In vitro potential cytotoxicity of an adhesive system to alveolar macrophages

The purpose of this study was to evaluate the potential cytotoxicity of Adper Single Bond 2 (SB) simplified etch-and-rinse adhesive system in alveolar macrophage cultures, as a function of the post-polymerization time and duration of immersion in the culture medium for preparation of extracts, by observing the levels of nitric oxide (NO) release and cell survival rate (MTT assay). Wistar rat alveolar macrophages were exposed to 200 μL of extracts obtained from 24- or 72-h immersion of adhesive samples in culture medium (RPMI), immediately or 24 h after polymerization. Fresh RPMI and E. coli lipopolysaccharides were used as negative and positive controls, respectively. The cells were placed in a humidified incubator for 24 h. The results were analyzed by the Student's-t test (α=5 percent). The amount of NO produced and viable cells were significantly different (p<0.05) between the experimental and the control groups, showing that, irrespective of the post-polymerization time and duration of immersion in the culture medium, the adhesive system caused intense cytotoxicity to the macrophages. The cytotoxic effects were not statistically different (p<0.05) among the experimental groups. In conclusion, chemical components released from SB in aqueous environment were highly toxic to cell culture and thus an inflammatory pulpal response should be considered during the clinical application of dental adhesives.

O objetivo deste estudo foi avaliar o potencial de citotoxicidade do sistema adesivo Adper Single Bond 2 (SB), em função dos tempos pós-polimerização e imersão no meio de cultura para preparação dos extratos, observando-se os níveis de liberação de óxido nítrico (NO) e taxa de sobrevivência celular (MTT assay). Macrófagos alveolares de ratos Wistar foram expostos a 200 μL de extratos obtidos a partir da imersão de amostras do adesivo em meio de cultura (RPMI), imediatamente ou 24 h após sua polimerização, onde permaneceram durante 24 ou 72 h. RPMI puro e lipopolissacarídeos de E. coli foram utilizados como controles negativo e positivo, respectivamente. As células foram levadas à incubadora umidificada por 24 h. Os resultados foram submetidos ao teste "t" de Student (α=5 por cento). As quantidades de NO produzido e células sobreviventes foram significativamente diferentes entre os grupos experimentais e grupos controle, mostrando que, independente do tempo pós-polimerização e tempo de elaboração dos extratos, o sistema adesivo causou uma intensa citotoxicidade sobre os macrófagos. Os efeitos citotóxicos não foram estatisticamente diferentes entre os grupos experimentais. Componentes químicos do SB liberados em meio aquoso podem ser altamente citotóxicos para as células em cultura e, portanto, uma resposta inflamatória pulpar deve ser considerada durante a aplicação clínica de adesivos dentinários.

Estudo imunohistoquímico do remodelamento pulmonar em camundongos expostos à fumaça de cigarro / Immunohistochemical study of lung remodeling in mice exposed to cigarette smoke

OBJETIVO: Muitos estudos sobre enfisema são realizados com exposição de animais à fumaça de cigarro durante um longo tempo, focando o tipo de célula envolvida no desequilíbrio protease/antiprotease e a degradação da matriz extracelular. A expressão aumentada de metaloproteinases no enfisema está associado com citocinas e evidências sugerem um papel importante da metaloproteinase de matriz-12 (MMP-12). Nosso objetivo foi estudar a detecção de inibidor tissular de metaloproteinase-2 (TIMP-2), fator de necrose tumoral alfa (TNF-α) e interleucina-6 (IL-6) por métodos imunohistoquímicos no pulmão de camundongos. MÉTODOS: Camundongos C57BL/6 machos foram expostos 3 vezes ao dia a fumaça de 3 cigarros por um período de 10, 20, 30 ou 60 dias através de uma câmara de inalação (grupos CS10, CS20, CS30 e CS60, respectivamente). O grupo controle foi exposto às mesmas condições ao ar ambiente. RESULTADOS: Nós observamos um aumento progressivo de macrófagos alveolares no lavado broncoalveolar dos grupos expostos. O diâmetro alveolar médio, um indicador de destruição alveolar, aumentou em todos os grupos expostos quando comparado ao grupo controle. O índice imunohistoquímico (II) para MMP-12 aumentou nos grupos CS10, CS20 e CS30 em paralelo a uma redução do II para TIMP-2 nos grupos CS10, CS20 e CS30. O II para as citocinas TNF-α e IL-6 aumentou em todos os grupos expostos quando comparado ao grupo controle. Enfisema foi observado no grupo CS60, com alterações na densidade de volume de fibras colágenas e elásticas. CONCLUSÕES: Estes achados sugerem que a fumaça de cigarro induz enfisema com uma participação importante do TNF-α e da IL-6 sem a participação de neutrófilos.

