Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.(AU)

Fungal infection control by garlic extracts (Allium sativum L) and modulation of peritoneal macrophages activity in murine model of sporotrichosis / Controle da infecção fúngica pelos extratos de alho (Allium sativum L) e modulação da atividade de macrófagos peritoneais em modelo murino de esporotricose

Abstract Garlic (Allium sativum L.) is grown all over the world as seasoning and medicinal vegetable since 3,000 BC. Allicin is the main component of garlic, being attributed to it the most of its biological activities, such as bactericidal, antifungal and antiviral actions. However, other compounds of garlic present antioxidant, hypocholesterolemic, vasodilator activities, protective action against different types of cancer, and immunomodulatory. Fungal infections are important causes of morbidity and mortality in people mainly in immunosuppressed ones. Sporothrix schenckii, the causing agent of Sporotrichosis (most common subcutaneous mycosis in Latin America), is dimorphic fungus, of saprophytic life in soil or plants, infecting people and animals mainly through skin injuries and bruises. The main of this work was to evaluate the influence of garlic consuming on immune modulation of healthy and infected Swiss mice in induced way by S. schenckii, since these animals functioning of peritoneal macrophages as well as the nitric oxide and cytokines' production (IL-1β, IL-10 and IL-12) and to evaluate the antifungal potential of garlic with S. schenckii through minimum inhibitory concentration test and colony-forming units. The results showed that garlic offers antifungal potential with S. schenckii. The oral taking of garlic extracts influences the releasing of cytokines by macrophages, regular consuming shows anti-inflammatory effect, and its acute use may take to an inflammatory response. Mice that consumed garlic responded more effectively to fight against the infection.

Resumo O alho (Allium sativum L.) é cultivado em todo o mundo como hortaliça condimentar e medicinal desde 3.000 a. C. A alicina é o principal componente do alho, sendo atribuída a ela a maior parte das suas atividades biológicas, dentre elas as ações bactericida, antifúngica e antiviral. Porém, outros compostos do alho apresentam atividade antioxidante, hipocolesterolemiante, vasodilatadora, ação protetora contra diversos tipos de câncer e imunomoduladora. As infecções por fungos são causas importantes de morbidade e mortalidade no homem principalmente em indivíduos imunossuprimidos. O Sporothrix schenckii, agente causal da esporotricose (micose subcutânea mais comum na América Latina), é fungo dimórfico, de vida saprofítica no solo ou em vegetais, infectando homens e os animais principalmente através de lesões e arranhões na pele. O objetivo desse trabalho foi avaliar a influência do consumo de alho na imunomodulação de camundongos Swiss saudáveis e infectados de forma induzida por S. schenckii, a partir do estado funcional dos macrófagos peritoneais desses animais quanto à produção de óxido nítrico e das citocinas (IL-1β, IL-10 e IL-12) e avaliar o potencial antifúngico do alho frente ao S. schenckii por meio de teste de concentração inibitória mínima e unidades formadoras de colônia. Os resultados demonstraram que o alho apresenta potencial antifúngico frente S. schenckii. A administração oral de extratos de alho influencia a liberação de citocinas por macrófagos, o consumo regular apresenta efeito anti-inflamatório, e seu uso agudo pode gerar uma resposta inflamatória. Camundongos que consumiram alho responderam de forma mais efetiva no combate da infecção.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

Modulatory effects of α- linolenic acid on generation of reactive oxygen species in elaidic acid enriched peritoneal macrophages in rats.

Fatty acids are known to influence the ability of macrophages to generate reactive oxygen species (ROS). However the effect of elaidic acid (EA, 18:1 trans fatty acid) on ROS generation is not well studied. Rat peritoneal macrophages were enriched with elaidic acid by incubating the cells with 80 µM EA. The macrophages containing EA generated higher amounts of superoxide anion (O2·-), hydrogen peroxide (H2O2) and nitric oxide (NO˙) by 54, 123 and 237%, respectively as compared to control cells which did not contain EA. To study the competition of other C18 fatty acids with EA macrophages were incubated with EA along with stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and α- linolenic acid (ALA, 18:3). ALA significantly reduced the incorporation of EA into macrophage lipids. This also significantly reduced the generation of O2· -, H2O2, NO˙ by macrophages. Studies were also conducted by feeding rats with diet containing partially hydrogenated vegetable fat (PHVF) as a source for EA and linseed oil (LSO) as a source for ALA. The rats were fed AIN-93 diet containing PHVF with 17% EA and incremental amounts of linseed oil for 10 weeks. The peritoneal macrophages from rats fed partially hydrogenated vegetable fat generated higher levels of O2·-, H2O2, NO˙ by 46, 161 and 76% respectively, when compared to rats fed control diets containing ground nut oil. Macrophages from rats fed PHVF with incremental amounts of LSO produced significantly lower levels ROS in a dose dependent manner. Thus ALA reduces the higher levels of ROS generated by macrophages containing EA.

