C) 2007 John Wiley & Sons, Ltd.

Blocking systemic nitric oxide production alters neuronal activation in brain structures involved in cardiovascular regulation during polymicrobial sepsis

In a previous study, we concluded that overproduction of nitric oxide (NO) by inducible nitric Oxide synthase (iNOS) in the late phase of sepsis prevents hypothalamic activation, blunts vasopressin secretion and contributes to hypotension, irreversible shock and death. The aim of this follow-up study was to evaluate if the same neuronal activation pattern happens in brain structures related to cardiovascular functions. Male Wistar rats received intraperitoneal injections of aminoguanidine, an iNOS inhibitor, or saline 30 min before cecal ligation and puncture (CLP) or sham surgeries. The animals were perfused 6 or 24 h after the surgeries and the brains were removed and processed for Fos immunocytochemistry We observed an increase (P < 0.001) in c-fos expression 6 h after CLP in the area postrema (AP), nucleus of he tractus solitarius (NTS), ventral lateral medulla (VLM), locus coeruleus (LC) and parabrachial nucleus (PB). At 24 h after CLP, however, c-fos expression was strongly decreased in all these nuclei (P < 0.05), except for the VLM. Aminoguanidine reduced c-fos expression in the AP and NTS at 6 h after CLR but showed an opposite effect at 24 h, with an increase in the AP, NTS, and also in the VLM. No such effect was observed in the LC and PB at 6 or 24 h. In all control animals, c-fos expression was minimal or absent. We conclude that in the early phase of sepsis iNOS-derived NO may be partially responsible for the activation of brain structures related to cardiovascular regulation. During the late phase, however, this activation is reduced or abolished. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Effects of Ethanol on the Surface and Bulk Properties of a Microwave-Processed PMMA Denture Base Resin

Purpose: This study evaluated the effect of different concentrations of ethanol on hardness, roughness, flexural strength, and color stability of a denture base material using a microwave-processed acrylic resin as a model system. Materials and Methods: Sixty circular (14 x 4 mm) and 60 rectangular microwave-polymerized acrylic resin specimens (65 x 10 x 3 mm(3)) were employed in this study. The sample was divided into six groups according to the ethanol concentrations used in the immersion solution, as follows: 0% (water), 4.5%, 10%, 19%, 42%, and 100%. The specimens remained immersed for 30 days at 37 degrees C. The hardness test was performed by a hardness tester equipped with a Vickers diamond penetrator, and a surface roughness tester was used to measure the surface roughness of the specimens. Flexural strength testing was carried out on a universal testing machine. Color alterations (Delta E) were measured by a portable spectrophotometer after 12 and 30 days. Variables were analyzed by ANOVA/Tukey`s test (alpha = 0.05). Results: For the range of ethanol-water solutions for immersion (water only, 4.5%, 10%, 19.5%, 42%, and 100%), the following results were obtained for hardness (13.9 +/- 2.0, 12.1 +/- 0.7, 12.9 +/- 0.9, 11.2 +/- 1.5, 5.7 +/- 0.3, 2.7 +/- 0.5 VHN), roughness (0.13 +/- 0.01, 0.15 +/- 0.07, 0.13 +/- 0.05, 0.13 +/- 0.02, 0.23 +/- 0.05, 0.41 +/- 0.19 mu m), flexural strength (90 +/- 12, 103 +/- 18, 107 +/- 16, 90 +/- 25, 86 +/- 22, 8 +/- 2 MPa), and color (0.8 +/- 0.6, 0.8 +/- 0.3, 0.7 +/- 0.4, 0.9 +/- 0.3, 1.3 +/- 0.3, 3.9 +/- 1.5 Delta E) after 30 days. Conclusions: The findings of this study showed that the ethanol concentrations of tested drinks affect the physical properties of the investigated acrylic resin. An obvious plasticizing effect was found, which could lead to a lower in vivo durability associated with alcohol consumption.