OBJECTIVE: Various studies of emphysema involve long-term exposure of animals to cigarette smoke, focusing on the cell type involved in the protease/antiprotease imbalance and on extracellular matrix degradation. In emphysema, increased expression of metalloproteinases is associated with cytokines, and evidence suggests that the matrix metalloproteinase-12 (MMP-12) plays an important role. Our objective was to investigate tissue inhibitor of metalloproteinase-2 (TIMP-2), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) detection by immunohistochemical methods in mouse lung. METHODS: Male C57BL/6 mice were exposed 3 times a day to smoke of 3 cigarettes over a period of 10, 20, 30 or 60 days in an inhalation chamber (groups CS10, CS20, CS30 and CS60, respectively). Controls were exposed to the same conditions in room air. RESULTS: A progressive increase in the number of alveolar macrophages was observed in the bronchoalveolar lavage fluid of the exposed mice. The mean linear intercept, an indicator of alveolar destruction, was greater in all exposed groups when compared to control group. In the CS10, CS20 and CS30 mice, the immunohistochemical index (II) for MMP-12 increased in parallel with a decrease in II for TIMP-2 in the CS10, CS20 and CS30 mice. The II for the cytokines TNF-α and IL-6 was greater in all exposed groups than in the control group. Emphysema, with changes in volume density of collagen and elastic fibers, was observed in the CS60 group. CONCLUSIONS: These findings suggest that cigarette smoke induces emphysema with major participation of TNF-α and IL-6 without participation of neutrophils.

Lysozyme plays a dual role against the dimorphic fungus Paracoccidioides brasiliensis / A lisozima desempenha um papel duplo contra o fungo dimórfico Paracoccidioides brasiliensis

In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-α-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27 percent more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependant fungal transition (p < 0.0001). When yeast cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subcellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrilar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.

Com a finalidade de determinar o papel da lisozima, um peptídeo antimicrobiano que pertence ao sistema imune inato, contra o fungo dimórfico Paracoccidioides brasiliensis, foram feitas co-culturas de uma linha de macrófagos alveolares murinos (MH-S) com as conídias do fungo na presença ou não do TNF-α e/ou um inibidor da lisozima; também foram feitos ensaios que avaliaram o efeito das concentrações fisiológicas e inflamatórias de lisozima diretamente sobre o ciclo de vida do fungo. Observamos que os macrófagos ativados com a citoquina tiveram um efeito significativo na inibição da transição conídia/levedura (p = 0,0043) e exerceram um efeito fungicida importante (p = 0,0044), matando mais de 27 por cento das propágulas do fungo em comparação com os macrófagos não ativados. No entanto, após ser o inibidor seletivo da lisozima adicionado, o efeito fungicida foi revertido. Quando os propágulos do fungo foram expostos diretamente a diferentes concentrações da lisozima, um duplo efeito foi observado. Assim, as concentrações fisiológicas da enzima facilitaram o processo de transição conídia-levedura (p < 0,05). Contrariamente, as concentrações inflamatórias prejudicaram a transição fúngica (p < 0,0001). Quando as leveduras foram expostas a qualquer concentração de lisozima, sua capacidade de multi-brotação foi gravemente prejudicada (p < 0,0001). Além disso, mudanças ultra-estruturais, como a sub degradação, a fusão dos vacúolos dos lípidos, estruturas lamelares e interrupção da camada fibrilar foram observadas em conídios expostos à lisozima. Estes resultados sugerem que a lisozima poderia exercer um duplo papel no mecanismo antifúngico contra P. brasiliensis.