Sympathetic glial cells and macrophages develop different responses to Trypanosoma cruzi infection or lipopolysaccharide stimulation

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagas disease and the proximity of parasitised glial cells and neurons in damaged myenteric ganglia is a frequent finding. Glial cells have crucial roles in many neuropathological situations and are potential sources of NO. Here, we investigate peripheral glial cell response to Trypanosoma cruzi infection to clarify the role of these cells in the neuronal lesion pathogenesis of Chagas disease. We used primary glial cell cultures from superior cervical ganglion to investigate cell activation and NO production after T. cruzi infection or lipopolysaccharide (LPS) exposure in comparison to peritoneal macrophages. T. cruzi infection was greater in glial cells, despite similar levels of NO production in both cell types. Glial cells responded similarly to T. cruzi and LPS, but were less responsive to LPS than macrophages were. Our observations contribute to the understanding of Chagas disease pathogenesis, as based on the high susceptibility of autonomic glial cells to T. cruzi infection with subsequent NO production. Moreover, our findings will facilitate future research into the immune responses and activation mechanisms of peripheral glial cells, which are important for understanding the paradoxical responses of this cell type in neuronal lesions and neuroprotection.

Comparative study of the effect of LPS on the function of BALB/c and C57BL/6 peritoneal macrophages

Macrophages influence their environment and surrounding immune cells as soon as stimulators affect them. Different sources of macrophages induce different reactions in their neighboring immune cells,which result in non-uniform immunologic outcomes. In this experimental research, we compare the behavior of peritoneal macrophages to lipopolysaccharide [LPS] stimulation from BALB/cmice as an indicator of a type 2 immune response and from C57BL/6 mice as an indicator of a type 1 immune response. In this experimental study, peritoneal macrophages prepared from thioglycolate stimulated BALB/c and C57BL/6 micewere treated with 1microg/ml LPS. At different time points after LPS treatment, nitric oxide [NO], interferon gamma [IFN-gamma]. interleukin 4 [IL-4],transforming growth factor beta[1] [TGF-beta[1]], interleukin 17 [IL-17], and interleukin 10[IL-10] production were measured in the supernatants of all macrophage cultures.Indoleamine 2, 3 dioxygenase [IDO] and phagocytic activitywere analyzed in the different experimental groups. The supernatant effects of LPS-treated macrophages on splenocyte proliferation was assessed by the colorimetric method using a 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide [MTT] reagent. According to cytokine analysis, different mouse strains show different cytokine patterns in response to LPS. C57BL/6 macrophages produced more IL-17, IL-10, and IFN-gamma while BALB/c macrophages produced more TGF-beta[1]., and IL-4. There was no significant difference in IDO activity between strains [p

Investigation into in vitro anti-leishmanial combinations of calcium channel blockers and current anti-leishmanial drugs

The need for drug combinations to treat visceral leishmaniasis (VL) arose because of resistance to antimonials, the toxicity of current treatments and the length of the course of therapy. Calcium channel blockers (CCBs) have shown anti-leishmanial activity; therefore their use in combination with standard drugs could provide new alternatives for the treatment of VL. In this work, in vitro isobolograms of Leishmania (Leishmania) chagasi using promastigotes or intracellular amastigotes were utilised to identify the interactions between five CCBs and the standard drugs pentamidine, amphotericin B and glucantime. The drug interactions were assessed with a fixed ratio isobologram method and the fractional inhibitory concentrations (FICs), sum of FICs (ΣFICs) and the overall mean ΣFIC were calculated for each combination. Graphical isobologram analysis showed that the combination of nimodipine and glucantime was the most promising in amastigotes with an overall mean ΣFIC value of 0.79. Interactions between CCBs and the anti-leishmanial drugs were classified as indifferent according to the overall mean ΣFIC and the isobologram graphic analysis.

In vitro evaluation of cytotoxicity of denture adhesives.