Lucigenin chemiluminescence assay as an adjunctive tool for assessment of various stages of inflammation: a study of quiescent inflammatory cells.

A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have distinct advantages due to its cost benefits and relative savings in time. Furthermore, a bioassay to differentiate acute and chronic in vivo pulmonary reactions could have potential value as predictors of fibrogenicity and pathogenicity. In this study we examined the potential use of lucigenin as a probe to evaluate the correlation between chemiluminescence (CL) generated by alveolar macrophages with the known cytotoxicity and patho genicity by conventional bioassays. In this study, we used small doses of dust (20 microg) to minimize cellular overload and to maintain homeostasis. Crystalline silica a highly fibrogenic dust was used as positive control and results are compared with those for bentonite, kaolin and talc. Among the three minerals compared with silica, bentonite was more reactive (27%) in CL assay and declined sharply compared to other minerals. This sudden decline in bentonite CL is caused by cytotoxicity leading to cell death. CL-induced by talc was comparable to silica and declines slowly. Kaolin on the other hand produced relatively a weaker (25%) CL compared to silica. Our data using relatively low doses of dust suggest that the CL assay may have a better predictive value in cytotoxicity evaluations compared to conventional toxicity assays.

Cytotoxicity of yellow sand in lung epithelial cells.

The present study was carried out to observe the cytotoxicity of yellow sand in comparison with silica and titanium dioxide in a rat alveolar type II cell line (RLE-6TN). Yellow sand (China Loess) was obtained from the loess layer in the Gunsu Province of China. The mean particle diameter of yellow sand was about 0.003 +/- 0.001 mm. Major elements of yellow sand were Si(27.7 +/- 0.6%), Al(6.01 +/- 0.17%), and Ca(5.83 +/- 0.23%) in that order. Silica and yellow sand significantly decreased cell viability and increased [Ca2+]i. All three particles increased the generation of H2O2. TiO2 did not change Fenton activity, while silica induced a slight increase of Fenton activity. In contrast, yellow sand induced a significant increase of Fenton activity. Silica, yellow sand and TiO2 induced significant nitrite formations in RLE-6TN cells. Silica showed the highest increase in nitrite formation, while yellow sand induced the least formation of nitrite. Silica and yellow sand increased the release of TNF-a. Based on these results, we suggest that yellow sand can induce cytotoxicity in RLE-6TN cells and reactive oxygen species, Fenton activity and reactive nitrogen species might be involved in this toxicity.

Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica.

Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages (AMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 mg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18-24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h after ex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were approximately 20% AMs and approximately 80% PMNs. After density gradient centrifugation of BAL cells from LPS- or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (approximately 60%) or PMNs (approximately 90%). The amounts of NOx produced by the AM-enriched fractions from LPS- or silica-treated rats were approximately 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0-24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.

Time Course Study of Cytokine mRNA Expression in LPS-Stimulated Porcine Alveolar Macrophages

The kinetics of cytokine mRNA expression was studied in porcine alveolar macrophages using an RT-PCR assay. The expression levels of IFN- gamma, IL-2, IL-4, IL-6, GM-CSF, IL-12 p35, and IL-12 p40 were examined after 2, 4, 14, 24, 48, and 72 h of incubation in unstimulated control and LPS-stimulated cells. The expression contents of IFN-gamma, IL-2, and IL-4 were not detected in both unstimulated and LPS-stimulated cells. On the other hand, the expression levels of IL-6, GM-CSF, and IL-12 in LPS-stimulated cells were almost always higher than those in control cells. Among those cytokines, IL-6 exhibited the predominant expression, and GM-CSF, IL-12 p40, and IL-12 p35 followed in the descending order. The times to reach the peak expression levels for IL-6, and GM-CSF, IL-12 p35, and IL-12 p40 were 14, and 24 h, respectively. After reaching the peak expression point, the expression levels of IL-6, GM-CSF, and IL-12 p40 reduced to the baseline by 72 h after stimulation, however, IL-12 p35 still kept a substantial expression by the same time. This study demonstrates that porcine alveolar macrophages primarily respond to express IL-6, GM-CSF, and IL-12 by LPS-stimulation and have a cytokine-specific expression profile during the stimulation time.