Aim: The aim of the study is to assess and compare the cytotoxicity of commercially available four denture adhesives ex-vivo. Materials and Methods: Four commercially available denture adhesives namely Metrodent powder, Fixon powder, Dentiro powder and Fixon cream were selected. Normal saline was used in control group. To evaluate the cytotoxicity of denture adhesives, macrophages were isolated from peritoneal cavity of Swiss albino mice and cell integrity/cell viability method was done by using trypan blue dye. Results: Viable cells were counted and subjected to statistical analysis. ANOVA, F and 't' test were performed, which showed statistically significant values (P < 0.001). The mean percentage of viable cells was highest in the control group (95%) and lowest in Fixon powder (55.66%), with Dentiro powder the mean percentage of viable cells was 63.66%, with Metrodent powder 67.6% while with Fixon cream it was 69.33%. Conclusion: All tested denture adhesives showed varied degree of cytotoxicity that is statistically significant. The degree of toxicity was more in Fixon powder followed by Dentiro powder and Metrodent powder with least in Fixon cream.

In vitro studies on inhibitory effect of proanthocyanidins in modulation of neutrophils and macrophages.

The role of proanthocyanidins (PC), a novel flavonoid extracted from grape seeds was studied in vitro in the modulation of neutrophil and macrophage function. We attempted to assess the levels of non-enzymatic and enzymatic mediators in the presence or absence of PC in 4-phorbol-12--myristate-13-acetate (PMA)-stimulated neutrophils isolated from humans and rats, E. coli endotoxin-stimulated macrophages and macrophages isolated from E. coli endotoxin-induced experimental periodontitis in rats. Addition of PC at a concentration of 50 µg/ml effectively blocked the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and exhibited a marked inhibition of myeloperoxidase (MPO) and lysosomal enzymes (p<0.001), as compared to PMA-stimulated neutrophils (human and rats) and neutrophils isolated from experimental periodontitis in rats. The levels of ROS, RNS and lysosomal enzymes were found to be elevated (p<0.001) and addition of PC significantly (p<0.001) reduced these levels as compared to those from E. coli endotoxin-stimulatedmacrophages from rats and macrophages isolated from experimental periodontitis in rats (p<0.001). Thus, the study demonstrated that PC decreased the levels of ROS and RNS and also inhibited the MPO and lysosomal enzymes activities in experimental periodontitis in rats. In addition, this study clearly indicated that PC could be developed as an effective antiinflammatory agent.

Chemistry, cytotoxicity and antileishmanial activity of the essential oil from Piper auritum

Leishmaniasis is one of the most important parasitic infections, but current treatments are unsatisfactory due to their toxicity, cost and resistance. Therefore, the development of new antileishmanial compounds is imperative. Many people who live in endemic areas use plants as an alternative to treat the disease. In this paper, we characterised the essential oil from Piper auritum, evaluated its cytotoxicity and determined its antileishmanial activity. The chromatogram obtained by gas chromatography revealed 60 peaks and we found that safrole was the most abundant compound, composing 87 percent of the oil. The oil was active against the promastigotes of Leishmania major, Leishmania mexicana, Leishmania braziliensis and Leishmania donovani with a favourable selectivity index against peritoneal macrophages from BALB/c mice. The Piper-oil inhibited the growing of intracellular amastigotes of L. donovani with an IC50 value of 22.3 ± 1.8 ìg/mL. This study demonstrates the usefulness of the essential oils as a promising alternative to treat leishmaniasis.

Phagocytic responses of peritoneal macrophages and neutrophils are different in rats following prolonged exercise

OBJECTIVE: To analyze the effects of exhausting long-duration physical exercise (swimming) sessions of different durations and intensities on the number and phagocytic capacity of macrophages and neutrophils in sedentary rats. INTRODUCTION: Exercise intensity, duration and frequency are important factors in determining immune response to physical effort. Thus, the effects of exhausting long-duration exercise are unclear. METHODS: Wistar rats were divided into two groups: an untreated group (macrophage study) and oyster glycogen-treated rats (neutrophil study). In each group, the animals were subdivided into five groups (10 rats per group): unexercised controls, an unadapted low-intensity exercise group, an unadapted moderate-intensity exercise group, a preadapted low-intensity exercise group and a preadapted moderate-intensity exercise group. All exercises were performed to exhaustion, and preadaptation consisted of 5, 15, 30 and 45 min sessions. RESULTS: Macrophage study: the number of peritoneal macrophages significantly decreased (9.22 ± 1.78 x 10(6)) after unadapted exercise but increased (21.50 ± 0.63 x 10(6)) after preadapted low-intensity exercise, with no changes in the moderate-intensity exercise group. Phagocytic capacity, however, increased by more than 80 percent in all exercise groups (low/moderate, unadapted/preadapted). Neutrophil study: the number of peritoneal neutrophils significantly decreased after unadapted (29.20 ± 3.34 x 10(6)) and preadapted (50.00 ± 3.53 x 10(6)) low-intensity exercise but increased after unadapted (127.60 ± 5.14 x 10(6)) and preadapted (221.80 ± 14.85 x 10(6)) moderate exercise. Neutrophil phagocytic capacity decreased by 63 percent after unadapted moderate exercise but increased by 90 percent after corresponding preadapted sessions, with no changes in the low-intensity exercise groups. CONCLUSION: Neutrophils and macrophages of sedentary rats respond differently to exercise-induced stress. Adaptation sessions reduce exercise-induced stress on the immune system.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsatPC, respectively), both used at concentrations of 32 and 64 ìM. The treatment of peritoneal macrophages with 64 ìM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 ìM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 ìM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 ìM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 ìM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 ìM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Legionella lipoprotein activates toll-like receptor 2 and induces cytokine production and expression of costimulatory molecules in peritoneal macrophages

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.

Induction of nitric oxide & tumour necrosis factor-alpha by Psoralea corylifolia.

BACKGROUND & OBJECTIVES: Psoralea corylifolia (PC) is an herb widely used in medicine for the treatment of a variety of ailment. PC is also known to have immunomodulatory activity. However, its mechanism of action is not known. In the present study we investigated effect of PC on nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) production in mouse peritoneal macrophages and also examined the mechanism by which PC regulates NO production. METHODS: MTT assay performed for cell viability test and nitrite concentration was measured by using Griess reagent. The amount of TNF-alpha secreted by the cells was measured by a modified enzyme-linked immunosorbent assay (ELISA). Expression of iNOS was investigated by western blot analysis. RESULTS: PC in combination with recombinant interferon-gamma (rIFN-gamma) showed a marked co-operative induction of NO production, with no effect on NO production by itself. The increased production of NO from rIFN-gamma plus PC-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). Furthermore, treatment of peritoneal macrophages with rIFN-gamma plus PC caused a significant increase in tumour necrosis factor-alpha (TNF-alpha) production. PDTC also decreased the effect of PC on TNF-alpha production significantly. INTERPRETATION & CONCLUSION: As NO and TNF-alpha play an important role in immune function and host defense, PC treatment could modulate several aspects of host defense mechanisms due to stimulation of the inducible nitric oxide synthase.

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.

Cytotoxicity evaluation of four endodontic sealers

This study evaluated in vitro the cytotoxicity of four root canal sealers (Topseal, EndoRez, TubliSeal and Kerr Pulp Canal Sealer E.W.T.) and their effects on reactive oxygen/nitrogen intermediate induction by mouse peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 mL 10 mM phosphate-buffered saline, washed twice and resuspended (106 cells/mL) in appropriate medium for each test. Cytotoxicity was determined by the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. Sealer suspensions were obtained in two different concentrations from each material: 18 mg/mL and 9 mg/mL, established according to compatibility parameters following MTT assay. Comparing the sealers, H2O2 release at concentrations of 9 mg/mL and 18 mg/mL was similar: Topseal > positive control (medium + cells + 5 mg/mL zimozan solution) > EndoRez > TubliSeal > Kerr Pulp E.W.T. > negative control (medium + cells). NO release at concentration of 9 mg/mL was: positive control (medium + cells + 10 µg/mL LPS solution) > Topseal > Kerr Pulp E.W.T. > TubliSeal = EndoRez > negative control (medium + cells); at concentration of 18 mg/mL was: positive control > Topseal > Kerr Pulp E.W.T > TubliSeal > EndoRez > negative control. Based on the results, it may be concluded that Topseal presented the highest cytotoxicity among the tested sealers, releasing higher concentrations of NO and H2O2 in macrophage culture.

Este estudo avaliou in vitro a citotoxicidade de quatro cimentos obturadores (Topseal, EndoRez, TubliSeal e Kerr Pulp Canal Sealer E.W.T) e seus efeitos na liberação de reativos intermediários do oxigênio e do nitrogênio em cultura de macrófagos peritoniais de ratos.Tioglicolato foi utlizado para se obter células peritoneias de camundongos. A cavidade peritoneal foi irrigada com 5 mL de solução salina 10 mM. As células foram lavadas duas vezes e foi feita uma suspensão (106 células/mL) em meio apropriado para cada um dos testes. A citotoxicidade dos cimentos foi determinada pela presença de peróxido de hidrogênio (H2O2) e óxido nítrico (NO) pela oxidação peroxidase-dependente do vermelho fenol e pela reação de Griess, respectivamente. Suspensões de cimento foram obtidas em duas diferentes concentrações para cada material: 18 mg/mL e 9 mg/mL, estabelecidas previamente pelo teste de viabilidade celular MTT. Comparando os cimentos, a liberação de H2O2 foi similar nas duas concentrações: Topseal > controle positivo (meio + células + Zimozan a 5mg/mL ) > EndoRez > TubliSeal > Kerr Pulp E.W.T. > controle negativo (meio + células). A liberação de NO na concentração de 9 mg/mL foi: de 9 mg/mL foi: controle positivo (meio + células + solução de LPS a 10 »g/mL) > Topseal > Kerr Pulp E.W.T. > TubliSeal = EndoRez > controle negativo (meio + células); e na concentração de 18 mg/mL; e na concentração de 18 mg/mL: controle positivo > Topseal > Kerr Pulp E.W.T > TubliSeal > EndoRez > controle negativo. Baseado nos resultados, pode-se concluir que o Topseal apresentou a maior citotoxicidade dentre os cimentos avaliados, liberando as mais altas concentrações de NO e H2O2 em cultura de macrófagos.

Cell adhesion by aqueous extract of human placenta used as wound healer.

Aqueous extract of human placenta, used as wound healer, has shown significant cell adhesion property on mouse peritoneal macrophages and P388D1 cultured macrophage cell line. This property was offered primarily by fibronectin type III like peptide present in the extract and is comparable to fibronectin on a molar basis. The peptide induce adhesion of cell through cell surface receptors having K(d) = 2.8 +/- 0.9 x 10(-5) M suggesting weak binding. This is in support of integrins receptors that typically exhibit low affinities. Cell adhesion was partially inhibited by Arg-Gly-Asp (RGD) peptide, anti-beta1 integrin suggesting that integrin beta1 receptors have roles to play in the process.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 æM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56 percent, respectively) in comparison with non-activated or untreated Mthetas (80 percent). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

O ferro é elemento essencial para o crescimento de microrganismos e sua limitação é um dos mecanismos usados por macrófagos para controlar a multiplicação microbiana. Paracoccidioides brasiliensis, o agente da paracoccidioidomicose, uma das micoses sistêmicas mais importantes na América Latina, é inibido em sua conversão de conídia-à-levedura na ausência do ferro. Estudamos a participação do ferro no mecanismo fungicida mediado pelo óxido nítrico (NO) na sua interação com as conídias do fungo. Macrófagos peritoneais murinos ativados com 50U/mL de IFN-gama ou tratados com 35 æM Deferoxamina (DEX) e infectados com conídias do P. brasiliensis foram co-cultivados e incubados por 96 h na presença de concentrações diferentes de holotransferrina (HOLO) e FeS0(4). Os sobrenadantes foram retirados a fim de avaliar a produção de NO2 pelo método de Griess. Os macrófagos eram fixados, corados e observados ao microscópio. A porcentagem da transição de conídia-à-levedura foi estimada contando 200 propágulos intracelulares. Os macrófagos ativados com citocina ou tratados com DEX apresentaram inibição marcada da conversão de conídia-à-levedura (19 e 56 por cento, respectivamente) em comparação com macrófagos controle (80 por cento). Os macrófagos ativados com IFN-gama produziram elevação nos níveis de NO em comparação com macrófagos não-tratados ou não-activados. Adicionalmente, quando as monocapas ativadas ou tratadas foram suplementadas com doadores do ferro (HOLO ou FeSO4), a ação inibitória foi revertida embora a produção de NO permanecesse intacto. Estes resultados sugerem que o mecanismo fungicida mediado pelo NO exercido por macrófagos ativados com IFN-gama contra conídias do P. brasiliensis é dependente de uma interação do ferro.

Semecarpus anacardium L, nuts inhibit lipopolysaccharide induced NO production in rat macrophages along with its hypolipidemic property.

Traditionally S. anacardium is used for rejuvenation, rheumatoid arthritis, fever and neurological disorders. In the present study it was observed that a fraction of S. anacacrdium at dose of 1 mg/100 g body wt, significantly reduced serum cholesterol from 378.87 mg/dl in the rats fed with atherogenic diet (AD) to 197.99 mg/dl (45-52%) in the rats fed with AD diet and increased serum HDL-cholesterol (33-37%). The same fraction also inhibited LPS induced NO production in the culture activated rat peritoneal macrophages in the dose dependent manner with IC50 value at 50 ng/ml of the culture medium. The drug in the above doses was completely safe and non-toxic, (no change in the enzymes), to liver and kidney functions.